Lydiane Funkelstein

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.

MAJOR ROLE OF CATHEPSIN L FOR PRODUCING THE PEPTIDE HORMONES ACTH, β-ENDORPHIN, AND α-MSH, ILLUSTRATED BY PROTEASE GENE KNOCKOUT AND EXPRESSION

The pituitary hormones adrenocorticotropic hormone (ACTH), β-endorphin, and α-melanocyte stimulating hormone (α-MSH) are synthesized by proteolytic processing of their common proopiomelanocortin (POMC) precursor. Key findings from this study show that cathepsin L functions as a major proteolytic enzyme for the production of POMC-derived peptide hormones in secretory vesicles. Specifically, cathepsin L knock-out mice showed major decreases in ACTH, β-endorphin, and α-MSH that were reduced to 23, 18, and 7% of wild-type controls (100%) in pituitary. These decreased peptide levels were accompanied by increased levels of POMC consistent with proteolysis of POMC by cathepsin L. Immunofluorescence microscopy showed colocalization of cathepsin L with β-endorphin and α-MSH in the intermediate pituitary and with ACTH in the anterior pituitary. In contrast, cathepsin L was only partially colocalized with the lysosomal marker Lamp-1 in pituitary, consistent with its extralysosomal function in secretory vesicles. Expression of cathepsin L in pituitary AtT-20 cells resulted in increased ACTH and β-endorphin in the regulated secretory pathway. Furthermore, treatment of AtT-20 cells with CLIK-148, a specific inhibitor of cathepsin L, resulted in reduced production of ACTH and accumulation of POMC. These findings demonstrate a prominent role for cathepsin L in the production of ACTH, β-endorphin, and α-MSH peptide hormones in the regulated secretory pathway.

Human iPSC Neurons Display Activity-Dependent Neurotransmitter Secretion: Aberrant Catecholamine Levels in Schizophrenia Neurons

Summary This study investigated human-induced pluripotent stem cell (hiPSC) -derived neurons for their ability to secrete neurotransmitters in an activity-dependent manner, the fundamental property required for chemical neurotransmission. Cultured hiPSC neurons showed KCl stimulation of activity-dependent secretion of catecholamines—dopamine (DA), norepinephrine (NE), and epinephrine (Epi)—and the peptide neurotransmitters dynorphin and enkephlain. hiPSC neurons express the biosynthetic enzymes for catecholamines and neuropeptides. Because altered neurotransmission contributes to schizophrenia (SZ), we compared SZ to control cultures of hiPSC neurons and found that SZ cases showed elevated levels of secreted DA, NE, and Epi. Consistent with increased catecholamines, the SZ neuronal cultures showed a higher percentage of tyrosine hydroxylase (TH)-positive neurons, the first enzymatic step for catecholamine biosynthesis. These findings show that hiPSC neurons possess the fundamental property of activity-dependent neurotransmitter secretion and can be advantageously utilized to examine regulation of neurotransmitter release related to brain disorders.

Cathepsin L Participates in the Production of Neuropeptide Y in Secretory Vesicles, Demonstrated by Protease Gene Knockout and Expression

Neuropeptide Y (NPY) functions as a peptide neurotransmitter and as a neuroendocrine hormone. The active NPY peptide is generated in secretory vesicles by proteolytic processing of proNPY. Novel findings from this study show that cathepsin L participates as a key proteolytic enzyme for NPY production in secretory vesicles. Notably, NPY levels in cathepsin L knockout (KO) mice were substantially reduced in brain and adrenal medulla by 80% and 90%, respectively. Participation of cathepsin L in producing NPY predicts their colocalization in secretory vesicles, a primary site of NPY production. Indeed, cathepsin L was colocalized with NPY in brain cortical neurons and in chromaffin cells of adrenal medulla, demonstrated by immunofluorescence confocal microscopy. Immunoelectron microscopy confirmed the localization of cathepsin L with NPY in regulated secretory vesicles of chromaffin cells. Functional studies showed that coexpression of proNPY with cathepsin L in neuroendocrine PC12 cells resulted in increased production of NPY. Furthermore, in vitro processing indicated cathepsin L processing of proNPY at paired basic residues. These findings demonstrate a role for cathepsin L in the production of NPY from its proNPY precursor. These studies illustrate the novel biological role of cathepsin L in the production of NPY, a peptide neurotransmitter, and neuroendocrine hormone.

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.

Cathepsin L Plays a Major Role in Cholecystokinin Production in Mouse Brain Cortex and in Pituitary AtT-20 Cells: Protease Gene Knockout and Inhibitor Studies

Cholecystokinin (CCK) is a peptide neurotransmitter whose production requires proteolytic processing of the proCCK precursor to generate active CCK8 neuropeptide in brain. This study demonstrates the significant role of the cysteine protease cathepsin L for CCK8 production. In cathepsin L knockout (KO) mice, CCK8 levels were substantially reduced in brain cortex by an average of 75%. To evaluate the role of cathepsin L in producing CCK in the regulated secretory pathway of neuroendocrine cells, pituitary AtT-20 cells that stably produce CCK were treated with the specific cathepsin L inhibitor, CLIK-148. CLIK-148 inhibitor treatment resulted in decreased amounts of CCK secreted from the regulated secretory pathway of AtT-20 cells. CLIK-148 also reduced cellular levels of CCK9 (Arg-CCK8), consistent with CCK9 as an intermediate product of cathepsin L, shown by the decreased ratio of CCK9/CCK8. The decreased CCK9/CCK8 ratio also suggests a shift in the production to CCK8 over CCK9 during inhibition of cathepsin L. During reduction of the PC1/3 processing enzyme by siRNA, the ratio of CCK9/CCK8 was increased, suggesting a shift to the cathepsin L pathway for the production of CCK9. The changes in ratios of CCK9 compared to CCK8 are consistent with dual roles of the cathepsin L protease pathway that includes aminopeptidase B to remove NH2-terminal Arg or Lys, and the PC1/3 protease pathway. These results suggest that cathepsin L functions as a major protease responsible for CCK8 production in mouse brain cortex, and participates with PC1/3 for CCK8 production in pituitary cells.

Cathepsin L participates in dynorphin production in brain cortex, illustrated by protease gene knockout and expression

Dynorphin opioid neuropeptides mediate neurotransmission for analgesia and behavioral functions. Dynorphin A, dynorphin B, and alpha-neoendorphin are generated from prodynorphin by proteolytic processing. This study demonstrates the significant role of the cysteine protease cathepsin L for producing dynorphins. Cathepsin L knockout mouse brains showed extensive decreases in dynorphin A, dynorphin B, and alpha-neoendorphin that were reduced by 75%, 83%, and 90%, respectively, compared to controls. Moreover, cathepsin L in brain cortical neurons was colocalized with dynorphins in secretory vesicles, the primary site of neuropeptide production. Cellular coexpression of cathepsin L with prodynorphin in PC12 cells resulted in increased production of dynorphins A and B. Comparative studies of PC1/3 and PC2 convertases showed that PC1/3 knockout mouse brains had a modest decrease in dynorphin A, and PC2 knockout mice showed a minor decrease in alpha-neoendorphin. Overall, these results demonstrate a prominent role for cathepsin L, jointly with PC1/3 and PC2, for production of dynorphins in brain.

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.

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.

Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters

Background: Proteases are required to generate peptide neurotransmitters. Results: Human cathepsin V participates in the production of enkephalin and NPY peptide neurotransmitters illustrated by gene expression and silencing. Conclusion: Human cathepsin V participates in producing enkephalin and NPY neurotransmitters in secretory vesicles. Significance: Elucidation of human-specific proteases participating in peptide neurotransmitter production is essential for understanding human health and disease. Proteases are required for processing precursors into active neuropeptides that function as neurotransmitters for cell-cell communication. This study demonstrates the novel function of human cathepsin V protease for producing the neuropeptides enkephalin and neuropeptide Y (NPY). Cathepsin V is a human-specific cysteine protease gene. Findings here show that expression of cathepsin V in neuroendocrine PC12 cells and human neuronal SK-N-MC cells results in production of (Met)enkephalin from proenkephalin. Gene silencing of cathepsin V by siRNA in human SK-N-MC cells results in reduction of (Met)enkephalin by more than 80%, illustrating the prominent role of cathepsin V for neuropeptide production. In vitro processing of proenkephalin by cathepsin V occurs at dibasic residue sites to generate enkephalin-containing peptides and an ∼24-kDa intermediate present in human brain. Cathepsin V is present in human brain cortex and hippocampus where enkephalin and NPY are produced and is present in purified human neuropeptide secretory vesicles. Colocalization of cathepsin V with enkephalin and NPY in secretory vesicles of human neuroblastoma cells was illustrated by confocal microscopy. Furthermore, expression of cathepsin V with proNPY results in NPY production. These findings indicate the unique function of human cathepsin V for producing enkephalin and NPY neuropeptides required for neurotransmission in health and neurological diseases.