Mutation in human CLPX elevates levels of δ- aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria

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 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.

Imipridone Anticancer Compounds Ectopically Activate the ClpP Protease and Represent a New Scaffold for Antibiotic Development

“…This result suggests that CLPX may be degraded adventitiously by active CLPP and/or that CLPP activation may normally be self-limited by degradation of its physiologically obligate cofactor. CLPX also performs chaperone functions independently of CLPP, as it promotes heme biosynthesis and enhances the DNA-binding activity of TFAM, a mitochondrial transcription factor (Kasashima et al 2012;Yien et al 2017). TFAM itself was also depleted in response to ONC212, in agreement with previous studies in other cell lines (Greer et al 2018;Graves et al 2019).…”

From Synthesis to Utilization: The Ins and Outs of Mitochondrial Heme

“…Unlike in ALAS1, N-terminal HRMs present in ALAS2 protein do not render mitochondrial import sensitive to heme [54]. Post-translational events that impact enzyme activity include the binding of ALAS2 and the AAA+ unfoldase, CLPX, a complex which facilitates both PLP binding and ALAS2 degradation [62], as well as hydroxylation of the ALAS2 C-terminal extension and the associated proteosomal degradation of the protein [63].…”

“…Clearly any mutations in these interacting proteins that cause dysfunction in heme synthesis may contribute to porphyrias and anemais. One example is a recently discovered CLPX mutation, which has been implicated in porphyria [62]. Additional studies on the function and interactions among the protein components will further our understanding of the regulation of heme synthesis and, thus, the pathophysiology of the anemias and porphyrias.…”

The molecular genetics of sideroblastic anemia

“…21 ALAS2 is subject to multiple negative regulatory mechanisms: (1) the mRNA is subject to translational downregulation in response to iron deficiency through an iron-responsive element (IRE) located in the 59 untranslated region (UTR), (2) enzymatic activity is negatively regulated by a C-terminal inhibitory domain, and (3) protein levels are regulated by the CLPX/CLPP mitochondrial quality control protease. [22][23][24] Indeed, C-terminal truncations of ALAS2 in humans and deletion of the IRE-binding protein IRP2 in mice lead to excessive protoporphyrin IX production (erythropoietic protoporphyria [EPP]) by upregulating ALAS2 activity and protein, respectively. 22,[25][26][27] This "opposite phenotype" (ie, EPP) resulting from loss of ALAS2 regulatory factors highlights the therapeutic potential for interfering with these mechanisms in XLSA to increase mutated ALAS2 expression, activity, or stability as a therapeutic strategy.…”