REVIEW ARTICLE Mitochondrial in human diseases Maria Gomez-Fabra Gala1,2,3 and Friederike-Nora Vogtle€ 1,4

1 Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Germany 2 Faculty of Biology, University of Freiburg, Germany 3 Spemann Graduate School of Biology and Medicine, University of Freiburg, Germany 4 CIBSS—Centre for Integrative Biological Signalling Studies, University of Freiburg, Germany

Correspondence Mitochondria contain more than 1000 different proteins, including several F.-Nora Vogtle,€ Institute of Biochemistry and proteolytic . These mitochondrial proteases form a complex system Molecular Biology, Stefan-Meier-Str. 17, that performs limited and terminal proteolysis to build the mitochondrial pro- 79104 Freiburg, Germany teome, maintain, and control its functions or degrade mitochondrial proteins Tel: +49 761 2035270 and peptides. During protein biogenesis, presequence proteases cleave and (Received 16 October 2020, revised 17 degrade mitochondrial targeting signals to obtain mature functional proteins. December 2020, accepted 18 December Processing by proteases also exerts a regulatory role in modulation of mito- 2020, available online 3 February 2021) chondrial functions and quality control enzymes degrade misfolded, aged, or superfluous proteins. Depending on their different functions and substrates, doi:10.1002/1873-3468.14039 defects in mitochondrial proteases can affect the majority of the mitochon- drial proteome or only a single protein. Consequently, mutations in mitochon- Edited by Peter Rehling drial proteases have been linked to several human diseases. This review gives an overview of the components and functions of the mitochondrial proteolytic machinery and highlights the pathological consequences of dysfunctional mitochondrial protein processing and turnover.

Keywords: cancer; cardiomyopathy; mitochondrial proteases; mitochondrial protein biogenesis; mitochondrial protein quality control; neurodegeneration; proteostasis

Mitochondria are essential eukaryotic organelles with into four subcompartments: the outer membrane important roles in energy conversion, synthesis of (OM), intermembrane space (IMS), inner membrane iron-sulfur clusters, biosynthesis of amino acids and (IM), or matrix [1–4]. Imbalances in the proteome lipids, or regulation of . To fulfill these result in mitochondrial defects with potential deleteri- diverse functions mitochondria contain a proteome of ous consequences for cellular survival [5–8]. Mitochon- more than 1000 different proteins that are distributed drial proteases play a key role in building and

Abbreviations AD, Alzheimer’s disease; AFG3L2, AFG3-like subunit 2; ALAS, d-aminolevulinate synthase; CLPP, caseinolytic peptidase subunit P; CLPX, caseinolytic peptidase subunit X; CODAS, cerebral, ocular, dental, auricular and skeletal; CRC, colorectal cancer; Cym1, cytosolic metallopro- tease 1; DOA, dominant optic atrophy; FRDA, Friedreich’s ataxia; FXN, frataxin; GTS, Gilles de la Tourette syndrome; HTRA2, high tempera- ture requirement A2; i-AAA, ATPases associated with various cellular activities (facing the IMS); Icp55, intermediate cleaving peptidase; IMP, inner membrane peptidase; LHON, Leber hereditary optic neuropathy; LONP, Lon ; LVNC, left-ventricular noncompaction; m- AAA, ATPases associated with various cellular activities (facing the matrix); MIP, mitochondrial intermediate peptidase; MPP, mitochondrial processing protease; NPHP, nephronophthisis; Oct1, octapeptidyl 1; OMA1, overlapping activity with m-AAA protease; OPA1, optic atrophy 1; PARL, presenilin-associated rhomboid like; PD, Parkinson’s disease; PINK1, PTEN-induced putative kinase 1; PMPCA, -subunit of MPP; PMPCB, -subunit of MPP; PreP, presequence protease; SNP, single nucleotide polymorphism; SPG7, paraplegin; TIM23, of the inner mitochondrial membrane; TOM, translocase of the outer mitochondrial membrane; XPNPEP3, mitochondrial x-prolyl aminopeptidase; YME1L, yeast mtDNA escape 1-like.

FEBS Letters 595 (2021) 1205–1222 ª 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of 1205 Federation of European Biochemical Societies. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Mitochondrial proteases in human diseases M. Gomez-Fabra Gala and F. N. Vogtle€ maintenance of the mitochondrial proteome, but are (PTEN-induced putative kinase 1) and PARKIN, an also involved in the response to mitochondrial stress E3 [24]. The mitochondrial proteases MPP and and in the adaptation of mitochondrial functions to PARL (presenilin-associated rhomboid like) are in changing cellular demands (reviewed in Ref. [9,10]). turn involved in processing of PINK1 to prevent label- As the vast majority of mitochondrial proteins are ing of healthy mitochondria for destruction, so that encoded in the nuclear DNA, mitochondrial protein only damaged mitochondria, for example, with a low biogenesis requires the synthesis of precursor proteins membrane potential, are removed [25,26]. in the and their subsequent transfer into the Mitochondrial proteases have also been implicated organelle [11–13]. Most precursor proteins possess an in mitochondrial signaling. For instance, the inner N-terminal presequence that is removed upon import membrane protease OMA1 was proposed to cleave a to obtain functional mitochondrial proteins [14,15] mitochondrial protein upon mitochondrial defects (Fig. 1). Processing by the mitochondrial processing releasing it into the cytosol to induce the integrated protease MPP that removes the presequence is often stress response [27,28]. Mitochondrial integrity is also followed by a second cleavage event by the mitochon- a key regulator of programmed cell death, in which drial intermediate peptidase [MIP, octapeptidyl mitochondrial proteases are implicated in diverse regu- aminopeptidase 1 (Oct1) in yeast] or the intermediate latory functions (reviewed in Ref. [9]). cleaving peptidase (Icp55 in yeast, encoded by Taken together, mitochondrial proteases not only XPNPEP3 in humans) [15–17]. The sequential cleav- play important roles in protein biogenesis and protein age generates mature proteins with stabilizing N-termi- quality control (PQC), but also in the regulation of nal amino acids [15,16,18]. The cleaved presequences key mitochondrial functions. Due to these diverse and octapeptides are degraded by matrix localized pep- roles, alterations in the activity of mitochondrial pro- tidases, among which the oligopeptidase PreP is the teases cause mitochondrial dysfunctions that can give most prominent one [19]. Defects in PreP cause accu- rise to a variety of human diseases including cardio- mulation of presequence peptides that in turn induce vascular and neurological disorders, or cancer feedback inhibition of MPP and MIP, demonstrating (Table 1). that presequence degradation and presequence process- ing are functionally coupled processes [20,21]. Proteases involved in mitochondrial Peptides accumulating in mitochondria are not only protein biogenesis derived from presequence processing but also from ter- minal degradation of nonfunctional, superfluous, or The majority of mitochondrial proteins is imported aged mitochondrial proteins. Four ATP-dependent as precursors from the cytosol, which is coordinated proteases are mainly responsible for quality control of by targeting and sorting signals that often require mature proteins in human mitochondria. Their mecha- protein maturation in form of a proteolytic process- nism of action is conserved throughout evolution and ing upon import into the organelle. Mitochondrial consists of an ATP-dependent step in which damaged presequence proteases recognize and cleave these tar- or aberrantly folded proteins are unfolded by the geting signals. AAA+ domain followed by their transfer into the pro- teolytic core for degradation [22] (Fig. 2). Mitochondrial processing protease (MPP) Proteases are also playing a role in mitochondrial morphology, which is balanced by fission and fusion Most mitochondrial precursor proteins require prote- events. Mitochondrial dynamics are regulated by dyna- olytic removal of their targeting signals (presequences) min-like GTPases that are localized in the OM and upon import into mitochondria, a task performed by IM [23]. The IM GTPase OPA1 (optic atrophy 1) is the essential MPP [29,30]. MPP localizes to the matrix, processed by the IM proteases YME1L (yeast mtDNA which is also the final localization of the majority of escape 1-like) and OMA1 (overlapping activity with presequence-targeted precursors. Proteins destined to m-AAA protease), which generate different ratios of the inner membrane, IMS, or outer membrane are the long and short OPA1 form and regulate mitochon- mostly using noncleavable and internal targeting sig- drial morphology. nals and are therefore not dependent on MPP for mat- Damaged mitochondria can be degraded by mito- uration. MPP consists of two subunits, which are in phagy, the sequestration of the whole organelle in human cells the catalytic subunit PMPCB (b-subunit autophagosomes that fuse with the lysosome for turn- of MPP), a zinc metalloprotease, and PMPCA (a-sub- over. Mitochondria destined for mitophagy are labeled unit of MPP) that is proposed to have a role in sub- by a mechanism that involves the kinase PINK1 strate recognition. Crucial for PMPCA function is a

1206 FEBS Letters 595 (2021) 1205–1222 ª 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. M. Gomez-Fabra Gala and F. N. Vogtle€ Mitochondrial proteases in human diseases

Fig. 1. Mitochondrial proteases in protein biogenesis. Mitochondrial proteins are synthesized as precursors on cytosolic ribosomes and imported post-translationally into the organelle. The majority of precursors use N-terminal presequences as targeting signals that are recognized by the TOM and TIM23 and that import the precursor into the matrix. MPP, a heterodimer composed of the PMPCB and PMPCA subunit, cleaves off the presequences, which are subsequently degraded by PreP. MPP processing generates either mature proteins or processing intermediates that are processed a second time to generate the stable mature proteins. This second processing is carried out by MIP removing an octapeptide or by Icp55 (XPNPEP3 in human) that cleaves off a single amino acid. Precursor proteins destined for the inner membrane contain a sorting signal that results in lateral release from TIM23 into the inner membrane. The sorting signal can be cleaved by IMP to release the protein into the IMS. IMP consists of two subunits in human cells, IMMP1L and IMMP2L. Analysis in yeast revealed that both have proteolytic activity. glycine-rich loop that participates in recognition of the variants in the presequence of MnSOD have been presequence and subsequent substrate transfer into the associated with an increased risk for some cancers catalytically of PMPCB [31–34]. MPP recog- [38,39] and a mutation in the presequence of YME1L nizes and cleaves presequences in an extended confor- has been described to cause a mitochondriopathy with mation [35]. Global N-proteomic analyses of infantile onset [40], see below). In contrast, mutations mitochondrial protein N termini in different model in one of the MPP subunits are likely to affect a organisms revealed that in position À2 of the broader substrate spectrum and single amino acid MPP cleavage site is the predominant recognition exchanges in both MPP subunits have been identified motif [15,36,37]. Mutations within the presequence can in patients that present with a predominantly neuro- result in loss of MPP recognition and processing logical phenotype. Point mutations in PMPCA result affecting only this particular substrate protein (e.g., in non- or slowly progressive cerebellar ataxia,

FEBS Letters 595 (2021) 1205–1222 ª 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of 1207 Federation of European Biochemical Societies. Mitochondrial proteases in human diseases M. Gomez-Fabra Gala and F. N. Vogtle€

Fig. 2. Mitochondrial proteases in quality control and regulation of mitochondrial functions. The mitochondrial matrix and inner membrane (IM) harbor four ATP-dependent proteases that are important for quality control of mitochondrial proteins. LONP and CLPXP reside in the matrix. CLPXP consists of the catalytic subunit CLPP and the adapter protein CLPX that is involved in substrate recognition. Two AAA proteases are localized in the IM, and their catalytic sites are exposed to the two opposing sites: the m-AAA faces the matrix and can be a homo-oligomer of AFG3L2 or a hetero-oligomer of AFG3L2 and SPG7 subunits. The i-AAA protease is active toward the IMS and consists of six subunits of YME1L. Both proteases can extract and degrade IM localized proteins. The i-AAA protease is also processing the long form of the fusion protein OPA1 (l-OPA1), generating the short (s-OPA1) form. l-OPA1 can also be processed by the protease OMA1 that is embedded in the inner membrane, with its catalytic activity also facing the IMS. The inner membrane protease PARL is a rhomboid intramembrane protease. PARL cleaves its substrates within the lipid bilayer and contributes to the dual localization of several proteins that exist as integrated IM proteins and soluble IMS or cytosolic proteins. The HTRA2 is localized in the IMS, where it degrades soluble proteins. OM, outer mitochondrial membrane.

characterized by abnormal gross motor development [35,41,42]. Mutations in PMPCB were recently identified in infancy together with hypotonia, that is followed by in patients with neurodegeneration and cerebellar atro- a lack of muscle control or reduced capacity for volun- phy, which was progressive in the majority of cases [43]. tary coordination of movement (ataxia). Interestingly, The course of disease in PMPCB patients was more sev- modeling of human MPP using the crystal structure of ere than in PMPCA patients and several PMPCB yeast MPP indicates that mutations in close proximity of patients died in early childhood, while diagnosed or within the glycine-rich loop presented with a more sev- PMPCA patients were also in their late adulthood. On a ere phenotype, a rather progressive course of disease and molecular level, the mutations in PMPCB and partially resulted in reduced protein steady state level of PMPCA in PMPCA resulted in a reduced protein level in patient

1208 FEBS Letters 595 (2021) 1205–1222 ª 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. eeaino uoenBohmclSocieties. Biochemical European of Federation Letters FEBS V N. F. and Gala Gomez-Fabra M. 595 22)1205–1222 (2021)

Table 1. List of mitochondrial proteases, subunits of proteases and peptidases discussed in this review. References refer to publications showing disease relevance of the protein of interest. ª ogtle € 01TeAtos ESLtespbihdb onWly&Sn t nbhl of behalf on Ltd Sons & Wiley John by published Letters FEBS Authors. The 2021 Yeast Protein homologue Class Function Human disease References

AFG3L2 AFG3L2 Yta12 Metallo PQC, protein biogenesis Spinocerebellar ataxia (SCA28), spastic ataxia 5, DOA [75,76,77,78] CLPP CLPP – Serin PQC, ribosome assembly Perrault syndrome, acute myeloid leukemia [105,106,134,135,136] CLPX CLPX Mcx1 – PQC, ribosome assembly Erythropoietic protoporphyria [107] HTRA2 HTRA2 – Serin PQC, apoptosis, mitophagy PD, essential tremor, infantile neurodegeneration [110,111,112,113,114,115,116,126, and 3-methylglutaconic aciduria, pediatric 127,128,129,130,132] nephroblastoma, ovarian cancer, lung cancer, hepatocellular carcinoma IMMP1L IMMP1L Imp1 Serin Protein biogenesis –– IMMP2L IMMP2L Imp2 Serin Protein biogenesis GTS [64, 65,66,67, 68, 69, 70, 71] LONP1 LONP1 Pim1 Serin PQC, mtDNA maintenance/ CODAS syndrome, cervical cancer [100,101,117,118,119,120,121] transcription, hypoxia adaptation MIP MIPEP Oct1 Metallo Protein biogenesis Cardiomyopathy (LVNC) [49] OMA1 OMA1 Oma1 Metallo Stress signaling Amyotrophic lateral sclerosis [97] PARL PARL Pcp1 Serin PQC PD, Leber hereditary optic neuropathy, [87,88,89,90,91] increased risk for leprosy Paraplegin SPG7 Afg3 Metallo PQC, protein biogenesis Spastic paraplegia, external ophthalmoplegia, DOA [72,73,74,75]

(Yta10) diseases human in proteases Mitochondrial PMPCA PMPCA Mas2 – Protein biogenesis Cerebellar ataxia [41,42,44] PMPCB PMPCB Mas1 Metallo Protein biogenesis Neurodegeneration, cerebellar atrophy [43] PreP PITRM1 Cym1 Metallo Peptide degradation Amyloidotic neurodegeneration, cerebellar atrophy [52,53,21] XPNPEP3 XPNPEP3 Icp55 Metallo Protein biogenesis NPHP, CRC [50,124] YME1L1 YME1L1 Yme1 Metallo PQC, mitochondrial biogenesis Ataxia, optic atrophy, PDAC [40,125] 1209 Mitochondrial proteases in human diseases M. Gomez-Fabra Gala and F. N. Vogtle€

fibroblasts and lymphoblastoid cell cultures, while the their stability, which indicated the presence of an level of the not mutated MPP subunit was not affected organellar N-end rule that correlates the half-life of [41,43,44]. As readout for MPP catalytic activity steady- a protein to its N-terminal residues [16,18,48]. state protein levels of six model substrates were analyzed Mutations in MIP have been identified in patients via western blotting and revealed alteration in processing that share as predominant clinical features left-ventric- of only one tested protein, Frataxin (FXN) [43]. FXN is ular noncompaction (LVNC), developmental delay, an unusual MPP substrate, as it undergoes two sequen- seizures, and hypotonia [49]. LVNC is a cardiomyopa- tial cleavage reactions by MPP, forming first an interme- thy characterized by the failure to develop compact diate and then the mature protein [45]. In all MPP myocardium during early embryonic development, patient samples analyzed the ratios of intermediate to which resulted in infantile death in most identified mature FXN were altered toward an accumulation of patients [49]. Sequencing revealed single nucleotide intermediate and a decrease in mature protein. Re-ex- variants in MIPEP resulting in single amino acid pression of PMPCA in PMPCA patient fibroblasts res- exchanges in MIP in the affected individuals. Intro- cued FXN processing [42]. The severity of disease might duction of the mutated amino acids at the corre- be correlating to the residual mutant protein level and sponding positions in the yeast MIP homologue Oct1 also to FXN processing, in which the absence of mature enabled functional analysis in vivo. Yeast cells harbor- protein could indicate a more severe course of disease. ing MIP patient mutations displayed a temperature- FXN was the only abnormally processed MPP substrate sensitive growth defect at elevated temperature on res- identified so far, and analysis of patient samples sug- piratory growth conditions, when mitochondrial respi- gested an early decrease in the activity of iron-sulfur clus- ration is essential for cell viability [49]. Analysis of ter biogenesis, in which FXN has an essential role [46]. protein steady state levels revealed a clear processing While this points into the direction of a role of the Fe-S- defect in all analyzed Oct1 substrate proteins with cluster biogenesis in disease development upon MPP accumulation of processing intermediates after initial dysfunction, FXN is unlikely the only affected protein in MPP processing pointing toward a global decrease in MPP patients. Decreased FXN protein levels cause Oct1 catalytic activity. Based on the analysis in yeast, Friedreichs ataxia (FRDA) [47]. FRDA patients are lar- it is likely that the patient mutations impact on the gely asymptomatic during the first 5–10 years of life, and protease function of MIP and as a consequence affect then develop progressive neurological symptoms, car- several mitochondrial functions, for example, respira- diac disease, and muscle weakness and often die in the tory chain, mitochondrial ribosome, or protein bio- second or third decade of life. In contrast to mutations in genesis. The predominant cardiac symptoms might be MPP, FRDA patients have a global depletion of all due to a high expression level of MIP in heart tissue; FXN isoforms due to decreased protein expression [47]. however, our understanding of tissue-specific effects of FXN is likely a very sensitive substrate to detect ubiquitously expressed proteins is still very rudimen- decreased MPP catalytic activity, due to its two-step pro- tary. cessing. Global N-terminal profiling, for example, by N- proteomics of patient samples might identify further Aminopeptidase XPNPEP3 (ICP55) affected MPP substrates that could shed light on the pathophysiology of the disease. It is interesting to specu- The yeast XPNPEP3 (mitochondrial x-prolyl late that yet unknown sequentially processed MPP sub- aminopeptidase) homologue, Icp55, is cleaving a single strates could in particular impact on the course of amino acid from MPP generated intermediates. By the disease and that they might vary in different cell types or removal of tyrosine, phenylalanine, or leucine, the pro- tissues. cessing intermediates are matured by appearance of a stable N-terminal amino acid according to the N-end rule [15,18]. Increased half-life of fully processed Icp55 Mitochondrial intermediate peptidase (MIP) substrates was demonstrated in yeast and plant cells The name of the metalloprotease MIP indicates its [15,36]. Deduced from these results, it can be proposed function: the processing of intermediate proteins. that the human metalloprotease XPNPEP3 is also hav- Analyses mainly performed in the model organism ing a role in processing of MPP-generated intermedi- yeast identified that Oct1 (yeast homologue of MIP) ates. However, experimental analysis in human cells is removes an octapeptide from MPP generated pro- still missing. cessing intermediates that have a phenylalanine, leu- Mutations in human XPNPEP3 have been linked to cine or isoleucine as N-terminal residue. Intermediate the autosomal recessive kidney disease nephronophthi- and mature proteins were found to often differ in sis (NPHP), which is the most frequent genetic cause

1210 FEBS Letters 595 (2021) 1205–1222 ª 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. M. Gomez-Fabra Gala and F. N. Vogtle€ Mitochondrial proteases in human diseases of end-stage kidney disease within the first three dec- degradation of the mutated protein, which is also ades of life [50]. XPNPEP3 patients reported with prone to aggregation upon heat-shock [21].A NPHP-related symptoms, but some in addition also PITRM1+/À mouse model developed progressive ataxia displayed tremor, seizures, mental or developmental and accumulation of amyloid-beta deposits [52]. Inter- delay, and cardiomyopathy indicative of a mitochon- estingly, PreP is able to degrade amyloid-beta peptides driopathy. NPHP has been described as ‘ciliopathies’ accumulating in mitochondria of Alzheimer’s disease as all so far identified causative NPHP are local- (AD) patients [54]. Cerebral organoids that were gen- ized to primary cilia, basal bodies, and centrosomes. erated from PITRM1-knockout induced pluripotent Icp55 has been localized to the mitochondrial matrix stem cells developed pathological features resembling and to a minor degree to the nucleus [15,51]. Kidney AD, for example, accumulation of protein aggregates, homogenate from mice separated into mitochondrial tau pathology and neuronal cell death [55]. Increased and cytosolic fractions implied dual localization of expression of PreP in cortical neurons of an AD XPNPEP3 to both compartments [50]. However, an mouse model attenuated mitochondrial amyloid XPNPEP3-GFP fusion protein did not show ciliary pathology and synaptic mitochondrial dysfunction localization. Analysis of XPNPEP3 in zebrafish [56]. Analysis of brain mitochondria from AD mice revealed that loss of XPNPEP3 phenocopied the devel- showed accumulation of amyloid-beta peptides in opmental phenotype observed in other ciliopathy mitochondria and defective MPP processing [20].Itis mutants and the defects were rescued by expression of likely that mitochondrial amyloid-beta peptides satu- XPNPEP3 that lacked its mitochondrial targeting sig- rate the proteolytic capacity of PreP, which is also nal. However, mitochondrial parameters were not yet declining upon aging, and that this causes accumula- analyzed. This would point into the direction that tion of presequence peptides. These in turn induce XPNPEP3 is dually localized and that the cytosolic inhibition of the essential protease MPP, which can be form might be processing substrates involved in cilia detected by accumulation of unprocessed precursor formation. However, identification of these ciliary sub- proteins within mitochondria. Indeed, analysis of mito- strates of human XPNPEP3 is still missing. Molecular chondria isolated from postmortem obtained temporal analysis of a possible role of cytosolic and mitochon- cortex detected nonprocessed precursors only in AD drial XPNPEP3 forms in disease development will patient samples [20]. While PreP seems to be the domi- shed light on their contribution to the various kidney-, nant peptidase in human mitochondria [21], the matrix heart-, and brain-related symptoms observed in of yeast mitochondria contains three peptidases: the XPNPEP3 patients. PreP homologue Cym1, Ste23, and Prd1 [3,57]. Ste23 is the homologue of human insulin degrading (IDE) that has also been associated with AD. It will Presequence protease (PreP) be interesting to investigate a potential contribution of The mitochondrial matrix harbors several peptidases IDE in mitochondrial decline in AD. that degrade peptides accumulating from cleavage of mitochondrial targeting signals or protein degradation. Inner membrane peptidase (IMP) The presequence protease (PreP) is the main peptidase in human mitochondria and—as its name suggests— The mitochondrial inner membrane peptidase (IMP) also responsible for the degradation of free prese- cleaves hydrophobic sorting signals of incoming pre- quences [19]. Deletion of PreP or its yeast homologue cursor proteins, often after initial processing by MPP. Cym1 (cytosolic metalloprotease 1) results in feedback The substrates are subsequently mostly released into inhibition of MPP by accumulation of cleaved prese- the IMS. IMP has so far been mainly studied in the quence peptides revealing a functional coupling model organism Saccharomyces cerevisiae and revealed between presequence degradation and presequence composition of two catalytic subunits, Imp1 and Imp2, processing [20,21]. with nonoverlapping substrate specificity and the small Point mutations in PITRM1 encoding PreP have protein Som1, required for Imp1 catalytic activity [58– been identified in patients with mental retardation, 60]. Imp1 can process N- and C-terminal targeting sig- spinocerebellar ataxia, cognitive decline, and psychosis nals [61]. Recently, the participation of IMP in a novel and a slowly progressive course of disease [52,53]. mitochondrial protein biogenesis route was identified, Brain imaging revealed severe progressive cerebellar in which the protein Mcp3 is imported via the translo- atrophy in some patients [53]. Both described muta- case of the outer membrane (TOM) and the inner tions resulted in a strong reduction of PreP protein membrane (TIM23) and following IMP processing is steady-state levels [52,53], caused by an accelerated integrally inserted into the outer membrane [62]. Imp2

FEBS Letters 595 (2021) 1205–1222 ª 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of 1211 Federation of European Biochemical Societies. Mitochondrial proteases in human diseases M. Gomez-Fabra Gala and F. N. Vogtle€ processes cytochrome c1 and glycerol-3-phosphate proteins, a role for these proteases in regulation of dehydrogenase in mammals, both important for mito- mitochondrial functions through processing of a par- chondrial energy metabolism [63]. Furthermore, Imp2 ticular substrate protein has emerged (for review, see is required for stable and functional expression of Ref. [10]). Among them the four ATP-dependent pro- Imp1 [59]. teases [mAAA, ATPases associated with various cellu- Gilles de la Tourette syndrome (GTS) is a chronic lar activities (facing the matrix); i-AAA, ATPases neurodevelopmental disorder characterized by multiple associated with various cellular activities (facing the motor and vocal tics (sudden, repetitive, and non- IMS); LONP1, Lon protease; CLPP, caseinolytic pep- rhythmic movements or sounds) with onset in child- tidase subunit P] that are exerting quality control and hood [64]. Only few genes have been associated with cleave their substrates at several sites to generate Tourette syndrome, including IMMP2L [65–67]. Previ- oligopeptides, the inner membrane peptidase PARL ous studies associated IMMP2L also with attention- and the IMS protease HTRA2 (high temperature deficit hyperactivity disorder (ADHD) and autism requirement A2) have been associated with various spectrum disorders, and interestingly, the majority of human disorders. The IM protease OMA1 processes GTS patients also showed other symptoms, for exam- substrates localized in the IMS and has been associ- ple, ADHD, obsessive-compulsory disorder, or Asper- ated with amyotrophic lateral sclerosis (ALS). ger syndrome, suggesting that alterations in IMMP2L may be a shared genetic factor in neurodevelopmental m-AAA disorders [64]. Patient mutations in IMMP2L resulted in chromosome aberrations or deletions rather than The m-AAA protease localizes to the inner membrane base-pair exchanges [64,68]. Mitochondria isolated and exposes its catalytic activity toward the matrix from various tissues of an IMMP2LÀ/À mouse model site. It forms homohexameric complexes composed of showed elevated levels of superoxide [69], and the mice AFG3L2 subunits or hetero-oligomeric complexes that developed early-onset ataxia and age-dependent neu- consist of AFG3L2 and paraplegin (SPG7), both met- rodegeneration of cerebellar granule neurons [70]. allo proteases [10]. Based on these results, it was suggested that defective Mutations in AFG3L2 and SPG7 result in different cerebellar IMMP2L might play a role in GTS patho- neurodegenerative phenotypes, which might be due to genesis. However, analysis of various mitochondrial the formation of two different m-AAA protease com- parameters (superoxide levels, membrane potential, plexes with different tissue-specific expression patterns mitochondrial respiratory chain) in fibroblasts from of the two subunits. Deletions and mutations in SPG7 GTS patients with and without IMMP2L deletions did have been identified in patients with hereditary spastic not detect significant changes within the analyzed paraplegia, a neurodegenerative disease characterized groups [71]. Involvement of IMMP2L in the pathogen- by progressive weakness and spasticity of the lower esis of Tourette syndrome is therefore unclear and limbs due to the degeneration of corticospinal axons needs further analyses, as IMMP2L deletions might [72]. Analysis of muscle fibers identified mitochondrial have a specific effect on cells with high-energy DNA deletions and defects in respiratory chain com- demand, for example, neurons. Taken together, given plexes [73]. Mitochondrial DNA deletions are also the variety of neurobiological and neuropsychiatric associated with progressive external ophthalmoplegia, symptoms observed in IMMP2L patients, deletions or an eye disorder characterized by the inability to move duplications in the IMMP2L gene might be considered eyes and eyebrows, and mutations in SPG7 have as general risk factors for neurological disorders rather recently been identified in several progressive external than be related specifically to GTS. ophthalmoplegia patients that also developed spastic ataxia or a progressive ataxic disorder [74]. Mutations Proteases involved in quality control in both m-AAA subunits SPG7 and AFG3L2 cause and regulation of mitochondrial dominant optic atrophy (DOA), characterized by functions degeneration of the optic nerves and central visual loss [75]. Interestingly, DOA patients with AFG3L2 vari- While presequence proteases are involved in building a ants did not display symptoms of spinocerebellar and functional mitochondrial proteome, several further spastic ataxia, which are also caused by mutations in mitochondrial proteases are functioning in quality con- AFG3L2. The variants identified in DOA patients trol of the organelle. While these enzymes have previ- were localized to different domains than the ones iden- ously been viewed as promiscuous degradation tified in ataxia patients [75]. Spinocerebellar ataxia machineries for damaged, superfluous or aged type 28 caused by dominant mutations in AFG3L2

1212 FEBS Letters 595 (2021) 1205–1222 ª 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. M. Gomez-Fabra Gala and F. N. Vogtle€ Mitochondrial proteases in human diseases affect cerebellar purkinje cells, in which AFG3L2 is PARL highly expressed [76]. Recessive mutations in AFG3L2 PARL (Presenilin-associated rhomboid-like) is an are associated with spastic ataxia 5, a neurological syn- intramembrane serine protease localizing in the mito- drome characterized by early onset of cerebellar atax- chondrial inner membrane. The active site of rhomboid ia, spasticity, deficient eye movement, dystonia, and proteases is embedded in the lipid bilayer so that they myoclonic epilepsy [77]. Several mice models have been cleave their substrates primarily within transmembrane generated to investigate the molecular mechanisms segments [81]. PARL activity was reported to be regu- underlying the neurodegenerative consequences of m- lated by N-terminal processing and phosphorylation [81] AAA mutations and are reviewed, for example, in and to have a role in diverse mitochondrial functions, Patron et al. [78]. for example, programmed cell death, lipid trafficking, coenzyme Q biosynthesis, or mitophagy [82–86]. Of the i-AAA known PARL substrates, PINK1 has been studied most [81]. In healthy mitochondria, PINK1 is imported into The i-AAA protease is also located in the IM, but in the organelle, where it is cleaved by MPP and PARL contrast to the m-AAA protease, its catalytic activity and released back into the cytosol for rapid degradation faces toward the IMS site. The protease is built from six by the proteasome [25]. Dysfunctional mitochondria subunits of the zinc metalloprotease YME1L1 [10]. accumulate PINK1 on the outer membrane, which sig- YME1L1 together with OMA1 is processing OPA1, nals degradation of the organelle by mitophagy, a pro- thereby regulating the balance between mitochondrial cess that involves the E3 ubiquitin ligase PARKIN [26]. fusion and fission. OPA1 mediates fusion and eight dif- Next to its role in PINK1 processing, PARL is emerging ferent isoforms exist that are expressed tissue-specifi- as protease involved in the regulation of dually localized cally. OPA1 is initially processed by MPP upon import proteins [10]. Cleavage within the transmembrane into mitochondria and the resulting long-OPA1 is inte- domains of these inner membrane proteins results in the grated into the inner membrane. Long-OPA1 can release of a soluble form to the IMS or export to the undergo a secondary cleavage by either YME1L or cytosol [10]. PARL knockout mice develop a severe OMA1 (see below) resulting in a short-OPA1 form that encephalomyelopathy that resembles Leigh syndrome is soluble. Mitochondrial morphology depends on the and results in neurodegeneration and early death [84]. amount of OPA1 and the ratio of its long and short pro- PARL has been associated with Parkinson’s disease cessing forms [10]. A homozygous mutation in YME1L (PD) as missense mutations in PARL were identified that results in a missense mutation within its prese- in PD patients [87]. However, subsequent studies indi- quence was identified in a family with four affected chil- cated that mutations in PARL are a rare cause of PD dren that developed an early-onset mitochondriopathy so that the role of PARL in PD development remains with developmental delay, muscle weakness, ataxia, and unclear [88]. Single nucleotide polymorphisms (SNPs) optic nerve atrophy [40]. While the mutant protein was in PARL were identified in patients with Leber heredi- still targeted to mitochondria, in vitro processing assays tary optic neuropathy (LHON) [89]. While the initial indicated that MPP did not recognize and cleave the identification was in a patient cohort from Thailand, mutated presequence. Mutant YME1L was rapidly no association between SNPs in PARL and LHON degraded and caused proliferation defects and fragmen- was identified in a study from China [90]. SNPs in tation of the mitochondrial network due to changes in PARL were reported to confer a risk to leprosy, a processing of OPA1. Altered OPA1 processing was also chronic and neurological diseases cause by Mycobac- observed upon cardiac-specific ablation of Yme1l in terium leprae [91]. A recent study using a proteomic mice [79]. Loss of Yme1l enhanced OMA1-dependent approach has identified novel PARL substrates, which processing of OPA1, tipping the balance toward might shed light on the contribution of PARL in PD increased mitochondrial fission, fragmentation of the and other disease pathogenesis [82]. mitochondrial network, and resulted in dilated car- diomyopathy and heart failure in mice, which was res- cued by ablation of Oma1 [79]. Loss of YME1L in the OMA1 nervous system similarly impaired mitochondrial mor- phology and in addition caused axonal degeneration OMA1 is a conserved metalloprotease anchored in the [80]. Interestingly, while deletion of Oma1 restored the inner membrane and assumed to form a homo-oligo- aberrant morphology, it did not rescue axonal degenera- meric complex [92]. The active site is oriented to the tion, demonstrating that loss of the i-AAA protease IMS. The protease seems to be in a dormant state under impacts differently on different tissues [80]. normal conditions and becomes rapidly active upon

FEBS Letters 595 (2021) 1205–1222 ª 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of 1213 Federation of European Biochemical Societies. Mitochondrial proteases in human diseases M. Gomez-Fabra Gala and F. N. Vogtle€ stress conditions, for example, loss of membrane poten- CLPX (caseinolytic peptidase subunit X) subunits that tial, heat or oxidative stress [93–95]. This activation has function in substrate recognition, ATP-dependent been suggested to be triggered by autocatalytic cleavage unfolding of the substrate, and its translocation into within the C-terminal part of the protease [93,95],but the proteolytic CLPP chamber [102]. CLPP turns over also a role of YME1L in stress-mediated degradation peptides and proteins, but has recently also been impli- and thereby regulation of OMA1 activity has been pro- cated in regulation of mitochondrial functions, for posed [96]. OMA1 processes the IM protein OPA1 that example, degradation of the putative rRNA chaperone mediates mitochondrial fusion. Processing by OMA1 (or ERAL1 by CLPP coordinates assembly of the mito- by the i-AAA protease YME1L that can also cleave chondrial ribosome and degradation of the N-module OPA1) releases the short OPA1-form into the IMS. A of respiratory chain complex I by CLPXP safeguards balance in the ratio of short- and long-OPA1 forms by the cells against accumulation of dysfunctional com- proteolytic processing is required to maintain the mito- plex I [103,104]. chondrial network [10]. Mutations in CLPP cause Perrault syndrome, which Missense mutations in OMA1 have been identified is characterized by sensorineural hearing loss and in patients with sporadic ALS [97]. ALS is a devastat- ovarian failure. In severe courses of disease, additional ing neurodegenerative disorder that is characterized by symptoms can include ataxia, neuropathies, and intel- the progressive degeneration of motor neurons in the lectual disability [105,106]. The so far identified muta- spinal cord, brain stem, and cerebral cortex [98]. tions cluster either near the for ClpX or Patients develop muscle weakness, a progressive paral- are adjacent to the active site of the protease. Analysis ysis and spasticity, and die within 2–5 years after dis- of the molecular consequences of the single mutations ease onset. Most ALS cases are sporadic (90%) and revealed a wide range of consequences from no mitochondrial dysfunctions are proposed as a central changes to a strong decrease in proteolytic activity, factor in ALS pathogenesis, for example, caused by loss of oligomeric assembly to an increased peptidase mutations in the superoxide dismutase 1 (SOD1). activity [106]. Interestingly, mutations in the noncat- Sequencing of the OMA1 gene in more ALS patients alytic subunit CLPX display a different phenotype and will be required to determine whether OMA1 plays a promote erythropoietic protoporphyria [107]. ClpX role in ALS pathogenesis. stimulates mitochondrial heme biogenesis by activation of the key enzyme d-aminolevulinate synthase (ALAS). Mutations in ClpX inactivate the ATPase activity, LONP which increases the stability of ALAS and causes the LONP1 resides in the matrix and forms homohexam- accumulation of the heme biosynthesis intermediate eric complexes. The ATP-dependent serine protease protoporphyrin IV. degrades misfolded, unassembled, or oxidatively dam- aged proteins. LONP1 is also involved in regulation HTRA2 of mitochondrial DNA copy numbers and mitochondrial transcription by selective degradation The serine protease HTRA2 is localized to the IMS of mitochondrial transcription factor A, a DNA and is also known as Omi. In addition to its prote- binding protein required for mtDNA maintenance olytic domain, HTRA family members have one or [99]. two PDZ domains that serve as protein-protein inter- Mutations in LONP1 have been identified in patients action site and preferentially bind C-terminal peptides with CODAS syndrome, a multisystemic developmen- of the target protein [108]. HTRA2 functions in PQC tal disorder with cerebral, ocular, dental, auricular, and in caspase-dependent and caspase-independent and skeletal abnormalities [100,101]. The identified apoptosis pathways [108,109]. mutations clustered in the ATP-binding and proteolytic Mice with a homozygous mutation in Htra2/Omi domain and resulted in defects in substrate proteolysis. (Ser276Cys, Mnd2 mice, motor neuron degeneration The mutations caused an aberrant mitochondrial ultra- 2), which strongly reduces its catalytic activity, devel- structure and defects in respiratory chain complexes, oped a Parkinsonian phenotype with degeneration of labeling CODAS as a mitochondrial disease. striatal neurons and muscle wasting and death after 40 days of age [110]. Similarly, mice with a targeted deletion of Htra2/Omi1 also developed a neurodegen- CLPXP erative disorder with decreased coordination and The matrix enzyme CLPXP is composed of seven mobility and development of tremor and died around CLPP subunits that possess proteolytic activity and six 30 days of birth [111]. SNPs in HTRA2 that result in

1214 FEBS Letters 595 (2021) 1205–1222 ª 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. M. Gomez-Fabra Gala and F. N. Vogtle€ Mitochondrial proteases in human diseases single amino acid substitutions were identified in initiation of sporadic colorectal cancer (CRC), and patients with PD. The mutations affected the prote- therefore, the elucidation of the Wnt/b-catenin-target olytic activity of HTRA2 and induced mitochondrial genes is of immense interest [123]. Gene expression dysfunction and altered mitochondrial morphology analysis of CRC samples with activated Wnt/b-catenin [112]. A further identified mutation in HTRA2 resulted signaling identified elevated expression of XPNPEP3 in hereditary essential tremor and homozygotes for the [124]. Canonical activation of Wnt/b-catenin signaling allele developed PD [113]. The observation that Htra2 in three human cell lines also resulted in increased is phosphorylated in a PINK1-dependent manner at XPNPEP3 transcript levels suggesting XPNPEP3 as an amino acid that is next to one of the positions novel Wnt/b-catenin transcriptional target. Ectopic mutated in PD patients suggested that PINK1-depen- overexpression of XPNPEP3 induced cell proliferation dent phosphorylation of Htra2 might modulate its pro- and analyses of XPNPEP3 transcription in various teolytic activity [114]. Recently, patients were reported cancers (e.g., anaplastic astrocytoma, breast carci- with missplicing mutations and small deletions in noma, soldering myeloma) showed a significant HTRA2 that result in infantile neurodegeneration and increase and correlated with poor survival [124].Of 3-methylglutaconic aciduria. The HTRA2 protein was interest, Wnt signaling is a positive regulator of ciliary not detectable in patient-derived fibroblasts, which development and XPNPEP3 has been implied to play showed impaired growth and increased sensitivity to a role in a NPHP -like ciliopathy (see above). It will apoptosis. These defects are reminiscent of the pheno- be interesting to follow up on this link and to investi- type observed in Htra2 knockout mice and highlight gate which isoform of the dually localized XPNPEP3 the important role of HTRA2 in modulating apoptosis (cytosolic or mitochondrial) is predominantly [115,116]. expressed in CRC. Also the i-AAA protease YME1L has recently been Emerging role of mitochondrial reported to play a role in tumor development, specifi- proteases in cancer cally in pancreatic ductal carcinoma (PDAC) [125]. Hypoxia or amino acid starvation inhibit mTORC1 Given that changes in cellular metabolism and bioen- and enhanced YME1L-dependent proteolysis. The ergetics, disabled apoptosis, and oxidative stress are resulting degradation of key proteins for mitochondrial hallmarks of cancer development, (de-)regulation of biogenesis rewires the organellar proteome. Similar mitochondrial functions is emerging to have an impor- changes were also detected in PDAC tumor tissues tant role in carcinogenesis. Several mitochondrial pro- and suggest that YME1L might be relevant in tumor teases show altered expression profiles in malignant pathophysiology [125]. cells, and increasing data suggest a role for some mito- The serine protease HTRA2 has been associated chondrial proteases in cancer progression. with several cancers, including ovarian cancer, lung The protease LONP has been reported to be upregu- cancer, or hepatocellular carcinoma [126–128]. lated in several tumors, promoting a more aggressive Decreased HTRA2 protein levels correlated with a phenotype of the cancer and being associated with a poor prognosis, chemoresistance and increased inva- worsened prognosis [117–119]. Downregulation of sion [126–129]. HTRA2 has been identified as binding LONP1 suppresses cell proliferation in cervical cancer partner of the Wilms’ tumor suppressor protein WT1. and LON silencing by shRNA causes alterations in the WT1 is a transcriptional regulator that controls genes mitochondrial proteome and induces cell death in involved in cellular growth, cell death, and differentia- colon cancer cells [120,121]. A possible link between tion [130]. While WT1 acts as a tumor suppressor in LONP and tumor development might be its role in the Wilms’ tumors (pediatric nephroblastoma), analysis of adaptation of the respiratory chain upon changes in WT1 expression levels in a variety of other tumors oxygen availability. Reduced oxygen levels induce suggests also an oncogenic function (reviewed in Ref. expression of a specific subunit of complex IV of the [131]). Treatment of cells with cytotoxic drugs trig- respiratory chain COX4-2 and of LONP1 via the gered WT1 degradation by HTRA2 resulting in hypoxia-inducible factor 1 (HIF-1). LONP1 degrades changes in gene expression that enhance apoptosis COX4-1, which is replaced by COX4-2 that adapts res- [130]. HTRA2 has also been implicated in prevention piration to the changed oxygen concentrations [122]. of cell invasion [132]. Oncogenic Ras is known to Wnt/b-catenin signaling is an evolutionary con- induce cellular transformation, but also promotes served pathway that plays an important role in embry- tumor cell invasion by actin reorganization, which can onic development and tissue regeneration. Aberrant be suppressed by p53. Upon Ras transformation p53 activation of Wnt signaling is a major reason for accumulates in the cytosol, which results in the

FEBS Letters 595 (2021) 1205–1222 ª 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of 1215 Federation of European Biochemical Societies. Mitochondrial proteases in human diseases M. Gomez-Fabra Gala and F. N. Vogtle€ activation of HTRA2. Concomitant Ras-induced frag- Acknowledgements mentation of mitochondria releases HTRA2 into the cytosol, where it cleaves b-actin in turn leading to fila- The work in the FNV laboratory is supported by mentous actin disassembly thereby suppressing inva- grants from the Deutsche Forschungsgemeinschaft siveness [132]. Besides HTRA2, also OMA1 is (DFG) under Germany’s Excellence Strategy (CIBSS responsive to p53 accumulation. Cisplatin treatment of —EXC-2189—Project ID 390939984), the SFB 1381 ovarian and cervical cancer cells increases OMA1 via (Project ID 403222702), the GRK 2606, and the p53, which results in processing of L-OPA1 and mito- Emmy-Noether Programm. chondrial fragmentation [133]. While HTRA2 and OMA1 are reacting to changes References in p53, the mitochondrial protease CLPP has been implicated in induction of cancer cell lethality in a 1 Sickmann A, Reinders J, Wagner Y, Joppich C, € p53-independent manner. Hyperactivation of CLPP Zahedi R, Meyer HE, Schonfisch B, Perschil I, selectively kills cancer, but not nonmalignant cells, by Chacinska A, Guiard B et al. (2003) The proteome of degradation of mitochondrial respiratory chain sub- Saccharomyces cerevisiae mitochondria. Proc Natl Acad Sci USA 100, 13207–13212. units, in turn disrupting mitochondrial structure and 2 Pagliarini DJ, Calvo SE, Chang B, Sheth SA, Vafai function [134]. On the other hand, it was reported that SB, Ong SE, Walford GA, Sugiana C, Boneh A, Chen CLPP is overexpressed in acute myeloid leukemia and WK et al. (2008) A mitochondrial protein compendium that CLPP inhibition decreases viability of the leuke- elucidates complex I disease biology. Cell 134, 112– mic cells [135]. In line with this, downregulation of 123. CLPP made human cancer cells more susceptible to 3Vogtle€ FN, Burkhart JM, Gonczarowska-Jorge H, cisplatin treatment [136]. While the role of CLPP in Kuc€ ukk€ ose€ C, Taskin AA, Kopczynski D, Ahrends R, cancer requires analysis of the pathways and molecular Mossmann D, Sickmann A, Zahedi RP et al. (2017) mechanisms and its impact on different tumors, the Landscape of submitochondrial protein distribution. manipulation of CLPP activity might represent a Nat Commun 8, 290. potential therapeutic target and imipridones, allosteric 4 Morgenstern M, Stiller SB, Lubbert€ P, Peikert CD, CLPP agonists that bind to and induce proteolysis by Dannenmaier S, Drepper F, Weill U, Ho߀ P, CLPP are currently in clinical trials [134]. Feuerstein R, Gebert M et al. (2017) Definition of a high-confidence mitochondrial proteome at quantitative scale. Cell Rep 19, 2836– Conclusions and Perspectives 2852. Mitochondrial proteases are emerging as critical regu- 5 Topf U, Wrobel L and Chacinska A (2016) Chatty lators of mitochondrial proteostasis and mitochondrial mitochondria: keeping balance in cellular protein functions. Novel enzymes have been discovered by homeostasis. Trends Cell Biol 26, 577–586. € proteomic approaches and new functions of already 6 Andreasson C, Ott M and Buttner S (2019) known proteases have been revealed. The importance Mitochondria orchestrate proteostatic and metabolic of mitochondrial proteases is underlined by the large stress responses. EMBO Rep 20, e47865. and growing number of human diseases that are asso- 7 Suomalainen A and Battersby BJ (2018) Mitochondrial diseases: the contribution of organelle ciated with defects in these enzymes. To reveal and stress responses to pathology. Nat Rev Mol Cell Biol understand the pathological mechanisms underlying 19,77–92. these diverse disorders, the comprehensive substrate 8 Eisenberg-Bord M and Schuldiner M (2017) Ground spectrum of the individual mitochondrial proteases control to major TOM: mitochondria-nucleus needs to be known in each affected tissue and mass communication. FEBS J 284, 196–210. spectrometric approaches, such as N-proteomics, have 9 Lebeau J, Rainbolt TK and Wiseman RL (2018) already proven valuable [82,137,138]. Previously con- Coordinating mitochondrial biology through the stress- sidered as mostly constitutively active, protease activity responsive regulation of mitochondrial proteases. Int might be regulated by certain stress stimuli or meta- Rev Cell Mol Biol 340,79–128. bolic changes, thus being a point for future studies. A 10 Deshwal S, Fiedler KU and Langer T (2020) deepened understanding of the function and regulation Mitochondrial proteases: multifaceted regulators of of mitochondrial proteases will give insight into dis- mitochondrial plasticity. Annu Rev Biochem 89, 501– ease pathomechanisms and might open the possibility 528. for therapeutic targeting of mitochondrial proteases in 11 Hansen KG and Herrmann JM (2019) Transport human diseases. of proteins into mitochondria. Protein J 38, 330–342.

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