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Abstract: Mutations in the ATP13A2 gene were identified as the cause of Kufor-Rakeb syndrome (KRS), a juvenile-onset form of Parkinson’s disease (PD). Developing relevant and predictive models for the rare PD forms is necessary to understand the pathological mechanisms and validate therapeutic strategies. Herein, we aimed to comprehensively characterize the first transgenic Atp13a2 knockout rat model. Behavioral assessment demonstrated specific developmental deficits in animals with deletion of Atp13a2. Further analysis revealed that Atp13a2 knockout rats displayed age-dependent fine motor skills deficits and impaired locomotor habituation similar to those observed in PD patients at the early stage of motor symptoms. In contrast, no change in the nigrostriatal integrity was observed. An extended investigation on heavy metals homeostasis, autophagy-related markers, and lipofuscin accumulation showed significant changes reminiscent of KRS. Finally, we tested whether inducing pathology by viral-mediated overexpression of human α-synuclein or human tyrosinase exacerbated the onset or extent of pathological changes. This Atp13a2 KO rat model could help better understand autophagy in PD pathogenesis and open new therapeutic validation opportunities.

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Abstract: Marine picocyanobacteria are essential drivers of global biogeochemical cycles, playing a crucial role in ecosystem functioning. Their significant phylogenetic and functional diversity allow them to thrive across various marine regions. However, the mechanisms underpinning their adaptation to diverse oceanic niches remain poorly understood, especially with changing nutrient landscapes driven by climate change and anthropogenic activities. A critical factor in their success are nutrient uptake systems, particularly ATP-binding cassette (ABC) transporters, which account for over half of their transport capabilities. This thesis explores the substrate specificity, and ecological roles of substrate-binding proteins (SBPs) associated with these transporters, highlighting how these systems aid niche adaptation in marine picocyanobacteria. SBPs are key to ABC transporter function, binding substrates with high specificity and affinity, facilitating their cellular uptake. This study integrates structural biology, genomics, biophysical studies, and environmental genomics to elucidate the functions and ecological roles of these proteins, providing insights into the adaptive strategies of marine picocyanobacteria. Functional characterisation of three phosphate binding protein homologs (PstS), three urea binding protein homologs (UrtA) and two glycine-betaine binding protein homologs (ProX) from Synechococcus strains inhabiting distinct oceanic niches was conducted. The study of PstS homologs in WH8102 revealed that PstS1b, unique to Synechococcus clade III strains, is the highest affinity binding protein, providing a competitive advantage in ultraoligotrophic environments. Analysis of UrtA homologs in CC9311 and WH8102 revealed UrtA from CC9311 had higher binding affinity to urea compared to both UrtA proteins from WH8102. This is intriguing as CC9311 originates from a high-nutrient mesotrophic environment, contrasting with WH8102’s habitat in a nutrient-poor oligotrophic region. Investigation of ProX homologs from MITS9220 and WH8102 revealed a functional distinction between the two proteins, with MITS9220_ProX likely behaving as a generalist solute-binding protein, while WH8102_ProX acts as a specialist binding protein for higher salinity environments. The findings of this thesis highlight the versatility and adaptability of SBP-dependent ABC transporters in marine picocyanobacteria. The study demonstrates that these transport systems modulate their substrate affinities and specificities in response to environmental conditions, facilitating niche adaptation. This research is part of a larger project aimed at characterising the full repertoire of SBPs in marine picocyanobacteria, enhancing our understanding of nutrient acquisition mechanisms in these important microorganisms. The integration of various scientific approaches in this study provides a comprehensive framework for investigating the physiological and ecological significance of ABC uptake systems, offering valuable insights into the adaptive strategies of marine microbes in the face of changing oceanic conditions.

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Abstract: The evolutionary history of retrotransposons and their hosts shapes the dynamics of transposition and restriction. The Pseudoviridae of yeast includes multiple Ty1 LTR-retrotransposon subfamilies. Saccharomyces cerevisiae prevents uncontrolled retrotransposition of Ty1 subfamilies using distinct mechanisms: canonical Ty1 is inhibited by a self-encoded restriction factor, p22/p18, whereas Ty1’ is inhibited by an endogenized restriction factor, Drt2. The minimal inhibitory fragment of both restriction factors (p18m and Drt2m) is a conserved C-terminal capsid domain. Here, we use biophysical and genetic approaches to demonstrate that p18m and Drt2m are highly specific to their subfamilies. Although the crystal structures of p18m and Drt2m are similar, three divergent residues found in a conserved hydrophobic interface direct restriction specificity. By mutating these three residues, we re-target each restriction factor to the opposite transposon. Our work highlights how a common lattice-poisoning mechanism of restriction evolved from independent evolutionary trajectories in closely related retrotransposon subfamilies. These data raise the possibility that similar capsid-capsid interactions may exist in other transposons/viruses and that highly specific inhibitors could be engineered to target capsid interfaces.

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Abstract: Since the first paper published by Susan Cole in 1990 detailing multidrug resistance mediated by ABCC1/MRP1, research into the C-subfamily of ATP-binding cassette transporters has continued to uncover a wide range of functionally divergent proteins. However, several orphan transporters remain in the C-subfamily, and the physiological function and substrates of ABCC5, ABCC11, and ABCC12 remain elusive. This review explores the emerging understanding of human ABCC5. Unlike other ABC transporters with well-defined drug export functions, ABCC5’s physiological roles remain only partially understood. While it is known for its involvement in multidrug resistance in cancers, recent studies suggest broader implications in development, metabolism, neurobiology, and male fertility. ABCC5 exports various endogenous substrates, including cyclic nucleotides (cAMP and cGMP), glutamate conjugates like NAAG, and possibly haem. Knockout models in mice, zebrafish, and sea urchins reveal ABCC5’s role in gut formation, brain function, eye development, and iron metabolism. In mice, its deletion results in lower adipose tissue mass, enhanced insulin sensitivity, and neurobehavioral changes resembling schizophrenia, highlighting its role in glutamatergic signalling and circadian regulation. Functionally, ABCC5 appears to impact adipocyte differentiation and GLP-1 release, implicating it in type 2 diabetes susceptibility in humans. Structural studies using human ABCC5 revealed a novel autoinhibitory mechanism involving a peptide segment (C46–S64) that blocks substrate binding, offering new potential for selective inhibitor development. However, this review emphasises caution in targeting ABCC5 for cancer therapy due to its underappreciated physiological function(s), particularly in the brain and male reproductive system. Understanding ABCC5’s substrate specificity, regulatory mechanisms, and functional redundancy with its paralog ABCC12 remains critical for successful therapeutic strategies in humans.

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Abstract: Mutations in the E3 ubiquitin ligase Parkin gene have been linked to early onset Parkinson’s disease. Besides many other roles, Parkin is involved in clearance of damaged mitochondria via mitophagy—a process of particular importance in dopaminergic neurons. Upon mitochondrial damage, Parkin accumulates at the outer mitochondrial membrane and is activated, leading to ubiquitination of many mitochondrial substrates and recruitment of mitophagy effectors. While the activation mechanisms of autoinhibited Parkin have been extensively studied, it remains unknown how Parkin recognizes its substrates for ubiquitination. Here, we characterize a conserved region in the flexible linker between the Ubl and RING0 domains of Parkin, which is indispensable for Parkin interaction with the mitochondrial GTPase Miro1. Our results may explain fast kinetics of Miro1 ubiquitination by Parkin in recombinant assays and provide a biochemical explanation for Miro1-dependent Parkin recruitment to the mitochondrial membrane observed in cells. Our findings are important for understanding mitochondrial homeostasis and may inspire new therapeutic avenues for Parkinson’s disease.