We found that LonP1 and ClpP are highly upregulated in most human cancers and correlated with poor survival rate of cancer patients. LONP1 and ClpP were specifically overexpressed in prostate cancer cell lines compared to a normal cell. Therefore, we hypothesized that activities of LONP1 and ClpP are critical for mitochondrial stress response to adapt to different tumor microenvironmental stress.
APEX and mass analysis reveal the LONP1 and ClpP substrates associated with metabolic pathways.
Background
- Metabolic Reprogramming in Cancer
- Relevance between Mitochondria and Cancer
- Mitochondrial Proteases
- LONP1
- ClpP
- Figures
- References
Since ClpP is a well-conserved protein, the role of ClpP degradation in mammalian cells is also well established.14 ClpP deficiency in mice causes hair loss and infertility. These characteristics were also found in three different human families. 16 In the AML cancer cell line they show a higher level of ClpP expression. Interestingly, not only ClpP-inhibited cells go through apoptosis, ClpP hyperactivation also selectively kills cancer cells.17 Recently, Imipridone ONC21, the approved drug for solid tumor treatment, is known to specifically induce hyperactivation of ClpP. ClpP without any side effects. in non-malignant cells by targeting distinct cancer metabolism.14 ClpP enzymatic activity demonstrates strong correlation with cancer cell survival.
Human mitochondrial ClpP is a stable heptamer that assembles into a tetradecamer in the presence of ClpX.
Mitoproteases LONP1 and ClpP regulates mitochondrial protein homeostasis for cancer
Introduction
Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit (ClpP) and Lon protease1 (LonP1), a soluble AAA+ mitochondrial protease, are in mitochondrial matrix and contribute to maintain the mitochondrial protein homeostasis (proteostasis). , infertility and cancer cell apoptosis.12,26,27 LONP1 and ClpP are key mitoproteases among numerous proteases, many of which are associated with mitochondrial protein import. In this study, we found the important role of LONP1 and ClpP required for cancer proliferation and survival. Genomic analyzes show most human cancer types show the overexpression of LONP1 and ClpP.
Interestingly, the highest expression level is of ClpP and the third highest expression level of LONP1 in prostate cancer. Also, LONP1 and ClpP show that the co-overexpression in various cancer types and the deletion of each gene synergistically promotes the growth of tumor cells and induces the apoptosis of prostate cancer cells. By applying APEX and mass spectrometry, we further studied the substrates of both LONP1 and ClpP and subsequently found several candidate target proteins associated with metabolic pathways.
Finally, Protein folding assay confirms SHMT2 and LRPPRC, putative target proteins, are bona fide substrates of LONP1 and ClpP.
Materials and Methods
5% (w/v) skim milk was used for blocking and primary antibodies, which referred, were incubated overnight at 4°C. After washing with Tris-buffered saline containing Tween 20 (TBS-T) for 3 times with an interval of 10 minutes, the membranes were incubated for 1 hour with horseradish peroxidase (HRP)-labeled secondary antibodies and visualized by ECL and ECL prime reagents (GE Healthcare) , RPN2232). For re-blotting of different primary antibodies, membranes were incubated with stripping buffer (Intron biotech, IBS-BS018a) for 30 minutes at 55°C and re-blocked with 5%.
DU145 cells were plated at a density of 4x105 cells/well in 6-well plates and treated 24 hours later with pRREP, pCas9 (ToolGen, Seoul, South Korea) and DNA construct expressing ClpP single guide RNA (sgRNA) (exon1 : 5'- CCGGTACCGTCTGCTCCACCACGAT-3') using jetPEI, DNA transfection reagent (Polyplus, 101-10N). LNCaP cells were transfected overnight with the mitochondrial matrix targeting APEX using jetPEI RNA transfection reagent (Polyplus, 101-10N). To stop biotinylation, APEX-transfected cells were washed with a quencher solution containing 10 mM sodium azide (NaN 3 ), 10 mM sodium ascorbate and 5 mM Trolox in DPBS (Dulbecco's Phosphate Buffered Saline) three times.
For staining, mitotracker was added to RPMI culture media for 1 h and then washed out with culture media. Transfected cells with reference siRNA were detached with trypsin-EDTA and centrifuged at 720 rpm, 2 min once. Cells were stained with propidium iodide staining solution including 3.8 mM sodium citrate, 50 µg/ml PI (Sigma, P4170), 10 µg/ml RNase A (Worthington Biochemcials, RASE LS005649, LS005650) in PBS incubate for at least 4 h or overnight over.
The mitochondrial fraction of control LNCaP cells treated with siRNA, siLONP1, or siClpP (20 nM for 48 h) was isolated and suspended in an equal volume of mitochondrial fractionation buffer containing CHAPS or 2.5%). Samples were incubated for 20 min on ice and detergent-insoluble protein aggregates were recovered by centrifugation (20,000 g ) for 20 min.
Results
Indeed, standard apoptotic protein markers, cleaved PARP, were increased when LONP1 and ClpP were deleted. Because the deletion of LONP1 and ClpP induces cancer cell apoptosis, we speculated that the deletion of LONP1 and ClpP might induce mitochondrial dysfunction and that causes cell apoptosis. Moreover, LC3-II was also increased (Fig. 2.4b), which discharges cells into the catabolic metabolism instead of anabolic metabolism to maintain in stress state.31 Thus, upregulation of LONP1 and ClpP in cancer maintains mitochondrial protein quality and has a significant role for mitochondrial function.
The above results have suggested that LONP1 and ClpP may be required for residual mitochondrial integrity and cancer cell survival. In contrast to the importance of LONP1 and ClpP that has been highlighted, only some of their putative targets remain elucidated. To validate the exact targets of LONP1 and ClpP, we performed Engineered Ascorbate Peroxidase (APEX) labeling technology.
We established the hypothetical model that, compared with the normal condition, deletion of LONP1 and ClpP causes the accumulation of misfolded target proteins and that improper quality control of the target protein could cause the dysfunctional mitochondria and mitophagy (Fig. 2.5c). Compared with the control group, approximately 138 of the putative target proteins LonP1 and ClpP were increased. However, only 6 proteins were overlapping targets of LONP1 and ClpP. Fig. 2.5d) Serine hydroxy-methyltransferase2 (SHMT2) Leucine-rich pentatrico peptide repeat-containing (LRPRPC), pyruvate dehydrogenase kinase 1 (PDK1), ATP synthase subunit beta (ATP5B), SSBP1 (single-stranded binding protein 1), fumarate hydratase (FH), Tu Translation Elongation Factor (TUFM), which is closely related to mitochondrial genomic maturation and mitochondrial metabolism (Table.
The presence of LONP1 and ClpP degraded the aggregated proteins, but upon knockdown of LONP1 and ClpP, the target proteins exhibit detergent resistance under the same conditions as CHAPS. Overall, we suggest that LRPPRC and SHMT2 may be specific targets of LONP1 and ClpP.
Discussion
Using the APEX annotation method allows us to list twenty non-overlapping target proteins and six overlapping target proteins. Most of the candidate target proteins were part of the OXPHOS cycle, TCA and other metabolic pathways (Table 1). The protein folding assay further confirms the key target proteins SHMT2 and LRPPRC demonstrating detergent resistance at the same detergent concentration in LONP1 and ClpP deletion state.
Mutation of LRPPRC also demonstrates embryonic lethality36 which is also shown in homozygous deletion of LONP1. 41 We shed light on those mitochondrial RNA-binding proteins because of their biological role to maintain the integrity of mitochondrial respiratory chain proteins that are essential for cancer metabolism. Therefore, we suggest that LONP1 and ClpP are overexpressed in cancer, which may be to maintain well-folded and well-functioning LRPPRC.
Other target proteins, SHMT2, are involved in serine/threonine one-carbon metabolism, which is required for normal cell proliferation, as well as cancer cell proliferation in a low-glucose environment.42 SHMT2 is generally increased by the transcription factor , Myc and HIF-1a in ischemic conditions, such as in a subset of cancer stem cells (CSCs) and the necrotic pseudopalisade region of glioblastoma or during tumor development.17,18 Several studies have shown that overexpression of SHMT2 is associated with aggressive characteristics and poor prognosis.43,44 Isotope tracing shows that cancer cells rely mainly on SHMT2 to promote serine catabolism to support dTTP, one of the key elements, for the rapid proliferation of cancer cells.45 It has also been reported that SHMT2 reduces the activities of pyruvate kinase isozyme 2 (PKM2) which serve as an important element. gateway for oxidative phosphorylation. Therefore, SHMT2 provides advantages to the cancer cell in ischemic tumor regions by limiting oxygen consumption rates in hypoxia, limiting the TCA cycle entry ratio.46 In short, we suggest that SHMT2 may be one of the targets of LONP1 and ClpP that make the cancer cell stay in hypoxia. region by reprogramming the metabolism of cancer cells. This is to preserve the specific target proteins of each LONP1 and ClpP, which also have a crucial role in cancer.
As we targeted LONP1 and ClpP, target proteins could not retain their protein properties and accumulate in mitochondrial matrix possibly causing the mitophagy and apoptosis. Therefore, we suggest that LONP1, ClpP and its substrates are deeply related to cancer cell metabolic reprogramming and survival, and also LONP1 and ClpP may be useful therapeutic targets for the treatment of cancer.
Figures
Most cancers show a higher expression level of LONP1 and ClpP compared to normal tissue. Immunoblots of LONP1 and ClpP indicate the higher expression level compared to its counterparts in prostate cell lines. Hypothetical model showing how LONP1 and ClpP control mitochondrial protein homeostasis (D) List putative target substrates of LONP1 and ClpP and their expression level.
Metabolic reprogramming of cancer cells via LONP1 and ClpP (A) General role of LONP1 and ClpP in mitochondria. B) Targeting LONP1 and ClpP induces inappropriate degradation of substrates and induces mitochondrial dysfunction. Biological role of candidate LONP1 and ClpP substrates and relevance to cancer Gene name Functions of target proteins Relevance to cancer.
The mitochondrial unfolded protein response as a non-oncogenic addiction to support adaptation to stress during transformation in cancer and beyond. Perrault syndrome is caused by recessive mutations in CLPP, which encodes a mitochondrial ATP-dependent ventricular protease. Inhibition of heat shock proteins HSP90 and HSP70 induces oxidative stress and inhibits cotton fiber development.
Serine catabolism by SHMT2 is required for proper initiation and maintenance of mitochondrial translation of formylmethionyl-tRNA. High-throughput synthetic lethality screen reveals a tumorigenic role of adenylate cyclase in fumarate hydratase-deficient cancer cells. SSBP1 suppresses TGF beta-driven epithelial-to-mesenchymal transition and metastasis in triple-negative breast cancer by regulating mitochondrial retrograde signaling.
Downregulation of mitochondrial single-stranded DNA-binding protein (SSBP1) induces mitochondrial dysfunction and increases the radiosensitivity in non-small cell lung cancer cells. TUFM downregulation induces epithelial-mesenchymal transition and invasion in lung cancer cells via a mechanism involving AMPK-GSK3 beta signaling.
