REVIEW ARTICLE Mitochondrial proteases 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 enzymes. 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 apoptosis. 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 protease; 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 aminopeptidase 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, translocase 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 ligase [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 cytosol 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