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Laboratory | Molecular mechanisms
The physiological and therapeutical relevance of Membrane Transport Proteins (MTPs) is reflected by the fact that MTPs comprise ~15 % of the FDA-approved human targets, and that ~18 % of total FDA-approved drugs target MTPs. We study the structure-function relationships of disease-related amino acid transporters and transporters encoded by malaria parasites. Our goal is to understand their biological role and pathophysiology, as well as to find new mechanisms for pharmaceutical intervention. To this end we combine structural biology, spectroscopy and biochemical tools, including protein engineering.
Group leader José-Luis Vazquez-IbarJoseLuis.VAZQUEZ-IBAR@cea.fr
Amino acid transporters are
essentials players in human physiology as they participate on protein
synthesis, energy metabolism, gene expression, redox balance or signal
transduction pathways. One of the largest families of amino acid transporters
is the L-amino acid transporter (LAT) family, where congenital mutatios of two
members of this family cause two pathological disorders: cystinuria and
lysinuric protein intolerance. In collaboration with Prof Manuel Palacín (IRB,
Barcelona, Spain) we solved the X-ray structure of a LAT prokaryotic homolog:
Adic (Kowalczyk, L
et al, 2011, Figure 1), revealing for the first time an induced-fit
mechanism during the first step of substrate-protein interaction. While two
structures of mammalian LATs have recently appeared, prokaryotic LATs are still
very valuable tools to continue deciphering the transport mechanism and
regulation of their mammalian homologs. Using a directed evolution strategy
(Rodriguez-Banqueri et al 2016), we have generated engineered versions of SteT
(a prokaryotic paradigm of LATs) trapped in different stages along the
catalytic cycle. Structure-function analyses of these mutants are expected to
improve our understanding of the transport mechanism and lipid regulation of
In collaboration with Dr Bruno Gasnier (Paris-Descartes) and Dr Liang Feng (Stanford University, USA), we are also studying the lysosomal cationic amino acid transporter PQLC2. PQLC2 activity is likely to be linked with mTORC1 signaling by tightly controlling intralysosomal arginine levels. In addition, PQLC2 mediates the recruitment of the C9orf72 complex to the lysosomal surface. Mutations in the gene encoding C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia. Our aims include unraveling the transport mechanism and regulation of PQLC2 as well as the study of the structural and functional consequences of PQLC2 interaction with the C9orf72 complex.
Due to the intracellular mode of living of Plasmodium parasites, MTPs encoded by these parasites are a source of potential candidates for drug targeting, although most of them are still considered as putative. Notably, a high proportion of transporters with predicted role in lipid transport and in the maintenance of membrane lipid asymmetry are essential for the parasite; like ATP2, the Plasmodium P4-ATPase (or lipid flippase), recently identified as the possible target of two antimalarial drug candidates. Recombinant ATP2 associates with two of the three Plasmodium-encoded Cdc50 proteins (Cdc50A and Cdc50B), and functional analysis of purified ATP2/Cdc50B complex have identified possible physiological phospholipid substrates of ATP2 (Lamy et al, 2020). Our next goals include a better understanding of the catalytic activity of ATP2 and the physiological meaning of ATP2 association with each of the Cdc50 subunits, and the finding of ATP2 inhibitors. We also aim at determining the 3D atomic structure of ATP2 in complex with these Cdc50 subunits.
MTPs are notoriously challenging proteins to produce in heterologous host. Also, their metastable nature is often an obstacle for their structural and functional characterization. We have developed new approaches and methodologies to circumvent some of these problems:
We developed a strategy to engineer stability in a LAT transporter combining random mutagenesis, and a split-GFP complementation assay as reporter of protein expression and membrane insertion (Rodriguez-Banqueri A et al, 2012), (Rodriguez-Banqueri A et al, 2016), (Errasti-Murugarren E et al, 2017).
We have used nanobodies (the heavy chain domains of camelide antibodies) against the GFP to purify the GFP-tagged ATP2 in complex with a Cdc50 beta-subunit, and to immobilize them in agarose beads for functional analysis (Lamy et al, 2020). In collaboration with Leandro Tabares and Sun Un, we are now aiming to extend the use of nanobodies for in vivo and in vitro structural characterization of MTPs (and complexes) using spectroscopic tools.
CEA is a French government-funded technological research organisation in four main areas: low-carbon energies, defense and security, information technologies and health technologies. A prominent player in the European Research Area, it is involved in setting up collaborative projects with many partners around the world.