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Schematic representation of mitochondrial ATP-dependent proteases. Mammalian mitochondria comprise four different proteases of the AAA+ superfamily for regulating protein quality control: The ClpXP complex and Lon protease 1 in the matrix and the i-AAA and m-AAA proteases in IM. Abbreviations: IMM, inner mitochondrial membrane, OMM, outer mitochondrial membrane; IMS: intermembrane space.
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Simple Summary
Alterations of cellular metabolism and bioenergetics, oxidative stress, and intracellular reactive oxygen species (ROS) levels are hallmarks of cancer development. Mitochondrial proteases, especially ATP-dependent proteases are essential to regulate mitochondrial function by maintaining protein quality. Emerging studies suggest the t...
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Mitochondrial proteolysis is an evolutionarily conserved quality-control mechanism to maintain proper mitochondrial integrity and function. However, the physiological relevance of stress-induced impaired mitochondrial protein quality remains unclear. Here, we demonstrate that LONP1, a major mitochondrial protease resides in the matrix, plays a role...
Citations
... Mitochondrial proteases are the central regulators of mitochondrial proteostasis. In addition to their role as quality control enzymes that remove damaged proteins and prevent their possible deleterious accumulation, mitochondrial proteases regulate the half-life of proteins, play roles in mitochondrial protein maturation, such as MPP, and occasionally act as scaffolds without proteolytic activity [90,118,119]. The four functional groups of mitochondrial proteases include ATP-dependent peptidases, oliogo-peptidases, processing peptidases, and other mitochondrial peptidases [118]. ...
... In addition to their role as quality control enzymes that remove damaged proteins and prevent their possible deleterious accumulation, mitochondrial proteases regulate the half-life of proteins, play roles in mitochondrial protein maturation, such as MPP, and occasionally act as scaffolds without proteolytic activity [90,118,119]. The four functional groups of mitochondrial proteases include ATP-dependent peptidases, oliogo-peptidases, processing peptidases, and other mitochondrial peptidases [118]. Several mitochondrial proteases regulate the turnover of frataxin protein in addition to MPP. ...
Friedreich’s ataxia (FRDA) is a progressive neurodegenerative disease caused in almost all patients by expanded guanine–adenine–adenine (GAA) trinucleotide repeats within intron 1 of the FXN gene. This results in a relative deficiency of frataxin, a small nucleus-encoded mitochondrial protein crucial for iron–sulfur cluster biogenesis. Currently, there is only one medication, omaveloxolone, available for FRDA patients, and it is limited to patients 16 years of age and older. This necessitates the development of new medications. Frataxin restoration is one of the main strategies in potential treatment options as it addresses the root cause of the disease. Comprehending the control of frataxin at the transcriptional, post-transcriptional, and post-translational stages could offer potential therapeutic approaches for addressing the illness. This review aims to provide a general overview of the regulation of frataxin and its implications for a possible therapeutic treatment of FRDA.
... Overall, it is necessary to validate whether the Clpp-KO mouse evidence holds true in other organisms, i.e., that the specific roles of CLPX and CLPP are defined by the granular compartments that they are monitoring. This would be analogous to most other peptidases in mitochondria, where MPP cleaves all precursor proteins at the import pore, m-AAA and i-AAA are responsible for protein quality control at either IMM face, PARL cleaves proteins within the IMM, and OMA1/HTRA2 performs surveillance in the intermembrane space [211][212][213][214]. Our proteome identification of specific factors whose abundance depends on CLPP will also be useful (more so than unspecific mitochondrial-respiratory assays) for comparing the efficacy of drugs that activate or inhibit CLPP. ...
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, especially 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 humans, despite their secondary role in 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 as first-aid systems near the inner mitochondrial membrane. Trimming within assemblies, CLPP rescues stalled processes in mitoribosomes, mitochondrial RNA granules and nucleoids, and the 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, as other mitochondrial peptidases: MPPs at precursor import pores, m-AAA and i-AAA at either IMM face, PARL within the IMM, and OMA1/HTRA2 in the intermembrane space.
... Overall, it is necessary to validate in other organisms if the Clpp-KO mouse evidence holds true -that the specific roles of CLPX and CLPP are defined by the granular compartments they are monitoring. This would be analogous to most other peptidases in mitochondria, where MPP cleaves all precursor proteins at the import pore, m-AAA and i-AAA are responsible for protein quality control at either IMM face, PARL cleaves proteins within the IMM, and OMA1/HTRA2 perform surveillance in the intermembrane space [211][212][213][214]. ...
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.
... A mitochondrial-associated proteasome would be a complement to known mitochondrial ATPases associated with diverse cellular activities (AAA+) proteases found within the intermembrane space (e.g., iAAA), inner membrane (e.g., SPG7/paraplegin), and matrix (e.g., LONP1 and ClpXP) (Feng et al., 2021). The AAA+ mitochondrial proteins have many non-peptolytic functions, in addition to protease activities that degrade preferential mitochondrial protein targets that are misfolded or oxidatively damaged. ...
Introduction
Loss of proteasome function, proteinopathy, and proteotoxicity may cause neurodegeneration across the human lifespan in several forms of brain injury and disease. Drugs that activate brain proteasomes in vivo could thus have a broad therapeutic impact in neurology.
Methods
Using pigs, a clinically relevant large animal with a functionally compartmental gyrencephalic cerebral cortex, we evaluated the localization and biochemical activity of brain proteasomes and tested the ability of small molecules to activate brain proteasomes.
Results
By Western blotting, proteasome protein subunit PSMB5 and PSMA3 levels were similar in different pig brain regions. Immunohistochemistry for PSMB5 showed localization in the cytoplasm (diffuse and particulate) and nucleus (cytoplasm < nucleus). Some PSMB5 immunoreactivity was colocalized with mitochondrial (voltage-gated anion channel and cyclophilin D) and cell death (Aven) proteins in the neuronal soma and neuropil in the neocortex of pig and human brains. In the nucleus, PSMB5 immunoreactivity was diffuse, particulate, and clustered, including perinucleolar decorations. By fluorogenic assay, proteasome chymotrypsin-like activities (CTL) in crude tissue soluble fractions were generally similar within eight different pig brain regions. Proteasome CTL activity in the hippocampus was correlated with activity in nasal mucosa biopsies. In pilot analyses of subcellular fractions of pig cerebral cortex, proteasome CTL activity was highest in the cytosol and then ~50% lower in nuclear fractions; ~15–20% of total CTL activity was in pure mitochondrial fractions. With in-gel activity assay, 26S-singly and -doubly capped proteasomes were the dominant forms in the pig cerebral cortex. With a novel in situ histochemical activity assay, MG132-inhibitable proteasome CTL activity was localized to the neuropil, as a mosaic, and to cell bodies, nuclei, and centrosome-like perinuclear satellites. In piglets treated intravenously with pyrazolone derivative and chlorpromazine over 24 h, brain proteasome CTL activity was modestly increased.
Discussion
This study shows that the proteasome in the pig brain has relative regional uniformity, prominent nuclear and perinuclear presence with catalytic activity, a mitochondrial association with activity, 26S-single cap dominance, and indications from small molecule systemic administration of pyrazolone derivative and chlorpromazine that brain proteasome function appears safely activable.
... Both the genetic and chemical inhibition of ClpXP and its overactivation by chemical and/or by mutation cause tumor cell death. In fact, on the one hand, its inhibition leads to the accumulation of misfolded and damaged respiratory chain proteins and impairs oxidative phosphorylation, resulting in the selective death of cancer cells [8]. Inhibitors of hClpP covalently modify the fourteen catalytic residues of Ser153 located within the lumen of the hClpP tetradecamer [9]. ...
... The overactivation of hClpP by its activators results in mitochondrial morphological damage and a decrease in oxidative phosphorylation, inducing tumor cell death [8]. Therefore, targeting AAA+ proteases like ClpXP could be a strategy against malignant cells sparing normal tissues. ...
... Activators binding to the allosteric hClpP site produce conformational changes in the enzyme complex, resulting in the enlargement of the axial proteolytic pore and the compaction of the protease. These changes lead to hClpP hyperactivation and increase substrate degradation in a selective and uncontrolled manner [8]. To date, it is not known whether the inhibition or activation of hClpP the preferred therapeutic strategy would be. ...
Diffuse intrinsic pontine glioma (DIPG), affecting children aged 4–7 years, is a rare, aggressive tumor that originates in the pons and then spreads to nearby tissue. DIPG is the leading cause of death for pediatric brain tumors due to its infiltrative nature and inoperability. Radiotherapy has only a palliative effect on stabilizing symptoms. In silico and preclinical studies identified ONC201 as a cytotoxic agent against some human cancer cell lines, including DIPG ones. A single-crystal X-ray analysis of the complex of the human mitochondrial caseinolytic serine protease type C (hClpP) and ONC201 (PDB ID: 6DL7) allowed hClpP to be identified as its main target. The hyperactivation of hClpP causes damage to mitochondrial oxidative phosphorylation and cell death. In some DIPG patients receiving ONC201, an acquired resistance was observed. In this context, a wide program was initiated to discover original scaffolds for new hClpP activators to treat ONC201-non-responding patients. Harmaline, a small molecule belonging to the chemical class of β-carboline, was identified through Fingerprints for Ligands and Proteins (FLAP), a structure-based virtual screening approach. Molecular dynamics simulations and a deep in vitro investigation showed interesting information on the interaction and activation of hClpP by harmaline.
... The dependency of tumor cells on particular mitochondrial chaperones is exemplified by the biological activity of small molecules targeting mitochondrial chaperones such as TRAP1 [4], HSPD1 [5], ClpX [6] and ClpP [7], all of which are promising anti-cancer compounds. Interestingly, it is not clear why particular cell types are sensitive to a chaperone's particular inhibitors. ...
Cancer cells alter the expression levels of metabolic enzymes to fuel proliferation. The mitochondrion is a central hub of metabolic reprogramming, where multiple chaperons service hundreds of clients, forming complex chaperone-client interaction networks (CCINs). Studying (i) how network structure varies across cancer types and (ii) the influence of structure on networks' robustness to chaperon targeting is key to developing cancer-specific drug therapy. We investigate these yet unaddressed questions using theory and methodology from network ecology. We show that CCINs are non-randomly structured across cancer types. Moreover, we identify groups of chaperones that interact with similar clients, providing specialization on clients but also redundancy in the case of chaperone removal on the other. Simulations of network robustness show that this modular structure strongly affects cancer-specific response to chaperon removal. Our results open the door for new hypotheses regarding the ecology and evolution of CCINs and can inform caner-specific drug development strategies.
... Matrix Quality control and biogenesis [15] YME1L Inner membrane Quality control and biogenesis [16] Processing peptidases ATP23 Intermembrane space Quality control [15] IMMP Inner membrane Protein import and activation [17] METAP1D Matrix Protein import and activation [17] MIP Matrix Protein import and activation [18] OMA1 Inner membrane Quality control, mitochondrial dynamics, and mitophagy [19] PARL Inner membrane Mitophagy and apoptosis [20] PMPCB Matrix Protein import and activation [21] XPNPEP3 Matrix Protein import and activation [17] Oligopeptidases MEP Intermembrane space Quality control [17] PITRM1 Matrix Quality control [15] Other mito-proteases HTRA2 Intermembrane space Quality control, mitophagy and apoptosis [22] LACTB Intermembrane space Mitochondrial biogenesis [17] targeting and transportation to the mitochondrial matrix. 31 As for its C-terminus, the 28-residue extension promotes the formation of an unstructured flexible loop, although the specific role is not clear, which probably assists the assembly of ClpP heptamer and increases the affinity with its partner molecule ClpX 32 ( Figure 2A). ...
... Matrix Quality control and biogenesis [15] YME1L Inner membrane Quality control and biogenesis [16] Processing peptidases ATP23 Intermembrane space Quality control [15] IMMP Inner membrane Protein import and activation [17] METAP1D Matrix Protein import and activation [17] MIP Matrix Protein import and activation [18] OMA1 Inner membrane Quality control, mitochondrial dynamics, and mitophagy [19] PARL Inner membrane Mitophagy and apoptosis [20] PMPCB Matrix Protein import and activation [21] XPNPEP3 Matrix Protein import and activation [17] Oligopeptidases MEP Intermembrane space Quality control [17] PITRM1 Matrix Quality control [15] Other mito-proteases HTRA2 Intermembrane space Quality control, mitophagy and apoptosis [22] LACTB Intermembrane space Mitochondrial biogenesis [17] targeting and transportation to the mitochondrial matrix. 31 As for its C-terminus, the 28-residue extension promotes the formation of an unstructured flexible loop, although the specific role is not clear, which probably assists the assembly of ClpP heptamer and increases the affinity with its partner molecule ClpX 32 ( Figure 2A). ...
... Matrix Quality control and biogenesis [15] YME1L Inner membrane Quality control and biogenesis [16] Processing peptidases ATP23 Intermembrane space Quality control [15] IMMP Inner membrane Protein import and activation [17] METAP1D Matrix Protein import and activation [17] MIP Matrix Protein import and activation [18] OMA1 Inner membrane Quality control, mitochondrial dynamics, and mitophagy [19] PARL Inner membrane Mitophagy and apoptosis [20] PMPCB Matrix Protein import and activation [21] XPNPEP3 Matrix Protein import and activation [17] Oligopeptidases MEP Intermembrane space Quality control [17] PITRM1 Matrix Quality control [15] Other mito-proteases HTRA2 Intermembrane space Quality control, mitophagy and apoptosis [22] LACTB Intermembrane space Mitochondrial biogenesis [17] targeting and transportation to the mitochondrial matrix. 31 As for its C-terminus, the 28-residue extension promotes the formation of an unstructured flexible loop, although the specific role is not clear, which probably assists the assembly of ClpP heptamer and increases the affinity with its partner molecule ClpX 32 ( Figure 2A). ...
Mitochondria, the main provider of energy in eukaryotic cells, contains more than 1000 different proteins and is closely related to the development of cells. However, damaged proteins impair mitochondrial function, further contributing to several human diseases. Evidence shows mitochondrial proteases are critically important for protein maintenance. Most importantly, quality control enzymes exert a crucial role in the modulation of mitochondrial functions by degrading misfolded, aged, or superfluous proteins. Interestingly, cancer cells thrive under stress conditions that damage proteins, so targeting mitochondrial quality control proteases serves as a novel regulator for cancer cells. Not only that, mitochondrial quality control proteases have been shown to affect mitochondrial dynamics by regulating the morphology of optic atrophy 1 (OPA1), which is closely related to the occurrence and progression of cancer. In this review, we introduce mitochondrial quality control proteases as promising targets and related modulators in cancer therapy with a focus on caseinolytic protease P (ClpP), Lon protease (LonP1), high‐temperature requirement protein A2 (HrtA2), and OMA‐1. Further, we summarize our current knowledge of the advances in clinical trials for modulators of mitochondrial quality control proteases. Overall, the content proposed above serves to suggest directions for the development of novel antitumor drugs.
... So far, varied mitochondrial proteases have been identified in different mitochondrial compartments (Quir os et al., 2015). The functions of these proteases have not been fully elucidated; some of which are only now emerging as prospective therapeutic targets for haematological malignancies (Castelli et al., 2019;Culp-Hill et al., 2021;Feng et al., 2021;Gomez-Fabra Gala & Vögtle, 2021). ...
Targeting cancer metabolism has emerged as an attractive approach to improve therapeutic regimens in acute myeloid leukaemia (AML). Mitochondrial proteases are closely related to cancer metabolism, but their biological functions have not been well characterized in AML. According to different categories, we comprehensively review the role of mitochondrial proteases in AML. This review highlights some ‘powerful’ mitochondrial protease targets, including their biological function, chemical modulators, and applicative prospect in AML.
... In turn, Hsp90 chaperoning stabilizes the multifunctional mitochondrial proteome in cancer, 19,20 including key metabolic regulators, 21 lowers ROS, 22 and prevents cell death. 17,23,24 Although targeting chaperone-directed proteostasis in mitochondria shows promising antitumor activity, 25,26 pharmacologically, this pathway escapes inhibition by smallmolecule Hsp90 antagonists with geldanamycin (GA) or non-GA backbones 27 as these agents fail to accumulate in mitochondria. 28 To overcome this conundrum, we generated Gamitrinib (GA mitochondrial matrix inhibitor), a first-inclass, mitochondrial-targeted inhibitor of organelle protein folding 25 that links the GA Hsp90 inhibitor 17-allylaminogeldanamyicn (17-AAG, Tanespimycin) 27 to an efficient mitochondrial-import carrier, triphenylphosphonium (TPP). ...
Mitochondria are key tumor drivers, but their suitability as a therapeutic target is unknown. Here, we report on the preclinical characterization of Gamitrinib (GA mitochondrial matrix inhibitor), a first-in-class anticancer agent that couples the Heat Shock Protein-90 (Hsp90) inhibitor 17-allylamino-geldanamycin (17-AAG) to the mitochondrial-targeting moiety, triphenylphosphonium. Formulated as a stable (≥24 weeks at −20°C) injectable suspension produced by microfluidization (<200 nm particle size), Gamitrinib (>99.5% purity) is heavily bound to plasma proteins (>99%), has intrinsic clearance from liver microsomes of 3.30 mL/min/g and minimally penetrates a Caco-2 intestinal monolayer. Compared to 17-AAG, Gamitrinib has slower clearance (85.6 ± 5.8 mL/min/kg), longer t1/2 (12.2 ± 1.55 h), mean AUC0-t of 783.1 ± 71.3 h∙ng/mL, and unique metabolism without generation of 17-AG. Concentrations of Gamitrinib that trigger tumor cell killing (IC50 ~1-4 µM) do not affect cytochrome P450 isoforms CYP1A2, CYP2A6, CYP2B6, CYP2C8 or ion channel conductance (Nav1.5, Kv4.3/KChIP2, Cav1.2, Kv1.5, KCNQ1/mink, HCN4, Kir2). Twice weekly IV administration of Gamitrinib to Sprague-Dawley rats or beagle dogs for up to 36 d is feasible. At dose levels of up to 5 (rats)- and 12 (dogs)-fold higher than therapeutically effective doses in mice (10 mg/kg), Gamitrinib treatment is unremarkable in dogs with no alterations in clinical-chemistry parameters, heart function, or tissue histology, and causes occasional inflammation at the infusion site and mild elevation of serum urea nitrogen in rats (≥10 mg/kg/dose). Therefore, targeting mitochondria for cancer therapy is feasible and well tolerated. A publicly funded, first-in-human phase I clinical trial of Gamitrinib in patients with advanced cancer is ongoing (ClinicalTrials.gov NCT04827810)
... The development of allosteric inhibitors and activators of LonP1 will be invaluable in elucidating its mechanistic and functional complexities and holds promise for chemotherapeutic benefit in treating cancers and age-associated disorders such as atherosclerosis and neurodegeneration. The overexpression of LonP1 has been observed in numerous solid tumors and blood cancers and is postulated to be a risk factor for promoting oncogenesis (4,6,(15)(16)(17)53). Emerging evidence suggests that inhibiting LonP1 or other quality control proteins in mitochondria (54,55) and endoplasmic reticulum (56) of cancer cells or immunosuppressor cells (57) is a potential strategy for disabling oncogenic progression. The endoplasmic reticulum (ER) and mitochondrial unfolded protein response pathways (UPR ER and UPR mt , respectively) have been postulated to impart an advantage to cancer cells, supporting cell survival, proliferation and evasion of immunosurveillance, and drug resistance (57)(58)(59)(60), by mitigating hostile conditions within the tumor microenvironment such as nutrient and oxygen deprivation, oxidative stress, and high metabolic demand. ...
... It is possible that CDDO derivatives may interfere with other AAA + proteins such as the mitochondrial matrix ClpXP protease. Recent work has identified small-molecule inhibitors and activators of ClpXP (65)(66)(67), which have shown chemotherapeutic potential in treating malignancies associated with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), breast cancer (54). Thus, experiments are being performed to test whether CDDO derivatives inhibit ClpXP as well as LonP1. ...
The mitochondrial protein LonP1 is an ATP-dependent protease that mitigates cell stress and calibrates mitochondrial metabolism and energetics. Bi-allelic mutations in the LONP1 gene are known to cause a broad spectrum of diseases, and LonP1 dysregulation is also implicated in cancer and age-related disorders. Despite the importance of LonP1 in health and disease, specific inhibitors of this protease are unknown. Here, we demonstrate that 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its -methyl and -imidazole derivatives reversibly inhibit LonP1 by a non-competitive mechanism, blocking ATP-hydrolysis and thus proteolysis. By contrast, we found that CDDO-anhydride inhibits the LonP1 ATPase competitively. Docking of CDDO derivatives in the cryo-EM structure of LonP1 shows these compounds bind a hydrophobic pocket adjacent to the ATP-binding site. The binding site of CDDO derivatives was validated by amino acid substitutions that increased LonP1 inhibition, and also by a pathogenic mutation that causes cerebral, ocular, dental, auricular and skeletal (CODAS) syndrome, which ablated inhibition. CDDO failed to inhibit the ATPase activity of the purified 26S proteasome, which like LonP1 belongs to the AAA⁺ superfamily of ATPases Associated with diverse cellular Activities, suggesting that CDDO shows selectivity within this family of ATPases. Furthermore, we show that non-cytotoxic concentrations of CDDO derivatives in cultured cells inhibited LonP1, but not the 26S proteasome. Taken together, these findings provide insights for future development of LonP1-specific inhibitors with chemotherapeutic potential.
