II. Mitoproteases LONP1 and ClpP regulates mitochondrial protein homeostasis for cancer
2.4. Discussion
The LONP1 and ClpP are known to degrade the misfolded proteins. Therefore, LONP1 and ClpP require for maintaining mitochondrial protein homeostasis. Despite of its important role, LONP1 and ClpP targets were poorly studied so far. Here, we identified LONP1 and ClpP substrates and show how important LONP1 and ClpP in cancer cell survival are and its potential function as degrading misfolded target proteins LRPPRC and SHMT2.
Improper protein quality control is one of the driving factors of several diseases including neurological, cardiovascular disorders, diabetes and even cancer. Impaired mitochondria protein homeostasis provokes cancer cell death. Therefore, Cancer cells show up-regulated chaperones and proteases expression, increased autophagy mechanism (Mitophagy) or induce cell death to maintain proper protein qulities.37,38 In this study, we have shown that LONP1 and ClpP, the key mitochondrial proteases are overexpressed in many human cancer types based on several data sets. Not only LONP1 and ClpP are overexpressed in several tumor types, also show the co-overexpressed pattern in numerous cancer types with meaningful p-values. We assume those co-overexpressed patterns of LONP1 and ClpP is for remaining extensive target protein qualities. Especially, LONP1 and ClpP were highly co-overexpressed in prostate adenocarcinoma exclude suspended cancer types.
Interference with LONP1 and ClpP expression suppresses tumor cell growth which show cell cycle independent manner. However, the deletion of LONP1 and ClpP shuts off cancer cell survival synergistically. This additive killing effects on cancer is thought to be simultaneous failures of both LONP1 and ClpP target protein quality control. Cells normally have a resilient to the threat of misfolded proteins up to a point.39 However, LONP1 and ClpP double deletion is inextricable damages on cells which extensively perturbate the mitochondrial protein homeostasis. Therefore, we demonstrate LONP1 and ClpP each are essential for cancer cell and deletion of both LONP1 and ClpP synergistically impacts on cancer. In addition, we have shown decreased cancer cell growth was induced by cellular stress induced mitophagy and apoptosis. Thus, our data support the concept that LONP1 and ClpP, mitochondrial proteases, are essential for cancer cell survival by maintaining mitochondrial proteostasis and function.
As far as the way LONP1 and ClpP acts on mitochondria, the simplest model is that LONP1 and ClpP expression increases on cancer cells, degrade misfolded/unfolded specific target proteins, and remain well-functioned mitochondria target proteins, in turn, prevent cell death. To find the LONP1 and ClpP target proteins, we interfered LONP1 and ClpP expression and conducted APEX.
Unlike conventional mitochondrial purification in vitro, APEX labeling method is exceptionally specific and distinguish the mitochondrial subspaces and label nearby proteins in living cells in a
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minute.33 Therefore, APEX have a lot more advantages compare to conventional method. Application of APEX labeling method make us to list up the twenty-un-overlapped target proteins, and six- overlapped target proteins. Majority of candidate target proteins were parts of OXPHOS, TCA cycle and other metabolic pathways (Table 1). Along with our expectation, the deletion of LONP1 and ClpP accumulated identified target proteins. Protein folding assay further validate the key target proteins SHMT2 and LRPPRC demonstrating detergent resistance in an identical detergent concentration upon LONP1 and ClpP deletion state.
LRPPRC are member of the pentatricopeptide repeat (PPR) protein family which have repeated 35 amino acid motifs involved in RNA binding and regulate the RNA life cycle from transcription to degradation. LRPPRC mainly function in mitochondrial RNA maturation and stabilization in post-transcriptional level.40 Knockdown of LRPPRC generally decreased the mitochondrial DNA copy number and the respiratory chin complexes.41 In fact, an amino-acid substitution of LRPPRC causes the French-Canadian type of Leigh syndrome (LSFC), a neuro- degenerative disorder characterized by complex IV deficiency. Mutation of LRPPRC also demonstrate the embryonic lethality36 which are also shown in homozygous deletion of LONP1. The importance of mitochondrial RNA binding proteins, LRPPRC, is proved by the variety of disorders caused by mutations in this protein, including neurological disorders, metabolic related diseases and cancer. 41 We also shed light on those mitochondrial RNA binding protein because of its biological role to maintain the integrity of mitochondrial respiratory chain proteins which are essential for cancer metabolism. Therefore, we suggest LONP1 and ClpP are overexpressed in cancer, that could be to preserve the well-folded and well-functioned LRPPRC.
Other target proteins, SHMT2, involves in serine/threonine one-carbon metabolism which required for normal cell proliferation, also cancer cell proliferation in a low-glucose environment.42 SHMT2 is generally elevated by transcription factor, Myc and HIF-1a in ischemic states, such as, in a subset of cancer stem cells (CSCs) and pseudopalisade necrotic 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 demonstrate cancer cells predominantly rely on SHMT2 to induce serine catabolism to support dTTP, one of the building block, for rapid cancer cell proliferation.45 It also have been reported that SHMT2 decrease the pyruvate kinase isozyme 2 (PKM2) activities which serve as an important gate way for oxidative phosphorylation. Therefore, SHMT2 provide the advantages to cancer cell in ischemic tumor regions by limiting oxygen consumption rates in hypoxia, limiting entry ratio of TCA cycle.46 In short, we suggest SHMT2 could be one of LONP1 and ClpP target which make cancer cell sustain in hypoxic region by reprogramming cancer cell metabolism.
In sum, these results provide a scheme of molecular level of cancer cell metabolic
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reprogramming and remain mitochondrial integrity through LONP1 and ClpP. In typical state, LONP1 and ClpP are overexpressed in several human cancer types. That is for a maintenance of specific target proteins of each LONP1 and ClpP, which also have a crucial role in cancer. As we targeted LONP1 and ClpP, target proteins failed to remain their protein qualities and accumulate in mitochondrial matrix which possibly induce the mitophagy and apoptosis. Therefore, we suggest LONP1, ClpP and its substrates are deeply related to cancer cell metabolic reprogramming and survival and, also LONP1 and ClpP might be actionable therapeutic targets for treating cancer. (Fig 2.7a and b)
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