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

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. Keywords: heme biosynthesis; porphyria; ALAS; protein unfoldases; AAA+ ATPaseNational Institutes of Health (U.S.) (Grant F32 DK095726)National Institutes of Health (U.S.) (Grant R01 GM049224

Mitochondrial ClpX activates an essential biosynthetic enzyme through partial unfolding

© 2020, eLife Sciences Publications Ltd. All rights reserved. Mitochondria control the activity, quality, and lifetime of their proteins with an autonomous system of chaperones, but the signals that direct substrate-chaperone interactions and outcomes are poorly understood. We previously discovered that the mitochondrial AAA+ protein unfoldase ClpX (mtClpX) activates the initiating enzyme for heme biosynthesis, 5-aminolevulinic acid synthase (ALAS), by promoting cofactor incorporation. Here, we ask how mtClpX accomplishes this activation. Using S. cerevisiae proteins, we identified sequence and structural features within ALAS that position mtClpX and provide it with a grip for acting on ALAS. Observation of ALAS undergoing remodeling by mtClpX revealed that unfolding is limited to a region extending from the mtClpX-binding site to the active site. Unfolding along this path is required for mtClpX to gate cofactor binding to ALAS. This targeted unfolding contrasts with the global unfolding canonically executed by ClpX homologs and provides insight into how substrate-chaperone interactions direct the outcome of remodeling

Structure of the Mitochondrial Aminolevulinic Acid Synthase, a Key Heme Biosynthetic Enzyme

5-Aminolevulinic acid synthase (ALAS) catalyzes the first step in heme biosynthesis. We present the crystal structure of a eukaryotic ALAS from Saccharomyces cerevisiae. In this homodimeric structure, one ALAS subunit contains covalently bound cofactor, pyridoxal 5′-phosphate (PLP), whereas the second is PLP free. Comparison between the subunits reveals PLP-coupled reordering of the active site and of additional regions to achieve the active conformation of the enzyme. The eukaryotic C-terminal extension, a region altered in multiple human disease alleles, wraps around the dimer and contacts active-site-proximal residues. Mutational analysis demonstrates that this C-terminal region that engages the active site is important for ALAS activity. Our discovery of structural elements that change conformation upon PLP binding and of direct contact between the C-terminal extension and the active site thus provides a structural basis for investigation of disruptions in the first step of heme biosynthesis and resulting human disorders. Brown et al. determine structures of ALAS, a heme biosynthetic enzyme, that reveal how its PLP cofactor orders the active site. These structures also reveal the positioning of the eukaryote-specific C-terminal extension, providing a framework for understanding the mechanism of erythroid disease-causing mutations.Burroughs Wellcome Postdoctoral Enrichment Program (Award 1015092)National Institutes of Health (Award F32DK095726)National Institutes of Health (Grant R01 DK115558

Nuclear localization and the 25 kDa C-terminus is essential for subsequent mitochondrial import of NOA1.

<p>(A) Immunofluorescence staining of different Flag tagged NOA1 proteins transfected into C2C12 cells reveals requirement of the NLS and the C-terminus of NOA1 for mitochondrial import. Deletion of the MTS abolishes mitochondrial localization leading to accumulation of NOA1 in the nucleus. Mutation of the NLS in the ΔMTS mutant confirms that the NLS is necessary for nuclear localization. Mitochondria were identified by co-staining for Tom20. (B) Quantitative evaluation of the subcellular dynamics of wild type and mutant NOA1 protein. Wild type NOA1 protein shows a predominant “Mitochondrial” localization. ΔMTS-NOA1 shows a predominant “nucleus” accumulation. NOA1-NLS mutants show a mixed localization as indicated by the term “aggregated”. Wild type NOA1 is dynamically distributed between nucleus and mitochondria. Leptomycin-B treatment prevents nuclear export and subsequent mitochondrial import of wild type NOA1. Mutation of the NLS prevents nuclear accumulation of NOA1 after leptomycin-B treatment. (C) N-terminal tagging prevents import of NOA1 into mitochondria of NIH 3T3 cells by masking the MTS. (D) A Western blot analysis of N-terminally (HA tag) and C-terminally (Flag tag) modified versions of NOA2 is shown. (E) Deletion of the RNA-binding domain containing C-terminus prevents mitochondrial import in NIH 3T3 cells. Western blot analysis demonstrated strongly reduced processing of the MTS after truncation of the C-terminus. Scale bars: 10 µm.</p

Model of the intracellular routing of NOA1.

<p>NOA1 is a nuclear encoded protein translated in the cytosol. The unprocessed precursor NOA1 protein (1) is imported into the nucleus in a NLS dependent manner mediated by the importin system, which requires GTP. (2). NOA1 localizes to the nucleolus and interacts with the UBF1 protein (3). NOA1 binds G-quadruplex RNA, which destabilizes interaction of NOA1 with the UBF1 protein complex followed by NES dependent Crm1 mediated nuclear export (4). Following nuclear export NOA1 is imported into the mitochondrial matrix where the mitochondrial targeting sequence is removed (5). The matrix protease complex ClpXP most likely mediates degradation of NOA1 in mitochondria (6) although the mammalian ClpXP complex was less efficient to degrade NOA1 compared to bacterial ClpXP.</p

NOA1 is a substrate of the mitochondrial matrix protease complex ClpXP.

<p>(A) The C-terminus of NOA1 contains three motifs resembling known ClpX recognition motifs found in proteins of <i>E.coli</i>. (B) Exemplary Western blot analysis revealing increased concentrations of NOA1 in C2C12 cells after mutation of lysin690 and lysin691 to alanines in the proximal ClpX recognition motif. (C) Quantitative analysis of Western blots demonstrating ca. 60% stabilization of the NOA1-KK690,691AA mutant compared to wild type NOA1. Densitometries from three independent experiments were normalized to Porin. (D) Overexpression of ClpX is sufficient to promote degradation of NOA1 independent of a C-terminal Flag tag in C2C12 cell lysates. (E) Recombinant bacterial ClpXP (<i>E.coli</i> ClpXP) (top left) and mammalian ClpXP (mouse ClpX, human ClpP) (top right) cleaves recombinant NOA1 (2.5 µM) <i>in vitro</i> in a time dependent manner. Mammalian ClpXP but not by <i>E. coli</i> ClpXP cleaves α-Casein (5 µM) in vitro. (F) Quantitation analysis of Western blots shown in (E) demonstrating the degradation of NOA1 by bacterial and mammalian ClpXP <i>in vitro</i>.</p

A fraction of NOA1 localizes in the nucleolus and interacts with UBF1.

<p>(A) Immunofluorescence staining of endogenous NOA1 in primary mouse myofibers reveals that a fraction of NOA1 is localized in the nucleolus. (B) Quantitative evaluation of confocal images of NIH 3T3 fibroblasts stained with antibodies against NOA1, UBF1 and Fibrillarin. NOA1 and UBF1 or NOA1 and Fibrillarin are co-localized in the nucleolus. Moving average smoothing was applied to fit data points into curves. (C) Pull-down assays demonstrating interaction of NOA1 and UBF1. Recombinant NOA1-His₆ protein was loaded on Ni-NTA beads and mixed with whole cell or nucleoli lysates from C2C12 cells to pull down interacting proteins. (D) Co-immunoprecipitation of NOA1-His₆ with endogenous UBF1 from C2C12 lysates. (E) Addition of RNAse H or RNAse A to pull-down assays increased the efficiency of the interaction between NOA1 and UBF1 while addition of DNAse I had no effect. Scale bars: 10 µm, Zoom scale bars: 1 µM.</p