Transcript Oxidative folding in mitochondria

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, PhD, (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, UoC
2007-now, Associate Prof, Dept Materials Science and Technology, UoC
1900s
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 mtDNA encoding only 13 proteins
>99% have to be imported
Protein import is the crucial mechanism
of
90% of the cells energy is provided
by mitochondria
-More than 300 mitochondrial diseases
-Involved in ageing, cancer, heart disease
-Key regulators of apoptosis
mitochondria biogenesis
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
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 mtDNA
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:
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, 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 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
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)