Evidence for the Existence in Arabidopsis thaliana of the Proteasome Proteolytic Pathway: ACTIVATION IN RESPONSE TO CADMIUM. Cécile Polge, Michel Jaquinod, Frances Holzer, Jacques Bourguignon, Linda Walling, Renaud Brouquisse To cite this version: Cécile Polge, Michel Jaquinod, Frances Holzer, Jacques Bourguignon, Linda Walling, et al.. Evi- dence for the Existence in Arabidopsis thaliana of the Proteasome Proteolytic Pathway: ACTIVA- TION IN RESPONSE TO CADMIUM.: Activation in response to cadmium. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2009, 284 (51), pp.35412-24. 10.1074/jbc.M109.035394. hal-00483927 HAL Id: hal-00483927 https://hal.archives-ouvertes.fr/hal-00483927 Submitted on 31 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Copyright THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 51, pp. 35412–35424, December 18, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Evidence for the Existence in Arabidopsis thaliana of the Proteasome Proteolytic Pathway ACTIVATION IN RESPONSE TO CADMIUM*□S Received for publication, June 18, 2009, and in revised form, October 9, 2009 Published, JBC Papers in Press, October 12, 2009, DOI 10.1074/jbc.M109.035394 Ce´cile Polge‡1, Michel Jaquinod§, Frances Holzer¶, Jacques Bourguignon‡, Linda Walling¶, and Renaud Brouquisse‡2 From the ‡Laboratoires de Physiologie Cellulaire Ve´ge´tale, CEA, IRTSV, UMR5168 CNRS/CEA/INRA, Universite´Joseph Fourier and §Etude de la Dynamique des Prote´omes, F-38054 Grenoble, France and the ¶Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, California 92521-0124 Downloaded from Heavy metals are known to generate reactive oxygen species metabolic activities. Cadmium toxicity involves production of that lead to the oxidation and fragmentation of proteins, which active oxygen species and free radicals, although the mecha- become toxic when accumulated in the cell. In this study, we nism is still obscure (3). Cadmium is not a transition metal and investigated the role of the proteasome during cadmium stress does not produce hydroxyl radicals through Fenton or Haber- http://www.jbc.org/ in the leaves of Arabidopsis thaliana plants. Using biochemical Weiss reactions, but it induces the production of superoxide and proteomics approaches, we present the first evidence of an anion, nitric oxide, and hydrogen peroxide, enhances lipid per- active proteasome pathway in plants. We identified and charac- oxidation and depletion of cellular glutathione, and finally gen- terized the peptidases acting sequentially downstream from the erates oxidative stress (3, 4). Active oxygen species can lead to proteasome in animal cells as follows: tripeptidyl-peptidase II, oxidation of side chains of amino acid residues and formation of thimet oligopeptidase, and leucine aminopeptidase. We investi- protein-protein covalent cross-linkage, which can lead to pro- at INRA Institut National de la Recherche Agronomique on June 14, 2018 gated the proteasome proteolytic pathway response in the leaves tein inactivation or denaturation (5–7). If they are not rapidly of 6-week-old A. thaliana plants grown hydroponically for 24, degraded, oxidatively modified proteins can undergo direct 48, and 144 h in the presence or absence of 50 ␮M cadmium. The fragmentation or can form large aggregates due to covalent gene expression and proteolytic activity of the proteasome and cross-linking and increased surface hydrophobicity, which lead the different proteases of the pathway were found to be up-reg- to cell death (5, 8). ulated in response to cadmium. In an in vitro assay, oxidized In animal cells, the proteasome has been shown to recognize bovine serum albumin and lysozyme were more readily and degrade mildly oxidized proteins in the cytosol, nucleus, degraded in the presence of 20 S proteasome and tripeptidyl- and endoplasmic reticulum, thus minimizing their cytotoxicity peptidase II than their nonoxidized form, suggesting that oxi- (5). From in vitro studies, it was shown that the 20 S proteasome dized proteins are preferentially degraded by the Arabidopsis recognizes and degrades oxidized proteins, probably through a 20 S proteasome pathway. These results show that, in response “by default” degradation mechanism (9), although the 26 S pro- to cadmium, the 20 S proteasome proteolytic pathway is up-reg- teasome does not (10–12). This may be explained by the fact ulated at both RNA and activity levels in Arabidopsis leaves and that a mild oxidative stress rapidly inactivates both the ubiq- may play a role in degrading oxidized proteins generated by the uitin-activating/conjugating system and 26 S proteasome activ- stress. ity in intact cells, but it does not affect 20 S proteasome activity (10, 12, 13). In mammals, a set of proteases has been identified acting Cadmium is a highly toxic and persistent environmental poi- sequentially downstream of the proteasome to peptide degra- son for plants, yeasts, and animals (1). Cadmium is released into dation and releasing free amino acids into the cytosol. The pep- the environment mainly through industrial wastes and is trans- tides issued from the proteasome range from 3 to 25 amino ferred to animals through the food chain (2). Cadmium inter- acids and have different features according to their size. The feres with many cellular functions mainly by complex forma- smallest products (2–6 amino acids) are directly degraded by tion with organic compounds such as proteins, lipids, and aminopeptidases, mainly leucine aminopeptidases (LAPs),3 nucleotides leading to the inhibition of gene expression and although larger peptides (Ͼ6 amino acids) are first cleaved by intermediate endopeptidases (14). The most important endopeptidases for these processes are the thimet oligopepti- * This work was supported by the French “Program Inter-organismes CEA CNRS INRA INSERM de Toxicologie Nucle´aire Environnementale.” □S The on-line version of this article (available at http://www.jbc.org) contains 3 The abbreviations used are: LAP, leucine aminopeptidase; TPPII, tripeptidyl- supplemental Figs. S1 and S2. peptidase II; TOP, thimet oligopeptidase; Mcc, 7-methoxycoumarin-3-car- 1 Present address: INRA and Human Nutrition Research Center of Clermont- boxyl; AMC, 7-amino-4-methylcoumarin; Suc, succinimidyl; RT, reverse Ferrand, UMR 1019, 63122 Ceyrat, France. transcription; BSA, bovine serum albumin; LC-MS/MS, liquid chromatogra- 2 To whom correspondence should be addressed: UMR, Interactions phy/tandem mass spectrometry; pNA, p-nitroanilide; MES, 4-morpho- Biotiques and Sante´Ve´ge´tale, Centre INRA de Sophia Antipolis, BP 167, lineethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)- F-06903 Sophia-Antipolis, France. E-mail: renaud.brouquisse@ ethyl]glycine; DNPH, dinitrophenylhydrazine; CMK, chloromethyl ketone; sophia.inra.fr. AAF, Ala-Ala-Phe; Dnp, 2,4-dinitrophenyl; FW, fresh weight. 35412 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 51•DECEMBER 18, 2009 Proteasome Pathway Activation in Response to Cadmium ␮ ␮ ␮ dase (TOP) (1) for peptides of 9–17 residues and tripeptidyl- (NH4)6Mo7O24, 3.6 M MnSO4,3 M ZnSO4, 9.25 M H3BO3, peptidase II (TPPII) for longer products (15–25 amino acids) and 785 nM CuSO4. The culture medium was renewed every 2 (15). The proteasome pathway (proteasome, TPPII, TOP, and days and was continuously sparged with air. When seedlings aminopeptidases) constitutes the main protein degradation were 6 weeks old, stress was induced by adding Cd(NO3)2 to the pathway in mammalian cells. It has been shown to play a central mineral solution at a 50 ␮M final concentration. No cadmium role in the degradation of many proteins in normal and disease was added to control cultures. Cadmium-containing solutions states (16) and also in antigen processing for major histocom- were renewed every 2 days. At 0, 24, 48, and 144 h of cadmium patibility complex class 1 presentation (17, 18). However, the exposure, leaves were harvested and quickly frozen in liquid N2, specific involvement of the proteasome pathway in the degra- Ϫ ground to a powder in liquid N2, and stored at 80 °C until dation of oxidized protein has not been investigated so far. analysis. In plants, the presence of the proteasome is now well estab- Preparation of Clarified Extracts—Five hundred mg of frozen lished. It was first identified in pea (19) and then in all species leaf powder were homogenized in a mortar at 4 °C with four investigated so far (20). Both the 20 S and 26 S proteasomes times their fresh weight of extraction medium (50 mM Tris-HCl Downloaded from were characterized at molecular (21, 22) and biochemical levels (pH 7.5), 5 mM ␤-mercaptoethanol, and 0.3% (w/v) insoluble (23) in Arabidopsis thaliana. As observed in animal and yeast polyvinylpyrrolidone). The homogenate was centrifuged for 20 cells, plant proteasome has been found to be up-regulated, at min at 16,000 ϫ g, and the supernatant was used for protease transcriptional or translational levels, in oxidative conditions purification and activity measurements. such as carbon starvation (24) or cadmium stress (25–28), and Chromatography Steps—The soluble leaf protein extract (6 http://www.jbc.org/ its involvement in the response to oxidative stress has been ml) was applied to a HiPrep 26/60 Sephacryl S-300 gel filtration postulated. In plants, the preferential involvement of the 20 S column (M 10,000–1,500,000; Amersham Biosciences) equil- proteasome in the degradation of oxidized proteins was r ibrated with 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl.