Biology subjects

Carlier, L.

Publications and source records attributed to Carlier, L..

3 recordsLinked to original sources

bioRxiv2023

Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70/80 heterodimer (Ku), XRCC4 in complex with DNA Ligase 4 (X4L4), and XLF form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have recently been obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here, we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at atomic resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs led to the formation of XLF and X4L4 condensates in vitro which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome editing strategies.

biophysics↗

bioRxiv2023

Plasmodium falciparum transmission in the highlands of Ethiopia is driven by closely related and clonal parasites

Malaria cases are frequently recorded in the Ethiopian highlands even at altitudes above 2,000 m. The epidemiology of malaria in the Ethiopian highlands, and in particular the role of importation by human migration from the highly endemic lowlands is not well understood. We characterized the parasite population structure and genetic relatedness by sequencing 159 P. falciparum samples from Gondar and an additional 28 samples from Ziway using a highly multiplexed droplet digital PCR (ddPCR)-based amplicon deep sequencing method targeting 35 microhaplotypes and drug resistance loci. Diversity was moderate (mean HE: 0.54), and infection complexity was low (74.9% single clone infections). A significant percentage of infections shared genomic haplotypes, even across transmission seasons, indicating persistent local and focal transmission. Multiple clusters of clonal or near-clonal infections were identified, highlighting the overall high genetic relatedness. Frequently, infections from travelers were the earliest observed cases, suggesting that parasites may have been imported and then transmitted locally. We observed population structure between Gondar and Ziway, although some haplotypes were shared between sites. 31.1% of infections carried pfhrp2 deletions and 84.4% pfhrp3 deletions, and 28.7% pfhrp2/pfhrp3 double deletions. Parasites with pfhrp2/3 deletions and wild-type parasites were genetically distinct. Mutations associated with resistance to sulfadoxine-pyrimethamine and lumefantrine were observed at near-fixation, but no mutations in pfk13 were found. In conclusion, genomic data corroborates local transmission and the importance of intensified control in the Ethiopian highlands.

genomics↗

bioRxiv2022

Structural transitions in TCTP tumor protein upon Mcl-1 binding

SummaryTranslationally Controlled Tumour Protein (TCTP) is a pro-survival factor in tumor cells. TCTP inhibits the mitochondrial apoptosis pathway by potentiating the anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL. Specifically, TCTP binds Bcl-xL and inhibits the Bax-dependent Bcl-xL-induced cytochrome c release and TCTP reduces Mcl-1 turnover by inhibiting its ubiquitinylation, thus resulting in decreased Mcl-1 mediated apoptosis. TCTP owns a BH3-like motif forming a {beta}-strand buried in the globular domain of the protein. The crystal structure of TCTP BH3-like peptide in complex with Bcl-xL highlighted the -helical conformation of TCTP BH3-like motif, suggesting major changes in TCTP structure upon complex formation. However, the structural impact of the interaction on the full-length TCTP and the structural description of TCTP/Mcl-1 interaction are still lacking. Here using biophysical/biochemical methods (NMR, SAXS, circular dichroism, limited proteolysis), we provide an in-depth description of the TCTP/Mcl-1 complex. We demonstrate that full length TCTP binds to the BH3 binding groove of Mcl-1 via its BH3-like motif which interconverts between different binding modes at the micro- to milli-second timescale. As a consequence of the engagement of the BH3-like motif in the interface, the TCTP globular domain is destabilized into a molten-globule state. We also establish that the residue D16 in TCTP BH3-like motif is crucial for the stability and dynamics of the intermolecular interface. As a conclusion, we reveal here in details the structural plasticity of TCTP and discuss its implications for TCTP biology and for future anticancer drug design strategies aiming at targeting TCTP complexes. ContactEwen Lescop, ewen.lescop@cnrs.fr. Supplementary InformationSupplementary figures, tables and files.

biochemistry↗