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Kostas Tokatlidis University of Crete and IMBB-FORTH Oxidative folding in mitochondria: From electron transfer reactions to cellular architecture and disease.

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Presentation on theme: "Kostas Tokatlidis University of Crete and IMBB-FORTH Oxidative folding in mitochondria: From electron transfer reactions to cellular architecture and disease."— Presentation transcript:

1 Kostas Tokatlidis University of Crete and IMBB-FORTH Oxidative folding in mitochondria: From electron transfer reactions to cellular architecture and disease

2 1988, BSc, Chemical Engineering, Aristotle Univ. Thessaloniki 1991, MChem, Chemical Engineering, Univ of Delaware, USA 1993, PhD, (Fulbright and EU Fellow) Chemical Engineering/Biochemistry Univ. of Delaware and Institut Pasteur France , postdoc (EU Fellow), Institut Pasteur France , postdoc (HFSP, EMBO, Roche Fellow) Biozentrum, Basel, Switzerland , Lecturer, Senior Lecturer, Lister Fellow, Univ. Manchester, UK now, Group Leader, IMBB-FORTH , Assistant Prof, Dept Chemistry, UoC 2007-now, Associate Prof, Dept Materials Science and Technology, UoC

3 1900s Ehrlich, ‘Magic bullet’ Goal: Tailored and efficient therapeutics Drug ? ? Critical need for drug delivery site-specifically at the subcellular and suborganellar Level

4 The Biological Problem 90% of the cells energy is provided by mitochondria -More than 300 mitochondrial diseases -Involved in ageing, cancer, heart disease -Key regulators of apoptosis -United Mitochondrial Disease foundation: a child born every 15 min suffers or will develop a mito disease by the age of 5 Mito facts: 1500 proteins own mtDNA encoding only 13 proteins >99% have to be imported  Protein import is the crucial mechanism of mitochondria biogenesis 1. Components? 2. Mechanisms? 3. Relevance in health and disease?

5 more than 30% of proteome are membrane proteins About 50% of drug targets in Pharma Industry are membrane proteins

6 Mitochondria are essential for life

7 Functional Complexity Respiration and ATP Synthesis Synthesis of heme, lipids, amino acids and nucleotides Intracellular homeostasis of inorganic ions Structural Complexity 5-15% of total cell protein 20% volume of eukaryotic cell IM is 1/3 of total cell membrane About 1000 different polypeptides (900 in yeast) Only a dozen encoded by mtDNA

8 Protein import is the major mechanism of mitochondria biogenesis Curiosity-driven research: How do proteins find their way to mitochondria?

9 Approach Isolated molecules In vitro Intact cells In vivo Isolated organelles In organello

10 Lithgow and Pfanner, 2005

11 By using … Protein purification Mutagenesis CD analysis Limited proteolysis Bioinformatics Mass spectrometry Chemical modification of thiol groups. in vivo thiol trapping Gel filtration Isothermal titration calorimetry ITC Analytical centrifugation Multi-angle light static scattering

12 NOVEL oxidative folding pathway operating in mitochondria in vivo

13 …closing the loop: CytC and the respiratory chain are the final acceptors of electrons from the imported precursor Allen et al., JMB, 2005; cover

14 Functional and Structural analysis of Mia40

15 Solution structure of ΜΙΑ40 by NMR Banci et al., Nature SMB, 2009

16 The hydrophobic cleft mediates non-covalent binding of the substrate Banci et al., Nature SMB, 2009

17 Structural basis for the binding of the ITS onto the cleft of Mia40 Sideris et al JCB

18 Mechanism of substrate recognition by Mia40: The sliding – docking model

19 Conclusions An oxidative folding pathway operates in mitochondria Docking of the substrate to the Mia40 represents a site specific event that is crucial step for the oxidative folding process The process is guided by a novel ITS that directs the first step of noncovalent recognition by Mia40 Mia40 represents structurally, functionally and mechanistically a new type of cellular oxidoreductase Selective publications Nature Structural and Molecular Biology 16(2), , 2009 J. Cell Biol. 187(7), , 2009 Proc Natl Acad. Sci USA 107(47), , 2010 Proc Natl Acad. Sci USA 108(12), , 2011

20 Future Directions: - Mechanism of protein-protein interactions and structural basis - Links to cytochrome C mediated cell-death pathways -Other substrates of Erv1 (ALS-linked SOD1, lifespan/aging determinants targeted to the IMS)

21 A new mechanism of peptide-based targeting in the oxidative folding pathway in mitochondria

22 Acknowledgements IMBB/UoCrete: Afroditi Hatzi (PhD) Emanouela Kalergi (PhD) Maria Andreadaki ( MSc) Nitsa Katrakili (BSc Chem Eng.) Alumni from Crete: - Dionisia(Jenny) Sideris (PhD 2009, postdoc Biology/MIT) -Eirini Lionaki (PhD student) Nikos Petrakis (PhD 2009, postdoc MAICH) -Paraskevi Kritsiligkou (Diploma 2009 MSc Biochemistry/ Oxford) - Fliss Alcock (PhD 2008, postdoc Monash, Australia) -Vasilia Balabanidou (MSc 2005, PhD IMBB) -Carine de Marcos (postdoc, Leeds UK) - Catherine Baud (postdoc, Toulouse France) Funding: GSRT, IMBB, UoC EU NMR, EU RegPot Collaborators CERM-Florence (NMR)


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