Functional coupling of presequence processing and degradation in human mitochondria

Abstract

The mitochondrial proteome is built and maintained mainly by import of nuclear‐encoded precursor proteins. Most of these precursors use N‐terminal presequences as targeting signals that are removed by mitochondrial matrix proteases. The essential mitochondrial processing protease MPP cleaves presequences after import into the organelle thereby enabling protein folding and functionality. The cleaved presequences are subsequently degraded by peptidases. While most of these processes have been discovered in yeast, characterization of the human enzymes is still scarce. As the matrix presequence peptidase PreP has been reported to play a role in Alzheimer's disease, analysis of impaired peptide turnover in human cells is of huge interest. Here, we report the characterization of HEK293T PreP knockout cells. Loss of PreP causes severe defects in oxidative phosphorylation and changes in nuclear expression of stress response marker genes. The mitochondrial defects upon lack of PreP result from the accumulation of presequence peptides that trigger feedback inhibition of MPP and accumulation of nonprocessed precursor proteins. Also, the mitochondrial intermediate peptidase MIP that cleaves eight residues from a subset of precursors after MPP processing is compromised upon loss of PreP suggesting that PreP also degrades MIP generated octapeptides. Investigation of the PrePR183Q patient mutation associated with neurological disorders revealed that the mutation destabilizes the protein making it susceptible to enhanced degradation and aggregation upon heat shock. Taken together, our data reveal a functional coupling between precursor processing by MPP and MIP and presequence degradation by PreP in human mitochondria that is crucial to maintain a functional organellar proteome.

Mitochondrial proteases in human diseases

“…The presequence protease (PreP) is the main peptidase in human mitochondria and-as its name suggestsalso responsible for the degradation of free presequences [19]. Deletion of PreP or its yeast homologue Cym1 (cytosolic metalloprotease 1) results in feedback inhibition of MPP by accumulation of cleaved presequence peptides revealing a functional coupling between presequence degradation and presequence processing [20,21].…”

Open questions on the mitochondrial unfolded protein response

“…Among them were the overexpression of misfolded proteins (OTCD) [30,32], treatment of cells with various toxins and inhibitors (e.g. arsenite [38], paraquat [32,39,40], oligomycin [35][36][37]41], antimycin A [35,37,41], piericidin [35], rotenone [40], actinonin [31], FCCP/CCCP [23,31,37], dinitrophenol [42], chloramphenicol [43], doxycycline [31,43], MitoBloCK-6 [31]), depletion of mtDNA/ethidium bromide treatment [39,41,42,44], mutations in the mitochondrial ribosome [24], knockdown or knockout of mitochondrial proteins by RNAi or CRISPR/Cas9 [TIM23 [32,39], SPG-7 [32,39,42,45], LON [34], presequence protease (PreP) [46], HSP70 [25,42,45], HSP60 [42], mitochondrial aspartyl-tRNA synthetase [47]], heat shock [42,45] or the use of temperature-sensitive alleles [e.g. mia40-4int, mas1, zc-32 (an unknown gene) …”

“…The role of ClpP was disputed by studies in mice, in which lack of ClpP did not affect mtUPR triggered by depletion of the mitochondrial aspartyl aminoacyl-tRNA synthetase (DARS2) implying that ClpP is not necessary for propagation of mtUPR in mammals [51]. Protein degradation in mitochondria is carried out by ClpP and several other proteases, for example LONP1, m-and i-AAA proteases and the identification of several matrix peptidases with partially redundant functions indicate that mitochondria have an efficient machinery to degrade peptides [8,9,46,52]. Mitochondria require this extensive proteolytic network as peptides continuously accumulate within the organelle not only due to protein degradation, but also in the form of cleaved targeting signals that 70% of all mitochondrial preproteins use for import into the organelle [7][8][9]53].…”

“…Mitochondria require this extensive proteolytic network as peptides continuously accumulate within the organelle not only due to protein degradation, but also in the form of cleaved targeting signals that 70% of all mitochondrial preproteins use for import into the organelle [7][8][9]53]. These free presequences need to be rapidly degraded as their accumulation inhibits the activity of the essential mitochondrial processing protease (MPP) and by this preprotein maturation [8,46,54].…”

“…Similarly, the activity of the presequence peptidase PreP also declines over time. Interestingly, PreP degrades amyloid-beta peptides that accumulate in mitochondria of Alzheimer's disease patients and these accumulating amyloid-beta peptides have been shown to induce feed-back inhibition of MPP and precursor accumulation within mitochondria [46,54,68]. Consequently, enhancement of mitochondrial proteostasis reduced amyloid-beta toxicity in AD mouse models indicating that mtUPR might be of relevance in AD pathogenesis [69].…”