1. A Combined N-terminomics and Shotgun Proteomics Approach to Investigate the Responses of Human Cells to Rapamycin and Zinc at the Mitochondrial Level
- Author
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Sarah Cianférani, Charlotte Macron, Sebastian Alvaro Vaca-Jacome, Catherine Aude-Garcia, Christine Carapito, Joanna Bons, Magali Rompais, Thierry Rabilloud, Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Protéomique, Métaux et Différenciation (ProMD ), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
- Subjects
Proteomics ,Cell biology ,Proteases ,Proteome ,Cellular organelles ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Mitochondrion ,Biochemistry ,Mass Spectrometry ,Analytical Chemistry ,Mitochondrial Proteins ,03 medical and health sciences ,Mitochondria function or biology ,[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Cluster Analysis ,Humans ,Citrate synthase ,Shotgun proteomics ,Molecular Biology ,030304 developmental biology ,Sirolimus ,0303 health sciences ,biology ,rapamycin ,Chemistry ,Research ,Succinate dehydrogenase ,zinc ,030302 biochemistry & molecular biology ,N-terminomics ,U937 Cells ,Metabolism ,Pyruvate dehydrogenase complex ,Mitochondria ,Enzymes ,Cytosol ,biology.protein - Abstract
International audience; All but thirteen mammalian mitochondrial proteins are encoded by the nuclear genome, translated in the cytosol and then imported into the mitochondria. For a significant proportion of the mitochondrial proteins, import is coupled with the cleavage of a presequence called the transit peptide, and the formation of a new N-terminus. Determination of the neo N-termini has been investigated by proteomic approaches in several systems, but generally in a static way to compile as many N-termini as possible. In the present study, we have investigated how the mitochondrial proteome and N-terminome react to chemical stimuli that alter mitochondrial metabolism, namely zinc ions and rapamycin. To this end, we have used a strategy that analyzes both internal and N-terminal peptides in a single run, the dN-TOP approach. We used these two very different stressors to sort out what could be a generic response to stress and what is specific to each of these stressors. Rapamycin and zinc induced different changes in the mitochondrial proteome. However, convergent changes to key mitochondrial enzymatic activities such as pyruvate dehydrogenase, succinate dehydrogenase and citrate synthase were observed for both treatments. Other convergent changes were seen in components of the N-terminal processing system and mitochondrial proteases. Investigations into the generation of neo-N-termini in mitochondria showed that the processing system is robust, as indicated by the lack of change in neo N-termini under the conditions tested. Detailed analysis of the data revealed that zinc caused a slight reduction in the efficiency of the N-terminal trimming system and that both treatments increased the degradation of mitochondrial proteins. In conclusion, the use of this combined strategy allowed a detailed analysis of the dynamics of the mitochondrial N-terminome in response to treatments which impact the mitochondria.
- Published
- 2019
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