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The N- and C-terminal fragments of ubiquitin are important for the antimicrobial activities¹

ANNE-ESTELLE KIEFFER^*, YANNICK GOUMON^*, OLIVIER RUH^*, SYLVETTE CHASSEROT-GOLAZ, GÉRARD NULLANS^*, CLAIRE GASNIER^*, DOMINIQUE AUNIS^* and MARIE-HÉLÉNE METZ-BOUTIGUE^*,2

^* INSERM Unité 575, IFR 37, Physiopathologie du Système Nerveux, Strasbourg Cedex, France; and
CNRS UPR 2356, IFR 37, Neurotransmission et Sécrétion Neuroendocrine, Strasbourg, France

²Correspondence: INSERM Unité 575, IFR 37, Physiopathologie du Système Nerveux, 5 rue Blaise Pascal 67084 Strasbourg Cedex, France. E-mail: metz{at}neurochem.u-strasbg.fr

SPECIFIC AIMS

Secretory granules of chromaffin cells of the adrenal medulla store catecholamines and a variety of antimicrobial peptides, which are coreleased in the extracellular medium upon exocytosis. We have shown that free ubiquitin (Ub) is stored in chromaffin granules and released into the circulation upon stimulation of chromaffin cells. Unexpectedly, we found that free Ub displays antibacterial and antifungal activities. Using different biochemical methods and confocal laser scan microscopy, we have (i) identified the domains of Ub that are important for the expression of the antifungal activity (Ub_1–34 and Ub_65–76), (ii) analyzed the effect of the corresponding synthetic peptides against fungal cell wall and membranes, and (iii) examined in vitro the calcineurin activity in the presence of free Ub and the synthetic lytic peptide Ub_65–76.

PRINCIPAL FINDINGS

1. Ub is an antimicrobial component present in secretions from stimulated adrenal medullary chromaffin cells
Chromaffin cells were washed four times with Locke’s solution to clean out dead cells or cell debris. Then, chromaffin cells were stimulated for 10 min with 20 µM nicotine in Locke’s, and their secretions were carefully collected, centrifuged at 800 gduring 10 min at 4°C to remove cells that might be present, and separated by high-pressure liquid chromatography (HPLC) on a reverse-phase column. In addition to catecholamines, secreted material contains numerous peptides with antimicrobial activity. We focused on the active fraction eluted at 37% acetonitrile. Automatic Edman degradation indicated the presence of a predominant sequence corresponding to the N-terminal end of bovine Ub. The mass spectrometry analysis, using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) technique, revealed an experimental molecular mass value of 8566 Da, in complete agreement with the theoretical molecular mass of Ub_1–76 (8565 Da). In control experiments, Ub was not detectable in the extracellular medium from nonstimulated chromaffin cells, revealing that Ub is not constitutively released. A more complete localization of free Ub in chromaffin cell has been achieved after Western blot by comparing its distribution with that of immunomarkers to cytoplasm (anti-phenylethanolamine N-methyltransferase), granule membrane (anti-dopamine-ß-hydroxylase), and intragranular matrix fraction [anti-chromogranin A (anti-CGA)]. Free Ub was immunodetected with a polyclonal anti-Ub in the soluble intragranular fraction, confirming its presence in secretions of stimulated chromaffin cells after exocytosis. Free Ub present in the granule matrix was evaluated to represent 12% of cytoplasmic-free Ub and 5% of the intragranular CGA.

2. Antimicrobial activities of bovine Ub and Ub_65–76
Free, Ub-enriched HPLC fraction displays antibacterial activity. The activities of Ub from bovine erythrocytes against bacteria and fungi were then tested. Our data showed that Ub inhibits the growth of Micrococcus luteus and Bacillus megaterium at a minimal inhibitory concentration (MIC) of 60 µM (Table 1 ). In addition, we observed that Ub inhibits the growth of Neurospora crassa at a MIC of 60 µM; this fungus is completely killed at a concentration of 100 µM. Then, we attempted to characterize the shortest active peptide derived from Ub, after proteolytic digestion with endoproteinase Glu-C and separation on reverse-phase HPLC. Automatic Edman degradation and MALDI-TOF analysis identified the peptide as Ub_65–76. The corresponding synthetic peptide was tested against several microorganisms (Table 1) . It is active against Gram-positive bacteria M. luteus and B. megaterium, with a MIC estimated to 5 and 4 µM, respectively. To complete the spectrum of the antimicrobial activity of the synthetic peptide, we also tested its ability to affect the growth of filamentous fungi and yeast cells. It is interesting that this peptide in the concentration range from 1 to 30 µM completely inhibits a variety of filamentous fungi (Table 1) . In addition, this synthetic peptide is active against several yeast forms, with a MIC varying from 15 to 20 µM. These experiments indicate a lytic effect of Ub_65–76 against Gram-positive bacteria and fungi, as the restart of microorganism growth could not be restored after 48 h. Furthermore, Ub_65–76 is unable to induce the lysis of sensitive mammalian cellssuch as erythrocytes at a concentration up to 50 µM.

3. Synergistic effect of Ub_1–34 on the antifungal activity of Ub_65–76
The three-dimensional structure of Ub reveals that the residue Glu₆₄ is located close to the N-terminal sequence surrounding the surface residue Phe₄. Furthermore, it has been reported that the N-terminal fragment (residues 1–34) adopts a ß-hairpin (residues 1–17), followed by an -helix (residues 23–34), which may be important for the interaction with membranes (Schematic diagram). Taking into account the structural features of the N-terminal fragment Ub_1–34, we decided to probe its antifungal activity. It displays a weak activity against N. crassa with a MIC at 100 µM. In contrast, when Ub_1–34 (10 µM) was added to Ub_65–76, the two peptides were found to act synergically to inhibit the growth of several filamentous fungi and yeasts (Table 1) .

View larger version (15K): [in this window] [in a new window]   Schematic diagram. Ub amino acid sequence is presented with the secondary structure (E, ß-sheet; H, -helix; T, ß-turn). The residues important for the different Ub functions are indicated: (i) the residues crucial for conjugation (V70–G76); (ii) the residue necessary for branched-chain multi-Ub adducts (K48); (iii) the residues implicated in proteasome degradation (L8I44V70 and V70–G76); (iv) the residues important for internalization (L8I44V70, Q2F4T12, V70–G76); and (v) the two clusters (Q2F4T12, L8R42I44G47K48) and the C-terminal fragment (V70–G76) required for life in yeasts. The flexible C-terminal tail (residues 70–76) is required for all known Ub functions. The two Ub-derived peptides displaying antifungal activities (Ub1–34 and Ub65–76) are indicated with their specific properties and their ability to act synergically.

4. Confocal laser scan analysis of the interaction of Ub_65–76 with A. fumigatus
We used confocal microscopy to analyze the interaction of the synthetic rhodamine-labeled Ub_65–76R peptide with fungal membranes (Fig. 1A ). By comparison with control experiments (Fig. 1A ,1), Ub_65–76R at 1 µM was visible at the level of cell wall and in the inner compartment after 2 min incubation (Fig. 1A ,2,3). As a control, the rhodaminated, inactive peptide CGB_602–626R (10 µM) was undetectable in A. fumigatus after 1 h treatment (Fig. 1A ,4). However, if A. fumigatus was first treated during 1 h with unlabeled Ub_65–76 (20 µM) and with CGB_602–626R (10 µM), an intense fluorescence was observed, indicating that Ub_65–76 destabilizes the cell wall, allowing the inactive peptide to penetrate into the fungi (Fig. 1A ,5).

View larger version (96K): [in this window] [in a new window]   Figure 1. Phase-contrast and fluorescence confocal laser micrographs of fungi in the presence of rhodaminated synthetic peptides Ub65–76R and Ub1–34R. A) A. fumigatus was incubated first, 21 h in cultured medium at 30°C (1), and then with 1 µM Ub65–76R for 2 min (2). At higher magnification (3), the core of fungi and unlabeled vacuoles are visible; note also the absence of fluorescence on the septum separating two cells. In two other experiments, after incubation in cultured medium, A. fumigatus was incubated with inactive CGB602–626R for 1 h at 10 µM (4) or with 20 µM Ub65–76 and 10 µM CGB602–626R for 1 h (5); note the fluorescence within cells as a result of CGB602–626R penetration because of membrane destabilization by Ub65–76. B) A. fumigatus was examined after 21 h in cultured medium at 30°C and then with 5 µM Ub1–34R for 15 min (1). N. crassa was incubated with 5 µM Ub1–34R for 15 min after a first incubation with cultured medium for 23 h (2); note the labeling along cell membrane of A. fumigatus and N. crassa. (3) A. fumigatus was incubated for 21 h in cultured medium and then with 20 µM Ub65–76 for 45 min before final incubation with 5 µM Ub1–34R for 15 min; note the fluorescence within cells as a result of Ub1–34R penetration because of membrane destabilization by Ub65–76.

5. Confocal laser scan analysis of the interaction of Ub_1–34 with A. fumigatus and N. crassa
A. fumigatus and N. crassa fungal spores were incubated for 15 min with 5 µM Ub_1–34R (Fig. 1B ,1,2). By comparison with control experiment (Fig. 1A ,1), Ub_1–34R was visible at the level of cell wall but not within cells. However, when A. fumigatus fungal spores were first treated during 45 min with unlabeled 20 µM Ub_65–76 and then with 5 µM Ub_1–34R, fluorescence was observed after a few minutes, indicating that Ub_65–76 allows the penetration of Ub_1–34R as a consequence of probable membrane destabilization (Fig. 1B ,3).

6. Inhibition of the calcineurin activity
In addition to the destabilization of fungal cell wall, Ub_65–76 may also exert activity on intracellular targets. It has been established that calcineurin (CaN), the calmodulin-activated phosphatase B, plays a crucial role in hyphal growth, morphology, and maintenance of the apical Ca²⁺ gradient in the filamentous fungus N. crassa. We examined the effect of Ub_65–76 on CaN phosphatase activity and found that 10 µM and 50 µM Ub_65–76 inhibited phosphatase activity by 43% and 85%, respectively. In similar conditions, 100 µM free Ub inhibited phosphatase activity by 25–30% only.

CONCLUSIONS

Ub is a protein that has been highly conserved throughout the course of evolution; it exists in cells as free and conjugated forms. It targets onto proteins as a signal for proteasome degradation and endocytosis. Here, we have detected free Ub in the matrix of secretory granules of the bovine chromaffin cell, upon stimulation, free Ub is released into the extracellular medium.

The presence of free Ub in the soluble intragranular material and in secretions from stimulated chromaffin cells could involve a mechanism including (i) the sorting of free Ub and Ub-conjugated proteins from the Golgi and (ii) the deubiquitination, once protein has been packed into granules, thus releasing free Ub. Indeed, the Ub C-terminal hydrolase is highly expressed in neuroendocrine cells and more particularly, rat chromaffin cells.

We have shown for the first time that Ub possesses antimicrobial properties and that the C-terminal peptide Ub_65–76 displays potent antifungal activities at µM range. In addition, we have shown that the N-terminal peptide Ub_1–34 is blocked at the level of the cell wall but acts synergically with Ub_65–76, killing fungi and yeasts. Furthermore, a pretreatment with Ub_65–76 helps it to penetrate into microorganisms. Furthermore, we found that Ub_65–76 is able to inhibit the phosphatase activity of CaN at the same concentration range as that required to inhibit fungal growth. To conclude, the two peptides Ub_65–76 and Ub_1–34 may be new agents, useful in the future, as potent therapeutic, antifungal agents.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0699fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0699fje

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