180 Chapter 6 and its level is dynamically regulated to adjust to the metabolic state through biosynthesis and turnover (Figure 5a)38. Next, we measured the relative gene expression of different genes involved in coenzyme A biosynthesis and total CoA levels (Figure 5b-c). This process occurs partly in the cytosol and partly in the mitochondria (Figure 5a). PANK2, encoding the mitochondrial pantothenate kinase, was slightly, but significantly downregulated in the MCADD organoids (Figure 5c). In addition to CoA biosynthesis, carnitine acyl-transferases and acyl-thioesterases (ACOT) also modulate the size of the CoA and acyl-CoA pools in different intracellular compartments. Peroxisomes also contain several enzymes involved in CoA recycling (Figure 5a), among them two carnitine acyl-transferases: carnitine octanoyl-transferase (CROT) and carnitine acetyl-transferase (CRAT), in charge of converting Acyl-CoAs into their respective acyl-carnitine form, with the concomitant release of free CoA. MCADD organoids showed substantially lower CROT and CRAT expression than healthy controls (Figure 5d). NUDT7, a gene that encodes a peroxisomal Nudix hydrolase, which hydrolyzes CoA and acyl-CoA species into 4’pantotheine and acyl-4’-pantotheine39,40 and also plays a role in the metabolism of dicarboxylic acids41,42 was clearly upregulated in MCADD organoids (Figure 5d). The total CoA pool (free CoA plus acyl-CoA) did not differ between MCADD and control Mat-EHOs (Figure 5b). Taken together, we observe a regulation of some genes involved in CoA metabolism, but an obvious compensatory response could not be identified.
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