Neuropeptidomic components generated by proteomic functions in secretory vesicles for cell-cell communication

Abstract

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.

Fig. 1

Neuropeptides for neuronal and endocrine cell–cell communication. a Neuropeptides, peptide neurotransmitters, in the central nervous system of brain. Brain neuropeptides function as peptide neurotransmitters to mediate chemical cell–cell communications among neurons. Neuropeptides are synthesized within secretory vesicles that are transported from the neuronal cell body via the axon to nerve terminals. The prohormone (also known as proneuropeptide) is packaged within the newly formed secretory vesicle in the cell body, and proteolytic processing of the precursor protein occurs during axonal transport and maturation of the secretory vesicle. Mature processed neuropeptides are contained within secretory vesicles at the synapse where activity-dependent, regulated secretion of neuropeptides occurs to mediate neurotransmission via neuropeptide activation of peptidergic receptors. b Neuropeptides, peptide neurotransmitters and peptide hormones, in the peripheral nervous system and endocrine systems for regulation of physiological organ functions. The peripheral nervous system regulates all organ systems, linking the central nervous system of the brain with peripheral neuronal control of physiological functions. In the body, neuropeptides also function as hormones that mediate endocrine cell–cell communication

Fig. 2

Prohormone precursors undergo proteolysis to generate active neuropeptides. Neuropeptides are synthesized as inactive preprohormone precursors that undergo removal of the N-terminal signal peptide sequence in the rough endoplasmic reticulum to generate the prohormone precursors (–11). The prohormones, also known as proneuropeptides, undergo proteolytic processing at dibasic and monobasic cleavage sites to liberate the active neuropeptides. The precursor proteins may contain one copy of the active neuropeptide, such as the proneuropeptides for NPY, galanin, CRF, and VIP. Some proneuropeptides such as proenkephalin contains multiple copies of the active neuropeptide; proenkephalin contains four copies of (Met)enkephalin (ME), one copy of (Leu)enkephalin (L), and the related opioid peptides ME-Arg-Phe (H) and ME-Arg-Gly-Cleu (O). Certain precursors contain different peptide hormones within the same precursor, such as the POMC precursor which gives rise to the distinct peptide hormones ACTH, α-MSH, and ß-endorphin. The presence of ACTH in anterior pituitary and the presence of α-MSH and ß-endorphin in intermediate pituitary illustrate that tissue-specific processing of the POMC prohormone occurs

Fig. 3

Neuropeptidomic analyses of human proenkephalin-derived peptides in secretory vesicles. Neuropeptidomic studies investigated endogenous peptides derived from human proenkephalin in chromaffin secretory vesicles. Endogenous peptides derived from human PE in human adrenal medullary secretory vesicles (purified from human pheochromocytoma tissue) are illustrated with respect to their location within PE. Peptides were identified by ion-trap and QTOF MS/MS, combined with InsPecT (Ins) and Spectrum Mill (SM) bioinformatic analyses of MS/MS data at 1% false discovery rate (FDR; with the exception of (Leu)enkephalin that was indicated at 5% FDR) (18). Peptides identified under each of these conditions were mapped to PE, illustrated by colored lines: QTOF MS/MS data analyzed by InsPect (Ins, orange) or Spectrum Mill (SM, yellow) and ion-trap (Trap) analyzed by InsPect (Ins, green) or SM (olive). Within PE, the active enkephalin neuropeptides sequences are shown in yellow. Dibasic cleavage sites are highlighted by boxes; in addition, monobasic residues within PE are shown. (Hyphens at the end of some lines indicate peptides that were split between two lines in the figure.)

Fig. 4

Neuropeptidomic analyses of human chromogranin-A-derived peptides in secretory vesicles. Neuropeptidomics studies investigated endogenous peptides derived from human CgA in chromaffin secretory vesicles. CgA-derived peptides in human adrenal medullary secretory vesicles (purified from human pheochromocytoma tissue) identified in neuropeptidomic studies are illustrated within the CgA precursor (18). Peptides were identified (as described in legend of Fig. 3) by ion-trap and QTOF MS/MS, combined with InsPecT (Ins) bioinformatic analyses of MS/MS data at 1% FDR. Peptides identified under each of these conditions were mapped to CgA, illustrated by colored lines: QTOF MS/MS data analyzed by InsPect (Ins, purple) and ion-trap (Trap) MS/MS data analyzed by InsPect (Ins, olive green). Within CgA, names of known peptide sequences are indicated. Dibasic cleavage sites are highlighted by boxes

Fig. 5

Multiple vasoactive peptide hormones regulated by ACE inhibitor drug therapeutics. The effects of an ACE inhibitor, captopril, on levels of plasma vasoactive peptides were analyzed in time course studies by nano-LC-MS/MS with quantitation using stable isotope-labeled internal peptide standards (29). ACE inhibitors are utilized as antihypertensive drugs. Chromatographic separation of target peptides and MRM provided quantitation of Ang I, Ang II, Ang1–7, BK 1–8, BK 2–9, and kallidin (KD). Results show significant reduction by the ACE inhibitor of the angiotensin peptides, with an interesting concomitant increase in plasma bradykinins and kallidin (potent vasodilators). The percent change in plasma concentration at different times after drug administration is shown in the table below the bar graph. Results illustrate the utility of simultaneous profiling of multiple peptides using mass spectrometry analysis to monitor drug-induced changes in vasoactive neuropeptides

Fig. 6

Protease pathways for neuropeptide production. Distinct cysteine protease and subtilisin-like protease pathways participate in prohormone processing (–11). The cysteine protease cathepsin L in secretory vesicles functions as a processing enzyme for the production of neuropeptides. Preference of cathepsin L to cleave at the NH₂-terminal side of dibasic residue processing sites yields peptide intermediates with NH₂-terminal residues, which are removed by Arg/Lys aminopeptidase that has been identified as aminopeptidase B. Cathepsin L also cleaves between the dibasic residues, resulting in peptide intermediates that then require both aminopeptidase and carboxypeptidase E to remove NH₂-terminal and C-terminal basic residues to generate active neuropeptides. The subtilisin-like protease pathway involves the prohormone convertases PC1/3 and PC2. The PC enzymes preferentially cleave at the COOH-terminal side of dibasic processing sites, which results in peptide intermediates with basic residue extensions at their COOH termini that are removed by carboxypeptidase E

Fig. 7

The secretory vesicle proteome for neuropeptidome biosynthesis and secretion. Proteins of the secretory vesicle, known as the neuroproteome, participate in the biosynthesis, storage, and regulated secretion of neuropeptides, known as the neuropeptidome. Thus, the neuropeptidome is generated and secreted by the neuroproteome of regulated secretory vesicles. The neuroproteome consists of soluble and membrane proteins that participate in secretory vesicle functions for providing neuropeptides for cell–cell communication in the nervous and endocrine systems. Proteomic studies of the soluble and membrane fractions of neuropeptide secretory vesicles isolated from adrenal medullary chromaffin cells of the sympathetic nervous system (bovine) indicate the protein systems participating in neuropeptide production and secretion (illustrated in the pie charts) that include neuropeptides and neurohumoral factors, proteases, neurotransmitters enzymes and transporters, receptors, enzymes, carbohydrate functions, lipids, reduction oxidation, ATPases and nucleotide metabolism, protein folding, signal transduction and GTP-binding proteins, vesicular trafficking and exocytosis, structural proteins, and cell adhesion proteins (48,49)