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Thesis (PhD(Medical Sciences)--University of South Australia, 2021. Includes bibliographical references (pages 140-153) This thesis examines the role of ubiquitination of Arrdc4 protein, an adaptor for Nedd4 family of ubiquitin ligases, in the release of extracellular vesicles (EV). As EVs play crucial functions in inter-cellular communication, it is important to understand how they are generated by the cells. Studies here report that Arrdc4, that has been previously shown to be involved in EV biogenesis, undergoes ubiquitination at several Lys (K) residues. Among these, ubiquitination at K270, involving K29-linked ubiquitin chains, is crucial for the function of Arrdc4 in EV biogenesis. The study also revealed the importance of Arrdc4-dependent EV release in reticulocyte maturation and identified potential interacting partners for Arrdc4. Overall, this study contributes novel findings towards ubiquitination-dependent regulation of EV biogenesis and provides new directions for further studies.

The transcriptional regulation of glucose homeostasis is a central tenet of biology. In metazoans, this function is performed by the Myc-related transcriptional activators, MondoA and ChREBP. ChREBP operates predominantly in liver and promotes the conversion of high levels of intracellular glucose to lipid for energy storage and membrane synthesis. MondoA operates predominantly in skeletal muscle and promotes the conversion of high levels of intracellular glucose to ATP for immediate use in this highly catabolic tissue type. The experiments presented here detail how MondoA:Mlx heterodimers sense and respond to glucose. The hexokinase-dependent phosphorylation of glucose to G6P stimulates nuclear accumulation and transcriptional activity of MondoA:Mlx, which in turn governs 75% of glucose-dependent transcription. In high glucose conditions, MondoA activates a negative regulator of glucose uptake, thioredoxin interacting protein (TXNIP) to downregulate further glucose uptake. This negative feedback loop is conserved in all cell types tested. MondoA:Mlx shuttles between mitochondria and the nucleus in high and low glucose conditions; G6P and heterodimerization with Mlx promote nuclear accumulation of the heterodimer. The Nterminal Mondo Conserved Regions (MCRs) of MondoA define the glucose-dependent character of the protein. In conjunction with the C-terminal DNA binding domain, the MCRs mediate activation of MondoA:Mlx heterodimers through three distinct steps: nuclear accumulation, promoter occupancy, and coactivator recruitment. Each of these The transcriptional regulation of glucose homeostasis is a central tenet of biology. In metazoans, this function is performed by the Myc-related transcriptional activators, MondoA and ChREBP. ChREBP operates predominantly in liver and promotes the conversion of high levels of intracellular glucose to lipid for energy storage and membrane synthesis. MondoA operates predominantly in skeletal muscle and promotes the conversion of high levels of intracellular glucose to ATP for immediate use in this highly catabolic tissue type. The experiments presented here detail how MondoA:Mlx heterodimers sense and respond to glucose. The hexokinase-dependent phosphorylation of glucose to G6P stimulates nuclear accumulation and transcriptional activity of MondoA:Mlx, which in turn governs 75% of glucose-dependent transcription. In high glucose conditions, MondoA activates a negative regulator of glucose uptake, thioredoxin interacting protein (TXNIP) to downregulate further glucose uptake. This negative feedback loop is conserved in all cell types tested. MondoA:Mlx shuttles between mitochondria and the nucleus in high and low glucose conditions; G6P and heterodimerization with Mlx promote nuclear accumulation of the heterodimer. The Nterminal Mondo Conserved Regions (MCRs) of MondoA define the glucose-dependent character of the protein. In conjunction with the C-terminal DNA binding domain, the MCRs mediate activation of MondoA:Mlx heterodimers through three distinct steps: nuclear accumulation, promoter occupancy, and coactivator recruitment. Each of theseThe transcriptional regulation of glucose homeostasis is a central tenet of biology. In metazoans, this function is performed by the Myc-related transcriptional activators, MondoA and ChREBP. ChREBP operates predominantly in liver and promotes the conversion of high levels of intracellular glucose to lipid for energy storage and membrane synthesis. MondoA operates predominantly in skeletal muscle and promotes the conversion of high levels of intracellular glucose to ATP for immediate use in this highly catabolic tissue type. The experiments presented here detail how MondoA:Mlx heterodimers sense and respond to glucose. The hexokinase-dependent phosphorylation of glucose to G6P stimulates nuclear accumulation and transcriptional activity of MondoA:Mlx, which in turn governs 75% of glucose-dependent transcription. In high glucose conditions, MondoA activates a negative regulator of glucose uptake, thioredoxin interacting protein (TXNIP) to downregulate further glucose uptake. This negative feedback loop is conserved in all cell types tested. MondoA:Mlx shuttles between mitochondria and the nucleus in high and low glucose conditions; G6P and heterodimerization with Mlx promote nuclear accumulation of the heterodimer. The Nterminal Mondo Conserved Regions (MCRs) of MondoA define the glucose-dependent character of the protein. In conjunction with the C-terminal DNA binding domain, the MCRs mediate activation of MondoA:Mlx heterodimers through three distinct steps: nuclear accumulation, promoter occupancy, and coactivator recruitment. Each of these steps is glucose-dependent, and requires the 12-amino acid MCRII domain. In sum, MondoA is essential for intracellular glucose homeostasis, MondoA activity is regulated at multiple glucose-dependent steps, and MondoA-dependent target genes control intracellular glucose availability.