Rôle de la protéine FAN dans la signalisation de récepteurs de la superfamille TNFR

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Voies de signalisation de l’apoptose médiées par les sphingolipides

Divers sphingolipides sont aujourd’hui reconnus comme étant doués de propriétés biologiques et/ou agissant comme seconds messagers. Parmi eux, le céra- mide (ou N-acylsphingosine) et la sphingosine se comportent comme des médiateurs généralement proapoptotiques. Ainsi, le céramide relaie le signal cytotoxique d’un certain nombre d’agents de stress qui, soit, stimulent sa biosynthèse de novo, soit, activent l’hydrolyse de sphingomyéline par des sphingomyélinases. Par exemple, la génération précoce de céramide induite par le TNF est médiée par une sphingomyélinase neutre dont l’activité est sous la dépendance de la protéine adaptatrice FAN, contrôlant ainsi l’activation des caspases et le processus apoptotique. De plus, l’activité de cette sphingomyélinase neutre semble être modulée négativement par la cavéoline, un constituant essentiel de certains microdomaines membranaires. Par ailleurs, la sphingosine kinase est une enzyme-clé du métabolisme des sphingolipides car elle contrôle les taux intracellulaires de deux molécules aux effets antagonistes, la sphingosine, pro-apoptotique, et la sphingosine 1-phosphate, anti-apoptotique. Le métabolisme du céramide et de la sphingosine est donc considéré actuellement comme au cœur d'un ensemble de voies de signalisation régulant le devenir de la cellule

LmABCB3, an atypical mitochondrial ABC transporter essential for Leishmania major virulence, acts in heme and cytosolic iron/sulfur clusters biogenesis

Abstract Background Mitochondria play essential biological functions including the synthesis and trafficking of porphyrins and iron/sulfur clusters (ISC), processes that in mammals involve the mitochondrial ATP-Binding Cassette (ABC) transporters ABCB6 and ABCB7, respectively. The mitochondrion of pathogenic protozoan parasites such as Leishmania is a promising goal for new therapeutic approaches. Leishmania infects human macrophages producing the neglected tropical disease known as leishmaniasis. Like most trypanosomatid parasites, Leishmania is auxotrophous for heme and must acquire porphyrins from the host. Methods LmABCB3, a new Leishmania major protein with significant sequence similarity to human ABCB6/ABCB7, was identified and characterized using bioinformatic tools. Fluorescent microscopy was used to determine its cellular localization, and its level of expression was modulated by molecular genetic techniques. Intracellular in vitro assays were used to demonstrate its role in amastigotes replication, and an in vivo mouse model was used to analyze its role in virulence. Functional characterization of LmABCB3 was carried out in Leishmania promastigotes and Saccharomyces cerevisiae. Structural analysis of LmABCB3 was performed using molecular modeling software. Results LmABCB3 is an atypical ABC half-transporter that has a unique N-terminal extension not found in any other known ABC protein. This extension is required to target LmABCB3 to the mitochondrion and includes a potential metal-binding domain. We have shown that LmABCB3 interacts with porphyrins and is required for the mitochondrial synthesis of heme from a host precursor. We also present data supporting a role for LmABCB3 in the biogenesis of cytosolic ISC, essential cofactors for cell viability in all three kingdoms of life. LmABCB3 fully complemented the severe growth defect shown in yeast lacking ATM1, an orthologue of human ABCB7 involved in exporting from the mitochondria a gluthatione-containing compound required for the generation of cytosolic ISC. Indeed, docking analyzes performed with a LmABCB3 structural model using trypanothione, the main thiol in this parasite, as a ligand showed how both, LmABCB3 and yeast ATM1, contain a similar thiol-binding pocket. Additionally, we show solid evidence suggesting that LmABCB3 is an essential gene as dominant negative inhibition of LmABCB3 is lethal for the parasite. Moreover, the abrogation of only one allele of the gene did not impede promastigote growth in axenic culture but prevented the replication of intracellular amastigotes and the virulence of the parasites in a mouse model of cutaneous leishmaniasis. Conclusions Altogether our results present the previously undescribed LmABCB3 as an unusual mitochondrial ABC transporter essential for Leishmania survival through its role in the generation of heme and cytosolic ISC. Hence, LmABCB3 could represent a novel target to combat leishmaniasis. Keywords Heme trafficking and metabolism Iron/sulfur clusters Trypanosomatid parasites Mitochondrial ABC transporter LeishmaniaThis work was supported by grants from the Spanish Ministerio de Economía y Competitividad SAF2011-28215 (JMPV) and Junta de Andalucia BIO1786 (JMPV) and by FEDER funds from the EU to JMPV. MMG was recipient of a FPI fellowship from the Spanish Ministerio de Economía y Competitividad; MCD was recipient of a FPU fellowship from the Spanish Ministerio de Educación, Cultura y Deporte; SMC was recipient of a JAE-DOC from the Spanish CSIC (Ministerio de Economía y Competitividad), cofounded by the Fondo Social Europeo.Peer reviewe

Trypanosomatid parasites rescue heme from endocytosed hemoglobin through lysosomal HRG transporters

Pathogenic trypanosomatid parasites are auxotrophic for heme and they must scavenge it from their human host. Trypanosoma brucei (responsible for sleeping sickness) and Leishmania (leishmaniasis) can fulfill heme requirement by receptor-mediated endocytosis of host hemoglobin. However, the mechanism used to transfer hemoglobin-derived heme from the lysosome to the cytosol remains unknown. Here we provide strong evidence that HRG transporters mediate this essential step. In bloodstream T. brucei, TbHRG localizes to the endolysosomal compartment where endocytosed hemoglobin is known to be trafficked. TbHRG overexpression increases cytosolic heme levels whereas its down-regulation is lethal for the parasites unless they express the Leishmania orthologue LmHR1. LmHR1, known to be an essential plasma membrane protein responsible for the uptake of free heme in Leishmania, is also present in its acidic compartments which colocalize with endocytosed hemoglobin. Moreover, LmHR1 levels modulated by its overexpression or the abrogation of an LmHR1 allele correlate with the mitochondrial bioavailability of heme from lysosomal hemoglobin. In addition, using heme auxotrophic yeasts we show that TbHRG and LmHR1 transport hemoglobin-derived heme from the digestive vacuole to the cytosol. Collectively, these results show that trypanosomatid parasites rescue heme from endocytosed hemoglobin through endolysosomal HRG transporters, which could constitute novel drug targets.We thank Ivan Hapala (IABG-SAS, Slovakia), Stephen M.Beverley (Washington University School of Medicine, USA)and Olivier Cagnac (EEZ-CSIC, Spain) for kindly providing,respectively, thehem1Dyeast strain, theLeishmaniaandthe yeast vectors used throughout this research work. Wethank Ignacio Perez-Victoria for the preparation of ApoHb.We are grateful to Jean Mathieu Bart (IPBLN-CSIC, Spain)for helpful discussions. This work was supported by Span-ish grants BIO1786 (JMPV) from the Junta de Andalucıa and SAF2011-28215 (JMPV) and BFU2014-55193-P (AE)from the Ministerio de Economıa y Competitividad and by FEDER funds from the EU to JMPV and AE. MCD was astudent of the PhD program “Biochemistry and MolecularBiology” of the University of Granada (Spain). MCD was recipient of a FPU fellowship from the Spanish Ministerio de Educacion, Cultura y Deporte; SMC was recipient of a JAE-DOC from the Spanish CSIC (Ministerio de Economıa y Competitividad), cofounded by the Fondo Social Europeo,LMOZ was recipient of a Colciencias fellowship from the Colombian Ministerio de Ciencia, Tecnologıa e Innovacion;MMG was recipient of a FPI fellowship from the Spanish Ministerio de Economıa y Competitividad. The authorsdeclare that they have no conflict of interestPeer reviewe

Additional file 5: Figure S3. of LmABCB3, an atypical mitochondrial ABC transporter essential for Leishmania major virulence, acts in heme and cytosolic iron/sulfur clusters biogenesis

The ScATM1 residues that interact with glutathione are conserved in LmABCB3. The alignment of the indicated amino acids of S. cerevisiae ScATM1, H. sapiens HsABCB7 and L. major LmABCB3 (ClustalW software) shows that LmABCB3 share the ScATM1 residues forming hydrogenen bonds with GSH (highlighted in yellow). The E433 residue of HsABCB7 mutated to lysine in XLSA/A patients is indicated by a red arrow. Other ScATM1 residues surrounding bound GSH are highlighted in green. Identical (*), strongly similar (:) and weakly similar (.) amino acids are coloured in red, green and blue, respectively. (TIF 379 kb