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J. Biol. Chem., Vol. 282, Issue 13, 9556-9563, March 30, 2007

This Article Full Text Full Text (PDF) All Versions of this Article: 282/13/9556    most recent M605510200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hwang, S.-R. Articles by Hook, V. Search for Related Content PubMed PubMed Citation Articles by Hwang, S.-R. Articles by Hook, V. Social Bookmarking                      What's this?

Cathepsin L Expression Is Directed to Secretory Vesicles for Enkephalin Neuropeptide Biosynthesis and Secretion^*

Shin-Rong Hwang, Christina Garza, Charles Mosier, Thomas Toneff, Eric Wunderlich, Paul Goldsmith^¶, and Vivian Hook¹

From the 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, the Buck Institute, Novato, California 94545, and the ^¶College of Pharmacy, Touro University, Vallejo, California 94592

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.

Received for publication, June 8, 2006 , and in revised form, December 27, 2006.

^* This research was supported by grants from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Skaggs School of Pharmacy and Pharmaceutical Sciences, 9500 Gilman Dr. MC 0744, University of California, San Diego, La Jolla, CA 92093-0744. Tel.: 858-822-6682; Fax: 858-822-6681; E-mail: vhook{at}ucsd.edu.

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