Mitochondrial inner membrane protein MPV17 is a protein of unknown function that is associated with mitochondrial DNA (mtDNA)-depletion syndrome (MDS). MPV17 loss-of-function has been reported to result in tissue-specific nucleotide pool imbalances, which can occur in states of perturbed folate-mediated one-carbon metabolism (FOCM), but MPV17 has not been directly linked to FOCM. FOCM is a metabolic network that provides one-carbon units for the de novo synthesis of purine and thymidylate nucleotides (e.g. dTMP) for both nuclear DNA (nuDNA) and mtDNA replication. In this study, we investigated the impact of reduced MPV17 expression on markers of impaired FOCM in HeLa cells. Depressed MPV17 expression reduced mitochondrial folate levels by 43% and increased uracil levels, a marker of impaired dTMP synthesis, in mtDNA by 3-fold. The capacity of mitochondrial de novo and salvage pathway dTMP biosynthesis was unchanged by the reduced MPV17 expression, but the elevated levels of uracil in mtDNA suggested that other sources of mitochondrial dTMP are compromised in MPV17-deficient cells. These results indicate that MPV17 provides a third dTMP source, potentially by serving as a transporter that transfers dTMP from the cytosol to mitochondria to sustain mtDNA synthesis. We propose that MPV17 loss-offunction and related hepatocerebral MDS are linked to impaired FOCM in mitochondria by providing insufficient access to cytosolic dTMP pools and by severely reducing mitochondrial folate pools.
Cancer metabolism is an intergrative ensemble of disrupted enzyme kinetics and dysregulated metabolite utilization leading to loss of normal cellular function that is the result of a multi-factorial yet coordinated breakdown in vascular, immune, cell cycle, apoptotic, and ECM components. In actively metabolizing cancer, the switch from mitochondrial OXPHOS to anaerobic glycolysis is very well characterized and understood. Global cellular changes in response to metabolic switch have either been overlooked or not been primary interest or relevance to cancer metabolism. We describe a novel systems biology/engineering approach encompassing cell models that are conditioned under various oncogenic perturbations or environments and then coupled with functional bioenergetic read out such as employing the XF24 Seahorse Bioscience analyzer, ATP assays, and ROS production. The OCR and ECAR measurements generated by XF24 analyzer enabled quantifying the switch from aerobic to the anerobic mode of energy metabolism. Cellular profiles were captured in the form of multi-omic (proteomic, genomic, proteomic) signatures using high-throughput mass spectrometry based protocols. Analyses were performed on oncogenic breast, prostate, liver, pancreatic, skin (melanoma, squamous cell carcinoma) and were compared to normal fibroblasts, keratinocytes, hepatocytes, kidney cells, adipocytes, and human aortic and endothelial cells. High throughput data cascades from various cancer states were integrated with the metabolic data from the XF24 analyzer using an AI-based data mining platform to generate causal network based on bayesian models (REFS™ model). The output enables the understanding of differential mechanisms that drive glycolysis and mitochondrial OXPHOS in a cancer versus normal environment. Further validation of prominent hub of activity as they partake as key drivers of metabolic end points by siRNA knockdown experiments followed by measurement using the XF24 analyzer confirmed the relevance of these hubs in cancer metabolism and their relevance as potential therapeutic targets and biomarkers for diagnostics development. The data output presented herein strongly suggest that the Interrogative Biology® platform is a key tool in deciphering differential network analysis pertinent to disease pathophysiology and bioenergetics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4933. doi:1538-7445.AM2012-4933
Mitochondrial DNA (mtDNA) synthesis is necessary for the replication and repair of damaged DNA; maintaining the proper balance of nucleotides is required to avoid mtDNA depletion and mutation. Mitochondrial deoxynucleotide triphosphates (dNTPs) are obtained from either salvage pathways or de novo biosynthesis within the mitochondria. In the mitochondria, de novo dTMP biosynthesis requires the enzymes serine hydroxymethyltransferase 2 (SHMT2), thymidylate synthase (TYMS), and dihydrofolate reductase‐like 1 (DHFRL1). In the nucleus, depletion of dTMP synthesis leads to misincorporation of deoxyuridine into nuclear DNA (nuDNA) resulting in genomic instability. SHMT2 in the mitochondria is responsible for transferring a one‐carbon unit from serine to tetrahydrofolate (THF), producing 5,10‐methyleneTHF and glycine in a reversible reaction; 5–10‐methyleneTHF is utilized by TYMS to create dTMP from dUMP.Our goal is to determine how mtDNA content is affected by disruptions of de novo mitochondrial dTMP synthesis, using two different approaches to decrease SHMT2 activity. First, SHMT2 expression in mammalian HeLa cells was decreased using siRNA technology. Second, HeLa cells were cultured in glycine concentrations ranging from 0mM to 10mM, which inhibits the SHMT2‐catalyzed synthesis of 5,10‐methyleneTHF. The mtDNA and nuDNA copy numbers were determined by qPCR using primers specific for the mitochondrial tRNALeu(UUR) gene and a single copy nuclear gene, β2‐microglobulin. Effects of SHMT2 inhibition on deoxyuridine misincorporation into mtDNA are being assessed.Our preliminary results suggest that mitochondrial de novo dTMP synthesis is affected by SHMT2 expression and glycine concentration. Knockdown of SHMT2 results in an increase (2.8 fold) in mtDNA copy number. Similarly, mtDNA copy number increases with increasing glycine concentration in culture media (4 fold). Supported by NIH R37DK58144.
Maintaining proper nucleotide balance is required to avoid genome instability, for both mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA). Mitochondrial deoxynucleotide triphosphates (dNTPs) are obtained from either salvage or de novo biosynthesis pathways. De novo biosynthesis of thymidylate and purines is folate‐dependent. De novo thymidylate (dTMP) biosynthesis has been shown to occur within mitochondria for mtDNA synthesis and the nucleus for nuDNA synthesis. Disruption of nuclear de novo dTMP biosynthesis affects genome integrity and increases uracil levels in DNA. In mitochondria, de novo dTMP biosynthesis requires serine hydroxymethyltransferase 2 (SHMT2), which transfers a one‐carbon unit from serine to tetrahydrofolate (THF), producing 5,10‐methyleneTHF and glycine; 5‐10‐methyleneTHF is then utilized by thymidylate synthase to generate dTMP from dUMP. In the nucleus, SHMT expression is limiting in de novo dTMP biosynthesis, and reduced SHMT expression elevates uracil content in nuDNA. We are investigating effects of reduced SHMT2 expression and alterations in the cellular serine/glycine ratio on mtDNA content, mitochondria mass, and uracil levels in mtDNA in HeLa cells. Preliminary data indicate that reduced SHMT2 expression and serine and glycine ratio affect mtDNA content and mtDNA integrity. Grant Funding Source: Supported by NIH R37DK58144
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