title: Cathepsin L expression is directed to secretory vesicles for enkephalin neuropeptide biosynthesis and secretion.
abstract: Proteases within secretory vesicles are required for conversion of neuropeptide precursors into active peptide neurotransmitters and hormones. This study demonstrates the novel cellular role of the cysteine protease cathepsin L for producing the (Met)enkephalin peptide neurotransmitter from proenkephalin (PE) in the regulated secretory pathway of neuroendocrine PC12 cells. These findings were achieved by coexpression of PE and cathepsin L cDNAs in PC12 cells with analyses of PE-derived peptide products. Expression of cathepsin L resulted in highly increased cellular levels of (Met)enkephalin, resulting from the conversion of PE to enkephalin-containing intermediates of 23, 18-19, 8-9, and 4.5 kDa that were similar to those present in vivo. Furthermore, expression of cathepsin L with PE resulted in increased amounts of nicotine-induced secretion of (Met)enkephalin. These results indicate increased levels of (Met)enkephalin within secretory vesicles of the regulated secretory pathway. Importantly, cathespin L expression was directed to secretory vesicles, demonstrated by colocalization of cathepsin L-DsRed fusion protein with enkephalin and chromogranin A neuropeptides that are present in secretory vesicles. In vivo studies also showed that cathepsin L in vivo was colocalized with enkephalin. The newly defined secretory vesicle function of cathepsin L for biosynthesis of active enkephalin opioid peptide contrasts with its function in lysosomes for protein degradation. These findings demonstrate cathepsin L as a distinct cysteine protease pathway for producing the enkephalin member of neuropeptides.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences and Departments of Pharmacology, Neuroscience, and Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093.
authors.forename: Shin-Rong
authors.initials: SR
authors.lastname: Hwang
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences and Departments of Pharmacology, Neuroscience, and Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093.
authors.forename: Christina
authors.initials: C
authors.lastname: Garza
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences and Departments of Pharmacology, Neuroscience, and Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093.
authors.forename: Charles
authors.initials: C
authors.lastname: Mosier
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences and Departments of Pharmacology, Neuroscience, and Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093.
authors.forename: Thomas
authors.initials: T
authors.lastname: Toneff
authors.affiliation: Buck Institute, Novato, California 94545.
authors.forename: Eric
authors.initials: E
authors.lastname: Wunderlich
authors.affiliation: College of Pharmacy, Touro University, Vallejo, California 94592.
authors.forename: Paul
authors.initials: P
authors.lastname: Goldsmith
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences and Departments of Pharmacology, Neuroscience, and Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093. Electronic address: vhook@ucsd.edu.
authors.forename: Vivian
authors.initials: V
authors.lastname: Hook
mesh_terms: D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology; D002417:Cattle; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology; D004745:Enkephalins / Q000096:biosynthesis* / Q000378:metabolism*; D004792:Enzyme Precursors / Q000502:physiology; D008247:Lysosomes / Q000201:enzymology; D016716:PC12 Cells; D011506:Proteins / Q000378:metabolism; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D004745:Enkephalins; D004792:Enzyme Precursors; D011506:Proteins; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534280:Ctsl protein, rat