On the hunt for mitochondrial proteolysis: Profiling changes in the mitochondrial N-terminal proteome during bacterial infection — ASN Events
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  2. Symposium Eight: Microbial Proteomics
  3. On the hunt for mitochondrial proteolysis: Profiling changes in the mitochondrial N-terminal proteome during bacterial infection

On the hunt for mitochondrial proteolysis: Profiling changes in the mitochondrial N-terminal proteome during bacterial infection (#202)

Natalie C Marshall , Michael P Thejomayen 1 2 , Theo Klein 3 , Niklas von Krosigk 2 , Nikolay Stoynov 4 , Leonard J Foster 4 5 , Christopher M Overall 5 6 , B. Brett Finlay 1 2 5
  1. Department of Microbiology & Immunology, University of British Columbia, Vancouver, BC, Canada
  2. Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada
  3. Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada
  4. Centre for High-Throughput Biology, University of British Columbia, Vancouver, BC, Canada
  5. Department of Biochemistry & Molecular Biology, University of British Columbia, Vancouver, BC, Canada
  6. Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada

Mitochondria mediate key cellular processes, including energy generation and cell death. Although mitochondrial proteases are important regulators of these processes, tools to study global proteolysis within mitochondria have not been developed.1,2 Our understanding of mitochondrial dynamics could be greatly improved with a proteomics technique that also identifies proteolytic events.

We have developed a new approach called ‘mito-TAILS’ that quantitatively profiles the mitochondrial proteome and proteolytic events. Mito-TAILS combines mitochondrial enrichment, stable isotope labeling by amino acids in cell culture (SILAC),3 and an amino (N)-terminal proteomics technique called terminal amine isotopic labeling of substrates (TAILS).4

In this study, mito-TAILS was used to characterize changes in the mitochondrial N-terminal proteome in response to infection with enteropathogenic Escherichia coli (EPEC). EPEC possesses several virulence factors that target mitochondria; they are imported, cleaved, and contribute to apoptosis of infected human cells via the mitochondrial pathway. Human epithelial (HeLa) cells were grown in either light or heavy isotope-coded SILAC medium and either infected with wild-type EPEC or mock infected in parallel. Cells were harvested, pooled, and homogenized. Mitochondria were then isolated from 95% of the cell homogenate; 5% was used for a complementary whole cell analysis. N-terminal proteomes were quantitatively compared using TAILS5 and tandem mass spectrometry. Data from three biological replicates and two cellular fractions were analyzed using programs developed in our lab.

Proteins from mitochondrial cellular compartments in the Gene Ontology were significantly enriched in the mitochondrial fractions from mito-TAILS. We identified changes in the overall abundance of mitochondrial proteins, changes in mitochondrial protein import, and known apoptotic events. Furthermore, novel proteolytic events were identified in mitochondrial proteins that regulate apoptosis and protein import. By profiling the cellular proteome alongside the mitochondrial proteome, this technique permits a more comprehensive investigation of cell death events in both mitochondria and cytoplasm.

Mito-TAILS is a valuable  approach to study mitochondrial proteome dynamics and proteolysis. To our knowledge, this is the first application of terminal proteomics to study mitochondria during infection and the first study of the mitochondrial proteome during infection. This method could easily be applied in other disciplines, including the study of mitochondrial diseases.

  1. Sekine, S., and Ichijo, H. (2014) Mitochondrial proteolysis: Its emerging roles in stress responses. Biochim. Biophys. Acta BBA - Gen. Subj. [Article in press]
  2. Anand, R., Langer, T., and Baker, M. J. (2013) Proteolytic control of mitochondrial function and morphogenesis. Biochim. Biophys. Acta 1833, 195–204
  3. Ong, S.-E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A., and Mann, M. (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics MCP 1, 376–386
  4. Kleifeld, O., Doucet, A., auf dem Keller, U., Prudova, A., Schilling, O., Kainthan, R. K., Starr, A. E., Foster, L. J., Kizhakkedathu, J. N., and Overall, C. M. (2010) Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products. Nat. Biotechnol. 28, 281–288
  5. Kleifeld, O., Doucet, A., Prudova, A., auf dem Keller, U., Gioia, M., Kizhakkedathu, J. N., and Overall, C. M. (2011) Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates. Nat. Protoc. 6, 1578–1611