CLPP-Null Eukaryotes with Excess Heme Biosynthesis Show Reduced L-arginine Levels, Probably via CLPX-Mediated OAT Activation

Abstract

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina, CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

Knockout Mouse Studies Show Mitochondrial CLPP Peptidase and CLPX Unfoldase to Act in Matrix Condensates Near IMM, as Fast Stress Response in Protein Assemblies for Transcript Processing, Translation, and Heme Production

“…In addition, the selective accumulation SFXN4 in Clpp-KO tissue, as a component of the complex-I assembly machinery that controls metal association, indicates that the biogenesis of complex-I may be altered [42]. It has to be mentioned, however, that none of the established complex-I subunits accumulate in Clpp-KO mouse tissues, and that proteome profiling in the CLPP-null fungal eukaryote P. anserina did reveal some accumulated complex-I subunits but not for any specific module [48]. Thus, we assume that complex-I assembly is not a primary and conserved target of CLPP.…”

“…Indeed, the effect of CLPP on cell growth is strong even under physiological conditions: its dysfunction leads to short stature of patients [15]. In the PRLTS3 mouse models, a reduction of weight up to 50% was observed, together with an underlying similar decrease of the nitrogen-storing amino acid L-arginine, which gets consumed in the maximized biosynthesis of heme instead of fueling growth [48]. In a preliminary meta-analysis of CLPP substrate trapping experiments and of CLPPnull proteome profiling in many organisms from bacteria to man, an enrichment of ribonucleoproteins was observed, with the unfoldase CLPX, the mitoribosomal translation factor GFM1 and the RNA degradation factor PNPT1 emerging as most consistent proteins that interact with CLPP and show accumulation upon its loss [36].…”

“…In particular, the tRNAs for valine (Val) and phenylalanine (Phe) exhibited higher aminoacylation in Clpp-KO heart [41]. Therefore, it is interesting that a very selective protein accumulation exists for mtLSU components like MRPL18 and MRPL38, which assemble with tRNA-Val/Phe in the central protuberance of mitoribosomes, and that this CLPP-null effect on central protuberance subunits of the mtLSU is conserved across eukaryotes until the ascomycete fungus Podospora anserina [42,48]. This selective impact on the mtLSU may also be relevant for mtDNA and the cytosolic stress response: MRPL38 influences the maintenance of the mitochondrial nucleoid at least in yeast [80].…”

“…In the Clpp-KO mouse, the consequent elevation of CLPX abundance will unfold also OAT (ornithine delta-aminotransferase) so that PLP binds it and triggers the consumption of L-arginine and L-ornithine via a delta-transaminase and delta-aminomutase reaction to produce GSA as precursor of heme. In parallel, a recruitment of L-glutamate occurs into maximized deltaALA generation via accumulation of the enzyme ALDH18A1 (also known as delta-1-pyrroline-5-carboxylate synthase) [48,52]. Thus, ALAS and OAT are both acquiring the ability to perform transaminations at the delta-carbon position, when CLPX unfolds them and enables them to bind PLP as cofactor [180].…”

Knockout Mouse Studies Show That Mitochondrial CLPP Peptidase and CLPX Unfoldase Act in Matrix Condensates near IMM, as Fast Stress Response in Protein Assemblies for Transcript Processing, Translation, and Heme Production

“…Indeed, the effect of CLPP is strong even under physiological conditions: its dysfunction leads to a short stature in patients [ 15 ], and it is unclear if this represents impaired cell growth, reduced proliferation rates, or developmental deficits. In the PRLTS3 mouse models, a reduction in weight of up to 50% was observed, together with an underlying similar decrease in the nitrogen-storing amino acid L-arginine, which is consumed in the maximized biosynthesis of heme instead of fueling growth [ 48 ]. This means that nitrogen availability in L-arginine could be the limiting factor for organism growth.…”