In humans, disruptions in the
heme biosynthetic pathway are associated with various types of
porphyrias, including
variegate porphyria that results from the decreased activity of
protoporphyrinogen oxidase IX (PPO; E.C.1.3.3.4), the
enzyme catalyzing the penultimate step of the
heme biosynthesis. Here we report the generation and characterization of human cell lines, in which PPO was inactivated using the CRISPR/Cas9 system. The PPO knock-out (PPO-KO) cell lines are viable with the normal proliferation rate and show massive accumulation of
protoporphyrinogen IX, the PPO substrate. Observed low
heme levels trigger a decrease in the amount of functional
heme containing respiratory complexes III and IV and overall reduced oxygen consumption rates. Untargeted proteomics further revealed dysregulation of 22 cellular
proteins, including strong upregulation of 5-aminolevulinic
acid synthase, the major regulatory
protein of the
heme biosynthesis, as well as additional ten targets with unknown association to
heme metabolism. Importantly, knock-in of PPO into PPO-KO cells rescued their wild-type phenotype, confirming the specificity of our model. Overall, our model system exploiting a non-erythroid human U-2 OS cell line reveals physiological consequences of the PPO ablation at the cellular level and can serve as a tool to study various aspects of dysregulated
heme metabolism associated with
variegate porphyria.