Proteases for processing proneuropeptides into peptide neurotransmitters and hormones

Abstract

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.

Figure 1. Peptide neurotransmitters in brain

Neuropeptides in brain function as peptide neurotransmitters to mediate chemical communications among neurons. Neuropeptides are synthesized within secretory vesicles that are transported from the neuronal cell body via the axon to nerve terminals. The proneuropeptide (or prohormone) is packaged with 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.

Figure 2. Peptide hormones in endocrine systems

Neuropeptides function as peptide hormones to mediate cell-cell communication in peripheral endocrine systems. For example, the hypothalamo-neurohypophyseal system regulates the pituitary-adrenal axis by secretion CRF from the hypothalamus region of brain to induce secretion of the peptide hormone ACTH from the pituitary. Released ACTH targets the adrenal cortex for stimulation of glucocorticoid production; resultant increases in plasma glucocorticoid participates in feedback inhibition of CRF and ACTH to maintain constant levels of glucocorticoid. Numerous peptide hormones regulate physiological functions.

Figure 3. Proneuropeptides: structural features for proteolytic processing

Neuropeptides are synthesized as proneuropeptide precursors, also known as prohormones, that require proteolytic processing to liberate the active neuropeptide. Proteolytic processing occurs at dibasic and monobasic sites, as well as at multibasic sites. The precursor proteins may contain one copy of the active neuropeptide, such as the proneuropeptides for NPY, galanin, CRF, and vasopressin. 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 (LE), 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.

Figure 4. Cysteine and subtilisin-like protease pathways for proneuropeptide processing

Distinct cysteine protease and subtilisin-like protease pathways have been demonstrated for proneuropeptide processing. Recent studies have identified secretory vesicle cathepsin L as an important processing enzyme for the production of the endogenous enkephalin opioid peptide. 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. The well-established subtilisin-like protease pathway involves several prohormone convertases (PC). PC1/3 and PC2 have been characterized as neuroendocrine processing proteases (processing in neuroendocrine tissues also involves PC5 (135)). 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/H.

Figure 5. Activity-based profiling for identification of proenkephalin cleaving activity as cathepsin L

Activity-based profiling (APB) utilizes the strategy of labeling the active site of active proteases, often with an inhibitor-related probe, to identify proteolytic activity. Inhibition of proenkephalin cleaving activity by the cysteine protease inhibitor E64c in isolated chromaffin secretory vesicles (also known as chromaffin granules) allowed affinity labeling of the 27 kDa active protease enzyme proteins by a biotinylated form of E64 known as DCG-04 (panel a). The inhibitor-labeled proteins were separated by 2-D gels (panel b) and subjected to peptide sequencing by mass spectrometry, revealing the identity of the proneuropeptide processing activity as cathepsin L.

Figure 6. Localization of cathepsin L to neuropeptide-containing secretory vesicles

(a) Colocalization of cathepsin L with enkephalin in chromaffin cells demonstrated by confocal immunofluorescence microscopy. Cathepsin L and (Met)enkephalin (green and red fluorescence, respectively) in chromaffin cells were visualized by immunofluorescence confocal microscopy. Excellent colocalization of cathepsin L and (Met)enkephalin was demonstrated by the merged images with colocalization indicated by yellow fluorescence. In chromaffin cells, the majority of cathepsin L is colocalized with (Met)enkephalin within secretory vesicles. (b) Immunoelectron microscopy demonstrates colocalization of cathepsin L with the (Met)enkephalin neuropeptide in secretory vesicles. Cathepsin L localization was indicated by labeling with 15 nm colloidal gold-conjugated anti-rabbit, and (Met)enkephalin (ME) was detected as 6 nm gold particles conjugated to anti-mouse. The presence of both 15- and 6-nm cold particles within these vesicles demonstrated the colocalization of cathepsin L with the enkephalin neuropeptide in secretory vesicles.

Figure 7. Proteomics reveals functional secretory vesicle protein systems for neuropeptide biosynthesis, storage, and secretion

To explore the in vivo protein environment and composition of neuropeptide-synthesizing secretory vesicles, chromaffin secretory vesicles (also known as chromaffin granules) were isolated and subjected to proteomic analyses of proteins in the soluble and membrane components of the vesicles. Based on the knowledge that the primary function of the secretory vesicle organelle is to produce, store, and release active neuropeptides, proteins obtained from proteomic data were organized into functional categories to represent formation of secretory vesicles, neuropeptide biosynthesis, and exocytotic mechanisms for regulated secretion. Protein systems in secretory vesicle function consisted of those for (1) production of hormones, neurotransmitters, and neuromodulatory factors, (2) generating selected internal vesicular conditions for reducing condition, acidic pH conditions maintained by ATPases, and chaperones for protein folding, and (3) vesicular trafficking mechanisms to allow mobilization of secretory vesicles for exocytosis, which utilizes proteins for nucleotide-binding, calcium regulation, and vesicle exocytosis. These protein systems are coordinated to allow the secretory vesicle to synthesize and release of neuropeptides for cell-cell communication in the control of neuroendocrine functions.