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)