Research


FUNDAMENTAL CELLULAR BIOCHEMISTRY

Intracellular protein sorting and mitochondria biogenesis
    
Our main interest is the intracellular protein trafficking in eukaryotic cells. About 30-50% of the human proteome consists of membrane proteins or proteins that have to be targeted to a site of action distant to where they are synthesized. This highlights the paramount importance of correct intracellular protein targeting, which is crucial for the architecture, organization and function of all cells. It is hence not surprising that more than 50% of the current Pharma Industry targets are against membrane proteins. Our focus is on the protein import mechanisms in mitochondria, the organelles that control several key cellular processes in normal physiology (ATP production, metal ion homeostasis) but are also involved in several medical conditions (neurological disorders, cancer) and cell death.

    As nearly all mitochondrial proteins (on average 10-15% of the proteome) are imported from the cytosol, these organelles have an elaborate and versatile protein machinery (translocases) to cope with these import processes. To study these, we use yeast mitochondria as
        (i) we can combine the power of yeast genetics (genome completely sequenced,
        all gene knockouts, tagged proteins, interactome databases etc are readily available)         with biochemical approaches,
        (ii) the yeast mitochondrial proteome is currently the most complete (95% of proteins         identified)
        (iii) isolated yeast mitochondria can be readily isolated to high homogeneity and in a
        functional form that can be kept for months.

    We combine three main methodologies ranging from in vivo studies (intact cells, about 6000 proteins), through in organello studies (isolated mitochondria and import assays, about 900 proteins)down to in vitro studies using isolated proteins and their complexes. In this context we use an interdisciplinary approach combining molecular biology (cloning, yeast genetics, mutagenesis), cell biology (cellular fractionation, confocal fluorescence microscopy), biochemistry (protein purification, chemical crosslinking, immunoprecipitations, several liquid chromatography methods, mass spectrometry), biophysics (circular dichroism, isothermal titration calorimetry, light scattering, surface plasmon resonance, fluorescence).

    The particular issues that we want to resolve include
        (i) how membrane proteins are targeted to the mitochondrial inner membrane
        (ii) how the TIM10 chaperone system for membrane proteins assembles and functions
        (iii) what is the molecular basis of a novel oxidative folding pathway that we have
         discovered in mitochondria and is essential for the assembly of a number of          intermembrane space proteins.
 

 

BIOTECHNOLOGY

Large scale membrane protein production of pharmaceutical interest

    We are interested in finding novel ways of facilitating the biotechnological production of membrane proteins of pharmaceutical interest. A bottleneck in the production of membrane proteins is usually their solubility and frequent aggregation upon overexpression. As one current means to overcome this problem, we develop the TIM10 chaperone that specifically recognizes membrane proteins as a way to facilitate their solubility and hence more efficient large scale production.

 

 

MEDICAL APPLICATIONS

A new mechanism underlying pathogenic bacteria-host interactions

    There is important but scattered evidence that several bacterial pathogens (H. pylori, S. aureus etc) inject pathogenic proteins into host cells upon infection that are eventually targeted intracellularly to mitochondria compromising thus their function and leading to cell lesions, cancer and cell death. There is increasing realisation that this may be a very efficient way for a pathogen to exert its deleterious effect on a host by affecting the host's centre of energy production that can lead to pleiotropic host cell defects. We are investigating whether hijacking of the mitochondrial import system by diverse bacterial pathogens is a general way of pathogenicity and determine (i) how this may be achieved by different proteins of the same pathogen and by different pathogens and (ii)what are the precise mitochondrial lesions that result in such cases of infection. To this end we make use of a focused effort using a welll defined bacterial pathogen imported in mitochondria, but also a more high throughput proteomics approach.