Mitochondrial ATP-dependent Protease Research Articles

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.

The mitochondrial protease LONP1 maintains oocyte development and survival by suppressing nuclear translocation of AIFM1 in mammals.

SummaryBackgroundOogenesis is a fundamental process of human reproduction, and mitochondria play crucial roles in oocyte competence. Mitochondrial ATP-dependent Lon protease 1 (LONP1) functions as a critical protein in maintaining mitochondrial and cellular homeostasis in somatic cells. However, the essential role of LONP1 in maintaining mammalian oogenesis is far from elucidated.MethodsUsing conditional oocyte Lonp1-knockout mice, RNA sequencing (RNA-seq) and coimmunoprecipitation/liquid chromatography–mass spectrometry (Co-IP/LC–MS) technology, we analysed the functions of LONP1 in mammalian oogenesis.FindingsConditional knockout of Lonp1 in mouse oocytes in both the primordial and growing follicle stages impairs follicular development and causes progressive oocyte death, ovarian reserve loss, and infertility. LONP1 directly interacts with apoptosis inducing factor mitochondria-associated 1 (AIFM1), and LONP1 ablation leads to the translocation of AIFM1 from the cytoplasm to the nucleus, causing apoptosis in mouse oocytes. In addition, women with pathogenic variants of LONP1 lack large antral follicles (>10 mm) in the ovaries, are infertile and present premature ovarian insufficiency.InterpretationWe demonstrated the function of LONP1 in regulating oocyte development and survival, and in-depth analysis of LONP1 will be crucial for elucidating the mechanisms underlying premature ovarian insufficiency.FundingThis work was supported by grants from the National Key Research and Development Program of China (2018YFC1004701), the National Nature Science Foundation of China (82001629, 81871128, 81571391, 81401166, 82030040), the Jiangsu Province Social Development Project (BE2018602), the Jiangsu Provincial Medical Youth Talent (QNRC2016006), the Youth Program of the Natural Science Foundation of Jiangsu Province (BK20200116) and Jiangsu Province Postdoctoral Research Funding (2021K277B).

Mitochondrial ATP-Dependent Proteases-Biological Function and Potential Anti-Cancer Targets.

Simple SummaryAlterations 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 therapeutic potential of targeting the matrix ATP-dependent protease ClpXP for a subset of malignancies. In this review, we summarize our current knowledge on the biological function and the anticancer effects of targeting ATP-dependent proteases with a focus on ClpXP.Cells must eliminate excess or damaged proteins to maintain protein homeostasis. To ensure protein homeostasis in the cytoplasm, cells rely on the ubiquitin-proteasome system and autophagy. In the mitochondria, protein homeostasis is regulated by mitochondria proteases, including four core ATP-dependent proteases, m-AAA, i-AAA, LonP, and ClpXP, located in the mitochondrial membrane and matrix. This review will discuss the function of mitochondrial proteases, with a focus on ClpXP as a novel therapeutic target for the treatment of malignancy. ClpXP maintains the integrity of the mitochondrial respiratory chain and regulates metabolism by degrading damaged and misfolded mitochondrial proteins. Inhibiting ClpXP genetically or chemically impairs oxidative phosphorylation and is toxic to malignant cells with high ClpXP expression. Likewise, hyperactivating the protease leads to increased degradation of ClpXP substrates and kills cancer cells. Thus, targeting ClpXP through inhibition or hyperactivation may be novel approaches for patients with malignancy.

The C3HC type zinc-finger protein (ZFC3) interacting with Lon/MAP1 is important for mitochondrial gene regulation, infection hypha development and longevity of Magnaporthe oryzae

BackgroundThe rice blast is a typical fungal disease caused by Magnaporthe oryzae, and the mitochondrial ATP-dependent Lon protease (MAP1) has been proven to be involved in blast development. We previously screened a C3HC type Zinc-finger domain protein (ZFC3), which is interacted with MAP1. The purpose of this research was to study the biological function of ZFC3 protein in M. oryzae.ResultsWe first confirmed that the ZFC3-RFP fusion protein is localized within the mitochondria. The deleted mutant strains of ZFC3 (∆ZFC3) showed the enhanced expression level of mtATP6, particularly mtATP8, and almost unchanged nATP9. ΔZFC3 produces more conidia and more tolerance to multiple stressors. The knock-out strain shows more melanin accumulation suggests the susceptibility to aging. ΔZFC3 displays faster early-stage hypha infiltration involved in MAP1-mediated pathogenicity in host rice.ConclusionThese results support the view that ZFC3 is a key regulator involved in gene regulation, stress response, cell wall integrity, longevity, conidiation, infection hypha development and MAP1-mediated pathogenicity in M. oryzae.

Mitochondrial LON protease-dependent degradation of cytochrome c oxidase subunits under hypoxia and myocardial ischemia

The mitochondrial ATP dependent matrix protease, Lon, is involved in the maintenance of mitochondrial DNA nucleoids and degradation of abnormal or misfolded proteins. The Lon protease regulates mitochondrial Tfam (mitochondrial transcription factor A) level and thus modulates mitochondrial DNA (mtDNA) content. We have previously shown that hypoxic stress induces the PKA-dependent phosphorylation of cytochrome c oxidase (CcO) subunits I, IVi1, and Vb and a time-dependent reduction of these subunits in RAW 264.7 murine macrophages subjected to hypoxia and rabbit hearts subjected to ischemia/reperfusion. Here, we show that Lon is involved in the preferential turnover of phosphorylated CcO subunits under hypoxic/ischemic stress. Induction of Lon protease occurs at 6 to 12 h of hypoxia and this increase coincides with lower CcO subunit contents. Over-expression of flag-tagged wild type and phosphorylation site mutant Vb and IVi1 subunits (S40A and T52A, respectively) caused marked degradation of wild type protein under hypoxia while the mutant proteins were relatively resistant. Furthermore, the recombinant purified Lon protease degraded the phosphorylated IVi1 and Vb subunits, while the phosphorylation-site mutant proteins were resistant to degradation. 3D structural modeling shows that the phosphorylation sites are exposed to the matrix compartment, accessible to matrix PKA and Lon protease. Hypoxic stress did not alter CcO subunit levels in Lon depleted cells, confirming its role in CcO turnover. Our results therefore suggest that Lon preferentially degrades the phosphorylated subunits of CcO and plays a role in the regulation of CcO activity in hypoxia and ischemia/reperfusion injury.

The Arabidopsis Mitochondrial Protease FtSH4 Is Involved in Leaf Senescence via Regulation of WRKY-Dependent Salicylic Acid Accumulation and Signaling.

Mitochondria and autophagy play important roles in the networks that regulate plant leaf senescence and cell death. However, the molecular mechanisms underlying the interactions between mitochondrial signaling and autophagy are currently not well understood. This study characterized the function of the Arabidopsis (Arabidopsis thaliana) mitochondrial AAA-protease gene FtSH4 in regulating autophagy and senescence, finding that FtSH4 mediates WRKY-dependent salicylic acid (SA) accumulation and signaling. Knockout of FtSH4 in the ftsh4-4 mutant resulted in severe leaf senescence, cell death, and high autophagy levels. The level of SA increased dramatically in the ftsh4-4 mutant. Expression of nahG in the ftsh4-4 mutant led to decreased SA levels and suppressed the leaf senescence and cell death phenotypes. The transcript levels of several SA synthesis and signaling genes, including SALICYLIC ACIDINDUCTION DEFICIENT2 (SID2), NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1), and NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1), increased significantly in the ftsh4-4 mutants compared with the wild type. Loss of function of SID2, NDR1, or NPR1 in the ftsh4-4 mutant reversed the ftsh4-4 senescence and autophagy phenotypes. Furthermore, ftsh4-4 mutants had elevated levels of transcripts of several WRKY genes, including WRKY40, WRKY46, WRKY51, WRKY60, WRKY63, and WRKY75; all of these WRKY proteins can bind to the promoter of SID2 Loss of function of WRKY75 in the ftsh4-4 mutants decreased the levels of SA and reversed the senescence phenotype. Taken together, these results suggest that the mitochondrial ATP-dependent protease FtSH4 may regulate the expression of WRKY genes by modifying the level of reactive oxygen species and the WRKY transcription factors that control SA synthesis and signaling in autophagy and senescence.

Mitochondrial Lon Protease and Cancer.

ATP-dependent Lon protease of mitochondrial matrix is encoded by nuclear DNA and highly evolutionarily conserved throughout all organisms, which is involved in the quality control of proteins by selective degradation of misfolded, oxidized, and short-lived regulatory proteins within mitochondrial matrix, maintenance of mitochondrial genome (mtDNA), and folding of mitochondria proteins. Various stimuli such as hypoxia and oxidative and ER stress lead to upregulation of Lon expression. Inhibition of protease activity or downregulation of Lon promotes cancer cell death and enhances sensitivity of cancer cells to anticancer drugs through metabolic reprogramming, thus reducing the viability of cancer cell in tumor microenvironment and epithelial to mesenchymal transition (EMT). Moreover, mitochondrial ATP-dependent Lon protease may serve as a potential biomarker for cancer diagnosis and novel target for the development of anticancer drugs and for predicting of the efficiency and effectiveness of chemotherapy of a variety of cancers.

Novel Mechanisms for Heme-dependent Degradation of ALAS1 Protein as a Component of Negative Feedback Regulation of Heme Biosynthesis

In eukaryotic cells, heme production is tightly controlled by heme itself through negative feedback-mediated regulation of nonspecific 5-aminolevulinate synthase (ALAS1), which is a rate-limiting enzyme for heme biosynthesis. However, the mechanism driving the heme-dependent degradation of the ALAS1 protein in mitochondria is largely unknown. In the current study, we provide evidence that the mitochondrial ATP-dependent protease ClpXP, which is a heteromultimer of CLPX and CLPP, is involved in the heme-dependent degradation of ALAS1 in mitochondria. We found that ALAS1 forms a complex with ClpXP in a heme-dependent manner and that siRNA-mediated suppression of either CLPX or CLPP expression induced ALAS1 accumulation in the HepG2 human hepatic cell line. We also found that a specific heme-binding motif on ALAS1, located at the N-terminal end of the mature protein, is required for the heme-dependent formation of this protein complex. Moreover, hemin-mediated oxidative modification of ALAS1 resulted in the recruitment of LONP1, another ATP-dependent protease in the mitochondrial matrix, into the ALAS1 protein complex. Notably, the heme-binding site in the N-terminal region of the mature ALAS1 protein is also necessary for the heme-dependent oxidation of ALAS1. These results suggest that ALAS1 undergoes a conformational change following the association of heme to the heme-binding motif on this protein. This change in the structure of ALAS1 may enhance the formation of complexes between ALAS1 and ATP-dependent proteases in the mitochondria, thereby accelerating the degradation of ALAS1 protein to maintain appropriate intracellular heme levels.

Emerging role of Lon protease as a master regulator of mitochondrial functions

Lon protease is a nuclear-encoded, mitochondrial ATP-dependent protease highly conserved throughout the evolution, crucial for the maintenance of mitochondrial homeostasis. Lon acts as a chaperone of misfolded proteins, and is necessary for maintaining mitochondrial DNA. The impairment of these functions has a deep impact on mitochondrial functionality and morphology. An altered expression of Lon leads to a profound reprogramming of cell metabolism, with a switch from respiration to glycolysis, which is often observed in cancer cells. Mutations of Lon, which likely impair its chaperone properties, are at the basis of a genetic inherited disease named of the cerebral, ocular, dental, auricular, skeletal (CODAS) syndrome. This article is part of a Special Issue entitled ‘EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2–6, 2016’, edited by Prof. Paolo Bernardi.

Proteins interacting with mitochondrial ATP-dependent Lon protease (MAP1) in Magnaporthe oryzae are involved in rice blast disease.

The ATP-dependent Lon protease is involved in many physiological processes. In bacteria, Lon regulates pathogenesis and, in yeast, Lon protects mitochondia from oxidative damage. However, little is known about Lon in fungal phytopathogens. MAP1, a homologue of Lon in Magnaporthe oryzae, was recently identified to be important for stress resistance and pathogenesis. Here, we focus on a novel pathogenic pathway mediated by MAP1. Based on an interaction system between rice and a tandem affinity purification (TAP)-tagged MAP1 complementation strain, we identified 23 novel fungal proteins from infected leaves using a TAP approach with mass spectrometry, and confirmed that 14 of these proteins physically interact with MAP1 in vivo. Among these 14 proteins, 11 candidates, presumably localized to the mitochondria, were biochemically determined to be substrates of MAP1 hydrolysis. Deletion mutants were created and functionally analysed to further confirm the involvement of these proteins in pathogenesis. The results indicated that all mutants showed reduced conidiation and sensitivity to hydrogen peroxide. Appressorial formations were not affected, although conidia from certain mutants were morphologically altered. In addition, virulence was reduced in four mutants, enhanced (with lesions forming earlier) in two mutants and remained unchanged in one mutant. Together with the known virulence-related proteins alternative oxidase and enoyl-CoA hydratase, we propose that most of the Lon-interacting proteins are involved in the pathogenic regulation pathway mediated by MAP1 in M. oryzae. Perturbation of this pathway may represent an effective approach for the inhibition of rice blast disease.

Targeting Lon Protease Activity Promotes Growth Inhibition and Apoptosis in Acute Myeloid Leukemia Cell Lines

INTRODUCTIONAcute myeloid leukemia (AML) is a progressive malignant disorder of the myeloid lineage of hematopoietic cells, where overall three-year survival is below 20% for patients above 65 years (1). Therapy resistance or relapsed are the main causes of mortality among these patients. The functional changes that affect mitochondria may contribute to the development of cancer (2). The mitochondrial Lon, ubiquitously expressed in human cells and tissues, seems to be involved in protein quality control and stress response pathways in mitochondria. Several stress conditions induce an upregulation of Lon that may promote tumorigenesis by inhibiting apoptosis and promoting cell proliferation, transformation and angiogenesis (3). The role of Lon in AML has not been described yet. Our study aims to investigate if the activity of the protease may be linked to AML leukemogenesis and drug resistance. In particular we tested Lon protease activity inhibitors to clarify a new pathogenetic mechanism in controlling drug responsiveness in this haematological disorder.METHODSAML cell lines Kasumi-1 (acute myeloblastic leukemia), MOLM-13 (acute monocytic leukemia) and HL-60 (acute promyelocytic leukemia) were used to perform the experiments. Lon expression was measured by RT-PCR and immunoblotting. Cell-growth was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Reactive oxygen species (ROS) were evaluated by Nitro Blue Tetrazolium (NBT) assay. For cell cycle analysis, cells were fixed with ethanol, resuspended in phosphate-buffered saline (PBS) containing 0.1mg/mL RNase A, NP40 0,15% and 100mg/mL of propidium iodide (PI) and analysed by using flow cytometry and Modfit software. The early and late apoptosis was assessed with Annexin V-FITC Apoptosis Detection kit. To measure the mitochondrial potential, JC-1 Mitochondrial Membrane Potential assay kit was used.RESULTSLon protease was expressed at high levels in AML cell lines compared with resting or activated peripheral blood mononuclear cells and monocytes, but less than other cancer cell lines such as K562 (human erythromyeloblastoid leukemia), HDLM-2 (Hodgkin lymphoma, nuclear sclerosing), SHSY5Y (neuroblastoma), MCF7 (breast carcinoma). Based on previous works (4), which show that 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivates inhibit the Lon protease activity, we evaluated the effect of two inhibitors on cell growth and the induction of apoptosis. The leukemic cell lines were treated with CDDO and CDDO-Me (CDDO methyl-ester) at different concentrations for 24, 48 and 72h. Both compounds inhibited the cell proliferation in a dose- and time-dependent manner and induced an increase of ROS. We observed a time-dependent increase in the percentage of Annexin V+ cells after treatment with CDDO (1,25µM) and CDDO-Me (0,25µM). The induction of apoptosis after CDDO exposure was important only at 72h, while we observed a percentage of apoptosis over 60% starting from 24h of CDDO-Me treatment. Interestingly, CDDO induced a time-dependent arrest in G2/M phase in MOLM-13 cells and in G0/G1 phase in Kasumi-1 and HL-60 cells. Instead the CDDO-Me induced an alteration of all cell cycle phases, probably for its cytotoxicity. Moreover CDDO-Me caused a higher decrease in mitochondrial potential (Dψm) than CDDO in all cell lines.CONCLUSIONIn conclusion, these findings show that Lon protease inhibitors promote growth inhibition and apoptosis and support the concept that mitochondrial Lon may represent a novel anticancer drug target.REFERENCES1. Burnett A, Wetzler M, Lowenberg B. Therapeutic advances in acute myeloid leukemia. J Clin Oncol. 2011, 29:487-494.2. Gogvadze V, Orrenius S, Zhivotovsky B. Mitochondria in cancer cells: what is so special about them? Trends Cell Biol. 2008, 18(4):165-73.3. Ngo JK, Pomatto LC, Davies KJ. Upregulation of the mitochondrial Lon Protease allows adaptation to acute oxidative stress but dysregulation is associated with chronic stress, disease, and aging. Redox Biol. 2013, 1(1):258-64.4. Bernstein SH, Venkatesh S, Li M, Lee J, Lu B, Hilchey SP, Morse KM, Metcalfe HM, Skalska J, Andreeff M, Brookes PS, Suzuki CK. The mitochondrial ATP-dependent Lon protease: a novel target in lymphoma death mediated by the synthetic triterpenoid CDDO and its derivatives. Blood. 2012, 119(14):3321-9. DisclosuresMartinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Bernstein SH, Venkatesh S, Li M, et al. The mitochondrial ATP-dependent Lon protease: a novel target in lymphoma death mediated by the synthetic triterpenoid CDDO and its derivatives. Blood. 2012;119(14):

Bernstein SH, Venkatesh S, Li M, et al. The mitochondrial ATP-dependent Lon protease: a novel target in lymphoma death mediated by the synthetic triterpenoid CDDO and its derivatives. Blood. 2012;119(14):

Perturbation of Auxin Homeostasis Caused by Mitochondrial FtSH4 Gene-Mediated Peroxidase Accumulation Regulates Arabidopsis Architecture

Reactive oxygen species and auxin play important roles in the networks that regulate plant development and morphogenetic changes. However, the molecular mechanisms underlying the interactions between them are poorly understood. This study isolated a mas (More Axillary Shoots) mutant, which was identified as an allele of the mitochondrial AAA-protease AtFtSH4, and characterized the function of the FtSH4 gene in regulating plant development by mediating the peroxidase-dependent interplay between hydrogen peroxide (H2O2) and auxin homeostasis. The phenotypes of dwarfism and increased axillary branches observed in the mas (renamed as ftsh4-4) mutant result from a decrease in the IAA concentration. The expression levels of several auxin signaling genes, including IAA1, IAA2, and IAA3, as well as several auxin binding and transport genes, decreased significantly in ftsh4-4 plants. However, the H2O2 and peroxidases levels, which also have IAA oxidase activity, were significantly elevated in ftsh4-4 plants. The ftsh4-4 phenotypes could be reversed by expressing the iaaM gene or by knocking down the peroxidase genes PRX34 and PRX33. Both approaches can increase auxin levels in the ftsh4-4 mutant. Taken together, these results provided direct molecular and genetic evidence for the interaction between mitochondrial ATP-dependent protease, H2O2, and auxin homeostasis to regulate plant growth and development.

Regulating mtDNA dynamics by TFAM phosphorylation and degradation (94.3)

Mitochondrial transcription factor A (TFAM or mtTFA) is a central regulator of mtDNA replication as well as mitochondrial transcription. TFAM is also a major component of mitochondrial nucleoids, which are protein complexes bound to mtDNA that orchestrate genome maintenance, expression and inheritance. Despite the key roles of TFAM in mtDNA metabolism, little is known about mechanisms for its post‐translational regulation. Using a combination of biochemistry, cell and structural biology as well as proteomics, we have demonstrated that cAMP‐dependent protein kinase (PKA) phosphorylates TFAM within its high mobility group box 1 (HMG1), which impairs DNA‐binding and leads to rapid degradation by the mitochondrial ATP‐dependent Lon protease. We propose that reversible phosphorylation of TFAM within HMG1 causes electrostatic repulsion of the DNA phosphate backbone, thus providing a mechanism for regulating cycles of mtDNA binding and release. In addition, we speculate that irreversible degradation of DNA‐free TFAM by Lon rapidly fine‐tunes the abundance and function of TFAM at the mitochondrial genome.

Mitochondrial ATP-dependent proteases in protection against accumulation of carbonylated proteins

Carbonylation is an irreversible oxidative modification of proteins induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) or by-products of oxidative stress. Carbonylation leads to the loss of protein function and is used as a marker of oxidative stress. Recent data indicate that carbonylation is not only an unfavorable chance process but may also play a significant role in the control of diverse physiological processes. In plants, carbonylated proteins have been found in all cellular compartments; however, mitochondria, one of the major sources of reactive species, show the highest levels of oxidatively modified proteins under normal or stress conditions. Carbonylated proteins tend to misfold and have to be removed to prevent the formation of harmful insoluble aggregates. Mitochondria have developed several pathways that continuously monitor and remove oxidatively damaged polypeptides, and the mitochondrial protein quality control (mtPQC) system, comprising chaperones and ATP-dependent proteases, is the first line of defense. The Lon protease has been recognized as a key protease involved in the removal of oxidized proteins in yeast and mammalian mitochondria, but not in plants. Recently, it has been reported that the inner-membrane human i-AAA and m-AAA and Arabidopsis i-AAA proteases are crucial components of the defense against accumulation of carbonylated proteins, but the molecular basis of their action is not yet clear. Altogether, the mitochondrial AAA proteases secure the mitochondrial proteome against accumulation of carbonylated proteins.

Down-Regulating Overexpressed Human Lon in Cervical Cancer Suppresses Cell Proliferation and Bioenergetics

The human mitochondrial ATP-dependent Lon protease functions in regulating the metabolism and quality control of proteins and mitochondrial DNA (mtDNA). However, the role of Lon in cancer is not well understood. Therefore, this study was undertaken to investigate the importance of Lon in cervical cancer cells from patients and in established cell lines. Microarray analysis from 30 cancer and 10 normal cervical tissues were analyzed by immunohistochemistry for Lon protein levels. The expression of Lon was also examined by immunoblotting 16 fresh cervical cancer tissues and their respective non-tumor cervical tissues. In all cases, Lon expression was significantly elevated in cervical carcinomas as compared to normal tissues. Augmented Lon expression in tissue microarrays did not vary between age, tumor-node-metastasis grades, or lymph node metastasis. Knocking down Lon in HeLa cervical cancer cells by lentivrial transduction resulted in a substantial decrease in both mRNA and protein levels. Such down-regulation of Lon expression significantly blocked HeLa cell proliferation. In addition, knocking down Lon resulted in decreased cellular bioenergetics as determined by measuring aerobic respiration and glycolysis using the Seahorse XF24 extracellular flux analyzer. Together, these data demonstrate that Lon plays a potential role in the oncogenesis of cervical cancer, and may be a useful biomarker and target in the treatment of cervical cancer. Lon; immunohistochemistry; cervical cancer; cell proliferation; cellular bioenergetics.

Impaired Folding of the Mitochondrial Small TIM Chaperones Induces Clearance by the i-AAA Protease

The intermembrane space of mitochondria contains a dedicated chaperone network-the small translocase of the inner membrane (TIM) family-for the sorting of hydrophobic precursors. All small TIMs are defined by the presence of a twin CX(3)C motif and the monomeric proteins are stabilized by two intramolecular disulfide bonds formed between the cysteines of these motifs. The conserved cysteine residues within small TIM members have also been shown to participate in early biogenesis events, with the most N-terminal cysteine residue important for import and retention within the intermembrane space via the receptor and disulfide oxidase, Mia40. In this study, we have analyzed the in vivo consequences of improper folding of small TIM chaperones by generating site-specific cysteine mutants and assessed the fate of the incompletely oxidized proteins within mitochondria. We show that no individual cysteine residue is required for the function of Tim9 or Tim10 in yeast and that defective assembly of the small TIMs induces their proteolytic clearance from mitochondria. We delineate a clearance mechanism for the mutant proteins and their unassembled wild-type partner protein by the mitochondrial ATP-dependent protease, Yme1 (yeast mitochondrial escape 1).

The mitochondrial ATP-dependent Lon protease: a novel target in lymphoma death mediated by the synthetic triterpenoid CDDO and its derivatives

AbstractSynthetic triterpenoids are multitarget compounds exhibiting promise as preventative and therapeutic agents for cancer. Their proposed mechanism of action is by forming Michael adducts with reactive nucleophilic groups on target proteins. Our previous work demonstrates that the 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivatives promote B-lymphoid cell apoptosis through a mitochondria-mediated pathway linked to mitochondrial protein aggregation. As one function of the Lon protease is to eliminate abnormal mitochondrial proteins, we hypothesized that CDDO-induced protein aggregation and lymphoma apoptosis occur by inactivating this enzyme. Here, we show that CDDO and its derivatives directly and selectively inhibit Lon. CDDO blocks Lon-mediated proteolysis in biochemical and cellular assays, but does not inhibit the 20S proteasome. Furthermore, a biotinylated-CDDO conjugate modifies mitochondrial Lon. A striking common phenotype of CDDO-treated lymphoma cells and Lon-knockdown cells is the accumulation of electron-dense aggregates within mitochondria. We also show that Lon protein levels are substantially elevated in malignant lymphoma cells, compared with resting or activated B cells. Finally, we demonstrate that Lon knockdown leads to lymphoma cell death. Together, these findings suggest that Lon inhibition plays a contributory role in CDDO-induced lymphoma cell death, and support the concept that mitochondrial Lon is a novel anticancer drug target.

Identification of Novel Oxidized Protein Substrates and Physiological Partners of the Mitochondrial ATP-dependent Lon-like Protease Pim1

ATP-dependent proteases are currently emerging as key regulators of mitochondrial functions. Among these proteolytic systems, Pim1, a Lon-like serine protease in Saccharomyces cerevisiae, is involved in the control of selective protein turnover in the mitochondrial matrix. In the absence of Pim1, yeast cells have been shown to accumulate electron-dense inclusion bodies in the matrix space, to lose integrity of mitochondrial genome, and to be respiration-deficient. Because of the severity of phenotypes associated with the depletion of Pim1, this protease appears to be an essential component of the protein quality control machinery in mitochondria and to exert crucial functions during the biogenesis of this organelle. Nevertheless, its physiological substrates and partners are not fully characterized. Therefore, we used the combination of different proteomic techniques to assess the nature of oxidized protein substrates and physiological partners of Pim1 protease under non-repressing growth conditions. The results presented here supply evidence that Pim1-mediated proteolysis is required for elimination of oxidatively damaged proteins in mitochondria.

The Triterpenoid 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic Acid and Its Derivatives Inhibit Mitochondrial Lon Protease Activity: A Potential Novel Pathway for the Induction of Lymphoma Cell Death.

Abstract 4142-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its C(28) imidazole and methylester derivatives are novel oleanane triterpenoids exhibiting promise as both therapeutic and preventative agents for cancer. We have previously shown that these synthetic triterpenoids promote lymphoma cell death through a novel mitochondria-mediated mechanism whereby mitochondrial protein thiols are modified resulting in the formation of mitochondrial protein aggregates leading to apoptotic cell death (Cancer Res. 2007; 67:4). As mitochondrial Lon is a critical quality control protease that prevents the toxic accumulation and aggregation of damaged mitochondrial proteins we hypothesized that one effect of the triterpenoids might be to inhibit Lon-mediated proteolysis. Herein we show that CDDO and its derivatives inhibit purified Lon protease. In particular, the imidazole and methylester derivatives of CDDO (CDDO-Im and CDDO-Me) are more effective inhibitors of Lon than CDDO. Interestingly, these derivatives are also more potent inducers of lymphoma cell death than CDDO. We also show that CDDO-Me inhibits cellular mitochondrial Lon, as determined by its ability to inhibit the degradation of the Mitochondrial Transcription Factor A (TFAM), an endogenous Lon protein substrate. To exclude the possibility that the triterpenoid mediated inhibition of TFAM degradation is mediated by the cytoplasmic proteasome, we show that the triterpenoids have no effect on proteasome-mediated proteolysis. To determine the mechanism through which the triterpenoids block Lon activity, we next used a biotinylated-CDDO derivative and showed that this derivative forms adducts with Lon, suggesting that the triterpenoids directly inhibit the protease in cells. In contrast, the biotinylated-CDDO derivative did not form adducts with other proteins, such as the mitochondrial ClpP protease, TFAM, or the endoplasmic reticulum protein BiP, suggesting a level of specificity for triterpenoid adduct formation. Indeed, the formation of protein adducts with Lon is consistent with the fact that the triterpenoids contain a Michael Addition moiety which allows it to form adducts with both protein and non-protein thiols. Consistent with the inhibitory effects of the triterpenoids on mitochondrial Lon protease activity, CDDO-treated lymphoma cells exhibit striking electron dense inclusions within mitochondria, but not the cytosol or other cellular compartments. Similar inclusions within mitochondria are also seen in a Lon knockdown cell line, in which a short hairpin RNA targeting Lon mRNA is inducibly expressed in LS174 colon cancer cells upon treatment with doxycycline. This finding supports the notion that the CDDO-induced inclusions are due, in part, to inhibition of Lon protease activity. Furthermore, we show that Lon protein expression levels are higher in both malignant lymphoma cell lines and patient derived biopsy specimens, as compared to that seen in resting or activated normal B cells. Taken together, these results suggest that; (a) the mitochondrial ATP-dependent Lon protease is a new target of synthetic triterpenoids, and; (b) Lon may be a novel and viable target for cancer therapeutics. Supported by an NCI SPORE grant in lymphoma 1P50 CA130805. Disclosures:Bernstein:millenium: Consultancy; genentech: Consultancy, Speakers Bureau; enzon: Consultancy.