More than 50% of the human spastic paraplegias (HSPs) depend on mutations of four endoplasmic reticulum (ER) resident proteins. Amongst them, are those specific to spastin, a protein with the ability to sever microtubules and therefore to regulate their dynamics. The localization and function of this protein suggest its primary role in determining the shape and distribution of ER and therefore its functions, but to date information is lacking to support these conclusions. Thanks to the different SPAST/SPG4 cell models available in the laboratory we will tackle the question regarding whether the defect in spastin expression or expression of mutated forms of this protein induce ER defects at morphological and functional level. Moreover we will investigate new potential molecular interactions of spastin that may help to understand the physio-pathological role of this protein. The results obtained will be valuable in the identification of possible cellular and molecular targets so as to come up with an effective therapeutic strategy for SPG4 linked-HSP and likely neurodegenerative diseases in which the ER is affected.
Investigate the role of spastin in the morphology and dynamics of the ER
Evaluate the level of ER-stress.
Analyze the role of spastin in the homeostasis of calcium concentration.
Validate the interaction of new molecular partners (Y2HS) of spastin in mammalian cells.
- Fibroblasts and cortical neurons derived from SPG4-KO mice and patients affected by SPAST mutation.
- Immunofluorescence, time lapse video-imaging, FRAP, calcium measurements.
- Western-blot, Co-immunoprecipitation.
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Molecular basis of neurodegenerative diseases: structural bioinformatics of protein-RNA complexes
As soon as they are transcribed, all the RNA molecules in a cell are bound by different sets of RNA-binding proteins (RBP) whose task is one of regulating their correct processing, transport, stability, and function/translation, all the way to degradation. RBPs are mostly nuclear proteins involved in RNA-splicing, in the regulation of translation and can associate with stress granules under stress conditions.They bind the nucleotides (RNA or DNA) through their cold shock domain (CSD).The misregulation of the genes involved in ARN metabolism or the autophagy/proteasome pathway plays a key role in the onset and the progression of several neurodegenerative diseases and of cancer.
Our interest is based on predicting and studying the physicochemical and biological properties of the systems at hand that are difficult to access by experimental means.
- Finding RNA sequence and structure preferences of the RBPs of interest to the laboratory.
- Generate accurate 3D molecular models of RNA-RBP complexes.
- Find the conformational changes that the RBPs and the RNAs undergo upon formation of the RBP-RNA complexes.
- Gain insight into the protein-protein and protein-nucleic acid interactions taking place.
Bioinformatics: sequence analysis and alignment, motif search, homology modeling, molecular simulationand docking,machine-learning algorithms, language programming (Python, R, …).
- The 3D solution structure of the CSD of the human protein YB-1, an RBP, is available in the protein 3D structure database (PDB). Several other CSDs from other proteins are available also in the PDB, just like the structures of several complexes between CSDs and oligonucleotides.These data will be used as starting points for modeling the complexes of interest.
- Machine-learning algorithms for identifying binding specificities of RBPs (RNAcontext, RNApredict) will be used and developed.
To understand the molecular mechanisms of action of the neurodegenerative diseases and cancer types studied in the laboratory, with important implications for the design and discovery of new drugs.
Note: The SABNP research laboratory (INSERM unit 1204), recently renewed, is part of Evry’s Genopole Biocluster. The Genopole is France’s first high-level research cluster entirely devoted to biotherapies, and to genetic, genomic, and post-genome research. SABNP is multidisciplinary and hosts, in addition to a structural biology platform (Atomic Force Microscopy and Nuclear Magnetic Resonance), cellular and molecular biology facilities, structural bioinformatics skills with access to a computer network including Linux clusters, and workstations and software dedicated to biological modeling and simulation. The PhD director will directly tutor the student.Experimental structural data coming from our NMR and AFM facilities will be complementary to the modeling of the specific RBP-RNA complexes.
For any application, send a letter of motivation and a CV to the following email address: firstname.lastname@example.org