Significance

Although heme synthesis is ubiquitous, specific regulatory mechanisms couple heme production to cellular demand and environmental conditions. The importance of these regulatory mechanisms is highlighted by clinical variability in porphyrias caused by loss-of-function mutations in heme synthesis enzymes. Heme synthesis is also controlled by the mitochondrial AAA+ unfoldase ClpX, which participates in both heme-dependent degradation of δ-aminolevulinate synthase (ALAS) and ALAS activation. This study reports a human familial mutation in CLPX that contributes to erythropoietic protoporphyria (EPP) by partially inactivating CLPX. Reduced CLPX activity increases ALAS post-translational stability, causing pathological accumulation of protoporphyrin IX (PPIX) in human patients. Our results thus identify an additional gene that promotes PPIX overproduction and EPP and highlight the complex gene network contributing to disorders of heme metabolism.

Abstract

Loss-of-function mutations in genes for heme biosynthetic enzymes can give rise to congenital porphyrias, eight forms of which have been described. The genetic penetrance of the porphyrias is clinically variable, underscoring the role of additional causative, contributing, and modifier genes. We previously discovered that the mitochondrial AAA+ unfoldase ClpX promotes heme biosynthesis by activation of δ-aminolevulinate synthase (ALAS), which catalyzes the first step of heme synthesis. CLPX has also been reported to mediate heme-induced turnover of ALAS. Here we report a dominant mutation in the ATPase active site of human CLPX, p.Gly298Asp, that results in pathological accumulation of the heme biosynthesis intermediate protoporphyrin IX (PPIX). Amassing of PPIX in erythroid cells promotes erythropoietic protoporphyria (EPP) in the affected family. The mutation in CLPX inactivates its ATPase activity, resulting in coassembly of mutant and WT protomers to form an enzyme with reduced activity. The presence of low-activity CLPX increases the posttranslational stability of ALAS, causing increased ALAS protein and ALA levels, leading to abnormal accumulation of PPIX. Our results thus identify an additional molecular mechanism underlying the development of EPP and further our understanding of the multiple mechanisms by which CLPX controls heme metabolism.

Porphyrias result from disorders of heme synthesis and are associated with mutations in numerous heme synthetic enzymes (1). The genetic penetrance of porphyria-causing alleles is variable, however (1⇓–3), often due to the presence of diverse environmental factors and the existence of modifier genes. In addition, some patients with porphyria who suffer from cutaneous photosensitivity and abnormal liver function have illnesses of unknown etiology, highlighting our incomplete understanding of this complex condition.

Erythropoietic protoporphyria (EPP) is a disorder characterized by the pathological accumulation of a late heme biosynthetic intermediate, protoporphyrin IX (PPIX), in erythroid cells. Approximately 90% of patients with EPP carry a partial deficiency in FECH(EC 4.99.1.1, OMIM 177000), the gene for the mitochondrial enzyme ferrochelatase, which catalyzes the insertion of iron into PPIX for the final step in heme production. In most cases, EPP is due to cosegregation of a heterogeneous, family-specific deleterious FECHallele in compound heterozygosity with the common, low-expression, c.315–48C FECHallele, which affects the use of a cryptic splice-acceptor site in FECH pre-mRNA (4⇓–6). A second, less common class of EPP results from gain-of-function mutations in ALAS2, the erythroid-specific gene for δ-aminolevulinate synthase (ALAS; EC 2.3.1.37, OMIM 300752), which catalyzes the initial step in heme biosynthesis. These dominant, gain-of-function ALAS2 alleles increase ALA production, which triggers the accumulation of downstream heme precursors, especially PPIX. The proportion of patients with EPP with an ALAS2mutation is <5% in Europe (4, 5) and <10% in the United States (2). Importantly, genetic analysis fails to detect FECH and ALAS2 mutations in 1–5% of families with a member with EPP (which are mostly homozygous for the WT FECH c.315–48T allele), suggesting involvement of loci other than FECH and ALAS2 in this important metabolic illness (3, 7).

Here, we report the identification of a third mechanism underlying EPP in an affected family. This EPP is promoted by a mutation in CLPX, which encodes an AAA+ (ATPases associated with various cellular activities) protein unfoldase. ClpX is widely conserved among bacteria and in the eukaryotic mitochondrion. ClpX works as a ring-shaped homohexamer and is best understood in its function in a proteasome-like complex with the peptidase ClpP (i.e., the ClpXP ATP-dependent protease). For well-studied examples of ClpXP substrates in bacteria, ClpX recognizes specific short sequence motifs in the substrate protein. ClpX unfolds the tertiary structure of substrate protein by ATP-powered polypeptide translocation through its central pore and then presents this unfolded sequence directly into the ClpP proteolytic chamber (reviewed in ref. 8). Recently, ClpX has been identified as a modulator of heme biosynthesis by activating ALAS (9), and also has been observed to mediate the turnover of ALAS under heme-replete conditions; this turnover requires a heme-binding motif in ALAS (10).

Here, we report the identification and analysis of a dominant heterozygous mutation that disables the ATPase activity of CLPX, causing accumulation of PPIX in red blood cells and promoting the development of EPP symptoms. Our experiments demonstrate how this impaired CLPX activity causes deleterious effects in heme metabolism, organismal physiology, and human health.

Results