Secretory granules of chromaffin cells contain catecholamines and several antimicrobial peptides derived from chromogranins and proenkephalin-A. These peptides are secreted in the extracellular medium following exocytosis. Here, we show that ubiquitin is stored in secretory chromaffin granules and released into the circulation upon stimulation of chromaffin cells. We also show that the C-terminal fragment (residues 65-76) of ubiquitin displays, at the micromolar range, a lytic antifungal activity. Using confocal laser scan microscopy and rhodamine-labeled synthetic peptides, we could demonstrate that the C-terminal peptide (residues 65-76) is able to cross the cell wall and the plasma membrane of fungi and to accumulate in fungi, whereas the N-terminal peptide (residues 1-34) is stopped at the fungal wall level. Furthermore, these two peptides act synergistically to kill filamentous fungi. Because of the interaction of the C-terminal sequence of ubiquitin with calmodulin, the synthetic peptide (residues 65-76) was tested in vitro against calmodulin-dependent calcineurin, an enzyme crucial for fungal growth. This peptide was found to inhibit the phosphatase activity of calcineurin. Our data show a new property of ubiquitin C-terminal-derived peptide (65-76) that could be used with N-terminal peptide (1-34) as a new potent antifungal agent.

In order to examine thoroughly the antifungal activity of chromofungin, confocal laser microscopy was used to demonstrate the ability of the rhodamine-labeled peptide to interact with the fungal cell wall, to cross the plasma membrane, and to accumulate in Aspergillus fumigatus, Alternaria brassicola, and Candida albicans. Our present data reveal that chromofungin inhibits calcineurin activity, extending a previous observation that the N-terminal region of chromogranin A interacts with calmodulin in the presence of calcium. Therefore, the destabilization of fungal wall and plasma membrane, together with the possible intracellular inhibition of calmodulin-dependent enzymes, is likely to represent the mechanism by which vasostatin-I and chromofungin exert antifungal activity.

The chromaffin granules have been shown to be an excellciit model to study the processing of proenkephalin-A and chromogranins. Recently, we reported a study dealing with the processing of chromogranin B/secretogranin 1 and the occurrence of the C-tertninal chromogranin B-derivcd peptide 614-626 which was shown to have antibacterial iictivity [Strub, J. M., Garcia-Sablonc, P., Looning, K., Taupenol, L., Hubert, P., Van Riochem. 229,. We also observed that this new antibacterial activity prcsent in chromaffin granules was associated with other endogenous protein-derived fragments yet to be characterizcd. The present study reports the isolation rind characterization of a pcptide which possesses antibacterial activity and which corresponds to the C-tcrniinal 209-237 sequence ol' procnkephalin-A. A detailcd study using niicroscquciicing and matrix-assisted-laser-desorptioii time-of-tlight Inass spectrometry (MALD-TOF MS) allowed us to correlate the antibacterial activity of this peptide named enkelytin (FAEPTBSEEEGE-SYSKEVPEMEKRYGGFM) with post-translational modifications. Endogenouc bisphosphorylated proenkephalin-A-(209 -237) was activc on Mirrocwcc.i~,r luteus and Bocillms negaterium killing bacteria in the 0.2-0.4 pM range but was inactive in similar conditions towards Excherichia coli. Eiikelytiii shares sequence and structiiral similarities with thc antibacterial C-terminal domain o l diazcpam-binding inhihitor. According to this similarity, a prediction of secondary structure is proposed for enkelylin and discussed in relation to its biological activity.KtJywor~1,s: bovinc adrenal medulla; chromaffin granules ; proenkephalin-A-derived fragments ; diazepamhinding inhibitor; antibacterial peptides.A large number of biologically active peptides are synthe-chrornaffin granules, these organelles havc been shown to reprcsized as part of much larger precursor inolecules (Mains ct al., sent iin excellent model LO study thc intragranular processing of 1983) and the conversion of thesc prohormones to active pep-thesc protcins. tides requires transfer from the rough endoplasmic reticulum to Chroiiiograniiis are widely distributed in endocrine cells and the Golgi apparatus, packaging into secretory granules (Gainer in neurons (Simon and Aunis, 1989). The function of each of the et d., 1985), limited proteolysis by highly specific proreascs different members is still elusive but thcy are iiow considered to &oh et al., 198s) and further dcgradation in the extracellular be precursor molecules (Dillen ct al., 1993). In bovine adrenal space after exocytotic rcleasc. The secretory granules of the bo-medulla, the major component, chrornogranin A represents 40%) vine adrenal medullary chromaffin cell contain a complex mix-of total soluble granulc proteins. Examination of the cDNA seture of secretory products which includc low-molecular-mass quence of human and bovine chromogranin A revcals thc presconstituents such as catecholamines, ascorbnte, nucltotidt.s, ence (if conservccl pairs of basic amino acids which represent ca...

The antifungal peptide named chromofungin is the most active vasostatin-I-derived peptide, corresponding to the sequence 47-66 of chromogranin A. (1)H-NMR analysis revealed that it adopts a helical structure. The mechanism implicated in the interaction of chromofungin with fungi and yeast cells was studied by penetration of monolayers and confocal laser microscopy. Chromofungin is able to interact with the cell wall, to cross the plasma membrane, to accumulate in the microorganism, and to inhibit calcineurin activity.