A Dominant Mutation in Mitochondrial Unfoldase CLPX Results in Erythropoietic Protoporphyria

Red cells synthesize large quantities of heme during terminal differentiation. Central to erythropoiesis is heme synthesis, which requires tight coordination between mitochondrial iron import and synthesis of protoporphyrin IX (PPIX).

Most individuals with erythropoietic porphyria carry loss of function mutations in FECH, or gain of function mutations in ALAS2, resulting in protoporphyrin IX accumulation. We performed whole exome sequencing to identify novel mutations in an individual exhibiting symptoms in whom FECH and ALAS2 mutations were absent (Fig. A, asterisk). We identified a novel CLPX point mutation in this individual (III.2), her father (II.4) and her paternal uncle (II.2), who also exhibited increased porphyrin levels relative to healthy individuals. The individual's mother was healthy and had a wild-type CLPX genotype (Fig. A). CLPX encodes a mitochondrial protein unfoldase that partially unfolds ALA synthase (ALAS) to allow efficient incorporation of its cofactor, pyridoxal phosphate (Kardon et al. 2015 Cell). This greatly stimulates the synthesis of d-aminolevulinic acid (ALA), the first step in heme biosynthesis.

To determine if the CLPX mutation was causative for porphyria, we expressed mutant CLPX in HEK293T embryonic kidney cells and Friend mouse erythroleukemia (MEL) cells. Mutant CLPX expression (MUT) caused a significant increase in ALAS1 (non-erythroid isoform) (Fig. B) and ALAS2 (erythroid isoform) (Fig. C) activity relative to control and wild-type CLPX expressing samples (WT). This increase in ALAS enzymatic activity translated to an increase in PPIX levels (Fig. D, E), consistent with the porphyria phenotype observed in the individuals in this study.

We observed that MUT-expressing samples had increased levels of ALAS1 and ALAS2. To determine if mutant CLPX altered ALAS protein stability, we transfected WT or MUT CLPX into HEK293T and MEL cells. Cells were treated with cycloheximide to block translation. We quantitated degradation rate of ALAS by western blot analysis of cell lysates obtained at several time points after cycloheximide treatment (chase). Expression of MUT CLPX stabilized both ALAS1 and ALAS2, accounting for the increase in ALAS protein levels, activity and downstream production of PPIX.

The control of ALAS enzymatic activity and protein stability by CLPX unveils a novel cause of protoporphyria and insights revealing the ways in which mitochondrial physiology and heme synthesis are interdependent. Our results reveal an important regulatory node where the mitochondrial protein quality control machinery intersects with a key step in heme synthesis and provides an important genetic tool for understanding the pathology of porphyrias.

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