94 Chapter 4 deprivation (Figure 1d and e). In electron micrographs, double-membrane bound electron-dense structures, with peroxisome-like structures inside, appeared in the organoids depleted of amino acids for either 48 or 96 hours (Figure 1f). These structures probably represent autophagosomes or autolysosomes, suggesting autophagy is occurring, most likely also including degradation of peroxisomes. These results are in agreement with previous observations in amino-acid deprived cell cultures33. We then wondered if amino acid restriction also affected the biogenesis of peroxisomes. Peroxins (PEX) are proteins involved in various aspects of peroxisome biogenesis. Gene expression of these peroxins was assessed by qPCR. PEX3, PEX6 and PEX19 are essential peroxins for the assembly of peroxisomal membranes. Targeting and insertion of membrane proteins involved PEX19 as a cytosolic receptor protein, PEX3 works as a membrane anchoring site for cargo-loaded PEX19 and PEX16 as possible receptor for PEX334. The mRNA level of PEX3 was significantly upregulated in amino-acid deprived organoids, while PEX19 showed a downwards trend and PEX16 remained unchanged. For the import of proteins into the peroxisome, PEX5 and PEX7 recognize cargo with Peroxisome Targeting Sequence 1 and 2 (PTS-1 and 2), respectively. Interestingly, mRNA level of PEX7 was significantly downregulated, while PEX5 was upregulated in amino-acid deprived organoids. The mRNA levels of PEX1 and PEX6, peroxins involved in the protein import, remained unchanged. The mRNA level of PEX11, involved in peroxisomal fission35, was increased upon amino acid removal (Figure 1g). These results indicate that amino-acid deprivation in hepatic organoids differently affects expression of mRNAs encoding peroxisomal biogenesis proteins.
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