Oxidative folding in mitochondria From electron transfer reactions

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

1988, BSc, Chemical Engineering, Aristotle Univ. Thessaloniki 1991, MChem, Chemical Engineering, Univ of Delaware, USA 1993, Ph. D, (Fulbright and EU Fellow) Chemical Engineering/Biochemistry Univ. of Delaware and Institut Pasteur France 1993 -1994, postdoc (EU Fellow), Institut Pasteur France 1994 -1998, postdoc (HFSP, EMBO, Roche Fellow) Biozentrum, Basel, Switzerland 1998 -2003, Lecturer, Senior Lecturer, Lister Fellow, Univ. Manchester, UK 2003 - now, Group Leader, IMBB-FORTH 2003 -2006, Assistant Prof, Dept Chemistry, Uo. C 2007 -now, Associate Prof, Dept Materials Science and Technology, Uo. C

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

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

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

Mitochondria are essential for life

Functional Complexity Structural Complexity Respiration and ATP Synthesis of heme, lipids, amino acids and nucleotides Intracellular homeostasis of inorganic ions 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 mt. DNA

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

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

Lithgow and Pfanner, 2005

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

NOVEL oxidative folding pathway operating in mitochondria in vivo

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

Functional and Structural analysis of Mia 40

Solution structure of ΜΙΑ 40 by NMR Banci et al. , Nature SMB, 2009

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

Structural basis for the binding of the ITS onto the cleft of Mia 40 Sideris et al. 2009 JCB

Mechanism of substrate recognition by Mia 40: The sliding – docking model

Conclusions An oxidative folding pathway operates in mitochondria Docking of the substrate to the Mia 40 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 Mia 40 represents structurally, functionally and mechanistically a new type of cellular oxidoreductase Selective publications 2009 -2011 Nature Structural and Molecular Biology 16(2), 198 -206, 2009 J. Cell Biol. 187(7), 1007 -1022, 2009 Proc Natl Acad. Sci USA 107(47), 20190 -20195, 2010 Proc Natl Acad. Sci USA 108(12), 4811 -4816, 2011

Future Directions: - Mechanism of protein-protein interactions and structural basis - Links to cytochrome C mediated cell-death pathways -Other substrates of Erv 1 (ALS-linked SOD 1, lifespan/aging determinants targeted to the IMS)

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

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