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

LONP1 is the principal AAA+ unfoldase and bulk protease in the mitochondrial matrix, so its deletion causes embryonic lethality. The AAA+ unfoldase CLPX and the peptidase CLPP also act in the matrix, conspicuously during stress periods, but their substrates are poorly defined. Mammalian CLPP deletion triggers infertility, deafness, growth retardation, and cGAS-STING activated cytosolic innate immunity. CLPX mutations impair heme biosynthesis and heavy metal homeostasis. CLPP and CLPX are conserved from bacteria to human, despite their secondary role for proteolysis. Based on recent proteomic-metabolomic evidence from knockout mice and patient cells, we propose that CLPP acts on phase-separated ribonucleoprotein granules, and CLPX on multi-enzyme condensates, near the inner mitochondrial membrane, as first-aid system. Trimming within assemblies, CLPP rescues stalled processes in mitoribosomes, in mitochondrial RNA granules and nucleoids, and in D-foci-mediated degradation of toxic double-stranded mtRNA / mtDNA. Unfolding multi-enzyme condensates, CLPX maximizes PLP-dependent delta-transamination, and rescues malformed nascent peptides. Overall, their actions occur in granules with multivalent or hydrophobic interactions, separated from the aqueous phase. Thus, the role of CLPXP in the matrix is compartment-selective, like peptidases MPP at precursor import pores, m-AAA and i-AAA at either IMM face, PARL within the IMM, and OMA1/HTRA2 in the intermembrane space.

Research on healthy ageing shows that lifespan reductions are often caused by mitochondrial dysfunction. Thus, it is very interesting that the deletion of mitochondrial matrix peptidase LonP1 was observed to abolish embryogenesis, while deletion of the mitochondrial matrix peptidase ClpP prolonged survival. To unveil the targets of each enzyme, we documented the global proteome of LonP1^+/- mouse embryonal fibroblasts (MEF), for comparison with ClpP^-/- depletion. Proteomic profiles of LonP1^+/- MEF generated by label-free mass spectrometry were further processed with the STRING webserver Heidelberg for protein interactions. ClpP was previously reported to degrade Eral1 as a chaperone involved in mitoribosome assembly, so ClpP deficiency triggers accumulation of mitoribosomal subunits and inefficient translation. LonP1^+/- MEF also showed Eral1 accumulation, but no systematic effect on mitoribosomal subunits. In contrast to ClpP^-/- profiles, several components of the respiratory complex I membrane arm were accumulated, whereas the upregulation of numerous innate immune defense components was similar. Overall, LonP1 as opposed to ClpP appears to have no effect on translational machinery, instead it shows enhanced respiratory dysfunction; this agrees with reports on the human CODAS syndrome caused by LonP1 mutations.