Steven Bark

Proteases for Processing Proneuropeptides into Peptide Neurotransmitters and Hormones

Peptide neurotransmitters and peptide hormones, collectively known as neuropeptides, are required for cell-cell communication in neurotransmission and for regulation of endocrine functions. Neuropeptides are synthesized from protein precursors (termed proneuropeptides or prohormones) that require proteolytic processing primarily within secretory vesicles that store and secrete the mature neuropeptides to control target cellular and organ systems. This review describes interdisciplinary strategies that have elucidated two primary protease pathways for prohormone processing consisting of the cysteine protease pathway mediated by secretory vesicle cathepsin L and the well-known subtilisin-like proprotein convertase pathway that together support neuropeptide biosynthesis. Importantly, this review discusses important areas of current and future biomedical neuropeptide research with respect to biological regulation, inhibitors, structural features of proneuropeptide and protease interactions, and peptidomics combined with proteomics for systems biological approaches. Future studies that gain in-depth understanding of protease mechanisms for generating active neuropeptides will be instrumental for translational research to develop pharmacological strategies for regulation of neuropeptide functions. Pharmacological applications for neuropeptide research may provide valuable therapeutics in health and disease.

Secretory vesicle aminopeptidase B related to neuropeptide processing: molecular identification and subcellular localization to enkephalin- and NPY-containing chromaffin granules

Biosynthesis of peptide hormones and neurotransmittters involves proteolysis of proprotein precursors by secretory vesicle cathepsin L. Cathepsin L generates peptide intermediates with basic residues at their NH2‐termini, indicating that Arg/Lys aminopeptidase is needed to generate the smaller biologically active peptide. Therefore, this study identified the Arg/Lys aminopeptidase that is present in secretory vesicles of adrenal medulla and neuroendocrine tissues, achieved by molecular cloning and localization in ‘model’ neuropeptide‐containing secretory vesicles (bovine). Molecular cloning of the bovine aminopeptidase B (AP‐B) cDNA defined its primary sequence that allowed selection of antisera for immunolocalization studies. AP‐B was present in secretory vesicles that contain cathepsin L with the neuropeptides enkephalin and neuropeptide Y. The AP‐B in several neuroendocrine tissues was detected by western blots. Recombinant bovine AP‐B showed preference for Arg‐methylcoumarinamide substrate. AP‐B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP‐B showed similar activities for Arg‐(Met)enkephalin (ME) and Lys‐ME neuropeptide substrates to generate ME, while rat AP‐B preferred Arg‐ME. Furthermore, AP‐B possesses an acidic pH optimum of 5.5–6.5 that is similar to the internal pH of secretory vesicles. The significant finding of the secretory vesicle localization of AP‐B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides.

Cysteine Cathepsins in the Secretory Vesicle Produce Active Peptides: Cathepsin L Generates Peptide Neurotransmitters and Cathepsin B Produces Beta-Amyloid of Alzheimer’s Disease

Recent new findings indicate significant biological roles of cysteine cathepsin proteases in secretory vesicles for production of biologically active peptides. Notably, cathepsin L in secretory vesicles functions as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell-cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in secretory vesicles has been recently identified as a β-secretase for production of neurotoxic β- amyloid (Aβ) peptides that accumulate in Alzheimers disease (AD), participating as a notable factor in the severe memory loss in AD. These secretory vesicle functions of cathepsins L and B for production of biologically active peptides contrast with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique secretory vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin functions. Features of the secretory vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Proteomics of dense core secretory vesicles reveal distinct protein categories for secretion of neuroeffectors for cell-cell communication.

Regulated secretion of neurotransmitters and neurohumoral factors from dense core secretory vesicles provides essential neuroeffectors for cell-cell communication in the nervous and endocrine systems. This study provides comprehensive proteomic characterization of the categories of proteins in chromaffin dense core secretory vesicles that participate in cell-cell communication from the adrenal medulla. Proteomic studies were conducted by nano-HPLC Chip MS/MS tandem mass spectrometry. Results demonstrate that these secretory vesicles contain proteins of distinct functional categories consisting of neuropeptides and neurohumoral factors, protease systems, neurotransmitter enzymes and transporters, receptors, enzymes for biochemical processes, reduction/oxidation regulation, ATPases, protein folding, lipid biochemistry, signal transduction, exocytosis, calcium regulation, as well as structural and cell adhesion proteins. The secretory vesicle proteomic data identified 371 proteins in the soluble fraction and 384 membrane proteins, for a total of 686 distinct secretory vesicle proteins. Notably, these proteomic analyses illustrate the presence of several neurological disease-related proteins in these secretory vesicles, including huntingtin interacting protein, cystatin C, ataxin 7, and prion protein. Overall, these findings demonstrate that multiple protein categories participate in dense core secretory vesicles for production, storage, and secretion of bioactive neuroeffectors for cell-cell communication in health and disease.

NeuroPedia: neuropeptide database and spectral library

SUMMARY Neuropeptides are essential for cell-cell communication in neurological and endocrine physiological processes in health and disease. While many neuropeptides have been identified in previous studies, the resulting data has not been structured to facilitate further analysis by tandem mass spectrometry (MS/MS), the main technology for high-throughput neuropeptide identification. Many neuropeptides are difficult to identify when searching MS/MS spectra against large protein databases because of their atypical lengths (e.g. shorter/longer than common tryptic peptides) and lack of tryptic residues to facilitate peptide ionization/fragmentation. NeuroPedia is a neuropeptide encyclopedia of peptide sequences (including genomic and taxonomic information) and spectral libraries of identified MS/MS spectra of homolog neuropeptides from multiple species. Searching neuropeptide MS/MS data against known NeuroPedia sequences will improve the sensitivity of database search tools. Moreover, the availability of neuropeptide spectral libraries will also enable the utilization of spectral library search tools, which are known to further improve the sensitivity of peptide identification. These will also reinforce the confidence in peptide identifications by enabling visual comparisons between new and previously identified neuropeptide MS/MS spectra. AVAILABILITY http://proteomics.ucsd.edu/Software/NeuroPedia.html CONTACT bandeira@ucsd.edu SUPPLEMENTARY INFORMATION Supplementary materials are available at Bioinformatics online.

Mass Spectrometry-Based Neuropeptidomics of Secretory Vesicles from Human Adrenal Medullary Pheochromocytoma Reveals Novel Peptide Products of Prohormone Processing

Neuropeptides are required for cell-cell communication in the regulation of physiological and pathological processes. While selected neuropeptides of known biological activities have been studied, global analyses of the endogenous profile of human peptide products derived from prohormones by proteolytic processing in vivo are largely unknown. Therefore, this study utilized the global, unbiased approach of mass spectrometry-based neuropeptidomics to define peptide profiles in secretory vesicles, isolated from human adrenal medullary pheochromocytoma of the sympathetic nervous system. The low molecular weight pool of secretory vesicle peptides was subjected to nano-LC-MS/MS with ion trap and QTOF mass spectrometry analyzed by different database search tools (InsPecT and Spectrum Mill). Peptides were generated by processing of prohormones at dibasic cleavage sites as well as at nonbasic residues. Significantly, peptide profiling provided novel insight into newly identified peptide products derived from proenkephalin, pro-NPY, proSAAS, CgA, CgB, and SCG2 prohormones. Previously unidentified intervening peptide domains of prohormones were observed, thus providing new knowledge of human neuropeptidomes generated from precursors. The global peptidomic approach of this study demonstrates the complexity of diverse neuropeptides present in human secretory vesicles for cell-cell communication.

Detecting low-abundance vasoactive peptides in plasma: Progress toward absolute quantitation using nano liquid chromatography–mass spectrometry

Profiling changes in the concentration of functionally related peptide hormones is critical to understanding the etiology of many diseases and therapies. We present novel data using nano liquid chromatography-mass spectrometry (LC-MS) to simultaneously measure a select group of vasoactive peptides (angiotensin, bradykinin, and related hormones) in 50-microl plasma samples, enabling repeated sampling in rodent models. By chromatographically resolving target peptides and using multiple reaction monitoring to enhance MS sensitivity, linear responses down to 10(-17) mol were achieved. Purification of plasma peptides by either methanol precipitation or off-line high-performance liquid chromatography (HPLC) fractionation enabled the detection of endogenous peptides and revealed approaches for enhancing recovery. As proof of principle, seven vasoactive peptides were profiled before, during, and after acute angiotensin-converting enzyme (ACE) inhibition in an anesthetized rat. Of note was an apparent 10-fold increase in vasodilatory bradykinin that reversed after drug infusion but relatively minor changes in angiotensin II levels. Targeted MS analysis used to profile functionally related peptides or other analytes will greatly enhance our ability to define the sequence of events regulating complex and dynamic physiological processes.

Neuropeptidomic Components Generated by Proteomic Functions in Secretory Vesicles for Cell–Cell Communication

Diverse neuropeptides participate in cell–cell communication to coordinate neuronal and endocrine regulation of physiological processes in health and disease. Neuropeptides are short peptides ranging in length from ~3 to 40 amino acid residues that are involved in biological functions of pain, stress, obesity, hypertension, mental disorders, cancer, and numerous health conditions. The unique neuropeptide sequences define their specific biological actions. Significantly, this review article discusses how the neuropeptide field is at the crest of expanding knowledge gained from mass-spectrometry-based neuropeptidomic studies, combined with proteomic analyses for understanding the biosynthesis of neuropeptidomes. The ongoing expansion in neuropeptide diversity lies in the unbiased and global mass-spectrometry-based approaches for identification and quantitation of peptides. Current mass spectrometry technology allows definition of neuropeptide amino acid sequence structures, profiling of multiple neuropeptides in normal and disease conditions, and quantitative peptide measures in biomarker applications to monitor therapeutic drug efficacies. Complementary proteomic studies of neuropeptide secretory vesicles provide valuable insight into the protein processes utilized for neuropeptide production, storage, and secretion. Furthermore, ongoing research in developing new computational tools will facilitate advancements in mass-spectrometry-based identification of small peptides. Knowledge of the entire repertoire of neuropeptides that regulate physiological systems will provide novel insight into regulatory mechanisms in health, disease, and therapeutics.

Linear and Accurate Quantitation of Proenkephalin-Derived Peptides by Isotopic Labeling with Internal Standards and Mass Spectrometry

Proenkephalin (PE) represents the precursor protein of the active peptide neurotransmitter enkephalin. Quantitative analysis of peptides and proteins is an objective of mass spectrometry-based studies of biological systems and will be important for studying the proteolytic conversion of proproteins to active enkephalin and neuropeptides. The goal of this study was to define and optimize quantitation of different amounts of tryptic peptides derived from PE using light (H4, 4 hydrogens) and heavy (D4, 4 deuteriums) succinic anhydride for isotopic labeling of peptides analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Comparisons were made between PE-derived peptides with and without internal standards. Importantly, incorporation of internal standards of known amounts of heavy isotope-labeled tryptic peptides of PE provided linear calibration plots with accurate quantitation. In contrast, comparison of light and heavy isotope-labeled peptides without internal standards produced variable and inaccurate nonlinear isotopic ratio comparisons of PE-derived peptides. These results demonstrate that use of internal standards composed of a defined amount(s) of standard peptides (PE-derived tryptic peptides) is necessary for high-quality linear quantitation of peptides by isotopic labeling and MS/MS.

The Protein Architecture of Human Secretory Vesicles Reveals Differential Regulation of Signaling Molecule Secretion by Protein Kinases

Secretory vesicles are required for release of chemical messengers to mediate intercellular signaling among human biological systems. It is necessary to define the organization of the protein architecture of the ‘human’ dense core secretory vesicles (DCSV) to understand mechanisms for secretion of signaling molecules essential for cellular regulatory processes. This study, therefore, conducted extensive quantitative proteomics and systems biology analyses of human DCSV purified from human pheochromocytoma. Over 600 human DCSV proteins were identified with quantitative evaluation of over 300 proteins, revealing that most proteins participate in producing peptide hormones and neurotransmitters, enzymes, and the secretory machinery. Systems biology analyses provided a model of interacting DCSV proteins, generating hypotheses for differential intracellular protein kinases A and C signaling pathways. Activation of cellular PKA and PKC pathways resulted in differential secretion of neuropeptides, catecholamines, and β-amyloid of Alzheimers disease for mediating cell-cell communication. This is the first study to define a model of the protein architecture of human DCSV for human disease and health.