PhD Defense Philémon VON GAUDECKER "Molecular determinants of WDR41 recognition by the lysosomal transceptor PQLC2" (eq. Lenoir/Vazquez - B3S dpt)

Philémon VON GAUDECKER - eq. Lenoir/Vazquez - B3S dpt

Title: Molecular determinants of WDR41 recognition by the lysosomal transceptor PQLC2

Jury members:
Agathe URVOAS (présidente)
Jean Michel JAULT (rapporteur)
Isabelle MUS-VETEAU (rapporteur)
Alex PERÁLVAREZ-MARÍN (examinateur)
José Luis VÁZQUEZ IBAR (directeur de thèse)

Abstract:
As lysosomes become more and more characterized as signaling hubs, new studies have revealed that the lysosomal cationic amino acid transporter PQLC2 (SLC66A1) [1] is involved in the regulation of an emerging signaling pathway. In addition to cationic amino acids export from the lysosomal lumen to the cytosol, PQLC2 responds to the cellular deprivation of its substrates by recruiting a three-protein complex at the lysosome: C9orf72-SMCR8-WDR41 (CSW). PQLC2 is thus a ‘transceptor’ combining both transporter and receptor function. The CSW complex is known for its GTPase-activating protein activity and implication in different cellular functions, including autophagy, vesicle trafficking and the regulation of mTORC1 kinase complex [2]. Importantly, mutations in the C9orf72 gene are also a major cause of familial forms of two neurodegenerative diseases, amyotrophic lateral sclerosis and fronto-temporal dementia [3]. The molecular basis of CSW’s recruitment by PQLC2 remain poorly characterized, as high-resolution structural data is missing. Pull-down assays in cell cultures suggested that the CSW recruitment occurs via interaction of a protruding flexible loop of WDR41 and PQLC2, where aromatic residues in both proteins seem to play essential roles [4]. The aim of my PhD thesis is to understand the transceptor role of PQLC2 by investigating the molecular mechanisms connecting amino acid sensing and CSW recruitment. In particular, my project has focused on characterizing “in vitro” and using purified proteins the structural and biochemical elements involving PQLC2/WDR41 recognition, key for the lysosomal recruitment of the CSW complex, and investigate how PQCL2 substrates modulate this interaction.  To obtain purified PQLC2 with optimal stability in detergent micelles, we constructed an engineered PQLC2 using a consensus-based mutagenesis approach. This engineered version of PQLC2 has allowed to gather preliminary structural information by Cryo-EM (collaboration with the Biofisika Institute,Bilbao, Spain) showing that PQLC2 assembles as both homodimers or homotrimers. In complement, SECMALS experiments attest that PQLC2 in detergent micelles is also forming dimers and trimers. To improve the yield of purification as well as the stability of WDR41 in solution, I engineered a ΔC WDR41 mutant by deleting its C-terminal end. This hydrophobic region is known to interact with SMCR8 in the CSW complex. Using two different techniques (pull-down and microscale thermophoresis), I demonstrated that the engineered ΔC WDR41 specifically binds to purified PQLC2 in detergent micelles. Moreover, competition experiments using synthetic peptides mimicking the WDR41 flexible loop and the use of WDR41 mutants in this loop, confirmed the essential role of aromatic residues in the flexible loop in mediating WDR41/PQLC2 recognition. In addition, aromatic residues predicted to sit in the cytosolic-open cavity of PQLC2 also participate on WDR41/PQLC2 interaction. Finally, using a N-terminal deleted WDR41 mutant I also discovered an unexpected stabilization role of the N-terminal region of WDR41 on PQLC2/WDR41 complex formation. Collectively, these results support a model of PQLC2/WDR41 association in which the flexible loop of WDR41 inserts into the cytosolic cavity of PQLC2, while the N-terminal stabilizes the complex, possibly by interacting with an adjacent PQLC2 protomer. In summary, by characterizing the molecular basis of PQLC2-WDR41 interaction using purified proteins, this thesis provides new insights towards understanding a novel and still understudied lysosome-mediated signaling pathway with possible implications in the pathogenesis of two major neurodegenerative diseases.

References:
1 A. Jézégou, et al. PNAS(2012) DOI : 10.1073/pnas.1300178110
2 J. Amick, et al. J Cell Bio (2019) DOI : 10.1083/jcb.201906076
3 M. DeJesus-Hernandez et al, Neuron (2011) DOI : 10,1016/j.neuron.201109011
4 G. Talaia, et al., PNAS(2021 ) DOI : /10.1073/pnas.2014941118