Involvement of Rad51 paralogs in Rad51 filament formation in DNA repair
Homologous recombination (HR) is a major repair mechanism for DNA double-strand breaks induced by ionizing radiation. A key step in HR is the formation of Rad51 nucleoprotein filaments on the single-stranded DNA generated from these breaks. We have shown that strict control of these filaments is essential, so that HR does not itself induce chromosomal rearrangements (eLife 2018, Cells 2021). In humans, functional homologs of control proteins are tumor suppressors. Thus, the control of HR appears to be as important as the HR mechanism itself. Our project involves the use of new molecular tools enabling a real breakthrough in the study of these controls. We will be using a functional fluorescent version of the Rad51 protein developed for the first time by our collaborators A. Taddei (Institut Curie), R. Guérois and F. Ochsenbein (I2BC, Joliot, CEA). This major advance will enable us to observe the influence of control proteins on DNA repair by microscopy in living cells. We have also developed highly accurate structural models of control protein megacomplexes in association with Rad51 filaments. This study also led to the identification of specific domains for each paralog protein, outside the structurally conserved Rad51-like core, that might define the specificity of each paralog proteins. We will use a multidisciplinary approach based on genetic, molecular biology, biochemistry, protein structure and live microscopy methods and yeast as model organism to study the consequences of the ablation of these specific domains. We will also search for proteins specifically binding these domains. Their identification would be crucial to understand the function of Rad51 paralog complexes and help to develop new therapeutic approaches.
Deciphering Complex Energy Landscape at Atomic Resolution of Human HSP90 using NMR and AI-Enhanced tools.
HSP90 is a human chaperone involved in the folding of a wide variety of client proteins, including many oncogenic proteins. This complex molecular machinery is known to undergo massive conformational rearrangements throughout its functional cycle. X-ray crystallography and cryoEM have provided high-resolution snapshot structures of this human machinery in complex with cochaperones and client proteins, but have failed to provide the kinetic and time-resolved information needed for a full understanding of its mechanism. We plan to use NMR experiments combined with a new AI-enhanced analysis tool to obtain a detailed picture of the energy landscape of this important drug target. This project will provide structural information on the different excited states of HSP90 and the conformational dynamics between these states. In collaboration with the pharmaceutical industry, we will exploit this new approach to reveal how ligands can modulate the energy landscape and population of different functional states. This information will be particularly useful for the design of new drugs capable of blocking the HSP90 chaperone in a single state, an important step towards the development of more specific and effective drugs.
Contribution of artificial intelligence (AI) to understand the modes of action of microRNAs, application to cancer
MicroRNAs have demonstrated importance in a large number of carcinogenesis processes ranging from initiation, propagation and the appearance of metastases. They raise many hopes as therapeutic treatment targets. However, the drug candidate MRX34 (which mimics a microRNA) proved to be a failure in patients because it was too toxic. It is therefore urgent to better understand the mode of action of microRNAs in order to design new therapeutic strategies.
The thesis project proposes to use two cutting-edge technologies for this: microRNA/mRNA co-sequencing data, at the single cell level, and artificial intelligence techniques (AI, including neural networks and XGBoost ). It will benefit from the contribution of two other projects, which end in 2025 (an overlap of a few months with the CFR thesis): a thesis financed by Pfizer-INSERM, and a multi-team project financed by the cancer plan. These two projects have already enabled rigorous statistical analysis of co-sequencing data at the single cell level, which will be used during the PhD work. A collaboration, already initiated, is planned with Gipsa-Lab, Grenoble, specialist in machine learning / AI.
Molecular dynamics and disorder in the viral replication machinery of SARS CoV 2
The nucleoprotein (N) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for genome replication, encapsidating the viral genome and regulating gene transcription. The central disordered domain is essential to the function of this highly dynamic protein, containing a number of important mutations that are responsible for enhanced viral fitness, and comprising a region that is hyperphosphorylated during the viral cycle. NMR spectroscopy is the tool of choice for studying the conformational behaviour of intrinsically disordered proteins, an abundant class of proteins that are functional in their disordered form. They represent 40% of the proteome and are too dynamic to be studied by crystallography or electron microscopy. The host lab has developed a large number of unique NMR-based tools to help understand the function of this class of proteins at atomic resolution. We will use NMR, paramagnetic NMR, small angle scattering, single molecule FRET and electron microscopy, in combination with molecular dynamics simulation, to describe the interactions of N with viral partner proteins and viral RNA. Post-translational modification, in particular phosphorylation, is thought to play an important functional role, that remains poorly understood, we will investigate the impact of phosphorylation on conformational dynamics and relate this to modifications in function. The results will be correlated with light end electron microscopy, carried out in collaboration.
[FeFe]-hydrogenase active site assembly machinery
To tackle the climate crisis, humanity urgently needs renewable and decarbonized energy sources. A promising solution lies in harnessing dihydrogen (H2), and enzymes known as [FeFe] hydrogenases can play a vital role in its production. These enzymes catalyze the reversible oxidation of dihydrogen, employing an active site called the "H-cluster," a complex organometallic structure. The intricate biosynthesis of this cluster involves three maturation proteins: HydG, HydE, and HydF. Despite recent progress, a full understanding of this process remains elusive due to the complexity of the chemical reactions involved. Our goal is to conduct a structural study combined with step-by-step reaction monitoring using spectroscopy. This approach aims to identify and characterize various reaction intermediates of one key enzyme in the process. This collaborative project involves two leading CEA teams specializing in the study of oxygen-sensitive metalloproteins. The doctoral student will benefit from an ideal scientific and technical environment to achieve this objective, crucially important for advancing hydrogen economy development.
Localization and dynamics of key nucleoid-associated proteins during stress-induced bacterial nucleoid remodeling.
Nucleoid remodeling, and in particular, nucleoid compaction, is a common stress response mechanism in bacteria that allows bacteria to rapidly respond to sudden changes in their environment. Using advanced optical microscopy approaches, we recently followed the changes in nucleoid shape and volume induced by exposure to intense UV-C light in the radiation resistant bacterium, Deinococcus radiodurans. This two-step process involves a rapid initial nucleoid condensation step followed by a slower decompaction phase to restore normal nucleoid morphology, before cell growth and division can resume. Nucleoid associated proteins (NAP) are known to be key players in this process, although the details of their implication remain largely elusive. We have started to shed light on the central role of the major NAP, the histone-like HU protein, in this process. The proposed PhD project will extend this work to the study of 5 additional NAPs involved in stress-induced nucleoid remodeling. The PhD student will perform biochemical studies to follow the abundance of these key factors, live cell imaging to map their distribution and single-particle tracking to determine their dynamics. This work will contribute to a better understanding of the fundamental processes that govern bacterial genome organisation and how they are affected by UV radiation and DNA damage.
The role of the signaling nucleotide ppGpp in plant resilience to climate change.
Amidst the growing challenges of climate change, crops face threats from rising temperatures and prolonged droughts, leading to reduced photosynthetic efficiency and the need for rapid stress acclimation. In this PhD project we will investigate the role of the nucleotide guanosine tetraphosphate (ppGpp) signalling pathway, a known regulator of plastid function and photosynthesis. Recent preliminary work from our and other labs indicate that ppGpp plays a pivotal role in plant stress acclimation, and we have indications that perturbation of ppGpp signalling affects plant responses to heat stress. This research aims to explore how ppGpp is involved in plant acclimation to heat and drought stress. Using a combination of physiological evaluations, biochemical techniques, transcriptomics, and biosensors this study will investigate the modulation of ppGpp levels under stress conditions, its impact on plastid genome expression, and its intersection with other signalling pathways. The ultimate goal is to enhance our understanding of ppGpp's role in plant acclimation, offering insights for improving crop resilience in a climate-challenged world.
Prion-like proteins in marine plankton: a quest towards new molecular factors of thermal adaptation
Climate change is reshaping the distribution of species on the planet and mechanisms for adaptation to thermal stress are then called upon. Recently, in terrestrial plants the role of prion-like proteins has been highlighted in flowering and vernalization mechanisms. However, these atypical proteins have not been characterized in the marine world where plankton plays an essential role in the biological carbon pump and the marine food web. To explore the world of prion-like proteins and their role in thermal adaptation of marine plankton species, we propose a three years PhD program in the computational biology team of the CEA-SEPIA in the François Jacob Institute of Biology located in Fontenay-aux-Roses, France. The first objective of the thesis is to identify and characterize the function of marine prion-like proteins and their biogeography in the world oceans. The student will also reconstruct the molecular evolution of these proteins across a wide spectrum of marine plankton species through gain/loss and adaptation signal analyses. The research approach will be based on comparative genomics and phylogeny on Tara Oceans metagenomic and metatranscriptomic data. Additionally, the student will identify prion-like proteins involved in the adaptation to temperature by integrating spatial and environmental data collected by the Tara Oceans expeditions. In a context of climate change, this research fits into the understanding of the molecular evolution of prion-like proteins, shedding light on their role in the thermal adaptation of species playing a key role in the marine food chain and geochemical cycles.
PPARy, a major player in bone marrow stromal homeostasis and a therapeutic target for myelofibrosis?
Myelofibrosis (MF) is the most severe of the Philadelphia-negative myeloproliferative neoplasias (MPNs), with a median survival of 5-6 years. Whether diagnosed de novo (Primary Myelofibrosis, PMF) or secondary to another MPN, the features of MF are similar. A subpopulation of haematopoietic cells derived from the pathological clone releases pro-inflammatory cytokines and growth factors into the bone marrow microenvironment. In response, the bone marrow microenvironment undergoes remodelling, resulting in osteosclerosis and fibrosis of the mesenchymal stromal cells (MSCs) associated with loss of haematopoietic support. The 2016 WHO classification includes a premyelofibrosis state to facilitate early diagnosis of patients at increased risk of progression. However, although major progress has been made in understanding the pathogenesis of the disease, notably with the description of the so-called "driver" mutations responsible for myeloproliferation (JAK2, CALR and MPL), apart from haematopoietic stem cell allotransplantation, which only concerns a minority of patients, current treatments are mainly symptomatic and have little influence on the natural history of MF.
Recently, we demonstrated that activation of the nuclear receptor PPARy (Peroxisome Proliferator-Activated Receptor-gamma) by its pharmacological ligands (Actos®) or (Pentaza®) reduced the development of osteosclerosis and reticulin fibrosis of the bone marrow (BM) and prevented anaemia resulting from bone marrow remodelling in three preclinical mouse models of MF (Lambert, Saliba et al. 2021). These results position PPARy agonists as interesting therapeutic candidates. However, before considering their therapeutic repositioning in the treatment of MF, it is imperative to characterise the status and function of PPARy within medullary MSCs both at the physiological stage and during the development of NMPs.
In this project, our initial results show that PPARy expression is decreased in murine and human MSCs at the MF stage. In contrast, no change in PPARy expression was observed in MSCs derived from other MPNs. Transcriptomic analyses also demonstrated that TGF-B, a major cytokine in the development of MF, is capable of negatively regulating PPARy expression in MSCs. In order to mimic this expression defect, we invalidated PPAR-y (KO) in two bone marrow MSC lines, the first murine (MS5), the second human (HS5, under characterisation). Under these conditions, basal expression of a panel of genes associated with MF is increased in MSC-KO to the level of wild-type lines stimulated by TGF-B. Expression of this panel was further increased in MSC-KO in the presence of TGF-B, indicating potentiation of the TGF-B-mediated signal in the absence of PPARy. This transcriptomic signature associated with KO-MSCs is found in murine MSCs from the thrombopoietin (TPOhigh) induced MF model as well as in human MSCs from patients with PMF. However, this expression profile was not found in MSCs from patients with another MPN, indicating that it is indeed a sign of a stage of MF.
Invalidation of PPARy does not affect the phenotypic signature of bone marrow MSCs, but their multipotent character is altered with a loss of adipocyte differentiation capacity associated with an increase in osteo-chondrocyte differentiation potential. These histological observations are corroborated by the decrease in the production of adipocyte factors by MSC-KO and an increase in the expression of the osteoblastic factor Runx-2. In addition, the supernatant of the KO line showed a marked increase in osteoprotegerin (OPG), a soluble molecule produced by osteoblasts that leads to apoptosis of osteoclasts. This deregulation of the osteoblast/osteoclast balance in KO conditions could explain the osteosclerosis observed in patients with MF. In addition, the production of CXCL12 (CXC motif Chemokine Ligand 12) and the bone marrow growth factor SCF (c-kit ligand) are greatly reduced in MSC-KO conditions, at both transcriptomic and protein levels. These data recapitulate the results described during transcriptomic analyses of MSC from patients with fibrosis. At the same time, the capacity of MSC-KO to support haematopoiesis, in both the short and long term, is significantly reduced, reflecting the cytopenias associated with MF.
In silico, RNA-Seq analyses were carried out on the MS5-WT and MS5-KO lines. Initial gene set enrichment analyses (GSEA) show that the pathways most significantly affected are inflammation, myogenesis (MSC to myofibroblast transition) and the cell cycle. Comprehensive analyses are now required to identify new therapeutic candidate genes and gain a better understanding of the development of bone marrow fibrosis.
These initial in vitro results support the key role of the PPARy receptor in the homeostasis of the bone marrow microenvironment and in the genesis of its remodelling during the development of myelofibrosis. However, in vitro approaches alone are unable to capture the full complexity of a disease involving multiple players including haematopoietic cells, immunological cells and all the cell types making up the bone marrow microenvironment. To integrate all these parameters, we have established a mouse model in which PPARy expression is reduced (haploinsufficiency) or invalidated (KO) in the medullary MSCs of animals. It is the study of this model that will form the core of the project. Initially, in vivo, it will be used to:
1) Characterise the role of PPARy in the homeostasis of the bone marrow microenvironment.
2) Assess the impact of reduced expression on the development of bone marrow fibrosis.
3) To validate the positioning of PPARy as a therapeutic target in the management of bone marrow fibrosis and to consider the repositioning of its pharmacological agonists (Actos®; Pentaza®) in this pathology.
The presence of medullary pre-fibrosis/fibrosis is a poor prognostic factor in MPN or acute myeloid leukaemia (AML). However, it is difficult to determine whether this condition is simply an indicator or whether it plays an active role in the development of haemopathies. The use of these models (Haplo-insufficient or KO for PPARy in MSCs) in association with preclinical mouse models of MPN (CML (BCR-ABL); PV (JAK2 V617F), ET (CALRDel52)) will allow, in a second phase, to determine whether:
1) The presence of a predisposition to bone marrow fibrosis influences the natural history of haemopathies.
2) In these diseases, which are purely haematopoietic in origin (mutation of the haematopoietic stem cell), it is appropriate to combine treatment targeting the malignant clone with treatment aimed at preventing the development of bone marrow fibrosis (activation of the PPARy receptor by its ligands in the haploinsufficiency condition).
This entire project is part of the Tomorrow's Biotechnologies (F) initiative, which aims to improve patient care through the development of personalised medicine.
Metal partitioning in coccolith-based calcite and biotechnological applications of metal-doped coccolith materials
Despite established cultures of coccolithophore microalgae and attainable large-scale production of coccolith biominerals (gram quantities of calcite mineral from litres of culture), the coccolith as an advanced functional material has made little progress in bionanotechnologies. This project will quantitatively describe metal doping into and on the surface of coccolith-based calcite for several transition metals, main group elements and lanthanides. Elucidating the potential of coccolithophore to incorporate metal ions into/onto biogenic calcite production will not only reveal the biotechnological possibilities for coccolith materials but will offer insights on the role of metals in the biomineralization process and the biological screening effect. Metal-doped coccolith materials will be subjected to physical and chemical characterization (focus placed on strategic metals that have enchanced incorporation into biogenic calcite or can be replaced/deposited on coccoltih surface). Select metal-doped coccolith candidates will be pursued for biotechnological application accordingly based on their physical and optical properties (e.g., catalytic activity for transition metals and photoluminescence for lanthanides).