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Inhibition of Mammalian Mitochondrial Protein Synthesis …
In conclusion, our aim was to establish linksbetween the altered metabolic phenotype of RCC and functionality ofthe TERE1 prenyltransferase. We have reported a TERE1-negativeexpression phenotype in a over half of the lesions from a tumormicroarray (TMA) set of human RCC tumor specimens, and demonstratedthat ectopic TERE1 expression profoundly decreased growth,suppressed colony forming ability, and increased caspase 3/7activity in a panel of RCC cell lines. We show TERE1 activatesmitochondrial activity using extra-cellular flux analysis and leadsto elevations in ROS/RNS. TERE1 and TBL2 reduced Caki-1 and Caki-2cell cholesterol and activated a common set of SXR target geneswith roles in cholesterol and lipid metabolism. We discuss severalhypotheses to relate possible TERE1/K-2/K-3 mediated mechanisms oftumor suppression to the altered metabolic phenotype of RCC. Tumorprogression depends on adaptations to maintain an elevatedoxidative stress level; however, tumor cells must manage oxidativestress levels below the apoptotic threshold (). In this regard, subversion ofapoptotic signaling by elevated mitochondrial cholesterol is highlyrelevant (–). The natural TERE1-mediatedtargeting of vitamin K-2 synthesis to mitochondria may represent aform of oxidative stress liability to tumor cell metabolism duringprogression. The loss of TERE1 expression in RCC may be a defect inmitochondrial to nuclear SXR signaling that tumors use to uncouplevitamin K-mediated oxidative stress signaling from apoptosis ornegative growth signaling by elevation of cholesterol.
We examined whether ectopic TERE1 expression wouldaffect cellular levels of nitric oxide in Caki-1 and Caki-2 RCCcells based on reports that vitamin K-2 could induce iNOS andincrease NO production in endothelial cells (), and NO production in zebrafishmodels for UBIAD1 (). Weconducted live cell imaging of Caki-1 and Caki-2 cells that hadbeen loaded with the DAF-FM-DA fluorogenic probe after infectionwith Ad-LACZ or Ad-TERE1. We also tested the effects of addition ofmenaquinone (K-2). DAF FM reacts with NO and RNS to form afluorescent benzotriazole. The graph in compares NO production in Caki-1(top panel) and Caki-2 (bottom panel) cells. TERE1 reduced thebasal cellular NO level in Caki-1 cells, but increased NO in Caki-2cells. Supplementing with vitamin K-2 increased NO production inAd-vector and Ad-TERE1 in both cell lines (not shown for Caki-1).Further studies will be needed to determine the basis for thedifference in basal NO production between Caki-1 and Caki-2 cells,e.g., whether there may be differences in INOS expression, or NOsecretion. Overall, the effects of TERE1 on NO in Caki-2 cells areconsistent with a TERE1-mediated K-2 enhancement of mitochondrialrespiratory chain to produce NO and other RNS (,,).Next, we turned our focus from mitochondria, to a mechanism ofretrograde signaling to the nucleus that is predicted byTERE1-mediated synthesis of K-2 and the activation of SXR nuclearreceptor target genes that play a role in lipid metabolism, andcholesterol efflux (,).
The process of mammalian mitochondrial protein synthesis
Next, we compared the proton production, ECARresponses of Ad-TERE1 and Ad-vector cells (left side of bottomplots in ). TERE1 increasedthe basal ECAR 1.5-fold (12/8=1.5) and the FCCP-induced ECAR1.4-fold (25/18=1.4), relative to Ad-vector cells. ECARmeasurements are generally indicative of lactic acid productionformed during glycolytic energy metabolism (), thus the TERE1-induced 1.5-fold ECARincrease suggests that only a portion of the 2-fold TERE1-inducedOCR is due to increase in metabolic flux due to glycolysis, theremainder likely is due to other components of OCR. Measurements ofOCR represent several concurrent factors including changes inoxidative phosphorylation, non-mitochondrial respiration includingoxidative stress, and mitochondrial proton leak, thus we examinedwhether TERE1 would elevate the level of oxidative stress in RCCcells.
Based on the paradigm of altered mitochondrialmetabolism in RCC, we analyzed two parameters of mitochondrialactivity in Caki-1 and Caki-2 RCC cells: oxygen consumption, OCR,and hydrogen production, ECAR. We found that TERE1 significantlyincreased the basal and maximal rates of oxygen consumption andhydrogen production. Our findings are consistent with the reportedrole of vitamin K-2 in mitochondrial electron transport and ATPproduction and the mitochondrial functionality of TERE1 we inferredvia co-localization with mitochondrial TBL2 (). Changes in OCR measurements mayreflect changes in oxidative phosphorylation, non-mitochondrialrespiration including oxidative stress, and mitochondrial protonleak (). Based on the fact thatthe electron transport chain is an abundant source of mitochondrialsuperoxide radicals (,,,),we examined ROS and RNS effects of TERE1.
Mitochondrial Protein Synthesis Adapts to Influx of …
We evaluated the growth of Caki-1, Caki-2 and A704renal carcinoma cell lines upon TERE1 expression to determine itspotential for growth inhibition as we had observed with TERE1 inbladder and prostate cancer cells (–).Caki-1, Caki-2 and A704 renal carcinoma cell lines were transducedwith Ad-TERE1 or Ad-LACZ virus (arrows) over 10 days, andproliferation determined via the Cell titer-Glo Luminescent Cellviability kit from Promega. Ectopic TERE1 expression inhibited cellgrowth up to 80% in all three of the cell lines after 10 days(). Ectopic TBL2 alsoinhibited A704 cell growth. TERE1 also caused significant decreasesin stable colony formation () in Caki-1 (80%), A498 (45%), ACHN (38%), and 786-O (57%)renal cancer cell lines after lentiviral transduction and selectionin blasticidin for 3 weeks, but only a slight decrease in A704colonies (6%). Methylene blue stained colonies shown for some celllines in . Conversely,TERE1 knockdown increased colony formation: in Caki-1 (1.9-fold),A704 (1.8-fold), A498 (1.5-fold), ACHN (1.7-fold), and 786-O(1.3-fold). A TERE1-mediated increase in caspase 3/7 activity wasobserved in Caki-1 (40% increase), 786-O (60% increase), and ACHN(20% increase) cell lines 4 days after Ad-TERE1 transduction. Thissuggests that a delayed apoptosis plays a role in theTERE1-mediated growth suppression in some of the RCC cells, thoughmay not account for all the growth inhibition. Based on theimportance of mitochondrial metabolism in the metabolic phenotypeof RCC (–), we turned our focus to looking ateffects of TERE1 on additional aspects of mitochondrialfunction.
There is important incentive to understand themechanisms by which TERE1 dosage affects metabolism, growthsignaling and tumor progression. Numerous reports describe K-2 andK-3 mediated inhibition of tumor cell growth and the basis of thedifferences in some of their effects; e.g., it has been reportedthat K-3 but not K-2 can arylate thiols (–,,).Research is focused on designing vitamin K analogs that maydistinguish the different mechanisms and offer therapeuticadvantage. Interestingly, more highly prenylated forms of K-2 werefound to be better SXR activators (,).These studies should guide the clarification of which activitiescontribute to the tumor suppressor activity of TERE1 in RCC. Onerelevant possibility concerns the proposed two-step mechanism ofTERE1-mediated conversion of phylloquinone, K-1, to menaquinone,K-2, with menadione, K-3, as an intermediate (). The presence of K-3 presents apossible mechanism for TERE1 to affect the glycolytic pyruvatekinase isoenzyme PKM2, which is highly relevant to the alteredmetabolic phenotype of RCC. PMK2 has an emerging role as a dominantregulator of tumor cell glycolysis and is the major pyruvate kinaseisoform in RCC (,,).PKM2 catalyzes the dephosphorylation of phosphoenolpyruvate topyruvate, hence, is responsible for oxygen-independent net ATPproduction that allows survival of the cells under hypoxicconditions as are often found in solid tumors. PMK2 can beinhibited by vitamin K-3, and to a lesser degree by K-2, as well asby ROS (,). This implies that a reduction inTERE1 levels in RCC could reduce the generation of the inhibitoryK-3 intermediate and may lead to a greater activity of PKM2,enhancing glycolytic flux and tumor growth. Considering the role ofmenaquinone in anaerobic organisms, an alternate possibility isthat tumor cells with low levels of TERE1 and menaquinone may beselected against in hypoxic environments and driven to invade.
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ATP synthase, H+ transporting, mitochondrial F1 …
Fredericks WJ, McGarvey T, Wang H, et al:The TERE1 (UBIAD1) bladder tumor suppressor protein interacts withmitochondrial TBL2: regulation of trans-membrane potential,oxidative stress and SXR signaling to the nucleus. J Cell Biochem. : : 2013.[Epub aheadof print].
Analysis of Mitochondrial Protein Synthesis in Yeast | …
Because mitochondria perform so many different functions in different tissues, there are literally hundreds of different mitochondrial diseases. Each disorder produces a spectrum of abnormalities that can be confusing to both patients and physicians in early stages of diagnosis. Because of the complex interplay between the hundreds of genes and cells that must cooperate to keep our metabolic machinery running smoothly, it is a hallmark of mitochondrial diseases that identical mtDNA mutations may not produce identical diseases. Genocopies are diseases that are caused by the same mutation but which may not look the same clinically.
Mitochondrial Protein Synthesis in Chlamydomonas reinhardtii …
Mitochondrial diseases are the result of either inherited or spontaneous mutations in mtDNA or nDNA which lead to altered functions of the proteins or RNA molecules that normally reside in mitochondria. Problems with mitochondrial function, however, may only affect certain tissues as a result of factors occurring during development and growth that we do not yet understand. Even when tissue-specific isoforms of mitochondrial proteins are considered, it is difficult to explain the variable patterns of affected organ systems in the mitochondrial disease syndromes seen clinically.
Protein Synthesis and Assembly in Mitochondrial …
Mitochondrial diseases are even more complex in adults because detectable changes in mtDNA occur as we age and, conversely, the aging process itself may result from deteriorating mitochondrial function. There is a broad spectrum of metabolic, inherited and acquired disorders in adults in which abnormal mitochondrial function has been postulated or demonstrated.
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