Kostas TokatlidisUniversity of Crete and IMBB-FORTH

Oxidative folding in mitochondria:

From electron transfer reactions to cellular architecture and disease

1988, BSc, Chemical Engineering, Aristotle Univ. Thessaloniki1991, MChem, Chemical Engineering, Univ of Delaware, USA1993, PhD, (Fulbright and EU Fellow)

Chemical Engineering/Biochemistry Univ. of Delaware and Institut Pasteur France

1993-1994, postdoc (EU Fellow), Institut Pasteur France1994-1998, postdoc (HFSP, EMBO, Roche Fellow) Biozentrum, Basel, Switzerland

1998-2003, Lecturer, Senior Lecturer, Lister Fellow, Univ. Manchester, UK

2003- now, Group Leader, IMBB-FORTH2003-2006, Assistant Prof, Dept Chemistry, UoC2007-now, Associate Prof, Dept Materials Science and Technology, UoC

1900s Ehrlich , ‘Magic bullet’

Goal: Tailored and efficient therapeutics

Drug

?

?

Critical need for drug delivery site-specifically at the subcellularand suborganellar Level

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 proteinsown 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?

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

Respiration and ATP Synthesis

Synthesis of heme, lipids, amino acids and nucleotides

Intracellular homeostasis of inorganic ions

Structural Complexity

5-15% of total cell protein20% volume of eukaryotic cellIM is 1/3 of total cell membrane

About 1000 different polypeptides(900 in yeast)

Only a dozen encoded by mtDNA

Protein import is the major mechanism

of mitochondriabiogenesis

Curiosity-driven research: How do proteins find their way to mitochondria?

Approach

Isolated molecules

In vitro

Intact cells

In vivo

Isolated organelles

In organello

Lithgow and Pfanner, 2005

By using …

Protein purificationMutagenesisCD analysisLimited proteolysisBioinformaticsMass spectrometryChemical modification of thiol groups. in vivo thiol trappingGel filtrationIsothermal titration calorimetry ITCAnalytical centrifugationMulti-angle light static scattering

NOVEL oxidative folding pathway operating in mitochondria in vivo

…closing the loop: CytC 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 Mia40

Solution structure of ΜΙΑ40 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 Mia40

Sideris et al. 2009 JCB

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

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 2009-2011

Nature Structural and Molecular Biology 16(2), 198-206, 2009 J. Cell Biol. 187(7), 1007-1022, 2009Proc Natl Acad. Sci USA 107(47), 20190-20195, 2010Proc 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 Erv1 (ALS-linked SOD1, lifespan/aging determinants targeted to the IMS)

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

in mitochondria

Acknowledgements

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

CollaboratorsCERM-Florence (NMR)