Mitochondrial Protease Research Articles

Readdressing the Localization of Apolipoprotein E (APOE) in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes (MAMs): An Investigation of the Hepatic Protein-Protein Interactions of APOE with the Mitochondrial Proteins Lon Protease (LONP1), Mitochondrial Import Receptor Subunit TOM40 (TOMM40) and Voltage-Dependent Anion-Selective Channel 1 (VDAC1).

As a component of circulating lipoproteins, APOE binds to cell surface receptors mediating lipoprotein metabolism and cholesterol transport. A growing body of evidence, including the identification of a broad variety of cellular proteins interacting with APOE, suggests additional independent functions. Investigating cellular localization and protein-protein interactions in cultured human hepatocytes, we aimed to contribute to the elucidation of hitherto unnoted cellular functions of APOE. We observed a strong accumulation of APOE in MAMs, equally evident for the two major isoforms APOE3 and APOE4. Using mass spectrometry proteome analyses, novel and previously noted APOE interactors were identified, including the mitochondrial proteins TOMM40, LONP1 and VDAC1. All three interactors were present in MAM fractions, which we think initially facilitates interactions with APOE. LONP1 is a protease with chaperone activity, which migrated to MAMs in response to ER stress, displaying a reinforced interaction with APOE. We therefore hypothesize that APOE may help in the unfolded protein response (UPR) by acting as a co-chaperone in cooperation with LONP1 at the interface of mitochondria and ER membranes. The interaction of APOE with the integral proteins TOMM40 and VDAC1 may point to the formation of bridging complexes connecting mitochondria with other organelles.

Abstract Tu118: The Mitochondrial LonP1 Is Indispensable For Cardiac Maturation And Function

Background: LonP1 is a major mitochondrial ATP-dependent protease critical for mitochondrial protein quality control, However its role in cardiac function is unclear. Hypothesis: We hypothesize that LonP1 is essential for postnatal cardiac maturation and function. Methods: We employed cardiac-specific LonP1 knock-out (LonP1cKO) and Cre-control mice, assessing their heart histology (HE, trichrome), cardiac function via echocardiography, mitochondrial function by measuring oxygen consumption rate (OCR), mitochondrial complexes (I-V) activity by (ETC complex maximal activity), Iron-Sulfur (Fe-S) clusters and free radical levels by electron paramagnetic resonance (EPR) spectroscopy. The significant difference between the groups was established at p<0.05 using the Student's 't' test. Results: We found that LonP1cKO mice were not embryonically lethal; however, they exhibited an early mortality rate, died at an average of 21 days after birth with significantly lower body weight (7.02±0.16 gm vs. control (11.41±0.25 gm), p<0.0001, n=24) and increased heart weight to the tibia length ratio (0.080±0.002 gm/cm vs. control (0.047±0.0014 gm/cm), p<0.001, n=24). Echocardiography analysis showed that LonP1ckO mice had severely decreased left ventricular ejection fraction (%EF) (14.06±1.82% vs. control (70±2.9%), p<0.0001, n=16) and fractional shortening (% FS) (6.05±0.8% vs. control (39.15±2.5%), p<0.0001, n=16). Histological analysis revealed severe ventricular dilation and increased cardiac fibrosis. Transmission electron microscope analysis showed a significant loss of cristae and accumulation of electron-dense aggregation within mitochondria of LonP1cKO but not in control mice. In addition, LonP1cKO hearts showed reduced Fe-S clusters level (10.70±0.85 a.u vs. 21.27±1.91, p<0.0001, n=6), complex I-V activities (p<0.001), and electron flow (152.2±4.04 nm/min/mg vs. 265.3±23.8, p<0.001, n=6) compared to control. Conclusion: Our findings indicate that the deletion of LonP1 makes the heart unable to transition from glycolysis to substrate oxidation during the early postnatal maturation process, leading to dilated cardiomyopathy and death. This highlights the pivotal role of LonP1 in both cardiac maturation and function.

Abstract Tu044: Cardiac Mitochondrial Dysfunction Induces Region-Specific Mitochondrial Stress Response In The Brain To Adapt Neuronal Changes

Background: The hippocampus and cortex are susceptible to changes in blood supply, metabolites, and oxygenation. However, how disrupted cardiac function affects these critical areas of the brain, leading to the cognitive and neurological consequences of heart failure, remains unclear. Hypothesis: We hypothesize that disrupted cardiac function cross-talks with the brain by inducing region-specific mitochondrial stress responses, leading to adaptive changes. Methods: We utilized cardiac-specific LonP1 knockout (LonP1cKO) mice to induce heart failure within 21 days of birth. Alongside cre- control mice, we assessed cerebral blood flow using Laser speckle contrast imaging, locomotor activity through a comprehensive laboratory animal monitoring system (CLAMS), and gene expression profiles in the hippocampus and cortex by RT-PCR. Student’s ‘t’ test determined statistical significance with a p-value<0.05. Results: At 21 days, LonP1cKO mice demonstrated a significant (p<0.0001) reduction in cerebral blood flow (587.1±33.68, n=7) compared to the control (1034±31.26, n=5). Additionally, there were notable decreases in both X and Y-axis ambulatory and total locomotor activities in LonP1cKO mice [XAMB (100.3±6.6 vs 224±13.6 n=16), YAMB) (67.9±4.7 vs 150±8.5 n=16), XTOT (351.8±18.1 vs 534.4±26.3 n=16) and YTOT (190.7±9.6 vs 354.9±17.07 n=16). RT-PCR analysis revealed significant upregulation of genes related to hypoxia and cellular stress (Hif1a, FGf2, Atf6), mitochondrial biogenesis (mtDNA, Tfam, mt-COI, mt-COII), mitochondrial stress response proteases (LonP1, Afg3l2, Spg7, Yme1l1), and downregulation in neurotrophic factors (Bdnf, Ngf, Gfap) in the hippocampus and cortex. Conclusion: The study highlights that cardiac dysfunction in LonP1cKO mice leads to mitochondrial and cellular stress response in the brain to adapt neuronal changes, suggesting a critical link between cardiac health and brain function regulated through mitochondrial stress response pathways.

Context-dependent roles of mitochondrial LONP1 in orchestrating the balance between airway progenitor versus progeny cells

While all eukaryotic cells are dependent on mitochondria for function, in a complex tissue, which cell type and which cell behavior are more sensitive to mitochondrial deficiency remain unpredictable. Here, we show that in the mouse airway, compromising mitochondrial function by inactivating mitochondrial protease gene Lonp1 led to reduced progenitor proliferation and differentiation during development, apoptosis of terminally differentiated ciliated cells and their replacement by basal progenitors and goblet cells during homeostasis, and failed airway progenitor migration into damaged alveoli following influenza infection. ATF4 and the integrated stress response (ISR) pathway are elevated and responsible for the airway phenotypes. Such context-dependent sensitivities are predicted by the selective expression of Bok, which is required for ISR activation. Reduced LONP1 expression is found in chronic obstructive pulmonary disease (COPD) airways with squamous metaplasia. These findings illustrate a cellular energy landscape whereby compromised mitochondrial function could favor the emergence of pathological cell types.

An adaptor for feedback regulation of heme biosynthesis by the mitochondrial protease CLPXP.

Heme biosynthesis is tightly coordinated such that essential heme functions including oxygen transport, respiration, and catalysis are fully supplied without overproducing toxic heme precursors and depleting cellular iron. The initial heme biosynthetic enzyme, ALA synthase (ALAS), exhibits heme-induced degradation that is dependent on the mitochondrial AAA+ protease complex CLPXP, but the mechanism for this negative feedback regulation had not been elucidated. By biochemical reconstitution, we have discovered that POLDIP2 serves as a heme-sensing adaptor protein to deliver ALAS for degradation. Similarly, loss of POLDIP2 strongly impairs ALAS turnover in cells. POLDIP2 directly recognizes heme-bound ALAS to drive assembly of the degradation complex. The C-terminal element of ALAS, truncation of which leads to a form of porphyria (XLDPP), is dispensable for interaction with POLDIP2 but necessary for degradation. Our findings establish the molecular basis for heme-induced degradation of ALAS by CLPXP, establish POLDIP2 as a substrate adaptor for CLPXP, and provide mechanistic insight into two forms of erythropoietic protoporphyria linked to CLPX and ALAS.

Differential physical interactions of wild-type and S59L mutant CHCHD10 with mitochondrial proteases

Background CHCHD10 is a small mitochondrial protein with a C-terminal coiled-coil-helix–coiled-coil-helix domain. The N-terminal region of CHCHD10 is mostly intrinsically disordered. Therefore, CHCHD10 has no catalytic activity other than protein-protein interactions through the CHCH domain or intrinsically disordered region. The S59L mutation in CHCHD10 has been identified as a genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. However, the disease-causing mechanisms of CHCHD10S59L are not yet fully understood. Our previous study showed that CHCHD10S59L induces PINK1 and Parkin accumulation. PINK1 stabilization in the mitochondria is dependent on proteolytic processing by mitochondrial proteases. It has also been reported that mutant CHCHD10 disrupts protein homeostasis within mitochondria. Therefore, we hypothesized that CHCHD10S59L physically interacts with mitochondrial proteases during the disease pathogenesis. Methods To test this hypothesis, we performed co-immunoprecipitation using transiently transfected wild-type and mutant S59L CHCHD10 in HeLa cells. We employed a dithiobis (succinimidyl propionate) cross-linker to detect transient physical interactions. Results We expected that CHCHD10S59L may activate mitochondrial protease processing or expression and that it interacts with more diverse mitochondrial proteases than the wild type. In general, however, there was no difference in the expression and processing levels of mitochondrial proteases and the proteases that interact with wild-type and CHCHD10S59L. LonP1 degrading unfolded proteins in the mitochondrial matrix was the major proteases that can bind to wild-type CHCHD10 and CHCHD10S59L. LonP1 and HtrA2 were significantly less strongly bound to CHCHD10S59L. Conclusions This result can be interpreted in many different ways, including the regulatory role of CHCHD10 in mitochondrial proteases. However, more precise biochemical and cell biological investigations are required to determine the role of CHCHD10 in the activity of mitochondrial proteases, mechanism of PINK1 stabilization, and disruption of mitochondrial proteostasis.

Enhancing mitochondrial proteolysis alleviates alpha-synuclein-mediated cellular toxicity

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized by mitochondrial dysfunction and accumulation of alpha-synuclein (α-Syn)-containing protein aggregates known as Lewy bodies (LB). Here, we investigated the entry of α-Syn into mitochondria to cause mitochondrial dysfunction and loss of cellular fitness in vivo. We show that α-Syn expressed in yeast and human cells is constitutively imported into mitochondria. In a transgenic mouse model, the level of endogenous α-Syn accumulation in mitochondria of dopaminergic neurons and microglia increases with age. The imported α-Syn is degraded by conserved mitochondrial proteases, most notably NLN and PITRM1 (Prd1 and Cym1 in yeast, respectively). α-Syn in the mitochondrial matrix that is not degraded interacts with respiratory chain complexes, leading to loss of mitochondrial DNA (mtDNA), mitochondrial membrane potential and cellular fitness decline. Importantly, enhancing mitochondrial proteolysis by increasing levels of specific proteases alleviated these defects in yeast, human cells, and a PD model of mouse primary neurons. Together, our results provide a direct link between α-synuclein-mediated cellular toxicity and its import into mitochondria and reveal potential therapeutic targets for the treatment of α-synucleinopathies.

DIPG-88. TARGETING THE ANTIOXIDANT RESPONSE ELEMENT AXIS SENSITIZES DIFFUSE MIDLINE GLIOMA CELLS TO ONC201

Abstract BACKGROUND Diffuse midline glioma (DMG) is a universally fatal CNS tumor, with a median overall survival of <12-months. Recent studies, including our own, have identified overexpression of the mitochondrial protease ClpP in DMG patient samples. ClpP plays a pivotal role in regulating energy production. Brain-penetrant ClpP agonists, including ONC201, ONC206, and TR107, induce non-specific degradation of the mitochondrial electron transport chain. However, there is considerable variability in the response of DMG cell lines to these therapies, with only about half reaching IC50. Notably, TP53-mutant cell lines exhibit reduced sensitivity. METHODS/RESULTS Further analysis using quantitative proteomics of TP53-mutant DMG cell lines exhibited hallmark activation of ClpP, increased mitochondrial stress and dysfunction. Importantly, ClpP agonism increased the activity of the transcription factor NRF2, driving expression of NQO1 (p=0.0017). NQO1 is a cytoprotective enzyme, promoting redox balance and cell survival. This highlights a potential adaptive response to ClpP agonist therapy in DMG cells. Analysis of DMG patient samples through publicly available RNA sequencing data identified ubiquitously high NRF2 expression compared to other pediatric CNS tumors. This led us to hypothesize that a clinically relevant, selective NQO1 inhibitor could elicit a strong response to ClpP agonism. Therefore, we examined ARQ761, a CNS-active therapy that disrupts NQO1 leading to the formation of an unstable hydroquinone, causing oxidative DNA damage and apoptosis. When ARQ761 was used concurrently with a ClpP agonist, the combination showed additive effects, average Bliss Score of 9.91 (n=4). Interestingly, administering ARQ761 24 h after ClpP agonist exposure combined synergistically, average Bliss Score of 15.87 (n=4). This synergy was further potentiated using ARQ761 48 h after ClpP agonism, when NQO1 expression peaked, resulting in an average Bliss Score of 20.30 (n=4). CONCLUSIONS This study has unveiled an intrinsic antioxidant defense mechanism in DMG, which facilitates evasion from treatments inducing oxidative stress. This defense mechanism appears particularly robust in cell lines harboring TP53-mutations, which are also known to exhibit resistance to radiotherapy.

Structural basis of substrate recognition and allosteric activation of the proapoptotic mitochondrial HtrA2 protease

The mitochondrial serine protease HtrA2 is a human homolog of the Escherichia coli Deg-proteins exhibiting chaperone and proteolytic roles. HtrA2 is involved in both apoptotic regulation via its ability to degrade inhibitor-of-apoptosis proteins (IAPs), as well as in cellular maintenance as part of the cellular protein quality control machinery, by preventing the possible toxic accumulation of aggregated proteins. In this study, we use advanced solution NMR spectroscopy methods combined with biophysical characterization and biochemical assays to elucidate the crucial role of the substrate recognizing PDZ domain. This domain regulates the protease activity of HtrA2 by triggering an intricate allosteric network involving the regulatory loops of the protease domain. We further show that divalent metal ions can both positively and negatively modulate the activity of HtrA2, leading to a refined model of HtrA2 regulation within the apoptotic pathway.

Mitochondrial Protease Oct1p Regulates Mitochondrial Homeostasis and Influences Pathogenicity through Affecting Hyphal Growth and Biofilm Formation Activities in Candida albicans.

Mitochondria, as the core metabolic organelles, play a crucial role in aerobic respiration/biosynthesis in fungi. Numerous studies have demonstrated a close relationship between mitochondria and Candida albicans virulence and drug resistance. Here, we report an octapeptide-aminopeptidase located in the mitochondrial matrix named Oct1p. Its homolog in the model fungus Saccharomyces cerevisiae is one of the key proteins in maintaining mitochondrial respiration and protein stability. In this study, we utilized evolutionary tree analysis, gene knockout experiments, mitochondrial function detection, and other methods to demonstrate the impact of Oct1p on the mitochondrial function of C. albicans. Furthermore, through transcriptome analysis, real-time quantitative PCR, and morphological observation, we discovered that the absence of Oct1p results in functional abnormalities in C. albicans, affecting hyphal growth, cell adhesion, and biofilm formation. Finally, the in vivo results of the infection of Galleria mellonella larvae and vulvovaginal candidiasis in mice indicate that the loss of Oct1p led to the decreased virulence of C. albicans. In conclusion, this study provides a solid theoretical foundation for treating Candida diseases, developing new targeted drugs, and serves as a valuable reference for investigating the connection between mitochondria and virulence in other pathogenic fungi.

Knockout Mouse Studies Show That Mitochondrial CLPP Peptidase and CLPX Unfoldase Act in Matrix Condensates near IMM, as Fast Stress Response in Protein Assemblies for Transcript Processing, Translation, and Heme Production.

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.

The mitochondrial protease ClpP is a druggable target that controls VSMC phenotype by a SIRT1-dependent mechanism

Vascular smooth muscle cells (VSMCs), known for their remarkable lifelong phenotypic plasticity, play a pivotal role in vascular pathologies through their ability to transition between different phenotypes. Our group discovered that the deficiency of the mitochondrial protein Poldip2 induces VSMC differentiation both in vivo and in vitro. Further comprehensive biochemical investigations revealed Poldip2's specific interaction with the mitochondrial ATPase caseinolytic protease chaperone subunit X (CLPX), which is the regulatory subunit for the caseinolytic protease proteolytic subunit (ClpP) that forms part of the ClpXP complex – a proteasome-like protease evolutionarily conserved from bacteria to humans. This interaction limits the protease's activity, and reduced Poldip2 levels lead to ClpXP complex activation. This finding prompted the hypothesis that ClpXP complex activity within the mitochondria may regulate the VSMC phenotype.Employing gain-of-function and loss-of-function strategies, we demonstrated that ClpXP activity significantly influences the VSMC phenotype. Notably, both genetic and pharmacological activation of ClpXP inhibits VSMC plasticity and fosters a quiescent, differentiated, and anti-inflammatory VSMC phenotype. The pharmacological activation of ClpP using TIC10, currently in phase III clinical trials for cancer, successfully replicates this phenotype both in vitro and in vivo and markedly reduces aneurysm development in a mouse model of elastase-induced aortic aneurysms. Our mechanistic exploration indicates that ClpP activation regulates the VSMC phenotype by modifying the cellular NAD+/NADH ratio and activating Sirtuin 1. Our findings reveal the crucial role of mitochondrial proteostasis in the regulation of the VSMC phenotype and propose the ClpP protease as a novel, actionable target for manipulating the VSMC phenotype.

Lon1 Inactivation Downregulates Autophagic Flux and Brassinosteroid Biogenesis, Modulating Mitochondrial Proportion and Seed Development in Arabidopsis.

Mitochondrial protein homeostasis is crucially regulated by protein degradation processes involving both mitochondrial proteases and cytosolic autophagy. However, it remains unclear how plant cells regulate autophagy in the scenario of lacking a major mitochondrial Lon1 protease. In this study, we observed a notable downregulation of core autophagy proteins in Arabidopsis Lon1 knockout mutant lon1-1 and lon1-2, supporting the alterations in the relative proportions of mitochondrial and vacuolar proteins over total proteins in the plant cells. To delve deeper into understanding the roles of the mitochondrial protease Lon1 and autophagy in maintaining mitochondrial protein homeostasis and plant development, we generated the lon1-2atg5-1 double mutant by incorporating the loss-of-function mutation of the autophagy core protein ATG5, known as atg5-1. The double mutant exhibited a blend of phenotypes, characterized by short plants and early senescence, mirroring those observed in the individual single mutants. Accordingly, distinct transcriptome alterations were evident in each of the single mutants, while the double mutant displayed a unique amalgamation of transcriptional responses. Heightened severity, particularly evident in reduced seed numbers and abnormal embryo development, was observed in the double mutant. Notably, aberrations in protein storage vacuoles (PSVs) and oil bodies were evident in the single and double mutants. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of genes concurrently downregulated in lon1-2, atg5-1, and lon1-2atg5-1 unveiled a significant suppression of genes associated with brassinosteroid (BR) biosynthesis and homeostasis. This downregulation likely contributes to the observed abnormalities in seed and embryo development in the mutants.

Abstract PO1-29-01: Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory triple negative breast cancer

Abstract Breast cancer remains a major global health concern, with triple-negative breast cancer (TNBC) being one of the most aggressive subtypes associated with limited targeted therapies. Chemotherapy resistance in TNBC patients poses a significant clinical challenge and is associated with poor progression-free and overall survival. Thus, exploring novel approaches to improve chemotherapeutic efficacy is of paramount importance. We previously demonstrated a role for mitochondrial fusion in supporting oxidative phosphorylation (oxphos) activity and cell survival in residual TNBC (PMID: 30996079 and 36813854) cells refractory to chemotherapy treatments. Based on these observations, we sought to inhibit mitochondrial function to improve chemotherapeutic efficacy. We used ONC206, a novel agonist of mitochondrial serine protease, ClpP (Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit) to induce mitochondrial proteotoxic stress. ONC201, a parent molecule of ONC206, has shown efficacy in TNBC as a single agent or when combined with a MEK inhibitor in vitro (PMID: 34680527). We demonstrate treatment of TNBC cell lines with ONC206 results in reduced mitochondrial fusion and OXPHOS. Furthermore, ONC206 co-treatment enhanced chemo-sensitivity in vitro. Based on these promising findings, we tested this in orthotopic patient-derived xenograft (PDX) mouse preclinical trials. Three PDX models of TNBC were tested, BCM-2665, BCM-5471, and BCM-0002. ONC206 as a single agent (twice weekly for 4 weeks, oral gavage, 100mg/kg) significantly decreased tumor growth in all three PDX models. Remarkably, treatment ONC206 achieved nearly complete tumor regression as a single agent or when combined with carboplatin in BCM-2665. Regressed tumors did re-grow, but this was significantly delayed when combined with carboplatin. Treatment with ONC206 in BCM-5471 resulted in a modest but significant reduction in tumor growth rate as a single agent. However, ONC206 did not improve carboplatin (weekly, i.p., 50mg/kg) nor docetaxel (weekly, i.p., 20mg/kg) efficacy in this model. ONC206 treatment of BCM-0002 resulted in prolonged tumor stasis and a slight enhancement of docetaxel and carboplatin efficacy. Interestingly, the efficacy of ONC206 in these PDX models correlates with their relative mRNA expression of ClpP. The mice demonstrated tolerance to ONC206 as a single agent or when combined with chemotherapy during the course of treatment. Our study underscores the potentially crucial role of mitochondria in driving chemotherapy resistance in TNBC and provides novel insights into mitochondria-targeted therapeutic approaches. We report a novel targeting option for TNBC and anticipate that these findings will stimulate further research into innovative combination therapies for TNBC patients, and may lead us to a viable inhibitor. Citation Format: Lily Baek, Lacey Dobrolecki, Stefanie Faucher, Christina Sallas, Nia Griffith, Varun Prabhu, Bora Lim, Michael Lewis, Gloria Echeverria. Single agent and combinatorial efficacy with imipridone ONC206 via inhibition of mitochondrial function in preclinical models of chemorefractory triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-29-01.

Polyphosphate and tyrosine phosphorylation in the N-terminal domain of the human mitochondrial Lon protease disrupts its functions.

Phosphorylation plays a crucial role in the regulation of many fundamental cellular processes. Phosphorylation levels are increased in many cancer cells where they may promote changes in mitochondrial homeostasis. Proteomic studies on various types of cancer identified 17 phosphorylation sites within the human ATP-dependent protease Lon, which degrades misfolded, unassembled and oxidatively damaged proteins in mitochondria. Most of these sites were found in Lon's N-terminal (NTD) and ATPase domains, though little is known about the effects on their function. By combining the biochemical and cryo-electron microscopy studies, we show the effect of Tyr186 and Tyr394 phosphorylations in Lon's NTD, which greatly reduce all Lon activities without affecting its ability to bind substrates or perturbing its tertiary structure. A substantial reduction in Lon's activities is also observed in the presence of polyphosphate, whose amount significantly increases in cancer cells. Our study thus provides an insight into the possible fine-tuning of Lon activities in human diseases, which highlights Lon's importance in maintaining proteostasis in mitochondria.

ANKZF1 knockdown inhibits glioblastoma progression by promoting intramitochondrial protein aggregation through mitoRQC

Protein homeostasis is fundamental to the development of tumors. Ribosome-associated quality-control (RQC) is able to add alanine and threonine to the stagnant polypeptide chain C-terminal (CAT-tail) when protein translation is hindered, while Ankyrin repeat and zinc-finger domain-containing-protein 1 (ANKZF1) can counteract the formation of the CAT-tail, preventing the aggregation of polypeptide chains. In particular, ANKZF1 plays an important role in maintaining mitochondrial protein homeostasis by mitochondrial RQC (mitoRQC) after translation stagnation of precursor proteins targeting mitochondria. However, the role of ANKZF1 in glioblastoma is unclear. Therefore, the current study was aimed to investigate the effects of ANKZF1 in glioblastoma cells and a nude mouse glioblastoma xenograft model. Here, we reported that knockdown of ANKZF1 in glioblastoma cells resulted in the accumulation of CAT-tail in mitochondria, leading to the activated mitochondrial unfolded protein response (UPRmt) and inhibits glioblastoma malignant progression. Excessive CAT-tail sequestered mitochondrial chaperones HSP60, mtHSP70 and proteases LONP1 as well as mitochondrial respiratory chain subunits ND1, Cytb, mtCO2 and ATP6, leading to mitochondrial oxidative phosphorylation dysfunction, membrane potential impairment, and mitochondrial apoptotic pathway activation. Our study highlights ANKZF1 as a valuable target for glioblastoma intervention and provides an innovative insight for the treatment of glioblastoma through the regulating of mitochondrial protein homeostasis.

Engineered Exosomes with ATF5-Modified mRNA Loaded in Injectable Thermogels Alleviate Osteoarthritis by Targeting the Mitochondrial Unfolded Protein Response.

Osteoarthritis (OA) progression is highly associated with chondrocyte mitochondrial dysfunction and disorders of catabolism and anabolism of the extracellular matrix (ECM) in the articular cartilage. The mitochondrial unfolded protein response (UPRmt), which is an integral component of the mitochondrial quality control (MQC) system, is essential for maintaining chondrocyte homeostasis. We successfully validated the pivotal role of activating transcription factor 5 (ATF5) in upregulating the UPRmt, mitigating IL-1β-induced inflammation and mitochondrial dysfunction, and promoting balanced metabolism in articular cartilage ECM, proving its potential as a promising therapeutic target for OA. Modified mRNAs (modRNAs) have emerged as novel and efficient gene delivery vectors for nucleic acid therapeutic approaches. In this study, we combined Atf5-modRNA (modAtf5) with engineered exosomes derived from bone mesenchymal stem cells (ExmodAtf5) to exert cytoprotective effects on chondrocytes in articular cartilage via Atf5. However, the rapid localized metabolization of ExmodAtf5 limits its application. PLGA-PEG-PLGA (Gel), an injectable thermosensitive hydrogel, was used as a carrier of ExmodAtf5 (Gel@ExmodAtf5) to achieve a sustained release of ExmodAtf5. In vitro and in vivo, the use of Gel@ExmodAtf5 was shown to be a highly effective strategy for OA treatment. The in vivo therapeutic effect of Gel@ExmodAtf5 was evidenced by the preservation of the intact cartilage surface, low OARSI scores, fewer osteophytes, and mild subchondral bone sclerosis and cystic degeneration. Consequently, the combination of ExmodAtf5 and PLGA-PEG-PLGA could significantly enhance the therapeutic efficacy and prolong the exosome release. In addition, the mitochondrial protease ClpP enhanced chondrocyte autophagy by modulating the mTOR/Ulk1 pathway. As a result of our research, Gel@ExmodAtf5 can be considered to be effective at alleviating the progression of OA.

Cytosolic retention of HtrA2 during mitochondrial protein import stress triggers the DELE1-HRI pathway

Mitochondrial stress inducers such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and oligomycin trigger the DELE1-HRI branch of the integrated stress response (ISR) pathway. Previous studies performed using epitope-tagged DELE1 showed that these stresses induced the cleavage of DELE1 to DELE1-S, which stimulates HRI. Here, we report that mitochondrial protein import stress (MPIS) is an overarching stress that triggers the DELE1-HRI pathway, and that endogenous DELE1 could be cleaved into two forms, DELE1-S and DELE1-VS, the latter accumulating only upon non-depolarizing MPIS. Surprisingly, while the mitochondrial protease OMA1 was crucial for DELE1 cleavage in HeLa cells, it was dispensable in HEK293T cells, suggesting that multiple proteases may be involved in DELE1 cleavage. In support, we identified a role for the mitochondrial protease, HtrA2, in mediating DELE1 cleavage into DELE1-VS, and showed that a Parkinson’s disease (PD)-associated HtrA2 mutant displayed reduced DELE1 processing ability, suggesting a novel mechanism linking PD pathogenesis to mitochondrial stress. Our data further suggest that DELE1 is likely cleaved into DELE1-S in the cytosol, while the DELE1-VS form might be generated during halted translocation into mitochondria. Together, this study identifies MPIS as the overarching stress detected by DELE1 and identifies a novel role for HtrA2 in DELE1 processing.

OMA1 protease eliminates arrested protein import intermediates upon mitochondrial depolarization.

Most mitochondrial proteins originate from the cytosol and require transport into the organelle. Such precursor proteins must be unfolded to pass through translocation channels in mitochondrial membranes. Misfolding of transported proteins can result in their arrest and translocation failure. Arrested proteins block further import, disturbing mitochondrial functions and cellular proteostasis. Cellular responses to translocation failure have been defined in yeast. We developed the cell line-based translocase clogging model to discover molecular mechanisms that resolve failed import events in humans. The mechanism we uncover differs significantly from these described in fungi, where ATPase-driven extraction of blocked protein is directly coupled with proteasomal processing. We found human cells to rely primarily on mitochondrial factors to clear translocation channel blockage. The mitochondrial membrane depolarization triggered proteolytic cleavage of the stalled protein, which involved mitochondrial protease OMA1. The cleavage allowed releasing the protein fragment that blocked the translocase. The released fragment was further cleared in the cytosol by VCP/p97 and the proteasome.

Abstract 7053: Serine phosphorylation marks proteins for degradation by the mitochondrial matrix protease, ClpXP

Abstract ClpXP is a conserved protein degradation system. In human mitochondria, ClpXP degrades damaged respiratory chain proteins and is vital for the survival of acute myeloid leukemia (AML) cells. This system consists of the barrel-like protease ClpP, capped by regulatory particles (RP) ClpX. In bacteria, ClpXP targets substrates with a co-translationally added SsrA sequence. However, in humans, the SsrA tag is absent, and the degradation markers for ClpXP remain unidentified. Notably, bacterial ClpXP homologues degrade substrates with phosphorylated arginine (pArg). We hypothesized that phosphorylated amino acids also mark substrates for ClpXP degradation in human mitochondria. We showed that ClpXP preferentially degraded phosphorylated α-casein substrate over dephosphorylated α-casein. In contrast, ClpP activated by ONC201, without ClpX, degraded phosphorylated and dephosphorylated α-casein with equal efficiency. Next, we screened a panel of phosphorylated amino acids and peptides for their ability to inhibit ClpXP-mediated degradation of α-casein. Phosphorylated serine (pSer) amino acids and peptides inhibited ClpXP protease activity, while phosphorylated tyrosine (pTyr) or pArg did not. Likewise, Ser and free phosphate did not inhibit ClpXP protease activity. Thermal shift assays showed that pSer amino acids and peptides bind to ClpX, not ClpP. Hydrogen/deuterium exchange mass spectrometry revealed that the addition of Ala-pSer-Ala (ApSA) peptide, but not ASA peptide induced conformational changes in the negatively-charged N terminus of ClpX, thus revealing a putative binding site for ApSA. Earlier, we established that ClpP interacts with the respiratory chain complex II subunit SDHA. Knocking down of ClpP in AML cells affected the respiratory chain and increased reactive oxygen species. Therefore, we tested how ClpXP knockdown impacts levels of phospho-serine SDHA (pSer-SDHA) in intact cells. Using shRNA, we knocked down ClpP and ClpX individually in OCI-AML2 cells. After target knockdown, we pulled down pSer proteins and probed for SDHA. Knockdown of both ClpP and ClpX increased the abundance of pSer-SDHA compared to control. We subsequently added recombinant ClpXP protein to total mitochondrial lysates and the pSer immunoprecipitated fraction from OCI-AML2 cells. This led to decreased levels of pSer-SDHA but did not alter levels of total SDHA, consistent with its preference for pSer-marked substrates suggesting that ClpXP degrades pSer-SDHA. Further analysis showed elevated pSer-SDHA levels only in the insoluble mitochondrial protein fraction after ClpX knockdown, suggesting these might be damaged proteins. In conclusion, our findings demonstrate that serine phosphorylation promotes the degradation of damaged mitochondrial proteins by ClpXP protease. Citation Format: Yue Feng, Monica M. Goncalves, Yulia Jitkova, Alexander Keszei, Chaitra Sarathy, Vincent Trudel, Jonathan St-Germain, Matthew Tcheng, Yongran Yan, Rose Hurren, Matthew Schultz, Brian Raught, Andrei Yudin, Mohammad Mazhab-Jafari, Siavash Vahidi, Aaron D. Schimmer. Serine phosphorylation marks proteins for degradation by the mitochondrial matrix protease, ClpXP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7053.