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Identification of peptides and proteins is a common task in mass spectrometry-based proteomics but often fails to deliver a comprehensive list of identifications. Downstream analysis, quantitative or qualitative, depends on the outcome of this process. Despite continuous improvement of computational methods, a large fraction of the screened peptides and/or proteins remains unidentified. We introduce here pacMASS, a method that de novo predicts the elemental composition of peptides and small proteins based on a single accurate mass, ie, the observed monoisotopic or average mass. This novel approach returns in a fast and memory efficient manner a limited number of elemental compositions per queried peptide or protein., (© 2019 John Wiley & Sons, Ltd.)

Identifying DPP-IV inhibitory peptides from dietary protein has attracted increased attention. In the present study, bovine α-lactalbumin hydrolysates were generated by alcalase for various hydrolysis times, and DPP-IV inhibitory activity of these hydrolysates was determined. The 4 h hydrolysates displayed the most potent DPP-IV inhibitory activity, with DPP-IV inhibition rate of 82.30 ± 1.39% at concentration of 1.0 mg/mL. DPP-IV inhibitory peptides were isolated from the 4 h-hydrolysates with gel filtration chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC). Using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS), two DPP-IV inhibitory peptides were identified, and their amino acid sequences were Glu-Leu-Lys-Asp-Leu-Lys-Gly-Tyr (ELKDLKGY) and Ile-Leu-Asp-Lys-Val-Gly-Ile-Asn-Tyr (ILDKVGINY), respectively. Furthermore, molecular docking analysis showed that peptides ELKDLKGY and ILDKVGINY could form hydrogen bonds, pi-cation interactions, and salt bridges with DPP-IV. These findings indicated that bovine α-lactalbumin may be a potential source of natural DPP-IV inhibitor.

Significant progress has been made in the past few years on the computational identification of biosynthetic gene clusters (BGCs) that encode ribosomally synthesized and post-translationally modified peptides (RiPPs). This is done by identifying both RiPP tailoring enzymes (RTEs) and RiPP precursor peptides (PPs). However, identification of PPs, particularly for novel RiPP classes remains challenging. To address this, machine learning has been used to accurately identify PP sequences. Current machine learning tools have limitations, since they are specific to the RiPPclass they are trained for and are context-dependent, requiring information about the surrounding genetic environment of the putative PP sequences. NeuRiPP overcomes these limitations. It does this by leveraging the rich data set of high-confidence putative PP sequences from existing programs, along with experimentally verified PPs from RiPP databases. NeuRiPP uses neural network archictectures that are suitable for peptide classification with weights trained on PP datasets. It is able to identify known PP sequences, and sequences that are likely PPs. When tested on existing RiPP BGC datasets, NeuRiPP was able to identify PP sequences in significantly more putative RiPP clusters than current tools while maintaining the same HMM hit accuracy. Finally, NeuRiPP was able to successfully identify PP sequences from novel RiPP classes that were recently characterized experimentally, highlighting its utility in complementing existing bioinformatics tools.

Tandem mass spectrometry has become the method of choice for high-throughput, quantitative analysis in proteomics. Peptide spectrum matching algorithms score the concordance between the experimental and the theoretical spectra of candidate peptides by evaluating the number (or proportion) of theoretically possible fragment ions observed in the experimental spectra without any discrimination. However, the assumption that each theoretical fragment is just as likely to be observed is inaccurate. On the contrary, MS2 spectra often have few dominant fragments. Using millions of MS/MS spectra we show that there is high reproducibility across different fragmentation spectra given the precursor peptide and charge state, implying that there is a pattern to fragmentation. To capture this pattern we propose a novel prediction algorithm based on hidden Markov models with an efficient training process. We investigated the performance of our interpolated-HMM model, trained on millions of MS2 spectra, and found that our model picks up meaningful patterns in peptide fragmentation. Second, looking at the variability of the prediction performance by varying the train/test data split, we observed that our model performs well independent of the specific peptides that are present in the training data. Furthermore, we propose that the real value of this model is as a preprocessing step in the peptide identification process. The model can discern fragment ions that are unlikely to be intense for a given candidate peptide rather than using the actual predicted intensities. As such, probabilistic measures of concordance between experimental and theoretical spectra will leverage better statistics.

Characterization of unknown monoclonal antibody (mAb) variants is important in order to identify their potential impact on safety, potency, and stability. Ion exchange chromatography (IEC) coupled with UV detection is frequently used to separate and quantify mAb variants in routine quality control (QC). However, characterization of the chromatographic peaks resulting from an IEC separation is an extremely time-consuming process, involving many cumbersome steps. Presented here is an online four-dimensional high performance liquid chromatography-mass spectrometry (4D HPLC/MS) approach, developed to circumvent these limitations. To achieve this, a 2D HPLC system was extended through the introduction of additional modules, hence enabling fully automated bioseparation of mAbs, fractionation of peaks, reduction, tryptic digestion, and reversed-phase (RP) separation of resulting peptides followed by MS detection. The entire separation and analytical process for an unknown peak is performed in less than 1.5 h, leading to a significant time savings, with comparable sequence coverage. To show the comparability with the traditional offline process, a proof of concept study with a previously characterized mAb1 is presented in this paper.

Biomarkers have become an essential component of Alzheimer disease (AD) research and because of the pervasiveness of AD pathology in the elderly, the same biomarkers are used in cognitive aging research. A number of current issues suggest that an unbiased descriptive classification scheme for these biomarkers would be useful. We propose the "A/T/N" system in which 7 major AD biomarkers are divided into 3 binary categories based on the nature of the pathophysiology that each measures. "A" refers to the value of a β-amyloid biomarker (amyloid PET or CSF Aβ42); "T," the value of a tau biomarker (CSF phospho tau, or tau PET); and "N," biomarkers of neurodegeneration or neuronal injury ([(18)F]-fluorodeoxyglucose-PET, structural MRI, or CSF total tau). Each biomarker category is rated as positive or negative. An individual score might appear as A+/T+/N-, or A+/T-/N-, etc. The A/T/N system includes the new modality tau PET. It is agnostic to the temporal ordering of mechanisms underlying AD pathogenesis. It includes all individuals in any population regardless of the mix of biomarker findings and therefore is suited to population studies of cognitive aging. It does not specify disease labels and thus is not a diagnostic classification system. It is a descriptive system for categorizing multidomain biomarker findings at the individual person level in a format that is easy to understand and use. Given the present lack of consensus among AD specialists on terminology across the clinically normal to dementia spectrum, a biomarker classification scheme will have broadest acceptance if it is independent from any one clinically defined diagnostic scheme., (© 2016 American Academy of Neurology.)

Syntenin has crucial roles in cell adhesion, cell migration and synaptic transmission. Its closely linked postsynaptic density-95, discs large 1, zonula occludens-1 (PDZ) domains typically interact with C-terminal ligands. We profile syntenin PDZ1-2 through proteomic peptide phage display (ProP-PD) using a library that displays C-terminal regions of the human proteome. The protein recognizes a broad range of peptides, with a preference for hydrophobic motifs and has a tendency to recognize cryptic internal ligands. We validate the interaction with nectin-1 through orthogonal assays. The study demonstrates the power of ProP-PD as a complementary approach to uncover interactions of potential biological relevance., (© 2015 Federation of European Biochemical Societies.)

Trichoplax adhaerens (phylum Placozoa) is a very simple organism that lacks a nervous system. However, its genome contains many genes essential for neuronal function and development. I report the results of regulatory peptide predictions for this enigmatic animal. Extensive transcriptome, genome, and predicted proteome mining allowed us to predict four insulins, at least five short peptide precursors, one granulin, one paracrine regulator of cell growth, and one complex temptin-attractin pheromone signaling system. The expression of three insulins, four short peptide precursors, granulin, and one out of the six temptin genes was detected. Five predicted regulatory peptide precursors could potentially release over 60 different mature peptides. Some of the predicted peptides are somewhat similar to anthozoan RW amides, Aplysia pedal peptide 3, and PRQFV amide. Other predicted short peptides could not readily be classified into established families. These data provide the foundation for the molecular, biochemical, physiological, and behavioral studies of one the most primitive animal coordination systems, and give unique insight into the origins and early evolution of the nervous system., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Neurokinin B (NKB) regulates the release of gonadotropin-releasing hormone (GnRH) via activation of the neurokinin-3 receptor (NK3R). We evaluated the biological stability of NK3R selective agonists to develop novel NK3R agonists to regulate reproductive functions. On the basis of degradation profiles, several peptidomimetic derivatives were designed. The modification of senktide with (E)-alkene dipeptide isostere generated a novel potent NK3R agonist with high stability and prolonged bioactivity.

The application of a proteotyping approach employing high resolution mass spectrometry based is shown to be able to determine the gene origin of all major viral proteins in a triple reassortant pandemic 2009 influenza strain. Key to this approach is the identification of unique swine-host-specific signature and indicator peptides that are characteristic of influenza viruses circulating in North American and Eurasian swine herds in the years prior to the 2009 influenza pandemic. These swine-and human pandemic-specific signatures enable the origins of viral proteins in a clinical virus specimen to be determined and such strains to be rapidly and directly differentiated from other co-circulating seasonal influenza viruses from the same period. The proteotyping strategy offers advantages over traditional RT-PCR-based approaches that are currently the mainstay of influenza surveillance at the molecular level., (Copyright © 2014 John Wiley & Sons, Ltd.)

Background: Amyloids are proteins capable of forming aberrant intramolecular contact sites, characteristic of beta zipper configuration. Amyloids can underlie serious health conditions, e.g. Alzheimer's or Parkinson's diseases. It has been proposed that short segments of amino acids can be responsible for protein amyloidogenicity, but no more than two hundred such hexapeptides have been experimentally found. The authors of the computational tool Pafig published in BMC Bioinformatics a method for extending the amyloid hexapeptide dataset that could be used for training and testing models. They assumed that all hexapeptides belonging to an amyloid protein can be regarded as amylopositive, while those from proteins never reported as amyloid are always amylonegative. Here we show why the above described method of extending datasets is wrong and discuss the reasons why the incorrect data could lead to falsely correct classification., Results: The amyloid classification of hexapeptides by Pafig was confronted with the classification results from different state of the art computational methods and the outputs of all methods were studied by clustering analysis. The clustering methods show that Pafig is an outlier with regard to other approaches. Our study of the statistical patterns of its training and testing datasets showed a strong bias towards STVIIE hexapeptide in their positive part. Different statistical patterns of seemingly amylo-positive and -negative hexapeptides allow for a repeatable classification, which is not related to amyloid propensity of the hexapetides., Conclusions: Our study on recognition of amyloid hexapeptides showed that occurrence of incidental patterns in wrongly selected datasets can produce falsely correct results of classification. The assumption that all hexapeptides belonging to amyloid protein can be regarded as amylopositive and those from proteins never reported as amyloid are always amylonegative is not supported by any other computational method. This is in line with experimental observations that amyloid propensity of a full protein can result from only one amyloidogenic fragment in this protein, while the occurrence of amyliodogenic part that is well hidden inside the protein may never lead to fibril formation. This leads to the conclusion that Pafig does not provide correct classification with regard to amyloidogenicity.

The milk of the one-humped camel (Camelus dromedarius) reportedly offers medicinal benefits, perhaps because of its unique bioactive components. Milk proteins were determined by (1) two-dimensional gel electrophoresis and peptide mass mapping and (2) liquid chromatography-tandem mass spectrometry (LC-MS/MS) following one-dimensional polyacrylamide gel electrophoresis. Over 200 proteins were identified: some known camel proteins including heavy-chain immunoglobulins and others exhibiting regions of exact homology with proteins from other species. Indigenous peptides were also identified following isolation and concentration by two strategies: (1) gel-eluted liquid fraction entrapment electrophoresis and (2) small-scale electrophoretic separation. Extracts were analyzed by LC-MS/MS and peptides identified by matching strategies, by de novo sequencing and by applying a sequence tag tool requiring similarity to the proposed sequence, but not an exact match. A plethora of protein cleavage products including some novel peptides were characterized. These studies demonstrate that camel milk is a rich source of peptides, some of which may serve as nutraceuticals., (Copyright © 2013 John Wiley & Sons, Ltd.)

Antimicrobial peptides or host defense peptides are fundamental components of human innate immunity. Recent and growing evidence suggests they have a role in a broad range of diseases, including cancer, allergies and susceptibility to infection, including HIV/AIDS. Antimicrobial peptide elicitors (APEs) are physical, biological or chemical agents that boost human antimicrobial peptide expression. The current knowledge of APEs and their potential use in the treatment of human infectious diseases are reviewed, and a classification system for APEs is proposed. The efficient use of APEs in clinical practice could mark the beginning of the urgently needed post-antibiotic era, but further trials assessing their efficacy and safety are required.

Objective: Drug-resistant (DR) HIV emerges during combined antiretroviral treatment (cART), creating concern about widespread transmission of DR-HIV as cART is expanded in resource-limited countries. The aim of this study was to determine the predominant HIV-1 subtypes and prevalence of transmitted DR mutations among antiretroviral-naïve patients in Iran., Design: To monitor transmission of DR HIV, a threshold surveillance based on the world health organization (WHO) guidelines was implemented in Iran., Methods: For this HIVDR threshold surveillance study, blood samples were collected from 50 antiretroviral-naïve HIV-1-infected patients. Antiretroviral-resistant mutations were determined by sequencing HIV-1 protease, reverse transcriptase and integrase regions. The HIV-1 subtype was determined by sequencing the p17 and C2-V5 regions of the gag and env genes, respectively., Results: Phylogenetic analyses of the sequenced regions revealed that 45 (95.7%) of 47 samples that were successfully obtained were CRF35_AD. The remaining two cases were subtype B (2.1%) and CRF01_AE (2.1%). Consistent results were obtained also from Env and Gag sequences. Regarding prevalence of transmitted DR viruses, two cases were found to harbor reverse transcriptase-inhibitor-resistant mutations (4.3%). In addition, although not in the WHO list for surveillance of transmitted mutations, 13 minor protease-inhibitor-resistant mutations listed in the International AIDS Society-USA panel of drug resistance mutations were found. No DR mutations were detected in the integrase region., Conclusions: Our study clarified that CRF35_AD is the major subtype among HIV-1-infected patients in Iran. According to the WHO categorization method of HIVDR threshold survey, the prevalence of transmitted drug resistant HIV in Iran was estimated as moderate (5-15%).

Formation of cyclic intermediates involving water or ammonia loss is a common occurrence in any reaction involving terminal amines or hydroxyl group containing species. Proteins that have both these functional groups in abundance are no exception, and presence of amino acids such as asparagine, glutamines, aspartic acids, and glutamic acids aid in formation of such intermediates. In the biopharma scenario, such intermediates lead to product- or process-related impurities that might be immunogenic. Mass spectroscopy is a powerful technique that is used to decipher the presence and physicochemical characteristics of such impurities. However, such intermediates can also form in situ during mass spectrometric analysis. We present here the detection of in-source and in-solution formation of succinimide and pyroglutamate in the protein granulocyte colony stimulating factor. We also propose an approach for quick differentiation of such in-situ species from the tangible impurities. We believe that this will not only reduce the time spent in unambiguous identification of succinimide- and/or pyroglutamate-related impurity in bio-pharmaceutics but also provide a platform for similar studies on other impurities that may form due to stabilized intermediates.

Fractionation of complex samples at the cellular, subcellular, protein, or peptide level is an indispensable strategy to improve the sensitivity in mass spectrometry-based proteomic profiling. This study revisits, evaluates, and compares the most common gel-based protein separation techniques i.e. 1D SDS-PAGE, 1D preparative SDS-PAGE, IEF-IPG, and 2D-PAGE in their performance as fractionation approaches in nano LC-ESI-MS/MS analysis of a mixture of protein standards and mitochondrial extracts isolated from rat liver. This work demonstrates that all the above techniques provide complementary protein identification results, but 1D SDS-PAGE and IEF-IPG had the highest number of identifications. The IEF-IPG technique resulted in the highest average number of detected peptides per protein. The 2D-PAGE was evaluated as a protein fractionation approach. This work shows that the recovery of proteins and resulting proteolytic digests is highly dependent on the total volume of the gel matrix. The performed comparison of the fractionation techniques demonstrates the potential of a combination of orthogonal 1D SDS-PAGE and IEF-IPG for the improved sensitivity of profiling without significant decrease in throughput., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The analysis of the cytosolic red blood cell (RBC) proteome is negatively affected by the high intracellular amount of hemoglobin complicating the detection of low-abundant cytosolic proteins. In this study, an alternative approach for the preparation of hemoglobin-depleted RBC lysates is presented, which was established in combination with downstream 2D PAGE analysis and Orbitrap MS. Hemoglobin removal was accomplished by using HemoVoid(TM) depletion reagent, which enabled a very efficient enrichment of low-abundant proteins by simultaneously reducing the hemoglobin concentration of the sample. After defining selected sample preparation protocol characteristics including specificity/selectivity, precision and linearity, a 2D reference map (pH 4-7) of the cytosolic RBC proteome was generated and a total of 189 different proteins were identified. Thus, the presented approach proved to be highly suitable to prepare reproducible high-resolution 2D protein maps of the RBC cytosol and provides a helpful tool for future studies investigating disease- or storage-induced changes of the cytosolic RBC proteome., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The interplay of modern molecular simulation and high-quality nuclear magnetic resonance (NMR) experiments has reached a fruitful stage for quantitative characterization of structural ensembles of disordered peptides. Amyloid-β 1-42 (Aβ42), the primary peptide associated with Alzheimer's disease, and fragments such as Aβ21-30 are both classified as intrinsically disordered peptides (IDPs). We use a variety of NMR observables to validate de novo molecular dynamics simulations in explicit water to characterize the tertiary structure ensemble of Aβ42 and Aβ21-30 from the perspective of their classification as IDPs. Unlike the Aβ21-30 fragment that conforms to expectations of an IDP that is primarily extended, we find that Aβ42 samples conformations reflecting all possible secondary structure categories and spans the range of IDP classifications from collapsed structured states to highly extended conformations, making it an IDP with a far more heterogeneous tertiary ensemble.

We demonstrate use of restricted access media with reversed phase functionality (RAM-RP) for analysis of low molecular weight proteins and peptides in mouse serum (75 μl) using a custom designed modular automated processing system (MAPS). RAM-RP fractionation with simultaneous removal of high molecular weight and high abundance proteins is integrated with a follow-on buffer exchange module (BE) to ensure compatibility with subsequent processing steps (trypsin digestion and intact peptide separation prior to mass spectrometric analysis). The high sample capacity afforded by chromatographic methods generates enough sample to achieve comprehensive serum peptidome identification (357 proteins) through tandem mass spectrometric analysis of both intact and digested peptides. Sample losses during transfer between modules are minimized through precise fluidic control; no clogging occurred over several months of serum processing in our low back pressure system. Computer controlled operation of both modules and thorough optimization yield excellent run-to-run reproducibility and protein/peptide overlap in analytical repeats. The robustness of our results demonstrate that the RAM-RP-BE workflow executed on our MAPS platform shows tremendous potential for high throughput peptidome processing, particularly with regard to direct analysis of small-volume serum samples., (Published by Elsevier B.V.)

This article is concerned with the reduction of protein concentration range differences by the peptide beads library technology (ProteoMiner™ or "equalizer" technology), which in principle allows the enrichment of proteins to the same concentration level (i.e. protein equalizer) regardless of the original protein abundance in a given biological fluid such as human serum, which is the subject of our investigation. After the equalization step, the captured proteins from human serum were fractionated on a series of tandem monolithic columns with surface-bound iminodiacetic acid ligands to which three different metal ions, namely, Zn²+, Ni²+ and Cu²+ were immobilized to yield the so-called immobilized metal affinity chromatography columns. These three monolithic columns were connected to a reversed-phase column packed with polystyrene divinyl benzene beads. Aliquots taken from the four collected fractions from the four tandem columns were subsequently fractionated by 2-DE. Also, aliquots from the four collected fractions were tryptically digested and analyzed by LC-MS/MS. The strategy of subsequent fractionation on the four tandem columns after equalization allowed the identification of more proteins than simply using the equalization by ProteoMiner™ . The equalizer technology was compared to the immuno-subtraction approach. While the ProteoMiner™ technology is superior in terms of the overall number of captured proteins, it only complements the immuno-subtraction approach since the latter can capture the proteins that were not captured by the former., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

"Binning" (or taxonomic classification) of DNA sequence reads is an initial step to analyzing an environmental biological sample. Currently, a homology-based tool, BLAST, is one of the most commonly used tools to label DNA reads, but it is argued that BLAST will quickly lose its classification ability as the genome databases grow. In this paper, we compare the accuracies of a naïve Bayes classifier (NBC) and statistical language model to BLAST for binning reads and demonstrate that NBC obtains good performance for the low cost of computational complexity. On the other hand, the back-off n-gram language model can improve accuracy when only partial training data is available (such as in-progress sequencing projects). NBC demonstrates comparable performance to BLAST and can also be optimized on partial training datasets by adjusting the word feature size. A fivefold cross validation is conducted to compare each method's accuracy for determining novel genomes at different taxonomic levels, with NBC outperforming BLAST for species-level classification but BLAST outperforming NBC for genus-level and phyla-level classification. In conclusion, the NBC is a competitive taxonomic classifier, and language models can improve performance when only partial training data is available.

Human urinary exosomes are 30-100 nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary track and may serve as a suitable noninvasive starting material for biomarker discovery relevant to a variety of renal disease. To comprehensively explore the low-abundance proteome, combinatorial peptide ligand libraries, combined with peptide OFFGEL electrophoresis were employed for the enrichment and separation of relatively low-abundant proteins in urinary exosomes. After analysis by nanoHPLC-chip-MS/MS, 512 proteins were identified, including a large number of proteins with extreme molecular weight or extreme pI value, which could not be well mapped by using traditional 2-D-gel-based separation methods. This in-depth analysis of low-abundant proteins in urinary exosomes led to an increased understanding of molecular composition of these little vesicles and may be helpful for the discovery of novel biomarker. Our work also provides an effective strategy of concentration and identification of low-abundance proteome from complex bio-samples.

In the field of proteomic investigation, the analysis of membrane proteins still faces many technical challenges. A fundamental question in this puzzle is how to maintain a proper solvent environment to allow the hydrophobic proteins to remain solubilized. We propose that the denaturation of membrane proteins in a highly concentrated urea solution enables them to be ionized such that ionic exchange chromatography can be employed to separate them. The membrane proteins prepared from the mouse liver were dissolved in 6M guanidine hydrochloride, 20mM Tris-HCl, pH 9.0, and loaded onto a tandem chromatography apparatus coupled with Q-Sepharose FF and Sephacryl S-200HR. These columns were able to adsorb 97.87% of the membrane protein preparations. Using a linear NaCl (0-1.0M) gradient, the bound proteins were eluted out at 0.1-1.0M NaCl, and examined by SDS-PAGE. Furthermore the protein bands underwent excision and digestion with trypsin, followed by reverse-phase chromatography for the separation of the digested peptides and ionic-trap mass spectrometry for the identification of the proteins. From the SDS-PAGE gels, the overlap between proteins from neighboring bands was only 21.34%, indicating that the anionic-size exclusion coupling chromatography efficiently separated these membrane proteins. Of a total of 392 proteins identified, 306 were membrane proteins or membrane-associated proteins. Based on the calculation of hydrophobicity, the GRAVY scores of 83 proteins are greater than, or equal to, 0.00. Taking all of this evidence together, our results revealed that this approach is satisfactory for studies on the membrane proteome from the mouse liver., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The objective of this project was to study the interaction between HR1 and HR2, the stability of the complex formed, and to characterize the antibodies produced against monomeric HR1 and HR2 peptides as well as the HR1-HR2 complex. In this work, HR1 was mimicked by peptide N36, and HR2 was mimicked by peptide C34L and its analogues C34M2, C34M3, and C34D. Whereas C34M2 and C34M3 are partially composed of D-amino acids, C34D has same sequence as C34L, but is assembled entirely of D-amino acids. Using CD analysis, SPR assays, and gel filtration chromatography, we demonstrate the physical interaction between N36 and C34L and its analogues C34M2 and C34M3, but not C34D. We show that the HR1-HR2 complex is formed rapidly (<1 min) and remains stable, as demonstrated by its inability, in contrast to each free peptide, to inhibit the formation of syncytia. To generate antibodies with predetermined specificity against the transiently exposed intermediate that corresponds to the six-helix bundle structure, purified preformed HR1-HR2 complex was used, in parallel with monomeric HR1 and HR2 peptides, as immunogens in mice. Although the produced antibodies recognize total HIV-1 envelope glycoproteins in ELISA, they are unable to neutralize HIV-1-mediated fusion at 37 °C. However, if the incubation with these antibodies is carried out at 27 °C, a temperature that allows stabilization of the transient intermediate complex, anti-peptide antibodies are able to bind their corresponding domains in HeLa cells expressing HIV-1 gp41 in co-culture with HeLa CD4-CCR5/CXCR4 during the dynamic mechanism of membrane fusion. In agreement with the latter results, these antibodies, if previously incubated for 2 h at 27 °C, are able to strongly neutralize HIV-1 entry by membrane fusion, as shown by their ability to block the formation of syncytia.

The ability of laserspray ionization (LSI) to produce multiply charged ions by laser ablation from the solid state, directly from a surface, and at atmospheric pressure allows protein analysis on an ion mobility spectrometry (IMS)-mass spectrometry (MS) instrument (SYNAPT G2) having a mass-to-charge limit of 8000. The matrix, 2,5-dihydroxyacetophenone, lowers the thermal requirements for desolvation of matrix/analyte clusters to produce the highly charged LSI ions under gentle conditions to retain structural integrity of the proteins. Examples include cytochrome C and lysozyme. The solvent-free IMS gas-phase separation is used to baseline separate in the drift time dimension the isomeric solubility restricted β-amyloid (1-42) from the reversed (42-1). The LSI process is shown to be sufficiently soft to preserve structural integrity and permit separation according to the different shapes. These results suggest that LSI-IMS-MS potentially combines speed of analysis and imaging capability common to matrix-assisted laser desorption/ionization and multiple charging with the potential for structural analysis common to electrospray ionization.

A proteomic-based method has been developed for the detection of chicken meat within mixed meat preparations. The procedure is robust and simple, comprising the extraction of myofibrillar proteins, enrichment of target proteins using OFFGEL isoelectric focusing, in-solution trypsin digestion of myosin light chain 3, and analysis of the generated peptides by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Using this approach, it was possible for example to detect 0.5% contaminating chicken in pork meat with high confidence. Quantitative detection of chicken meat was done by using AQUA stable isotope peptides made from the sequence of previously selected species-specific peptide biomarkers. Linearity was observed between the amount of the peptide biomarker and the amount of chicken present in the mixture; further independent replication is required now to validate the method. Apart from its simplicity, this approach has the advantage that it can be used effectively for the detection of both raw and cooked meat. The method is robust, reliable, and sensitive, representing a serious alternative to methods currently in use for these purposes. It is amenable to highly processed foods which can be particularly problematic, as the tertiary protein structure is often affected in processed food precluding immunoassays. In addition, this proteomic analysis will permit the determination of definitive discriminatory sequence, unlike the DNA PCR based methods used presently. The present article also demonstrates the translation of the technology to routine mass spectrometry equipment, making the methodology suitable for public analysts.

Gingival crevicular fluid (GCF) is a pathophysiological fluid that flows into the oral cavity. Human GCF was collected using sterile glass microcapillary tubes from inflamed periodontal sites in patients who had a history of periodontal disease and were in the maintenance phase of treatment. Samples from individual sites were analyzed using MS techniques both before and following HPLC. GCF samples were also pooled and subjected to SDS-PAGE, in-gel digestion and MS analyses using both MALDI-TOF/TOF MS and nanoLC-ESI-MS/MS. MS spectra were used to search human protein sequence databases for protein identification. With these approaches, 33 peptides and 66 proteins were positively identified in human GCF. All of the peptides discovered in this study are reported in GCF here for the first time. Forty-three of the identified proteins, such as actin and the actin binding proteins profilin, cofilin and gelsolin, have not been reported in GCF before.

Administration of the multipotent hematopoietic progenitor cell (HPC) line DKmix improved cardiac function after myocardial infarction and accelerated dermal wound healing due to paracrine mechanisms. The aim of this study was to analyse the secreted proteins of DKmix cells in order to identify the responsible paracrine factors and assess their relevance to the wide spectrum of therapeutic effects. A mass spectrometry (MS)-based approach was used to identify secreted proteins of DKmix cells. Serum free culture supernatants of DKmix-conditioned medium were collected and the proteins present were separated, digested by trypsin and the resulting peptides were then analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) MS. Overall 95 different proteins were identified. Among them, secretory proteins galectin-3 and gelsolin were identified. These proteins are known to stimulate cell migration and influence wound healing and cardiac remodelling. The remaining proteins originate from intracellular compartments like cytoplasm (69%), nucleus (12%), mitochondria (4%), and cytoplasmic membrane (3%) indicating permeable or leaky DKmix cells in the conditioned medium. Additionally, a sandwich immunoassay was used to detect and quantify cytokines and chemokines. Interleukin-6 (IL-6), interleukin-13 (IL-13), monocyte-chemoattractant protein-1 (MCP-1), monocyte-chemoattractant protein-3 (MCP-3), monocyte-chemoattractant protein-1alpha (MIP-1alpha) and monocyte-chemoattractant protein-1beta (MIP-1beta) were detected in low concentrations. This study identified a subset of proteins present in the DKmix-conditioned medium that act as paracrine modulators of tissue repair. Moreover, it suggests that DKmix-derived conditioned medium might have therapeutic potency by promoting tissue regeneration., (Copyright 2010 John Wiley & Sons, Ltd.)

Lewy bodies, alpha-synuclein (alpha-syn) immunopositive intracellular deposits, are the pathological hallmark of Parkinson's disease (PD). Interestingly, Lewybody-like structures have been identified in fetal tissue grafts about one decade after transplantation into the striatum of PD patients. One possible explanation for the accelerated deposition of alpha-syn in the graft is that the aggregation of alpha-syn from the host tissue to the graft is spread by a prion disease-like mechanism. We discuss here an in vitro model which might recapitulate some aspects of disease propagation in PD. We found here that in vitro-generated alpha-syn oligomers induce transmembrane seeding of alpha-syn aggregation in a dose- and time-dependent manner. This effect was observed in primary neuronal cultures as well as in neuronal cell lines. The seeding oligomers were characterized by a distinctive lithium dodecyl sulfate-stable oligomer pattern and could be generated in a dynamic process out of pore-forming oligomers. We propose that alpha-syn oligomers form as a dynamic mixture of oligomer types with different properties and that alpha-syn oligomers can be converted into different types depending on the brain milieu conditions. Our data indicate that extracellular alpha-syn oligomers can induce intracellular alpha-syn aggregation, therefore we hypothesize that a similar mechanism might lead to alpha-syn pathology propagation.

AlphaD-conotoxins are peptide inhibitors of nicotinic acetylcholine receptors (nAChRs) first described from Conus vexillum (alphaD-VxXIIA-C and renamed here to alphaD-VxXXA, alphaD-VxXXB, and alphaD-VxXXC). In this study, we report cDNA sequences encoding D-superfamily conopeptides identified in the Clade XII Conidae Conus vexillum, Conus capitaneus, Conus mustelinus, and Conus miles, together with partial sequences of corresponding peptides from this family. The D-superfamily signal peptide sequences display greater heterogeneity than reported for other conotoxin superfamilies. Phylogenetic analysis of the relationships among alphaD-conotoxin precursors reveals two distinct groups containing either an EMM or AVV signal peptide sequence motif. Homodimer and heterodimer combinations of predicted mature toxin sequences likely account for the partial amino acid sequences and mass values observed for several of the native dimeric peptide components identified in C. capitaneus, C. miles, and C. mustelinus venom. The discovery of the precursors and several novel conotoxins from different species defines this large conotoxin family and expands our understanding of sequence diversification mechanisms in Conus species.

The accumulation of aggregated protein in the cell is associated with the pathology of many diseases and constitutes a major concern in protein production. Intracellular aggregates have been traditionally regarded as nonspecific associations of misfolded polypeptides. This view is challenged by studies demonstrating that, in vitro, aggregation often involves specific interactions. However, little is known about the specificity of in vivo protein deposition. Here, we investigate the degree of in vivo co-aggregation between two self-aggregating proteins, Abeta42 amyloid peptide and foot-and-mouth disease virus VP1 capsid protein, in prokaryotic cells. In addition, the ultrastructure of intracellular aggregates is explored to decipher whether amyloid fibrils and intracellular protein inclusions share structural properties. The data indicate that in vivo protein aggregation exhibits a remarkable specificity that depends on the establishment of selective interactions and results in the formation of oligomeric and fibrillar structures displaying amyloid-like properties. These features allow prokaryotic Abeta42 intracellular aggregates to act as effective seeds in the formation of Abeta42 amyloid fibrils. Overall, our results suggest that conserved mechanisms underlie protein aggregation in different organisms. They also have important implications for biotechnological and biomedical applications of recombinant polypeptides.

The present study proposes a strategy for human in vivo acquired enamel pellicle (AEP) peptidome characterisation based on sequential extraction with guanidine and TFA followed by MALDI-TOF/TOF identification. Three different nanoscale analytical approaches were used: samples were subjected to tryptic digestion followed by nano-HPLC and mass spectrometry (MS and MS/MS) analysis. Undigested samples were analysed by LC-MS (both linear and reflector modes) and LC-MS/MS analysis, and samples were subjected to nano-HPLC followed by on-plate digestion and mass spectrometry (MS and MS/MS) analysis. The majority of the identifications corresponded to peptide/protein fragments of salivary protein, belonging to the classes: acidic PRPs, basic PRPs, statherin, cystatins S and SN and histatin 1 (all also identified in intact form). Overall, more than 90 peptides/proteins were identified. Results clearly show that peptides with acidic groups are enriched in the TFA fraction while peptides with no acidic or phosphate groups are prevalent on the guanidine extract. Also, phosphorylated peptides were observed mainly on the TFA fraction. Fragments present in the AEP show a predominance of cleavage points located at Arg, Tyr and Lys residues. Obtained data suggest that proteolytic activity could influence AEP formation and composition.

Deposition of amyloid beta (Abeta) fibrils has been suggested to play a central role in Alzheimer's disease. In clarifying the mechanism by which fibrils form and moreover in developing new treatments for amyloidosis, direct observation is important. Focusing on the interactions with surfaces at the early stages, we studied the spontaneous formation of Abeta(1-40) fibrils on quartz slides, monitored by total internal reflection fluorescence microscopy combined with thioflavin T, an amyloid-specific fluorescence dye. Self-assembly of Abeta(1-40), accelerated by a low concentration of sodium dodecyl sulfate, produced various remarkable amyloid assemblies. Densely packed spherulitic structures with radial fibril growth were typically observed. When the packing of fibrils was coarse, extremely long fibrils often protruded from the spherulitic cores. In other cases, a large number of wormlike fibrils were formed. Transmission electron microscopy and atomic force microscopy revealed relatively short and straight fibrillar blocks associated laterally without tight interaction, leading to random-walk-like fibril growth. These results suggest that, during spontaneous fibrillation, the nucleation occurring in contact with surfaces is easily affected by environmental factors, creating various types of nuclei, and hence variations in amyloid morphology. A taxonomy of amyloid supramolecular assemblies will be useful in clarifying the structure-function relationship of amyloid fibrils.

HIV-1 coreceptor usage and phenotype mainly determined by V3 loop are associated with the disease progression of AIDS. Predicting HIV-1 coreceptor usage and phenotype facilitates the monitoring of R5-to-X4 switch and treatment decision-making. In this study, we employed random forest to predict HIV-1 biological phenotype, based on 37 random features of V3 loop. In comparison with PSSM method, our RF predictor obtained higher prediction accuracy (95.1% for coreceptor usage and 92.1% for phenotype), especially for non-B non-C HIV-1 subtypes (96.6% for coreceptor usage and 95.3% for phenotype). The net charge, polarity of V3 loop and five V3 sites are seven most important features for predicting HIV-1 coreceptor usage or phenotype. Among these features, V3 polarity and four V3 sites (22, 12, 18 and 13) are first reported to have high contribution to HIV-1 biological phenotype prediction.

Ugl-Y (young age-related urinary glycoprotein) is an age-specific protein that we have previously identified in urine from healthy subjects under 18 years of age. Isoelectric focusing analysis of Ugl-Y gives a set of three bands, Y1, Y2 and Y3, in the pH region around 3. To determine the complete structure of Ugl-Y, purified Y1 and Y2 from pediatric urine were enzymatically cleaved, and the resulting peptides were analyzed by protein sequencing and/or MALDI-TOF MS. As a result, it was demonstrated that Y1 consists of 189 amino acid residues, and is identical to the region from A723 to R911 of fibronectin, whereas Y2 consists of 181 amino acid residues, and is identical to the region from A723 to R903. Electrophoretic analysis of the lysate prepared from COS-7 cells transfected with Y1- or Y2-expressing vectors gave specific bands corresponding to Y1 or Y2, respectively, showing the validity of the sequences determined. Partial purification of pediatric serum followed by western blotting revealed that Ugl-Y is derived from plasma. Furthermore, Ugl-Y was generated by in vitro digestion of fibronectin by acid protease in extracts of osteoclast cells. These findings suggest that Ugl-Y is probably produced by degradation of fibronectin comprising bone matrix during the process of vigorous bone resorption in children and adolescents. This is the first report on the identification and characterization of juvenile-specific fibronectin fragments excreted into urine.

Biomarkers for differential diagnosis of the three most frequent degenerative forms of dementia, Alzheimer's disease (AD), dementia with Lewy bodies (DLB) and frontotemporal dementias (FTD), are currently under intensive investigation, but disease-specific biomarkers for FTD and DLB are still lacking. We analyzed 303 cerebrospinal fluid (CSF) samples of 71 AD, 32 DLB and 36 FTD patients in comparison to 93 various other dementias (OD), 20 peripheral neurologic disease (PND) controls, 25 neurodegenerative disorders without dementia (ND) and 26 depressive cognitive complainers (DCC) for distinct CSF amyloid-beta (Abeta) peptide patterns, using the quantitative Abeta-SDS-PAGE/immunoblot. Additionally, the novel electrochemiluminescence technique (MSD) was used to validate the measures on Abeta1-38. The main outcome measures were a striking decrease of Abeta1-42 in AD (P=7.4 x 10(-19)), and most interestingly a pronounced decrease of Abeta1-38 in FTD (P=9.6 x 10(-7)). Moreover, a novel peptide that most probably represents an oxidized alpha-helical form of Abeta1-40 (Abeta1-40(ox)) displayed a highly significant increase in DLB (P=3.7 x 10(-3)) as compared to non-demented disease controls. The overall diagnostic accuracy of percentage Abeta peptide abundances (Abeta1-X%) was clearly superior to absolute CSF Abeta levels. Abeta1-42% and Abeta1-38% enabled contrasts of 85% or beyond to distinguish AD and FTD, respectively, from all other investigated subjects. Abeta1-40(ox)% yielded a diagnostic sensitivity and specificity of 88 and 73% for the detection of DLB among all other investigated patients. We found a strong correlation between Abeta1-38 levels as measured by the Abeta-SDS-PAGE/immunoblot and MSD, respectively. CSF Abeta peptides may reflect disease-specific impact of distinct neurodegenerative processes on Abeta peptide metabolism and represent a potential diagnostic biomarker for AD, FTD and DLB.

Motivation: The size of current protein databases is a challenge for many Bioinformatics applications, both in terms of processing speed and information redundancy. It may be therefore desirable to efficiently reduce the database of interest to a maximally representative subset., Results: The MinSet method employs a combination of a Suffix Tree and a Genetic Algorithm for the generation, selection and assessment of database subsets. The approach is generally applicable to any type of string-encoded data, allowing for a drastic reduction of the database size whilst retaining most of the information contained in the original set. We demonstrate the performance of the method on a database of protein domain structures encoded as strings. We used the SCOP40 domain database by translating protein structures into character strings by means of a structural alphabet and by extracting optimized subsets according to an entropy score that is based on a constant-length fragment dictionary. Therefore, optimized subsets are maximally representative for the distribution and range of local structures. Subsets containing only 10% of the SCOP structure classes show a coverage of >90% for fragments of length 1-4., Availability: http://mathbio.nimr.mrc.ac.uk/~jkleinj/MinSet., Supplementary Information: Supplementary data are available at Bioinformatics online.

The field of protein-structure prediction has been revolutionized by the application of "mix-and-match" methods both in template-based homology modeling and in template-free de novo folding. Consensus analysis and recombination of fragments copied from known protein structures is currently the only approach that allows the building of models that are closer to the native structure of the target protein than the structure of its closest homologue. It is also the most successful approach in cases in which the target protein exhibits a novel three-dimensional fold. This review summarizes the recent developments in both template-based and template-free protein structure modeling and compares the available methods for protein-structure prediction by recombination of fragments. A convergence between the "protein folding" and "protein evolution" schools of thought is postulated.

Amyloid beta 1-42 (Abeta42) and Abeta17-42 are major constituents of diffuse plaque in brains with Alzheimer's disease (AD). We demonstrate the potent cytotoxicity of Abeta42 and Abeta17-42, lesser toxicity of Abeta1-40 (Abeta40) and lack of toxicity of Abeta1-16 (Abeta16) in neuronal cells as measured by inhibition of cell proliferative response using thymidine incorporation assay and that this cytotoxicity can be reduced with Abeta16 and eight-residue Abeta derivatives such as Abeta1-8 and Abeta9-16. FACS analysis also revealed that Abeta16 could dramatically protect against the apoptosis induced by Abeta17-42 with over 80% viable cells. We determined the caspases involved in the Abeta-mediated apoptotic pathway using caspase-specific inhibitors in MTT assays. For all Abetas, the executor was caspase 3, while the initiator was caspase 9 for Abeta42 and caspase 8 for Abeta40 and Abeta17-42. Microscopic observation of lucifer-yellow-labeled neuronal cells demonstrated the occurrence of lysosomal membrane injury of the cells, corresponding to the severe cytotoxic effects of Abeta42. Our findings suggest that the apoptosis of neuronal cells due to Abeta42, Abeta40 and Abeta17-42 is mediated by the different caspase pathways and that this apoptosis can be reduced with the eight-residue Abeta-derived fragments Abeta1-8, Abeta9-16 and Abeta16.

Timely classification and identification of bacteria is of vital importance in many areas of public health. Mass spectrometry-based methods provide an attractive alternative to well-established microbiologic procedures. Mass spectrometry methods can be characterized by the relatively high speed of acquiring taxonomically relevant information. Gel-free mass spectrometry proteomics techniques allow for rapid fingerprinting of bacterial proteins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or, for high-throughput sequencing of peptides from protease-digested cellular proteins, using mass analysis of fragments from collision-induced dissociation of peptide ions. The latter technique uses database searching of product ion mass spectra. A database contains a comprehensive list of protein sequences translated from protein-encoding open reading frames found in bacterial genomes. The results of such searches allow the assignment of experimental peptide sequences to matching theoretical bacterial proteomes. Phylogenetic profiles of sequenced peptides are then used to create a matrix of sequence-to-bacterium assignments, which are analyzed using numerical taxonomy tools. The results thereof reveal the relatedness between bacteria, and allow the taxonomic position of an investigated strain to be inferred.

Motivation: Monte Carlo methods are the most effective means of exploring the energy landscapes of protein folding. The rugged topography of folding energy landscapes causes sampling inefficiencies however, particularly at low, physiological temperatures., Results: A hybrid Monte Carlo method, termed density guided importance sampling (DGIS), is presented that overcomes these sampling inefficiencies. The method is shown to be highly accurate and efficient in determining Boltzmann weighted structural metrics of a discrete off-lattice protein model. In comparison to the Metropolis Monte Carlo method, and the hybrid Monte Carlo methods, jump-walking, smart-walking and replica-exchange, the DGIS method is shown to be more efficient, requiring no parameter optimization. The method guides the simulation towards under-sampled regions of the energy spectrum and recognizes when equilibrium has been reached, avoiding arbitrary and excessively long simulation times., Availability: Fortran code available from authors upon request., Contact: m.j.parker@leeds.ac.uk.

Dexamethasone (DEX) suppresses the secretion of and responsiveness to growth hormone (GH). Here we aimed to assess the therapeutic effects of GH on the DEX-induced osteopenia. Female Wistar rats were treated for 2 weeks with DEX (200 microg/day) or saline as a control. DEX significantly decreased body weight gain, bone mineral density (BMD), growth plate thickness, area ratio of trabecular bone, and serum osteocalcin levels. DEX also elongated the tibia primary spongiosa and caused many tiny lipid droplets in the tibia marrow. These results indicated that DEX induced osteopenia in rats. We then assessed the effects of GH on the recovery of osteopenia after withdrawal of DEX. DEX-treated rats were subsequently treated for 1 week with GH (0.1 or 0.3 U/day) or saline, while saline-pretreated rats were treated for 1 week with saline as a control. GH (0.1 or 0.3 U/day)-treated rats showed a catch-up growth in various bone measurements by one week after DEX withdrawal, though most of them remained subnormal. GH treatment did not enhance the recovery of DEX-induced osteopenia. Therefore a short-term exposure to DEX significantly impaired the bone metabolism, which started to recover soon after withdrawal of DEX. Unfortunately, immediate administration of GH after withdrawal of DEX did not enhance the recovery process.

The influence of three C-terminal sequences and of transmembrane domain from amyloid precursor protein (APP) on the activity of G-proteins and of the coupled cAMP-signalling system in the postmortem Alzheimer's disease (AD) and age-matched control brains was compared. 10 microM APP(639-648)-APP(657-676) (PEP1) causes a fivefold stimulation in the [35S]GTPgammaS-binding to control hippocampal G-proteins. APP(657-676) (PEP2) and APP(639-648) (PEP4) showed less pronounced stimulation whereas cytosolic APP(649-669) (PEP3) showed no regulatory activity in the [35S]GTPgammaS-binding. PEP1 also showed 1.4-fold stimulatory effect of on the high-affinity GTPase and adenylate cyclase activity in control membranes, whereas in AD hippocampal membranes the stimulatory effect of PEP1 was substantially weaker. The PEP1 stimulation of the [35S]GTPgammaS-binding to the control membranes was significantly reduced by 1.5 mM glutathione, 0.5 mM antioxidant N-acetylcysteine and, in the greatest extent, by 0.01 mM of desferrioxamine. In AD hippocampus these antioxidants revealed no remarkable reducing effect on PEP1-induced stimulation. Our results suggest that C-terminal and transmembrane APP sequences possess receptor-like G-protein activating function in human hippocampus and that abnormalities of this function contribute to AD progression. The stimulatory action of these sequences on G-protein mediated signalling suggests the region-specific formation of reactive species.

Histidine-free variants of salivary histatin 5 have a broad antimicrobial activity against various bacteria. In relation to a possible therapeutic application, we were interested in the susceptibility of these small peptides (14 amino acids long) to microbial proteinases and whether this affects their antimicrobial activity. Analyses by SDS-PAGE of supernatants of peptide-bacteria incubation showed a reduction in protein bands within 15 minutes' incubation, as a result of cellular internalization. Degradation products of dhvar1 and dhvar2 appeared within one hour in the supernatants of Streptococcus mutans and Staphylococcus aureus. In contrast, the variants dhvar3 and dhvar4 were more resistant to degradation under the same conditions. MALDI-TOF analyses identified cleavage of dhvar1 and dhvar2 at Glu(6). The N-terminal peptide part (1-6) of dhvar1 and 2 showed no bactericidal activity, while peptide fragment (7-14) showed a highly reduced bactericidal activity.

We have shown previously that the protease-resistant and neurotoxic prion peptide fragment PrP[106-126] of human PrP incorporates into lipid bilayer membranes to form heterogeneous ion channels, one of which is a Cu(2+)-sensitive fast cation channel. To investigate the role of PrP[106-126]'s hydrophobic core, AGAAAAGA, on its ability to form ion channels and their regulation with Cu(2+), we used the lipid-bilayer technique to examine membrane currents induced as a result of PrP[106-126] (AA/SS) and PrP[106-126] (VVAA/SSSS) interaction with lipid membranes and channel formation. Channel analysis of the mutant (VVAAA/SSS), which has a reduced hydrophobicity due to substitution of hydrophobic residues with the hydrophilic serine residue, showed a significant change in channel activity, which reflects a decrease in the beta-sheet structure, as shown by CD spectroscopy. One of the channels formed by the PrP[106-126] mutant has fast kinetics with three modes: burst, open and spike. The biophysical properties of this channel are similar to those of channels formed with other aggregation-prone amyloids, indicating their ability to form the common beta sheet-based channel structure. The current-voltage (I-V) relationship of the fast cation channel, which had a reversal potential, E(rev), between -40 and -10 mV, close to the equilibrium potential for K(+) ( E(K) = -35 mV), exhibited a sigmoidal shape. The value of the maximal slope conductance (g(max)) was 58 pS at positive potentials between 0 and 140 mV. Cu(2+) shifted the kinetics of the channel from being in the open and "burst" states to the spike mode. Cu(2+) reduced the probability of the channel being open (P(o)) and the mean open time (T(o)) and increased the channel's opening frequency (F(o)) and the mean closed time (T(c)) at a membrane potential ( V(m)) between +20 and + 140 mV. The fact that Cu(2+) induced changes in the kinetics of this channel with no changes in its conductance, indicates that Cu(2+) binds at the mouth of the channel via a fast channel block mechanism. The Cu(2+)-induced changes in the kinetic parameters of this channel suggest that the hydrophobic core is not a ligand Cu(2+) site, and they are in agreement with the suggestion that the Cu(2+)-binding site is located at M(109) and H(111) of this prion fragment. Although the data indicate that the hydrophobic core sequence plays a role in PrP[106-126] channel formation, it is not a binding site for Cu(2+). We suggest that the role of the hydrophobic region in modulating PrP toxicity is to influence PrP assembly into neurotoxic channel conformations. Such conformations may underlie toxicity observed in prion diseases. We further suggest that the conversions of the normal cellular isoform of prion protein (PrP(c)) to abnormal scrapie isoform (PrP(Sc)) and intermediates represent conversions to protease-resistant neurotoxic channel conformations.

We investigated the functional interaction between neuropeptide Y (NPY) receptors using nerve terminals and cultured rat hippocampal neurons, and we evaluated the involvement of voltage-gated Ca(2+) channels (VGCCs) in NPY receptors-induced inhibition of Ca(2+) influx and glutamate release. The KCl-evoked release of glutamate from hippocampal synaptosomes was inhibited by 1 microM NPY and this effect was insensitive to either BIBP3226 (Y1 receptor antagonist) or L-152,804 (Y5 receptor antagonist), but was sensitive to BIIE0246 (Y2 receptor antagonist). We could also pharmacologically dissect the NPY receptors activity by using Y1, Y2 and Y5 receptor agonists ([Leu(31),Pro(34)]NPY, NPY13-36, NPY (19-23)-(Gly(1),Ser(3),Gln(4),Thr(6),Ala(31),Aib(32),Gln(34))-pancreatic polypeptide (PP), respectively), and in all the cases we observed that these agonists could inhibited the KCl-induced release of glutamate. However, the selective and specific co-activation of both Y1 and Y2 or Y2 and Y5 receptors resulted in non-additive inhibition, and this effect was prevented in the presence of the Y2 antagonist, but was insensitive to the Y1 or Y5 receptor antagonist. Moreover, as we previously showed for Y1 receptors, we also observed that the activation of Y5 receptors inhibited the glutamate release in the dentate gyrus and CA3 subregion, without significant effect in the CA1 subregion of the hippocampus. The same qualitative results were obtained when we investigated the role of NPY Y1 and Y2 receptors in modulating the changes in [Ca(2+)](i) due to KCl depolarisation in cultured hippocampal neurons. The inhibitory effect of nitrendipine (L-type VGCC blocker) or omega-conotoxin GVIA (omega-CgTx; N-type VGCC blocker) was not potentiated by the simultaneous activation of Y1 or Y2 receptors. Moreover, the exocytotic release of glutamate was inhibited by omega-agatoxin IVA (omega-Aga; P-/Q-type VGCC blocker), and this VGCC blocker did not potentiate Y1, Y2 or Y5 receptor-mediated inhibition of glutamate release. Also, the effect of ionomycin in inducing the exocytotic release of glutamate from hippocampal synaptosomes was insensitive to the activation of NPY receptors. In the present paper, we identified a role for NPY Y1, Y2 and Y5 receptors in modulating the exocytotic release of glutamate and the [Ca(2+)](i) changes in the rat hippocampus. In conditions of co-activation, there appears to exist a physiological cross-talk between Y1 and Y2 and also between Y2 and Y5 receptors, in which Y2 receptors play a predominant role. Moreover, we also show that Y1 and Y2 receptors exert their inhibitory action by directly modulating L-, N-, and P-/Q-type VGCCs, whereas the inhibition of glutamate release mediated by the Y5 receptors seems to involve P-/Q-type VGCCs.

Cell penetrating peptides (CPPs) have been postulated to carry macromolecules across cell plasma membranes without the need of receptors, transporters, endocytosis or any energy-consuming mechanism. We developed an assay to study lipid bilayer permeation of CPPs. HIV-1 TAT peptides were conjugated to N-(4-carboxy-3-hydroxyphenyl)maleimide (SAM) and incubated with Tb(3+)-containing liposomes. Upon chelation of Tb(3+) by an aromatic carboxylic acid, the fluorescence of Tb(3+) increases many fold. The CPP TAT(44-57)-SAM and TAT(37-53)-SAM, as a negative control, were unable to enter liposomes consisting of phosphatidylcholine (PC) or a mix of PC, negatively charged lipids and cholesterol. In parallel, cell entry of fluorescein-labeled TAT peptides was studied using confocal laser scanning microscopy (CLSM). TAT(44-57)-fluorescein did not enter Madin Darby canine kidney (MDCK) cells with intact plasma membranes but accumulated at their basal side. Only cells with impaired plasma membranes, as identified by nuclear staining with ethidium homodimer-1 (EthD-1), showed accumulation of TAT(44-57). Our findings change the perspectives of the potential use of TAT peptides as carriers for intracellular targeting. SAM- and fluorescein-labeled TAT(44-57) cannot penetrate lipid bilayers and intact plasma membranes of MDCK cells, respectively.

Motivation: The expanding protein sequence and structure databases await methods allowing rapid similarity search. Geometric parameters-dihedral angle between two sequential peptide bond planes (V) and radius of curvature (R) as they appear in pentapeptide fragments in polypeptide chains-are proposed for use in evaluating structural similarity in proteins (VeaR). The parabolic (empirical) function expressing the radius of curvature's dependence on the V-angle in model polypeptides is altered in real proteins in a form characteristic for a particular protein. This can be used as a criterion for judging similarity., Results: A structural comparison of proteins representing a wide spectrum of structures was assessed versus sequence similarity analysis based on the genetic semihomology algorithm. The term 'consensus structure', analogous to 'consensus sequence', was introduced for the serpine family., Availability: Semihom-sequence comparison freely available on request from J. Leluk. VeaR-structural comparison freely available on request from I. Roterman.