Publications

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Newman, L.E., Weiser Novak, S., Rojas, G.R., Tadepalle, N., Schiavon, C.R., Grotjahn, D.A., Towers, C.G., Tremblay, M.�.�., Donnelly, M.P., Ghosh, S., Medina, M., Rocha, S., Rodriguez-Enriquez, R., Chevez, J.A., Lemersal, I., Manor, U., Shadel, G.S. Mitochondrial DNA replication stress triggers a pro-inflammatory endosomal pathway of nucleoid disposal. (2024) Nature Cell Biology. DOI: 10.1038/s41556-023-01343-1

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Mitochondrial DNA (mtDNA) encodes essential subunits of the oxidative phosphorylation system, but is also a major damage-associated molecular pattern (DAMP) that engages innate immune sensors when released into the cytoplasm, outside of cells or into circulation. As a DAMP, mtDNA not only contributes to anti-viral resistance, but also causes pathogenic inflammation in many disease contexts. Cells experiencing mtDNA stress caused by depletion of the mtDNA-packaging protein, transcription factor A, mitochondrial (TFAM) or during herpes simplex virus-1 infection exhibit elongated mitochondria, enlargement of nucleoids (mtDNA-protein complexes) and activation of cGAS-STING innate immune signalling via mtDNA released into the cytoplasm. However, the relationship among aberrant mitochondria and nucleoid dynamics, mtDNA release and cGAS-STING activation remains unclear. Here we show that, under a variety of mtDNA replication stress conditions and during herpes simplex virus-1 infection, enlarged nucleoids that remain bound to TFAM exit mitochondria. Enlarged nucleoids arise from mtDNA experiencing replication stress, which causes nucleoid clustering via a block in mitochondrial fission at a stage when endoplasmic reticulum actin polymerization would normally commence, defining a fission checkpoint that ensures mtDNA has completed replication and is competent for segregation into daughter mitochondria. Chronic engagement of this checkpoint results in enlarged nucleoids trafficking into early and then late endosomes for disposal. Endosomal rupture during transit through this endosomal pathway ultimately causes mtDNA-mediated cGAS-STING activation. Thus, we propose that replication-incompetent nucleoids are selectively eliminated by an adaptive mitochondria-endosomal quality control pathway that is prone to innate immune system activation, which might represent a therapeutic target to prevent mtDNA-mediated inflammation during viral infection and other pathogenic states.

Victorelli, S., Salmonowicz, H., Chapman, J., Martini, H., Vizioli, M.G., Riley, J.S., Cloix, C., Hall-Younger, E., Machado Espindola-Netto, J., Jurk, D., Lagnado, A.B., Sales Gomez, L., Farr, J.N., Saul, D., Reed, R., Kelly, G., Eppard, M., Greaves, L.C., Dou, Z., Pirius, N., Szczepanowska, K., Porritt, R.A., Huang, H., Huang, T.Y., Mann, D.A., Masuda, C.A., Khosla, S., Dai, H., Kaufmann, S.H., Zacharioudakis, E., Gavathiotis, E., LeBrasseur, N.K., Lei, X., Sainz, A.G., Korolchuk, V.I., Adams, P.D., Shadel, G.S., Tait, S.W.G., Passos, J.F. Apoptotic stress causes mtDNA release during senescence and drives the SASP. (2023) Nature. DOI: 10.1038/s41586-023-06621-4

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Mangalhara, K.C., Varanasi, S.K., Johnson, M.A., Burns, M.J., Rojas, G.R., Esparza Moltó, P.B., Sainz, A.G., Tadepalle, N., Abbott, K.L., Mendiratta, G., Chen, D., Farsakoglu, Y., Kunchok, T., Hoffmann, F.A., Parisi, B., Rincon, M., Vander Heiden, M.G., Bosenberg, M., Hargreaves, D.C., Kaech, S.M., Shadel, G.S. Manipulating mitochondrial electron flow enhances tumor immunogenicity. (2023) Science. 381(6664):1316-1323. DOI: 10.1126/science.abq1053

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Lei, Y., VanPortfliet, J.J., Chen, Y.F., Bryant, J.D., Li, Y., Fails, D., Torres-Odio, S., Ragan, K.B., Deng, J., Mohan, A., Wang, B., Brahms, O.N., Yates, S.D., Spencer, M., Tong, C.W., Bosenberg, M.W., West, L.C., Shadel, G.S., Shutt, T.E., Upton, J.W., Li, P., West, A.P. Cooperative sensing of mitochondrial DNA by ZBP1 and cGAS promotes cardiotoxicity. (2023) Cell. DOI: 10.1016/j.cell.2023.05.039

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Al Khatib, I., Deng, J., Lei, Y., Torres-Odio, S., Rojas, G.R., Newman, L.E., Chung, B.K., Symes, A., Zhang, H., Huang, S.N., Pommier, Y., Khan, A., Shadel, G.S., West, A.P., Gibson, W.T., Shutt, T.E. Activation of the cGAS-STING innate immune response in cells with deficient mitochondrial topoisomerase TOP1MT. (2023) Human Molecular Genetics. DOI: 10.1093/hmg/ddad062

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Malik, N., Ferreira, B.I., Hollstein, P.E., Curtis, S.D., Trefts, E., Weiser Novak, S., Yu, J., Gilson, R., Hellberg, K., Fang, L., Sheridan, A., Hah, N., Shadel, G.S., Manor, U., Shaw, R.J. Induction of lysosomal and mitochondrial biogenesis by AMPK phosphorylation of FNIP1. (2023) Science. 380(6642):eabj5559. DOI: 10.1126/science.abj5559

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Newman, L.E., Shadel, G.S. Mitochondrial DNA Release in Innate Immune Signaling. (2023) Annual Review of Biochemistry. DOI: 10.1146/annurev-biochem-032620-104401

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Nassour, J., Aguiar, L.G., Correia, A., Schmidt, T., Mainz, L., Przetocka, S., Haggblom, C., Tadepalle, N., Williams, A., Shokhirev, M.N., Akincilar, S.C., Tergaonkar, V., Shadel, S., Karlseder, J. Telomere-to-mitochondria signalling by ZBP1 mediates replicative crisis (2023) Nature. DOI: https://doi.org/10.1038/s41586-023-05710-8

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Cobo, I., Tanaka, T.N., Chandra Mangalhara, K., Lana, A., Yeang, C., Han, C., Schlachetzki, J., Challcombe, J., Fixsen, B.R., Sakai, M., Li, R.Z., Fields, H., Mokry, M., Tsai, R.G., Bejar, R., Prange, K., de Winther, M., Shadel, G.S., Glass, C.K. DNA methyltransferase 3 alpha and TET methylcytosine dioxygenase 2 restrain mitochondrial DNA-mediated interferon signaling in macrophages. (2022) Immunity. DOI: 10.1016/j.immuni.2022.06.022

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Hancock-Cerutti, W., Wu, Z., Xu, P., Yadavalli , N., Leonzino, M., Tharkeshwar, A.K., Ferguson, S.M., Shadel, G.S., De Camilli, P. ER-lysosome lipid transfer protein VPS13C/PARK23 prevents aberrant mtDNA-dependent STING signaling (2022) Journal of Cell Biology. 221(7):e202106046. DOI: DOI: 10.1083/jcb.202106046

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Xian, H., Watari, K., Sanchez-Lopez, E., Offenberger, J., Onyuru, J., Sampath, H., Ying, W., Hoffman, H.M., Shadel, G.S., Karin, M. Oxidized DNA fragments exit mitochondria via mPTP- and VDAC-dependent channels to activate NLRP3 inflammasome and interferon signaling. (2022) Immunity. DOI: 10.1016/j.immuni.2022.06.007

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Hepokoski, M.L., Odish, M.F., Lam, M.T., Coufal, N.G., Rolfsen, M.L., Shadel, G.S., Moyzis, A.G., Sainz, A.G., Takiar, P.G., Patel, S., Leonard, A.J., Samandari, N., Hansen, E., Trescott, S., Nguyen, C., Jepsen, K., Cutter, G., Gillespie, M.N., Spragg, R.G., Sasik, R., Ix, J.H. Absolute quantification of plasma mitochondrial DNA by droplet digital PCR marks COVID-19 severity over time during intensive care unit admissions. (2022) Am J Physiol Lung Cell Mol Physiol. DOI: 10.1152/ajplung.00128.2022

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Shadel, G.S., Adams, P.D., Berggren, W.T., Diedrich, J.K., Diffenderfer, K.E., Gage, F.H., Hah, N., Hansen, M., Hetzer, M.W., Molina, A.J.A., Manor, U., Marek, K., O’Keefe, D.D., Pinto, A.F.M., Sacco, A., Sharpee, T.O., Shokriev, M.N., Zambetti, S. The San Diego Nathan Shock Center: tackling the heterogeneity of aging. (2021) Geroscience. DOI: 10.1007/s11357-021-00426-x

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Understanding basic mechanisms of aging holds great promise for developing interventions that prevent or delay many age-related declines and diseases simultaneously to increase human healthspan. However, a major confounding factor in aging research is the heterogeneity of the aging process itself. At the organismal level, it is clear that chronological age does not always predict biological age or susceptibility to frailty or pathology. While genetics and environment are major factors driving variable rates of aging, additional complexity arises because different organs, tissues, and cell types are intrinsically heterogeneous and exhibit different aging trajectories normally or in response to the stresses of the aging process (e.g., damage accumulation). Tackling the heterogeneity of aging requires new and specialized tools (e.g., single-cell analyses, mass spectrometry-based approaches, and advanced imaging) to identify novel signatures of aging across scales. Cutting-edge computational approaches are then needed to integrate these disparate datasets and elucidate network interactions between known aging hallmarks. There is also a need for improved, human cell-based models of aging to ensure that basic research findings are relevant to human aging and healthspan interventions. The San Diego Nathan Shock Center (SD-NSC) provides access to cutting-edge scientific resources to facilitate the study of the heterogeneity of aging in general and to promote the use of novel human cell models of aging. The center also has a robust Research Development Core that funds pilot projects on the heterogeneity of aging and organizes innovative training activities, including workshops and a personalized mentoring program, to help investigators new to the aging field succeed. Finally, the SD-NSC participates in outreach activities to educate the general community about the importance of aging research and promote the need for basic biology of aging research in particular.

Wu, Z., Sainz, A.G., Shadel, G.S. Mitochondrial DNA: cellular genotoxic stress sentinel. (2021) Trends in Biochemical Sciences. DOI: 10.1016/j.tibs.2021.05.004

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Tadepalle, N., Shadel, G.S. RNA reports breaking news from mitochondria. (2021) Molecular Cell. 81(9):1863-1865. DOI: 10.1016/j.molcel.2021.04.005

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Lei, Y., Zhang, J., Schiavon, C.R., He, M., Chen, L., Shen, H., Zhang, Y., Yin, Q., Cho, Y., Andrade, L., Shadel, G.S., Hepokoski, M., Lei, T., Wang, H., Zhang, J., Yuan, J.X., Malhotra, A., Manor, U., Wang, S., Yuan, Z.Y., Shyy, J.Y. SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE2. (2021) Circulation research. DOI: 10.1161/CIRCRESAHA.121.318902

Schwartz, R.E., Shokhirev, M.N., Andrade, L.R., Gutkind, J.S., Iglesias-Bartolome, R., Shadel, G.S. Insights into epithelial cell senescence from transcriptome and secretome analysis of human oral keratinocytes. (2021) Aging. 13. DOI: 10.18632/aging.202658

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Schiavon, C.R., Shadel, G.S., Manor, U. Impaired Mitochondrial Mobility in Charcot-Marie-Tooth Disease. (2021) Front Cell Dev Biol. 9:624823. DOI: 10.3389/fcell.2021.624823

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Schiavon, C.R., Zhang, T., Zhao, B., Moore, A.S., Wales, P., Andrade, L.R., Wu, M., Sung, T.C., Dayn, Y., Feng, J.W., Quintero, O.A., Shadel, G.S., Grosse, R., Manor, U. Actin chromobody imaging reveals sub-organellar actin dynamics. (2020) Nature Methods. DOI: 10.1038/s41592-020-0926-5

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Wu, Z., Oeck, S., West, A.P., Mangalhara, K.C., Sainz, A.G., Newman, L.E., Zhang, X.O., Wu, L., Yan, Q., Bosenberg, M., Liu, Y., Sulkowski, P.L., Tripple, V., Kaech, S.M., Glazer, P.M., Shadel, G.S. Mitochondrial DNA Stress Signalling Protects the Nuclear Genome. (2019) Nature Metabolism. 1(12):1209-1218. DOI: 10.1038/s42255-019-0150-8

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Gale, M., Li, Y., Cao, J., Liu, Z.Z., Holmbeck, M.A., Zhang, M., Lang, S.M., Wu, L., Do Carmo, M., Gupta, S., Aoshima, K., DiGiovanna, M.P., Stern, D.F., Rimm, D.L., Shadel, G.S., Chen, X., Yan, Q. Acquired resistance to HER2-targeted therapies creates vulnerability to ATP synthase inhibition. (2019) Cancer Research. DOI: 10.1158/0008-5472.CAN-18-3985

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Harel, M., Ortenberg, R., Varanasi, S.K., Mangalhara, K.C., Mardamshina, M., Markovits, E., Baruch, E.N., Tripple, V., Arama-Chayoth, M., Greenberg, E., Shenoy, A., Ayasun, R., Knafo, N., Xu, S., Anafi, L., Yanovich-Arad, G., Barnabas, G.D., Ashkenazi, S., Besser, M.J., Schachter, J., Bosenberg, M., Shadel, G.S., Barshack, I., Kaech, S.M., Markel, G., Geiger, T. Proteomics of Melanoma Response to Immunotherapy Reveals Mitochondrial Dependence. (2019) Cell. DOI: 10.1016/j.cell.2019.08.012

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Mangalhara, K.C., Shadel, G.S. A Mitochondrial-Derived Peptide Exercises the Nuclear Option. (2018) Cell Metabolism. 28(3):330-331. DOI: 10.1016/j.cmet.2018.08.017

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Newman, L.E., Shadel, G.S. Pink1/Parkin link inflammation, mitochondrial stress, and neurodegeneration. (2018) Journal of Cell Biology. 217(10):3327-3329. DOI: 10.1083/jcb.201808118

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Cox, C.S., McKay, S.E., Holmbeck, M.A., Christian, B.E., Scortea, A.C., Tsay, A.J., Newman, L.E., Shadel, G.S. Mitohormesis in Mice via Sustained Basal Activation of Mitochondrial and Antioxidant Signaling. (2018) Cell Metabolism. 28(5):776-786. DOI: 10.1016/j.cmet.2018.07.011

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Wu, L., Cao, J., Cai, W.L., Lang, S.M., Horton, J.R., Jansen, D.J., Liu, Z.Z., Chen, J.F., Zhang, M., Mott, B.T., Pohida, K., Rai, G., Kales, S.C., Henderson, M.J., Hu, X., Jadhav, A., Maloney, D.J., Simeonov, A., Zhu, S., Iwasaki, A., Hall, M.D., Cheng, X., Shadel, G.S., Yan, Q. KDM5 histone demethylases repress immune response via suppression of STING. (2018) PLOS Biology. 16(8):e2006134. DOI: 10.1371/journal.pbio.2006134

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Zhang, Y., Lee, J.H., Paull, T.T., Gehrke, S., D’Alessandro, A., Dou, Q., Gladyshev, V.N., Schroeder, E.A., Steyl, S.K., Christian, B.E., Shadel, G.S. Mitochondrial redox sensing by the kinase ATM maintains cellular antioxidant capacity. (2018) Science Signaling. 11(538). DOI: 10.1126/scisignal.aaq0702

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Kuszak, A.J., Espey, M.G., Falk, M.J., Holmbeck, M.A., Manfredi, G., Shadel, G.S., Vernon, H.J., Zolkipli-Cunningham, Z. Nutritional Interventions for Mitochondrial OXPHOS Deficiencies: Mechanisms and Model Systems. (2018) Annual Review of Pathology. 13(163-191):163-191. DOI: 10.1146/annurev-pathol-020117-043644

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West, A.P., Shadel, G.S. Mitochondrial DNA in innate immune responses and inflammatory pathology. (2017) Nature Reviews. Immunology. 17(6):363-375. DOI: 10.1038/nri.2017.21

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Schweppe, D.K., Chavez, J.D., Lee, C.F., Caudal, A., Kruse, S.E., Stuppard, R., Marcinek, D.J., Shadel, G.S., Tian, R., Bruce, J.E. Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry. (2017) Proceedings of the National Academy of Sciences of the United States of America. 114(7):1732-1737. DOI: 10.1073/pnas.1617220114

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Oran, A.R., Adams, C.M., Zhang, X.Y., Gennaro, V.J., Pfeiffer, H.K., Mellert, H.S., Seidel, H.E., Mascioli, K., Kaplan, J., Gaballa, M.R., Shen, C., Rigoutsos, I., King, M.P., Cotney, J.L., Arnold, J.J., Sharma, S.D., Martinez-Outschoorn, U.E., Vakoc, C.R., Chodosh, L.A., Thompson, J.E., Bradner, J.E., Cameron, C.E., Shadel, G.S., Eischen, C.M., McMahon, S.B. Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer. (2016) Oncotarget. 7(45):72395-72414. DOI: 10.18632/oncotarget.11718

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Kang, M.J., Shadel, G.S. A Mitochondrial Perspective of Chronic Obstructive Pulmonary Disease Pathogenesis. (2016) Tuberculosis and Respiratory Diseases. 79(4):207-213. DOI: 10.4046/trd.2016.79.4.207

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Holmbeck, M.A., Shadel, G.S. Mitochondria provide a ‘complex’ solution to a bacterial problem. (2016) Nature Immunology. 17(9):1009-10. DOI: 10.1038/ni.3534

Nouws, J., Goswami, A.V., Bestwick, M., McCann, B.J., Surovtseva, Y.V., Shadel, G.S. Mitochondrial Ribosomal Protein L12 Is Required for POLRMT Stability and Exists as Two Forms Generated by Alternative Proteolysis during Import. (2016) Journal of Biological Chemistry. 291(2):989-97. DOI: 10.1074/jbc.M115.689299

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McKay, S.E., Yan, W., Nouws, J., Thormann, M.J., Raimundo, N., Khan, A., Santos-Sacchi, J., Song, L., Shadel, G.S. Auditory Pathology in a Transgenic mtTFB1 Mouse Model of Mitochondrial Deafness. (2015) Am. J. Pathol. 185(12):3132-40. DOI: 10.1016/j.ajpath.2015.08.014

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Camell, C.D., Nguyen, K.Y., Jurczak, M.J., Christian, B.E., Shulman, G.I., Shadel, G.S., Dixit, V.D. Macrophage-specific de Novo Synthesis of Ceramide Is Dispensable for Inflammasome-driven Inflammation and Insulin Resistance in Obesity. (2015) Journal of Biological Chemistry. 290(49):29402-13. DOI: 10.1074/jbc.M115.680199

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Shadel, G.S., Horvath, T.L. Mitochondrial ROS signaling in organismal homeostasis. (2015) Cell. 163(3):560-9. DOI: 10.1016/j.cell.2015.10.001

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Longo, V.D., Antebi, A., Bartke, A., Barzilai, N., Brown-Borg, H.M., Caruso, C., Curiel, T.J., de Cabo, R., Franceschi, C., Gems, D., Ingram, D.K., Johnson, T.E., Kennedy, B.K., Kenyon, C., Klein, S., Kopchick, J.J., Lepperdinger, G., Madeo, F., Mirisola, M.G., Mitchell, J.R., Passarino, G., Rudolph, K.L., Sedivy, J.M., Shadel, G.S., Sinclair, D.A., Spindler, S.R., Suh, Y., Vijg, J., Vinciguerra, M., Fontana, L. Interventions to Slow Aging in Humans: Are We Ready? (2015) Aging Cell. 14(4):497-510. DOI: 10.1111/acel.12338

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Kang, M.J., Yoon, C.M., Kim, B.H., Lee, C.M., Zhou, Y., Sauler, M., Homer, R., Dhamija, A., Boffa, D., West, A.P., Shadel, G.S., Ting, J.P., Tedrow, J.R., Kaminski, N., Kim, W.J., Lee, C.G., Oh, Y.M., Elias, J.A. Suppression of NLRX1 in chronic obstructive pulmonary disease. (2015) Journal of Clinical Investigation. 125(6):2458-62. DOI: 10.1172/JCI71747

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Rongvaux, A., Jackson, R., Harman, C.C., Li, T., West, A.P., de Zoete, M.R., Wu, Y., Yordy, B., Lakhani, S.A., Kuan, C.Y., Taniguchi, T., Shadel, G.S., Chen, Z.J., Iwasaki, A., Flavell, R.A. Apoptotic caspases prevent the induction of type I interferons by mitochondrial DNA. (2014) Cell. 159(7):1563-77. DOI: 10.1016/j.cell.2014.11.037

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Clayton, D.A., Shadel, G.S. Isolation of mitochondria from cells and tissues. (2014) Cold Spring Harbor Protocols. 2014(10):pdb.top074542. DOI: 10.1101/pdb.top074542

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Clayton, D.A., Shadel, G.S. Purification of mitochondria by sucrose step density gradient centrifugation. (2014) Cold Spring Harbor Protocols. 2014(10):pdb.prot080028. DOI: 10.1101/pdb.prot080028

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Clayton, D.A., Shadel, G.S. Isolation of mitochondria from animal tissue. (2014) Cold Spring Harbor Protocols. 2014(10):pdb.prot080010. DOI: 10.1101/pdb.prot080010

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Clayton, D.A., Shadel, G.S. Isolation of mitochondria from tissue culture cells. (2014) Cold Spring Harbor Protocols. 2014(10):pdb.prot080002. DOI: 10.1101/pdb.prot080002

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Nouws, J., Shadel, G.S. microManaging mitochondrial translation. (2014) Cell. 158(3):477-8. DOI: 10.1016/j.cell.2014.07.010

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Shadel, G.S. Live longer on MARS: a yeast paradigm of mitochondrial adaptive ROS signaling in aging. (2014) Microbial Cell. 1(5):140-144. DOI: 10.15698/mic2014.05.143

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Mannam, P., Shinn, A.S., Srivastava, A., Neamu, R.F., Walker, W.E., Bohanon, M., Merkel, J., Kang, M.J., Dela Cruz, C.S., Ahasic, A.M., Pisani, M.A., Trentalange, M., West, A.P., Shadel, G.S., Elias, J.A., Lee, P.J. MKK3 regulates mitochondrial biogenesis and mitophagy in sepsis-induced lung injury. (2014) Am. J. Physiol. Lung Cell Mol. Physiol. 306(7):L604-19. DOI: 10.1152/ajplung.00272.2013

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Christian, B.E., Shadel, G.S. Aging: it’s SIRTainly possible to restore mitochondrial dysfunction. (2014) Current Biology. 24(5):R206-8. DOI: 10.1016/j.cub.2014.01.027

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Schroeder, E.A., Shadel, G.S. Crosstalk between mitochondrial stress signals regulates yeast chronological lifespan. (2014) Mech. Ageing Dev. 135:41-9. DOI: 10.1016/j.mad.2013.12.002

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Sharma, N.K., Lebedeva, M., Thomas, T., Kovalenko, O.A., Stumpf, J.D., Shadel, G.S., Santos, J.H. Intrinsic mitochondrial DNA repair defects in Ataxia Telangiectasia. (2014) DNA Repair. 13:22-31. DOI: 10.1016/j.dnarep.2013.11.002

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Schroeder, E.A., Raimundo, N., Shadel, G.S. Epigenetic silencing mediates mitochondria stress-induced longevity. (2013) Cell Metabolism. 17(6):954-64. DOI: 10.1016/j.cmet.2013.04.003

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Bestwick, M.L., Shadel, G.S. Accessorizing the human mitochondrial transcription machinery. (2013) Trends in Biochemical Sciences. 38(6):283-91. DOI: 10.1016/j.tibs.2013.03.006

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Surovtseva, Y.V., Shadel, G.S. Transcription-independent role for human mitochondrial RNA polymerase in mitochondrial ribosome biogenesis. (2013) Nucleic Acids Research. 41(4):2479-88. DOI: 10.1093/nar/gks1447

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Ocampo, A., Liu, J., Schroeder, E.A., Shadel, G.S., Barrientos, A. Mitochondrial respiratory thresholds regulate yeast chronological life span and its extension by caloric restriction. (2012) Cell Metabolism. 16(1):55-67. DOI: 10.1016/j.cmet.2012.05.013

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Longo, V.D., Shadel, G.S., Kaeberlein, M., Kennedy, B. Replicative and chronological aging in Saccharomyces cerevisiae. (2012) Cell Metabolism. 16(1):18-31. DOI: 10.1016/j.cmet.2012.06.002

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Lodeiro, M.F., Uchida, A., Bestwick, M., Moustafa, I.M., Arnold, J.J., Shadel, G.S., Cameron, C.E. Transcription from the second heavy-strand promoter of human mtDNA is repressed by transcription factor A in vitro. (2012) Proceedings of the National Academy of Sciences of the United States of America. 109(17):6513-8. DOI: 10.1073/pnas.1118710109

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Schroeder, E.A., Shadel, G.S. Alternative mitochondrial fuel extends life span. (2012) Cell Metabolism. 15(4):417-8. DOI: 10.1016/j.cmet.2012.03.011

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Raimundo, N., Song, L., Shutt, T.E., McKay, S.E., Cotney, J., Guan, M.X., Gilliland, T.C., Hohuan, D., Santos-Sacchi, J., Shadel, G.S. Mitochondrial stress engages E2F1 apoptotic signaling to cause deafness. (2012) Cell. 148(4):716-26. DOI: 10.1016/j.cell.2011.12.027

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Woo, D.K., Green, P.D., Santos, J.H., D’Souza, A.D., Walther, Z., Martin, W.D., Christian, B.E., Chandel, N.S., Shadel, G.S. Mitochondrial genome instability and ROS enhance intestinal tumorigenesis in APC(Min/+) mice. (2012) Am. J. Pathol. 180(1):24-31. DOI: 10.1016/j.ajpath.2011.10.003

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Malarkey, C.S., Bestwick, M., Kuhlwilm, J.E., Shadel, G.S., Churchill, M.E. Transcriptional activation by mitochondrial transcription factor A involves preferential distortion of promoter DNA. (2012) Nucleic Acids Research. 40(2):614-24. DOI: 10.1093/nar/gkr787

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Liu, L., Sanosaka, M., Lei, S., Bestwick, M.L., Frey, J.H., Surovtseva, Y.V., Shadel, G.S., Cooper, M.P. LRP130 protein remodels mitochondria and stimulates fatty acid oxidation. (2011) Journal of Biological Chemistry. 286(48):41253-64. DOI: 10.1074/jbc.M111.276121

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Surovtseva, Y.V., Shutt, T.E., Cotney, J., Cimen, H., Chen, S.Y., Koc, E.C., Shadel, G.S. Mitochondrial ribosomal protein L12 selectively associates with human mitochondrial RNA polymerase to activate transcription. (2011) Proceedings of the National Academy of Sciences of the United States of America. 108(44):17921-6. DOI: 10.1073/pnas.1108852108

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Baysal, B.E., McKay, S.E., Kim, Y.J., Zhang, Z., Alila, L., Willett-Brozick, J.E., Pacak, K., Kim, T.H., Shadel, G.S. Genomic imprinting at a boundary element flanking the SDHD locus. (2011) Human Molecular Genetics. 20(22):4452-61. DOI: 10.1093/hmg/ddr376

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Raimundo, N., Baysal, B.E., Shadel, G.S. Revisiting the TCA cycle: signaling to tumor formation. (2011) Trends in Molecular Medicine. 17(11):641-9. DOI: 10.1016/j.molmed.2011.06.001

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Pan, Y., Schroeder, E.A., Ocampo, A., Barrientos, A., Shadel, G.S. Regulation of yeast chronological life span by TORC1 via adaptive mitochondrial ROS signaling. (2011) Cell Metabolism. 13(6):668-78. DOI: 10.1016/j.cmet.2011.03.018

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West, A.P., Shadel, G.S., Ghosh, S. Mitochondria in innate immune responses. (2011) Nature Reviews. Immunology. 11(6):389-402. DOI: 10.1038/nri2975

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Chatenay-Lapointe, M., Shadel, G.S. Repression of mitochondrial translation, respiration and a metabolic cycle-regulated gene, SLF1, by the yeast Pumilio-family protein Puf3p. (2011) PLOS One. 6(5):e20441. DOI: 10.1371/journal.pone.0020441

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West, A.P., Brodsky, I.E., Rahner, C., Woo, D.K., Erdjument-Bromage, H., Tempst, P., Walsh, M.C., Choi, Y., Shadel, G.S., Ghosh, S. TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. (2011) Nature. 472(7344):476-80. DOI: 10.1038/nature09973

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Shutt, T.E., Bestwick, M., Shadel, G.S. The core human mitochondrial transcription initiation complex: It only takes two to tango. (2011) Transcription. 2(2):55-59. DOI: 10.4161/trns.2.2.14296

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Woo, D.K., Shadel, G.S. Mitochondrial stress signals revise an old aging theory. (2011) Cell. 144(1):11-2. DOI: 10.1016/j.cell.2010.12.023

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Lee, H.Y., Choi, C.S., Birkenfeld, A.L., Alves, T.C., Jornayvaz, F.R., Jurczak, M.J., Zhang, D., Woo, D.K., Shadel, G.S., Ladiges, W., Rabinovitch, P.S., Santos, J.H., Petersen, K.F., Samuel, V.T., Shulman, G.I. Targeted expression of catalase to mitochondria prevents age-associated reductions in mitochondrial function and insulin resistance. (2010) Cell Metabolism. 12(6):668-74. DOI: 10.1016/j.cmet.2010.11.004

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Chatenay-Lapointe, M., Shadel, G.S. Stressed-out mitochondria get MAD. (2010) Cell Metabolism. 12(6):559-60. DOI: 10.1016/j.cmet.2010.11.018

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Shutt, T.E., Lodeiro, M.F., Cotney, J., Cameron, C.E., Shadel, G.S. Core human mitochondrial transcription apparatus is a regulated two-component system in vitro. (2010) Proceedings of the National Academy of Sciences of the United States of America. 107(27):12133-8. DOI: 10.1073/pnas.0910581107

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Shutt, T.E., Shadel, G.S. A compendium of human mitochondrial gene expression machinery with links to disease. (2010) Environmental and Molecular Mutagenesis. 51(5):360-79. DOI: 10.1002/em.20571

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Pawar, V., Jingjing, L., Patel, N., Kaur, N., Doetsch, P.W., Shadel, G.S., Zhang, H., Siede, W. Checkpoint kinase phosphorylation in response to endogenous oxidative DNA damage in repair-deficient stationary-phase Saccharomyces cerevisiae. (2009) Mech. Ageing Dev. 130(8):501-8. DOI: 10.1016/j.mad.2009.06.002

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Shadel, G.S., Pan, Y. Multi-faceted regulation of mitochondria by TOR. (2009) Cell Cycle. 8(14):2143. DOI: 10.4161/cc.8.14.9117

Cotney, J., McKay, S.E., Shadel, G.S. Elucidation of separate, but collaborative functions of the rRNA methyltransferase-related human mitochondrial transcription factors B1 and B2 in mitochondrial biogenesis reveals new insight into mate (2009) Human Molecular Genetics. 18(14):2670-82. DOI: 10.1093/hmg/ddp208

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Griffiths, L.M., Doudican, N.A., Shadel, G.S., Doetsch, P.W. Mitochondrial DNA oxidative damage and mutagenesis in Saccharomyces cerevisiae. (2009) Methods in Molecular Biology. 554:267-86. DOI: 10.1007/978-1-59745-521-3_17

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Crouser, E.D., Shao, G., Julian, M.W., Macre, J.E., Shadel, G.S., Tridandapani, S., Huang, Q., Wewers, M.D. Monocyte activation by necrotic cells is promoted by mitochondrial proteins and formyl peptide receptors. (2009) Critical Care Medicine. 37(6):2000-9. DOI: 10.1097/CCM.0b013e3181a001ae

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Suissa, S., Wang, Z., Poole, J., Wittkopp, S., Feder, J., Shutt, T.E., Wallace, D.C., Shadel, G.S., Mishmar, D. Ancient mtDNA genetic variants modulate mtDNA transcription and replication. (2009) PLOS Genetics. 5(5):e1000474. DOI: 10.1371/journal.pgen.1000474

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Although the functional consequences of mitochondrial DNA (mtDNA) genetic backgrounds (haplotypes, haplogroups) have been demonstrated by both disease association studies and cell culture experiments, it is not clear which of the mutations within the haplogroup carry functional implications and which are “evolutionary silent hitchhikers”. We set forth to study the functionality of haplogroup-defining mutations within the mtDNA transcription/replication regulatory region by in vitro transcription, hypothesizing that haplogroup-defining mutations occurring within regulatory motifs of mtDNA could affect these processes. We thus screened >2500 complete human mtDNAs representing all major populations worldwide for natural variation in experimentally established protein binding sites and regulatory regions comprising a total of 241 bp in each mtDNA. Our screen revealed 77/241 sites showing point mutations that could be divided into non-fixed (57/77, 74%) and haplogroup/sub-haplogroup-defining changes (i.e., population fixed changes, 20/77, 26%). The variant defining Caucasian haplogroup J (C295T) increased the binding of TFAM (Electro Mobility Shift Assay) and the capacity of in vitro L-strand transcription, especially of a shorter transcript that maps immediately upstream of conserved sequence block 1 (CSB1), a region associated with RNA priming of mtDNA replication. Consistent with this finding, cybrids (i.e., cells sharing the same nuclear genetic background but differing in their mtDNA backgrounds) harboring haplogroup J mtDNA had a >2 fold increase in mtDNA copy number, as compared to cybrids containing haplogroup H, with no apparent differences in steady state levels of mtDNA-encoded transcripts. Hence, a haplogroup J regulatory region mutation affects mtDNA replication or stability, which may partially account for the phenotypic impact of this haplogroup. Our analysis thus demonstrates, for the first time, the functional impact of particular mtDNA haplogroup-defining control region mutations, paving the path towards assessing the functionality of both fixed and un-fixed genetic variants in the mitochondrial genome.

Lebedeva, M.A., Eaton, J.S., Shadel, G.S. Loss of p53 causes mitochondrial DNA depletion and altered mitochondrial reactive oxygen species homeostasis. (2009) Biochimica et Biophysica Acta. 1787(5):328-34. DOI: 10.1016/j.bbabio.2009.01.004

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Raimundo, N., Shadel, G.S. A “radical” mitochondrial view of autophagy-related pathology. (2009) Aging. 1(4):354-6. DOI: 10.18632/aging.100037

Tal, M.C., Sasai, M., Lee, H.K., Yordy, B., Shadel, G.S., Iwasaki, A. Absence of autophagy results in reactive oxygen species-dependent amplification of RLR signaling. (2009) Proceedings of the National Academy of Sciences of the United States of America. 106(8):2770-5. DOI: 10.1073/pnas.0807694106

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Pan, Y., Shadel, G.S. Extension of chronological life span by reduced TOR signaling requires down-regulation of Sch9p and involves increased mitochondrial OXPHOS complex density. (2009) Aging. 1(1):131-45. DOI: 10.18632/aging.100016

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Shadel, G.S. Into the wild frontier of mitochondrial disease and aging. (2008) Methods. 46(4):239-40. DOI: 10.1016/j.ymeth.2008.10.027

Bonawitz, N.D., Chatenay-Lapointe, M., Wearn, C.M., Shadel, G.S. Expression of the rDNA-encoded mitochondrial protein Tar1p is stringently controlled and responds differentially to mitochondrial respiratory demand and dysfunction. (2008) Current Genetics. 54(2):83-94. DOI: 10.1007/s00294-008-0203-0

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Shadel, G.S. Expression and maintenance of mitochondrial DNA: new insights into human disease pathology. (2008) Am. J. Pathol. 172(6):1445-56. DOI: 10.2353/ajpath.2008.071163

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Schleicher, M., Shepherd, B.R., Suarez, Y., Fernandez-Hernando, C., Yu, J., Pan, Y., Acevedo, L.M., Shadel, G.S., Sessa, W.C. Prohibitin-1 maintains the angiogenic capacity of endothelial cells by regulating mitochondrial function and senescence. (2008) Journal of Cell Biology. 180(1):101-12. DOI: 10.1083/jcb.200706072

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Eaton, J.S., Lin, Z.P., Sartorelli, A.C., Bonawitz, N.D., Shadel, G.S. Ataxia-telangiectasia mutated kinase regulates ribonucleotide reductase and mitochondrial homeostasis. (2007) Journal of Clinical Investigation. 117(9):2723-34. DOI: 10.1172/JCI31604

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Lebedeva, M.A., Shadel, G.S. Cell cycle- and ribonucleotide reductase-driven changes in mtDNA copy number influence mtDNA Inheritance without compromising mitochondrial gene expression. (2007) Cell Cycle. 6(16):2048-57. DOI: 10.4161/cc.6.16.4572

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Bonawitz, N.D., Shadel, G.S. Rethinking the mitochondrial theory of aging: the role of mitochondrial gene expression in lifespan determination. (2007) Cell Cycle. 6(13):1574-8. DOI: 10.4161/cc.6.13.4457

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Cotney, J., Wang, Z., Shadel, G.S. Relative abundance of the human mitochondrial transcription system and distinct roles for h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and gene expression. (2007) Nucleic Acids Research. 35(12):4042-54. DOI: 10.1093/nar/gkm424

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Bonawitz, N.D., Chatenay-Lapointe, M., Pan, Y., Shadel, G.S. Reduced TOR signaling extends chronological life span via increased respiration and upregulation of mitochondrial gene expression. (2007) Cell Metabolism. 5(4):265-77. DOI: 10.1016/j.cmet.2007.02.009

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Wang, Z., Cotney, J., Shadel, G.S. Human mitochondrial ribosomal protein MRPL12 interacts directly with mitochondrial RNA polymerase to modulate mitochondrial gene expression. (2007) Journal of Biological Chemistry. 282(17):12610-8. DOI: 10.1074/jbc.M700461200

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The core human mitochondrial transcription machinery comprises a single subunit bacteriophage-related RNA polymerase, POLRMT, the high mobility group box DNA-binding protein h-mtTFA/TFAM, and two transcriptional co-activator proteins, h-mtTFB1 and h-mtTFB2 that also have rRNA methyltransferase activity. Recapitulation of specific initiation of transcription in vitro can be achieved by a complex of POL-RMT, h-mtTFA, and either h-mtTFB1 or h-mtTFB2. However, the nature of mitochondrial transcription complexes in vivo and the potential involvement of additional proteins in the transcription process in human mitochondria have not been extensively investigated. In Saccharomyces cerevisiae, transcription and translation are physically coupled via the formation of a multiprotein complex nucleated by the binding of Nam1p to the amino-terminal domain of mtRNA polymerase (Rpo41p). This model system paradigm led us to search for proteins that interact with POLRMT to regulate mitochondrial gene expression in humans. Using an affinity capture strategy to identify POL-RMT-binding proteins, we identified mitochondrial ribosomal protein L7/L12 (MRPL12) as a protein in HeLa mitochondrial extracts that interacts specifically with POLRMT in vitro. Purified recombinant MRPL12 binds to POLRMT and stimulates mitochondrial transcription activity in vitro, demonstrating that this interaction is both direct and functional. Finally, from HeLa cells that overexpress FLAG epitope-tagged MRPL12, increased steady-state levels of mtDNA-encoded transcripts are observed and MRPL12-POLRMT complexes can be co-immunoprecipitated, providing strong evidence that this interaction enhances mitochondrial transcription or RNA stability in vivo. We speculate that the MRPL12 interaction with POLRMT is likely part of a novel regulatory mechanism that coordinates mitochondrial transcription with translation and/or ribosome biogenesis during human mitochondrial gene expression.

Zhang, H., Luo, Y., Zhang, W., He, Y., Dai, S., Zhang, R., Huang, Y., Bernatchez, P., Giordano, F.J., Shadel, G., Sessa, W.C., Min, W. Endothelial-specific expression of mitochondrial thioredoxin improves endothelial cell function and reduces atherosclerotic lesions. (2007) Am. J. Pathol. 170(3):1108-20. DOI: 10.2353/ajpath.2007.060960

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Lin, Z.P., Belcourt, M.F., Carbone, R., Eaton, J.S., Penketh, P.G., Shadel, G.S., Cory, J.G., Sartorelli, A.C. Excess ribonucleotide reductase R2 subunits coordinate the S phase checkpoint to facilitate DNA damage repair and recovery from replication stress. (2007) Biochem. Pharmacol. 73(6):760-72. DOI: 10.1016/j.bcp.2006.11.014

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Shutt, T.E., Shadel, G.S. Expanding the mitochondrial interactome. (2007) Genome Biology. 8(2):203. DOI: 10.1186/gb-2007-8-2-203

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Bonawitz, N.D., Clayton, D.A., Shadel, G.S. Initiation and beyond: multiple functions of the human mitochondrial transcription machinery. (2006) Molecular Cell. 24(6):813-25. DOI: 10.1016/j.molcel.2006.11.024

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Cotney, J., Shadel, G.S. Evidence for an early gene duplication event in the evolution of the mitochondrial transcription factor B family and maintenance of rRNA methyltransferase activity in human mtTFB1 and mtTFB2. (2006) J. Mol. Evol. 63(5):707-17. DOI: 10.1007/s00239-006-0075-1

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Bonawitz, N.D., Rodeheffer, M.S., Shadel, G.S. Defective mitochondrial gene expression results in reactive oxygen species-mediated inhibition of respiration and reduction of yeast life span. (2006) Molecular and Cellular Biology. 26(13):4818-29. DOI: 10.1128/MCB.02360-05

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O’Rourke, T.W., Doudican, N.A., Zhang, H., Eaton, J.S., Doetsch, P.W., Shadel, G.S. Differential involvement of the related DNA helicases Pif1p and Rrm3p in mtDNA point mutagenesis and stability. (2005) Gene. 354:86-92. DOI: 10.1016/j.gene.2005.03.031

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With the exception of base excision repair, conserved pathways and mechanisms that maintain mitochondrial genome stability have remained largely undelineated. In the budding yeast, Saccharomyces cerevisiae, Pif1p is a unique DNA helicase that is localized both to the nucleus and mitochondria, where it is involved in maintaining DNA integrity. We previously elucidated a role for Pif1p in oxidative mtDNA damage resistance that appears to be distinct from its postulated function in mtDNA recombination. Strains lacking Pif1p (pif1Delta) exhibit an increased rate of formation of petite mutants (an indicator of mtDNA instability) and elevated mtDNA point mutagenesis. Here we show that deletion of the RRM3 gene, which encodes a DNA helicase closely related to Pif1p, significantly rescues the petite-induction phenotype of a pif1Delta strain. However, suppression of this phenotype was not accompanied by a corresponding decrease in mtDNA point mutagenesis. Instead, deletion of RRM3 alone resulted in an increase in mtDNA point mutagenesis that was synergistic with that caused by a pif1Delta mutation. In addition, we found that over-expression of RNR1, encoding a large subunit of ribonucleotide reductase (RNR), rescued the petite-induction phenotype of a pif1Delta mutation to a similar extent as deletion of RRM3. This, coupled to our finding that the Rad53p protein kinase is phosphorylated in the rrm3Delta pif1Delta double-mutant strain, leads us to conclude that one mechanism whereby deletion of RRM3 influences mtDNA stability is by modulating mitochondrial deoxynucleoside triphosphate pools. We propose that this is accomplished by signaling through the conserved Mec1/Rad53, S-phase checkpoint pathway to induce the expression and activity of RNR. Altogether, our results define a novel role for Rrm3p in mitochondrial function and indicate that Pif1p and Rrm3p influence a common process (or processes) involved in mtDNA replication, repair, or stability.

Shadel, G.S. Mitochondrial DNA, aconitase ‘wraps’ it up. (2005) Trends in Biochemical Sciences. 30(6):294-6. DOI: 10.1016/j.tibs.2005.04.007

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Doudican, N.A., Song, B., Shadel, G.S., Doetsch, P.W. Oxidative DNA damage causes mitochondrial genomic instability in Saccharomyces cerevisiae. (2005) Molecular and Cellular Biology. 25(12):5196-204. DOI: 10.1128/MCB.25.12.5196-5204.2005

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Taylor, S.D., Zhang, H., Eaton, J.S., Rodeheffer, M.S., Lebedeva, M.A., O’rourke, T.W., Siede, W., Shadel, G.S. The conserved Mec1/Rad53 nuclear checkpoint pathway regulates mitochondrial DNA copy number in Saccharomyces cerevisiae. (2005) Molecular Biology of the Cell. 16(6):3010-8. DOI: 10.1091/mbc.e05-01-0053

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Shadel, G.S. Coupling the mitochondrial transcription machinery to human disease. (2004) Trends in Genetics. 20(10):513-9. DOI: 10.1016/j.tig.2004.08.005

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Shadel, G.S. A dual-function mitochondrial transcription factor tunes out deafness. (2004) Molecular Genetics and Metabolism. 82(1):1-3. DOI: 10.1016/j.ymgme.2004.02.003

McCulloch, V., Shadel, G.S. Human mitochondrial transcription factor B1 interacts with the C-terminal activation region of h-mtTFA and stimulates transcription independently of its RNA methyltransferase activity. (2003) Molecular and Cellular Biology. 23(16):5816-24.

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Rodeheffer, M.S., Shadel, G.S. Multiple interactions involving the amino-terminal domain of yeast mtRNA polymerase determine the efficiency of mitochondrial protein synthesis. (2003) Journal of Biological Chemistry. 278(20):18695-701. DOI: 10.1074/jbc.M301399200

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Seidel-Rogol, B.L., McCulloch, V., Shadel, G.S. Human mitochondrial transcription factor B1 methylates ribosomal RNA at a conserved stem-loop. (2003) Nature Genetics. 33(1):23-4. DOI: 10.1038/ng1064

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Coelho, P.S., Bryan, A.C., Kumar, A., Shadel, G.S., Snyder, M. A novel mitochondrial protein, Tar1p, is encoded on the antisense strand of the nuclear 25S rDNA. (2002) Genes & Development. 16(21):2755-60. DOI: 10.1101/gad.1035002

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O’Rourke, T.W., Doudican, N.A., Mackereth, M.D., Doetsch, P.W., Shadel, G.S. Mitochondrial dysfunction due to oxidative mitochondrial DNA damage is reduced through cooperative actions of diverse proteins. (2002) Molecular and Cellular Biology. 22(12):4086-93.

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Seidel-Rogol, B.L., Shadel, G.S. Modulation of mitochondrial transcription in response to mtDNA depletion and repletion in HeLa cells. (2002) Nucleic Acids Research. 30(9):1929-34.

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McCulloch, V., Seidel-Rogol, B.L., Shadel, G.S. A human mitochondrial transcription factor is related to RNA adenine methyltransferases and binds S-adenosylmethionine. (2002) Molecular and Cellular Biology. 22(4):1116-25.

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Bryan, A.C., Rodeheffer, M.S., Wearn, C.M., Shadel, G.S. Sls1p is a membrane-bound regulator of transcription-coupled processes involved in Saccharomyces cerevisiae mitochondrial gene expression. (2002) Genetics. 160(1):75-82.

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Shadel, G.S., Seidel-Rogol, B.L. Diagnostic assays for defects in mtDNA replication and transcription in yeast and humans. (2001) Methods in Cell Biology. 80:465-79. DOI: 10.1016/S0091-679X(06)80023-3

Seidel-Rogol, B.L., Shadel, G.S. Diagnostic assays for defects in mitochondrial DNA replication and transcription in yeast and human cells. (2001) Methods in Cell Biology. 65:413-27. DOI: 10.1016/s0091-679x(01)65024-6

Rodeheffer, M.S., Boone, B.E., Bryan, A.C., Shadel, G.S. Nam1p, a protein involved in RNA processing and translation, is coupled to transcription through an interaction with yeast mitochondrial RNA polymerase. (2001) Journal of Biological Chemistry. 276(11):8616-22. DOI: 10.1074/jbc.M009901200

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Devine, J.H., Shadel, G.S. Assay of autoinducer activity with luminescent Escherichia coli sensor strains harboring a modified Vibrio fischeri lux regulon. (2000) Meth. Enzymol. 305:279-87. DOI: 10.1016/s0076-6879(00)05494-x

Shadel, G.S., Buckenmeyer, G.A., Clayton, D.A., Schmitt, M.E. Mutational analysis of the RNA component of Saccharomyces cerevisiae RNase MRP reveals distinct nuclear phenotypes. (2000) Gene. 245(1):175-84. DOI: 10.1016/s0378-1119(00)00013-5

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Shadel, G.S. Yeast as a model for human mtDNA replication. (1999) American Journal of Human Genetics. 65(5):1230-7. DOI: 10.1086/302630

Wang, Y., Shadel, G.S. Stability of the mitochondrial genome requires an amino-terminal domain of yeast mitochondrial RNA polymerase. (1999) Proceedings of the National Academy of Sciences of the United States of America. 96(14):8046-51. DOI: 10.1073/pnas.96.14.8046

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Shadel, G.S., Clayton, D.A. Mitochondrial DNA maintenance in vertebrates. (1997) Annual Review of Biochemistry. 66:409-35. DOI: 10.1146/annurev.biochem.66.1.409

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Sitnikov, D.M., Shadel, G.S., Baldwin, T.O. Autoinducer-independent mutants of the LuxR transcriptional activator exhibit differential effects on the two lux promoters of Vibrio fischeri. (1996) Molecular & General Genetics. 252(5):622-5. DOI: 10.1007/bf02172408

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Shadel, G.S., Clayton, D.A. Isolation and characterization of vertebrate mitochondrial transcription factor A homologs. (1996) Meth. Enzymol. 264:149-58. DOI: 10.1016/s0076-6879(96)64016-6

Shadel, G.S., Clayton, D.A. Mapping promoters in displacement-loop region of vertebrate mitochondrial DNA. (1996) Meth. Enzymol. 264:139-48. DOI: 10.1016/s0076-6879(96)64015-4

Carrodeguas, J.A., Yun, S., Shadel, G.S., Clayton, D.A., Bogenhagen, D.F. Functional conservation of yeast mtTFB despite extensive sequence divergence. (1996) Gene Expr. 6(4):219-30.

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Dairaghi, D.J., Shadel, G.S., Clayton, D.A. Human mitochondrial transcription factor A and promoter spacing integrity are required for transcription initiation. (1995) Biochimica et Biophysica Acta. 1271(1):127-34. DOI: 10.1016/0925-4439(95)00019-z

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Dairaghi, D.J., Shadel, G.S., Clayton, D.A. Addition of a 29 residue carboxyl-terminal tail converts a simple HMG box-containing protein into a transcriptional activator. (1995) Journal of Molecular Biology. 249(1):11-28. DOI: 10.1006/jmbi.1995.9889

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Shadel, G.S., Clayton, D.A. A Saccharomyces cerevisiae mitochondrial transcription factor, sc-mtTFB, shares features with sigma factors but is functionally distinct. (1995) Molecular and Cellular Biology. 15(4):2101-8. DOI: 10.1128/mcb.15.4.2101

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Shadel, G.S., Clayton, D.A. Mitochondrial transcription initiation. Variation and conservation. (1993) Journal of Biological Chemistry. 268(22):16083-6.

Shadel, G.S., Baldwin, T.O. Positive autoregulation of the Vibrio fischeri luxR gene. LuxR and autoinducer activate cAMP-catabolite gene activator protein complex-independent and -dependent luxR transcription. (1992) Journal of Biological Chemistry. 267(11):7696-702.

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Shadel, G.S., Baldwin, T.O. Identification of a distantly located regulatory element in the luxD gene required for negative autoregulation of the Vibrio fischeri luxR gene. (1992) Journal of Biological Chemistry. 267(11):7690-5.

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Shadel, G.S., Baldwin, T.O. The Vibrio fischeri LuxR protein is capable of bidirectional stimulation of transcription and both positive and negative regulation of the luxR gene. (1991) J. Bacteriol. 173(2):568-74. DOI: 10.1128/jb.173.2.568-574.1991

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Regulation of the genes required for bioluminescence in the marine bacterium Vibrio fischeri (the lux regulon) is a complex process requiring coordination of several systems. The primary level of regulation is mediated by a positive regulatory protein, LuxR, and a small diffusible molecule, N-(3-oxo-hexanoyl)-homoserine lactone, termed autoinducer. Transcription of the luxR gene, which encodes the regulatory protein, is positively regulated by the cyclic AMP-CAP system. The lux regulon of V. fischeri consists of two divergently transcribed operons designated operonL and operonR. Transcription of the rightward operon (operonR; luxICDABE), consisting of the genes required for autoinducer synthesis (luxI) and light production (luxCDABE), is activated by LuxR in an autoinducer-dependent fashion. The leftward operon (operonL) consists of a single known gene, luxR. The LuxR protein has also been shown to decrease transcription of operonL through an autoinducer-dependent mechanism, thereby negatively regulating its own synthesis. In this paper we demonstrate that the autoinducer-dependent repression of operonL transcription requires not only LuxR but also DNA sequences within operonR which occur upstream of the promoter for operonL. In the absence of these DNA sequences, the LuxR protein causes an autoinducer-dependent activation of transcription of operonL. The lux operator, located in the control region between the two operons, was required for both the positive and negative autoinducer-dependent responses. By titration of high levels of LuxR supplied in trans with synthetic autoinducer, we found that low levels of autoinducer could elicit a positive response even in the presence of the negative-acting DNA sequences, while higher levels of autoinducer resulted in a negative response. Without these DNA sequences in operonR, LuxR and autoinducer stimulated transcription regardless of the level of autoinducer. These results suggest that a switch between stimulation and repression of operonL transcription is mediated by the levels of the LuxR-autoinducer complex, which in these experiments reflects the level of autoinducer in the growth medium.

Shadel, G.S., Young, R., Baldwin, T.O. Use of regulated cell lysis in a lethal genetic selection in Escherichia coli: identification of the autoinducer-binding region of the LuxR protein from Vibrio fischeri ATCC 7744. (1990) J. Bacteriol. 172(7):3980-7. DOI: 10.1128/jb.172.7.3980-3987.1990

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Shadel, G.S., Devine, J.H., Baldwin, T.O. Control of the lux regulon of Vibrio fischeri. (1990) J. Biolumin. Chemilumin. 5(2):99-106. DOI: 10.1002/bio.1170050205

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Devine, J.H., Shadel, G.S., Baldwin, T.O. Identification of the operator of the lux regulon from the Vibrio fischeri strain ATCC7744. (1989) Proceedings of the National Academy of Sciences of the United States of America. 86(15):5688-92. DOI: 10.1073/pnas.86.15.5688

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Escherichia coli that carry a recombinant plasmid bearing the Vibrio fischeri lux regulon express luminescence that mimics the luminescence of V. fischeri. The lux regulon consists of two divergently transcribed operons, the rightward operon (luxICDABE genes) and the leftward operon (luxR gene). The luxR and luxI genes and the control region separating the two operons supply the primary regulatory control over the lux regulon; the regulatory mechanisms result in a dramatic increase in the rate of luciferase synthesis after induction, apparently due to a unique autoregulatory positive feedback mechanism, and in an enormous difference (greater than 10(4] in levels of luminescence in cells before and after induction. The generally accepted model of primary regulation of bioluminescence in V. fischeri involves the interaction of the product of the luxR gene and N-(3-oxohexanoyl)homoserine lactone, the autoinducer produced by the enzyme encoded by luxI, the first gene of the rightward operon, with an operator sequence within the control region to stimulate transcription of the rightward operon in a positive feedback loop. We have used deletion mapping of a transcription reporter vector to determine the approximate location of the operator. By site-directed mutagenesis of the presumed operator, we have demonstrated that the 20-base-pair inverted repeat ACCTGTAGGA/TCGTA CAGGT (where the vertical line is the center of symmetry), which bears striking similarity to the recognition sequence for the pleiotropic repressor protein LexA, is the operator of the lux regulon. We also found that deletion of sequences upstream of the palindrome leads to increased transcription from the rightward promoter (PR), indicative of a cis-acting element that represses transcription in the absence of the LuxR-autoinducer complex. Modifications of the palindrome that eliminate stimulation by LuxR-autoinducer of transcription from PR have no effect on repression by the cis-acting mechanism(s), suggesting that the palindrome is not necessary for repression of the rightward operon. Thus, it appears that the large increase in transcription upon induction of the lux regulon is the result of at least two independent mechanisms, one positive and the other negative.

Baldwin, T.O., Devine, J.H., Heckel, R.C., Lin, J.W., Shadel, G.S. The complete nucleotide sequence of the lux regulon of Vibrio fischeri and the luxABN region of Photobacterium leiognathi and the mechanism of control of bacterial bioluminescence. (1989) J. Biolumin. Chemilumin. 4(1):326-41. DOI: 10.1002/bio.1170040145

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We have determined the complete nucleotide sequence of a 7622 base pair fragment of DNA from Vibrio fischeri strain ATCC7744 that contains all the information required to confer plasmid-borne, regulated bioluminescence upon strains of Escherichia coli. The lux regulon from V. fischeri consists of two divergently transcribed operons, L (left) and R (right), and at least seven genes, luxR (L operon) and luxICDABE (R operon) and the intervening control region. The luxA and luxB genes encode respectively the alpha and beta subunits of luciferase. The gene order luxCDABE seen in V. fischeri is the same as for V. harveyi. We have determined the sequence of the luxAB and flanking regions from Photobacterium leiognathi and have found upstream sequences homologous with luxC from the Vibrio species, but between luxB and luxE, there is an open reading frame encoding a protein of 227 amino acids (26,229 molecular weight) that is not found in this location in the Vibrio species. The amino terminal amino acid sequence of the encoded protein is nearly identical to that determined by O’Kane and Lee (University of Georgia) for the non-fluorescent flavoprotein from a closely related Photobacterium species (Dr Dennis O’Kane, personal communication). We have therefore designated this gene luxN. There is a 20-base inverted repeat ACCTGTAGGAxTCGTACAGGT, centred between bases 927 and 928 in the region between the two operons of V. fischeri. This region appears to fulfil two functions: it is critical for the LuxR protein to exert its effect and it is a consensus binding site for the E. coli LexA protein, a negative regulatory protein involved with the SOS response. There are sequences within the luxR coding region that appear to function in a cis-acting fashion to repress transcription from both the leftward and rightward promoters in the absence of the respective transcriptional activator proteins, thereby resulting in low basal levels of transcription. It now appears clear that there are multiple levels of control on the lux system allowing for a modulation of the intensity of bioluminescence of over four orders of magnitude.