Henriques,Bárbara Joana de AlmeidaDomingos,Ruy Manuel Soares de Albergaria2026-05-212026-05-212025http://hdl.handle.net/10400.5/118662Tese de mestrado, Bioquímica e Biomedicina, 2025, Universidade de Lisboa, Faculdade de CiênciasSuccinylation and Glutarylation, two negative charge acylations, have recently been proposed as important regulators of cellular and mitochondrial processes. One such process is mitochondrial fatty acid β-oxidation. However, little is known about how these acylations regulate this metabolic pathway. Additionally, mitochondrial fatty acid oxidation disorders (FAOD), due to metabolic decompensation upon fasting or prolonged infections, lead to dysregulated metabolite levels. This, in turn, could affect cellular succinylation and glutarylation levels, potentially contributing to symptom onset. Despite this, no concrete link between FAOD related metabolic alterations and negative charge acylations has been established.Taking this into account, we aimed to, firstly, shed light on mechanisms by which both suc-cinylation and glutarylation regulate β-oxidation. Secondly, we aimed to take preliminary steps towards characterizing succinylation and glutarylation profiles in cellular models of FAOD. These profiles could provide valuable insight into how these negative charge acylations are associated with symptom onset and disease progression in FAOD patients. In this work, we identified that medium chain acyl-CoA dehydrogenase (MCAD) was suscep-tible to in vitro succinylation and glutarylation. Additionally, these modifications regulated MCAD ac-tivity by modulating its substrate affinity, in a process that is controlled by the deacylase Sirtuin 5. Surprisingly, neither modification had significant structural implications on MCAD nor did they impede its interaction with electron transfer flavoprotein (ETF), the natural electron acceptor during dehydro-genation. Through cellular work, we also provide evidence that supports the link between metabolism and these modifications, by showing that activation of specific metabolic pathways positively modulates modification levels.These findings shed light on the mechanisms by which these modifications regulate fatty acid β-oxidation, by uncovering a novel regulatory mechanism of MCAD enzymatic activity. Furthermore, we provide evidence that succinylation and glutarylation levels are modulated by cellular metabolism, and could therefore be implicated in pathophysiological mitochondrial deregulation associated with FAOD.application/pdfengSuccinylationglutarylationmedium chain acyl-CoA dehydrogenasemitochondrial β-oxidationfatty acid oxidation disordersmaster thesis204173485