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Caractérisation structurale du recrutement de la protéine JIP1 par la chaîne légère (KLC) de la kinésine1

Abstract : AbstractKinesins are molecular motors involved in the intracellular transport of many cargos within the cell. Although the motility of kinesins is well understood, the molecular mechanisms underlying cargo recruitment are much less so.Kinesin1 plays various roles in neuronal cells, where it contributes to the spatial and temporal organization of many cellular components. It would play a role in various neurological pathologies, such as Alzheimer's disease. Understanding how kinesin1 recognizes and interacts with its cargos is important to decorticate its role, as well as that of its cargos, in normal and pathological cells. Kinesin1 is a heterotetramer consisting of two heavy chains (KHC) and two light chains (KLC), both of which are capable of recruiting cargo proteins. One of the first cargo proteins to have been identified is JIP1 (JNK-interacting protein 1) which is: (i) a scaffold protein for the signaling pathway of MAP kinases and (ii) an adaptor protein for transporting amyloid precursor protein (APP) responsible for Alzheimer's disease. In both cases, JIP1 regulates critical processes at the cell level, making it an interesting protein to study. Early studies have led to a better understanding of how JIP1 is recruited and transported by kinesin1. However, the detail of the interaction between KLC and JIP1 is not yet fully described and therefore understood.Objectives: My doctoral work aims at characterizing at the molecular level the interaction between KLC and JIP1. To do this, I had the following objectives: 1) to characterize the interaction domains of the two proteins alone, 2) to study the formation of the complex in solution by biophysical approaches, and 3) to determine the 3D structure of the complex by crystallography.Results: Initially, I characterized the TPR domain of KLC alone, contributing among others to the development of a molecular toolbox. I also participated in the determination of two crystallographic structures of the TPR domain of KLC1/2 that highlights the structural plasticity of the first helix of this domain (Nguyen et al, submitted). In a second step, I set up the conditions for the expression and purification of the PTB domain of JIP1 and carry out the structural characterization of this domain in solution. Although this domain of JIP1 is not necessary for interaction with KLC, I studied the impact of its presence on recruitment by KLC. Finally, I characterized the recruitment of JIP1 by KLC by confirming a number of information on the interaction between the KLC-TPR and the C-terminal region (Cter) of JIP1 at the molecular level. The numerous crystallization tests that I carried out did not make it possible to obtain crystals of the KLC: JIP1 complex. However, I was able to precisely map the interaction zone of JIP1-Cter with the KLC-TPR domain using the various KLC tools available by determining by ITC their affinity with JIP1-Cter (Nguyen et al., In preparation ).Conclusion: Thus, my PhD work allowed to better understand 1) the structural versatility of the KLC-TPR domain, 2) the impact of the JIP1-PTB domain for its KLC recruitment, and 3) the interaction mode of JIP1 by KLC . On the basis of these data, I will discuss the structural basis of the mode of binding of KLC with JIP1 and compare it with that of KLC with WD-motif cargo, such as SKIP and Alcadein-α.
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Submitted on : Monday, April 30, 2018 - 10:32:22 AM
Last modification on : Saturday, September 12, 2020 - 3:14:54 AM

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  • HAL Id : tel-01780347, version 2

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The Quyen Nguyen. Caractérisation structurale du recrutement de la protéine JIP1 par la chaîne légère (KLC) de la kinésine1. Biologie structurale [q-bio.BM]. Université Paris-Saclay, 2017. Français. ⟨NNT : 2017SACLS250⟩. ⟨tel-01780347v2⟩

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