Publications by authors named "Daniel Bogenhagen"

32 Publications

A549 cells contain enlarged mitochondria with independently functional clustered mtDNA nucleoids.

PLoS One 2021 25;16(3):e0249047. Epub 2021 Mar 25.

Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, United States of America.

Mitochondria are commonly viewed as highly elongated organelles with regularly spaced mtDNA genomes organized as compact nucleoids that generate the local transcripts essential for production of mitochondrial ribosomes and key components of the respiratory chain. In contrast, A549 human lung carcinoma cells frequently contain apparently swollen mitochondria harboring multiple discrete mtDNA nucleoids and RNA processing granules in a contiguous matrix compartment. While this seemingly aberrant mitochondrial morphology is akin to "mito-bulbs" previously described in cells exposed to a variety of genomic stressors, it occurs in A549 cells under typical culture conditions. We provide a detailed confocal and super-resolution microscopic investigation of the incidence of such mito-bulbs in A549 cells. Most mito-bulbs appear stable, engage in active replication and transcription, and maintain respiration but feature an elevated oxidative environment. High concentrations of glucose and/or L-glutamine in growth media promote a greater incidence of mito-bulbs. Furthermore, we demonstrate that treatment of A549 cells with TGFβ suppresses the formation of mito-bulbs while treatment with a specific TGFβ pathway inhibitor substantially increases incidence. This striking heterogeneity of mitochondrial form and function may play an important role in a variety of diseases involving mitochondrial dysfunction.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0249047PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7993880PMC
March 2021

The complicated role of mitochondria in the podocyte.

Am J Physiol Renal Physiol 2020 12 19;319(6):F955-F965. Epub 2020 Oct 19.

Division of Nephrology, Department of Medicine, Renaissance School of Medicine at Stony Brook University, Stony Brook, New York.

Mitochondria play a complex role in maintaining cellular function including ATP generation, generation of biosynthetic precursors for macromolecules, maintenance of redox homeostasis, and metabolic waste management. Although the contribution of mitochondrial function in various kidney diseases has been studied, there are still avenues that need to be explored under healthy and diseased conditions. Mitochondrial damage and dysfunction have been implicated in experimental models of podocytopathy as well as in humans with glomerular diseases resulting from podocyte dysfunction. Specifically, in the podocyte, metabolism is largely driven by oxidative phosphorylation or glycolysis depending on the metabolic needs. These metabolic needs may change drastically in the presence of podocyte injury in glomerular diseases such as diabetic kidney disease or focal segmental glomerulosclerosis. Here, we review the role of mitochondria in the podocyte and the factors regulating its function at baseline and in a variety of podocytopathies to identify potential targets for therapy.
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http://dx.doi.org/10.1152/ajprenal.00393.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7792691PMC
December 2020

Mitochondrial genetic variation is enriched in G-quadruplex regions that stall DNA synthesis in vitro.

Hum Mol Genet 2020 05;29(8):1292-1309

Laboratory of Molecular Gerontology, National Institute on Aging, Baltimore, MD 21224, USA.

As the powerhouses of the eukaryotic cell, mitochondria must maintain their genomes which encode proteins essential for energy production. Mitochondria are characterized by guanine-rich DNA sequences that spontaneously form unusual three-dimensional structures known as G-quadruplexes (G4). G4 structures can be problematic for the essential processes of DNA replication and transcription because they deter normal progression of the enzymatic-driven processes. In this study, we addressed the hypothesis that mitochondrial G4 is a source of mutagenesis leading to base-pair substitutions. Our computational analysis of 2757 individual genomes from two Italian population cohorts (SardiNIA and InCHIANTI) revealed a statistically significant enrichment of mitochondrial mutations within sequences corresponding to stable G4 DNA structures. Guided by the computational analysis results, we designed biochemical reconstitution experiments and demonstrated that DNA synthesis by two known mitochondrial DNA polymerases (Pol γ, PrimPol) in vitro was strongly blocked by representative stable G4 mitochondrial DNA structures, which could be overcome in a specific manner by the ATP-dependent G4-resolving helicase Pif1. However, error-prone DNA synthesis by PrimPol using the G4 template sequence persisted even in the presence of Pif1. Altogether, our results suggest that genetic variation is enriched in G-quadruplex regions that impede mitochondrial DNA replication.
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http://dx.doi.org/10.1093/hmg/ddaa043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7254849PMC
May 2020

Pulse-chase SILAC-based analyses reveal selective oversynthesis and rapid turnover of mitochondrial protein components of respiratory complexes.

J Biol Chem 2020 02 23;295(9):2544-2554. Epub 2020 Jan 23.

Department of Pathology, Stony Brook University, Stony Brook, New York 11794-8691; Proteomics Center, Stony Brook University, Stony Brook, New York 11794-8691.

Mammalian mitochondria assemble four complexes of the respiratory chain (RCI, RCIII, RCIV, and RCV) by combining 13 polypeptides synthesized within mitochondria on mitochondrial ribosomes (mitoribosomes) with over 70 polypeptides encoded in nuclear DNA, translated on cytoplasmic ribosomes, and imported into mitochondria. We have previously observed that mitoribosome assembly is inefficient because some mitoribosomal proteins are produced in excess, but whether this is the case for other mitochondrial assemblies such as the RCs is unclear. We report here that pulse-chase stable isotope labeling with amino acids in cell culture (SILAC) is a valuable technique to study RC assembly because it can reveal considerable differences in the assembly rates and efficiencies of the different complexes. The SILAC analyses of HeLa cells indicated that assembly of RCV, comprising F/F-ATPase, is rapid with little excess subunit synthesis, but that assembly of RCI ( NADH dehydrogenase) is far less efficient, with dramatic oversynthesis of numerous proteins, particularly in the matrix-exposed N and Q domains. Unassembled subunits were generally degraded within 3 h. We also observed differential assembly kinetics for individual complexes that were immunoprecipitated with complex-specific antibodies. Immunoprecipitation with an antibody that recognizes the ND1 subunit of RCI co-precipitated a number of proteins implicated in FeS cluster assembly and newly synthesized ubiquinol-cytochrome reductase Rieske iron-sulfur polypeptide 1 (UQCRFS1), the Rieske FeS protein in RCIII, reflecting some coordination between RCI and RCIII assemblies. We propose that pulse-chase SILAC labeling is a useful tool for studying rates of protein complex assembly and degradation.
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http://dx.doi.org/10.1074/jbc.RA119.011791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049976PMC
February 2020

Pulse SILAC Approaches to the Measurement of Cellular Dynamics.

Adv Exp Med Biol 2019 ;1140:575-583

Biological Mass Spectrometry Center, Stony Brook University School of Medicine, Stony Brook, NY, USA.

The global measurement of assembly and turnover of protein containing complexes within cells has advanced with the development of pulse stable isotope labelled amino acid approaches. Stable isotope labeling with amino acids in cell culture (SILAC) allows the incorporation of "light" 12-carbon amino acids or "heavy" 13-carbon amino acids into cells or organisms and the quantitation of proteins and peptides containing these amino acid tags using mass spectrometry. The use of these labels in pulse or pulse-chase scenarios has enabled measurements of macromolecular dynamics in cells, on time scales of several hours. Here we review advances with this approach and alternative or parallel strategies. We also examine the statistical considerations impacting datasets detailing mitochondrial assembly, to highlight key parameters in establishing significance and reproducibility.
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http://dx.doi.org/10.1007/978-3-030-15950-4_34DOI Listing
September 2019

Podocyte-Specific Loss of Krüppel-Like Factor 6 Increases Mitochondrial Injury in Diabetic Kidney Disease.

Diabetes 2018 11 16;67(11):2420-2433. Epub 2018 Aug 16.

Division of Nephrology, Department of Medicine, Stony Brook University, Stony Brook, NY

Mitochondrial injury is uniformly observed in several murine models as well as in individuals with diabetic kidney disease (DKD). Although emerging evidence has highlighted the role of key transcriptional regulators in mitochondrial biogenesis, little is known about the regulation of mitochondrial cytochrome c oxidase assembly in the podocyte under diabetic conditions. We recently reported a critical role of the zinc finger Krüppel-like factor 6 (KLF6) in maintaining mitochondrial function and preventing apoptosis in a proteinuric murine model. In this study, we report that podocyte-specific knockdown of increased the susceptibility to streptozotocin-induced DKD in the resistant C57BL/6 mouse strain. We observed that the loss of in podocytes reduced the expression of with resultant increased mitochondrial injury, leading to activation of the intrinsic apoptotic pathway under diabetic conditions. Conversely, mitochondrial injury and apoptosis were significantly attenuated with overexpression of in cultured human podocytes under hyperglycemic conditions. Finally, we observed a significant reduction in glomerular and podocyte-specific expression of KLF6 in human kidney biopsies with progression of DKD. Collectively, these data suggest that podocyte-specific KLF6 is critical to preventing mitochondrial injury and apoptosis under diabetic conditions.
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http://dx.doi.org/10.2337/db17-0958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6198342PMC
November 2018

Kinetics and Mechanism of Mammalian Mitochondrial Ribosome Assembly.

Cell Rep 2018 02;22(7):1935-1944

Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794-8651, USA.

Mammalian mtDNA encodes only 13 proteins, all essential components of respiratory complexes, synthesized by mitochondrial ribosomes. Mitoribosomes contain greatly truncated RNAs transcribed from mtDNA, including a structural tRNA in place of 5S RNA as a scaffold for binding 82 nucleus-encoded proteins, mitoribosomal proteins (MRPs). Cryoelectron microscopy (cryo-EM) studies have determined the structure of the mitoribosome, but its mechanism of assembly is unknown. Our SILAC pulse-labeling experiments determine the rates of mitochondrial import of MRPs and their assembly into intact mitoribosomes, providing a basis for distinguishing MRPs that bind at early and late stages in mitoribosome assembly to generate a working model for mitoribosome assembly. Mitoribosome assembly is a slow process initiated at the mtDNA nucleoid driven by excess synthesis of individual MRPs. MRPs that are tightly associated in the structure frequently join the complex in a coordinated manner. Clinically significant MRP mutations reported to date affect proteins that bind early on during assembly.
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http://dx.doi.org/10.1016/j.celrep.2018.01.066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855118PMC
February 2018

Extracellular Mitochondrial DNA Is Generated by Fibroblasts and Predicts Death in Idiopathic Pulmonary Fibrosis.

Am J Respir Crit Care Med 2017 12;196(12):1571-1581

4 Department of Pathology, Stony Brook University School of Medicine, Stony Brook, New York.

Rationale: Idiopathic pulmonary fibrosis (IPF) involves the accumulation of α-smooth muscle actin-expressing myofibroblasts arising from interactions with soluble mediators such as transforming growth factor-β1 (TGF-β1) and mechanical influences such as local tissue stiffness. Whereas IPF fibroblasts are enriched for aerobic glycolysis and innate immune receptor activation, innate immune ligands related to mitochondrial injury, such as extracellular mitochondrial DNA (mtDNA), have not been identified in IPF.

Objectives: We aimed to define an association between mtDNA and fibroblast responses in IPF.

Methods: We evaluated the response of normal human lung fibroblasts (NHLFs) to stimulation with mtDNA and determined whether the glycolytic reprogramming that occurs in response to TGF-β1 stimulation and direct contact with stiff substrates, and spontaneously in IPF fibroblasts, is associated with excessive levels of mtDNA. We measured mtDNA concentrations in bronchoalveolar lavage (BAL) from subjects with and without IPF, as well as in plasma samples from two longitudinal IPF cohorts and demographically matched control subjects.

Measurements And Main Results: Exposure to mtDNA augments α-smooth muscle actin expression in NHLFs. The metabolic changes in NHLFs that are induced by interactions with TGF-β1 or stiff hydrogels are accompanied by the accumulation of extracellular mtDNA. These findings replicate the spontaneous phenotype of IPF fibroblasts. mtDNA concentrations are increased in IPF BAL and plasma, and in the latter compartment, they display robust associations with disease progression and reduced event-free survival.

Conclusions: These findings demonstrate a previously unrecognized and highly novel connection between metabolic reprogramming, mtDNA, fibroblast activation, and clinical outcomes that provides new insight into IPF.
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http://dx.doi.org/10.1164/rccm.201612-2480OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754440PMC
December 2017

Scalable Isolation of Mammalian Mitochondria for Nucleic Acid and Nucleoid Analysis.

Methods Mol Biol 2016 ;1351:67-79

Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, 11794-8651, USA.

Isolation of mitochondria from cultured cells and animal tissues for analysis of nucleic acids and bona fide mitochondrial nucleic acid binding proteins and enzymes is complicated by contamination with cellular nucleic acids and their adherent proteins. Protocols presented here allow for quick isolation of mitochondria from a small number of cells and for preparation of highly purified mitochondria from a larger number of cells using nuclease treatment and high salt washing of mitochondria to reduce contamination. We further describe a method for the isolation of mitochondrial DNA-protein complexes known as nucleoids from these highly purified mitochondria using a combination of glycerol gradient sedimentation followed by isopycnic centrifugation in a non-ionic iodixanol gradient.
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http://dx.doi.org/10.1007/978-1-4939-3040-1_6DOI Listing
September 2016

Assignment of 2'-O-methyltransferases to modification sites on the mammalian mitochondrial large subunit 16 S ribosomal RNA (rRNA).

J Biol Chem 2014 Sep 29;289(36):24936-42. Epub 2014 Jul 29.

From the Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York 11794-8651

Advances in proteomics and large scale studies of potential mitochondrial proteins have led to the identification of many novel mitochondrial proteins in need of further characterization. Among these novel proteins are three mammalian rRNA methyltransferase family members RNMTL1, MRM1, and MRM2. MRM1 and MRM2 have bacterial and yeast homologs, whereas RNMTL1 appears to have evolved later in higher eukaryotes. We recently confirmed the localization of the three proteins to mitochondria, specifically in the vicinity of mtDNA nucleoids. In this study, we took advantage of the ability of 2'-O-ribose modification to block site-specific cleavage of RNA by DNAzymes to show that MRM1, MRM2, and RNMTL1 are responsible for modification of human large subunit rRNA at residues G(1145), U(1369), and G(1370), respectively.
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http://dx.doi.org/10.1074/jbc.C114.581868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4155661PMC
September 2014

Initial steps in RNA processing and ribosome assembly occur at mitochondrial DNA nucleoids.

Cell Metab 2014 Apr;19(4):618-29

Proteomics Center, Stony Brook University, Stony Brook, NY 11794-8691, USA.

Mammalian mitochondrial DNA (mtDNA) resides in compact nucleoids, where it is replicated and transcribed into long primary transcripts processed to generate rRNAs, tRNAs, and mRNAs encoding 13 proteins. This situation differs from bacteria and eukaryotic nucleoli, which have dedicated rRNA transcription units. The assembly of rRNAs into mitoribosomes has received little study. We show that mitochondrial RNA processing enzymes involved in tRNA excision, ribonuclease P (RNase P) and ELAC2, as well as a subset of nascent mitochondrial ribosomal proteins (MRPs) associate with nucleoids to initiate RNA processing and ribosome assembly. SILAC pulse-chase labeling experiments show that nascent MRPs recruited to the nucleoid fraction were highly labeled after the pulse in a transcription-dependent manner and decreased in labeling intensity during the chase. These results provide insight into the landscape of binding events required for mitochondrial ribosome assembly and firmly establish the mtDNA nucleoid as a control center for mitochondrial biogenesis.
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http://dx.doi.org/10.1016/j.cmet.2014.03.013DOI Listing
April 2014

Mitochondrial ribosomal RNA (rRNA) methyltransferase family members are positioned to modify nascent rRNA in foci near the mitochondrial DNA nucleoid.

J Biol Chem 2013 Oct 13;288(43):31386-99. Epub 2013 Sep 13.

From the Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York 11794-8651.

We have identified RNMTL1, MRM1, and MRM2 (FtsJ2) as members of the RNA methyltransferase family that may be responsible for the three known 2'-O-ribose modifications of the 16 S rRNA core of the large mitochondrial ribosome subunit. These proteins are confined to foci located in the vicinity of mtDNA nucleoids. They show distinct patterns of association with mtDNA nucleoids and/or mitochondrial ribosomes in cell fractionation studies. We focused on the role of the least studied protein in this set, RNMTL1, to show that this protein interacts with the large ribosomal subunit as well as with a series of non-ribosomal proteins that may be involved in coupling of the rate of rRNA transcription and ribosome assembly in mitochondria. siRNA-directed silencing of RNMTL1 resulted in a significant inhibition of translation on mitochondrial ribosomes. Our results are consistent with a role for RNMTL1 in methylation of G(1370) of human 16 S rRNA.
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http://dx.doi.org/10.1074/jbc.M113.515692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829452PMC
October 2013

Phosphorylation of human TFAM in mitochondria impairs DNA binding and promotes degradation by the AAA+ Lon protease.

Mol Cell 2013 Jan 29;49(1):121-32. Epub 2012 Nov 29.

Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA.

Human mitochondrial transcription factor A (TFAM) is a high-mobility group (HMG) protein at the nexus of mitochondrial DNA (mtDNA) replication, transcription, and inheritance. Little is known about the mechanisms underlying its posttranslational regulation. Here, we demonstrate that TFAM is phosphorylated within its HMG box 1 (HMG1) by cAMP-dependent protein kinase in mitochondria. HMG1 phosphorylation impairs the ability of TFAM to bind DNA and to activate transcription. We show that only DNA-free TFAM is degraded by the Lon protease, which is inhibited by the anticancer drug bortezomib. In cells with normal mtDNA levels, HMG1-phosphorylated TFAM is degraded by Lon. However, in cells with severe mtDNA deficits, nonphosphorylated TFAM is also degraded, as it is DNA free. Depleting Lon in these cells increases levels of TFAM and upregulates mtDNA content, albeit transiently. Phosphorylation and proteolysis thus provide mechanisms for rapid fine-tuning of TFAM function and abundance in mitochondria, which are crucial for maintaining and expressing mtDNA.
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http://dx.doi.org/10.1016/j.molcel.2012.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3586414PMC
January 2013

Mitochondrial DNA nucleoid structure.

Biochim Biophys Acta 2012 Sep-Oct;1819(9-10):914-20. Epub 2011 Nov 27.

Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA.

Eukaryotic cells are characterized by their content of intracellular membrane-bound organelles, including mitochondria as well as nuclei. These two DNA-containing compartments employ two distinct strategies for storage and readout of genetic information. The diploid nuclei of human cells contain about 6 billion base pairs encoding about 25,000 protein-encoding genes, averaging 120 kB/gene, packaged in chromatin arranged as a regular nucleosomal array. In contrast, human cells contain hundreds to thousands of copies of a ca.16 kB mtDNA genome tightly packed with 13 protein-coding genes along with rRNA and tRNA genes required for their expression. The mtDNAs are dispersed throughout the mitochondrial network as histone-free nucleoids containing single copies or small clusters of genomes. This review will summarize recent advances in understanding the microscopic structure and molecular composition of mtDNA nucleoids in higher eukaryotes. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.
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http://dx.doi.org/10.1016/j.bbagrm.2011.11.005DOI Listing
October 2012

Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction.

Mol Cell Biol 2011 Dec 17;31(24):4994-5010. Epub 2011 Oct 17.

Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147, USA.

A fundamental objective in molecular biology is to understand how DNA is organized in concert with various proteins, RNA, and biological membranes. Mitochondria maintain and express their own DNA (mtDNA), which is arranged within structures called nucleoids. Their functions, dimensions, composition, and precise locations relative to other mitochondrial structures are poorly defined. Superresolution fluorescence microscopy techniques that exceed the previous limits of imaging within the small and highly compartmentalized mitochondria have been recently developed. We have improved and employed both two- and three-dimensional applications of photoactivated localization microscopy (PALM and iPALM, respectively) to visualize the core dimensions and relative locations of mitochondrial nucleoids at an unprecedented resolution. PALM reveals that nucleoids differ greatly in size and shape. Three-dimensional volumetric analysis indicates that, on average, the mtDNA within ellipsoidal nucleoids is extraordinarily condensed. Two-color PALM shows that the freely diffusible mitochondrial matrix protein is largely excluded from the nucleoid. In contrast, nucleoids are closely associated with the inner membrane and often appear to be wrapped around cristae or crista-like inner membrane invaginations. Determinations revealing high packing density, separation from the matrix, and tight association with the inner membrane underscore the role of mechanisms that regulate access to mtDNA and that remain largely unknown.
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http://dx.doi.org/10.1128/MCB.05694-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3233019PMC
December 2011

Does mtDNA nucleoid organization impact aging?

Exp Gerontol 2010 Aug 11;45(7-8):473-7. Epub 2009 Dec 11.

Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA.

Somatic cells in tissue culture package several copies of mitochondrial DNA (mtDNA) in aggregates known as nucleoids that appear to be remarkably stable. The clustering of multiple mtDNA genomes in a single nucleoid complex may promote the progressive age-related accumulation of deletion and point mutations in mtDNA in many somatic tissues, particularly in post-mitotic cells. In contrast, oocytes appear to have the ability to select against deleterious mutations in mtDNA, at least in mice. This fundamental difference suggests that oocytes may be better able to detect and remove defective mtDNA genomes than somatic cells, possibly due in part to the simpler organization of the mtDNA in smaller nucleoids. These observations suggest the hypothesis that a complex nucleoid structure containing several mtDNA molecules may impair the ability of the cell to select against deleterious mtDNA mutations, thereby contributing to age-related mitochondrial dysfunction.
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http://dx.doi.org/10.1016/j.exger.2009.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2879461PMC
August 2010

Biochemical isolation of mtDNA nucleoids from animal cells.

Methods Mol Biol 2009 ;554:3-14

Department of Pharmacological Sciences, University at Stony Brook, Stony Brook, NY, USA.

Mitochondrial DNA (mtDNA) in animal cells is organized into clusters of 5-7 genomes referred to as nucleoids. Contrary to the notion that mtDNA is largely free of bound proteins, these structures are nearly as rich in protein as nuclear chromatin. While the purification of intact, membrane-bound mitochondria is an established method, relatively few studies have attempted biochemical purification of mtDNA nucleoids. In this chapter, two alternative methods are presented for the purification of nucleoids. The first method yields the so-called native nucleoids, using conditions designed to preserve non-covalent protein-DNA and protein-protein interactions. The second method uses formaldehyde to crosslink proteins to mtDNA and exposes nucleoids to treatment with harsh detergents and high salt concentrations.
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http://dx.doi.org/10.1007/978-1-59745-521-3_1DOI Listing
October 2009

Human DNA2 is a mitochondrial nuclease/helicase for efficient processing of DNA replication and repair intermediates.

Mol Cell 2008 Nov;32(3):325-36

Department of Radiation Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA 91010, USA.

DNA2, a helicase/nuclease family member, plays versatile roles in processing DNA intermediates during DNA replication and repair. Yeast Dna2 (yDna2) is essential in RNA primer removal during nuclear DNA replication and is important in repairing UV damage, base damage, and double-strand breaks. Our data demonstrate that, surprisingly, human DNA2 (hDNA2) does not localize to nuclei, as it lacks a nuclear localization signal equivalent to that present in yDna2. Instead, hDNA2 migrates to the mitochondria, interacts with mitochondrial DNA polymerase gamma, and significantly stimulates polymerase activity. We further demonstrate that hDNA2 and flap endonuclease 1 synergistically process intermediate 5' flap structures occurring in DNA replication and long-patch base excision repair (LP-BER) in mitochondria. Depletion of hDNA2 from a mitochondrial extract reduces its efficiency in RNA primer removal and LP-BER. Taken together, our studies illustrate an evolutionarily diversified role of hDNA2 in mitochondrial DNA replication and repair in a mammalian system.
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http://dx.doi.org/10.1016/j.molcel.2008.09.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2636562PMC
November 2008

Removal of oxidative DNA damage via FEN1-dependent long-patch base excision repair in human cell mitochondria.

Mol Cell Biol 2008 Aug 9;28(16):4975-87. Epub 2008 Jun 9.

Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, Massachusetts 02115, USA.

Repair of oxidative DNA damage in mitochondria was thought limited to short-patch base excision repair (SP-BER) replacing a single nucleotide. However, certain oxidative lesions cannot be processed by SP-BER. Here we report that 2-deoxyribonolactone (dL), a major type of oxidized abasic site, inhibits replication by mitochondrial DNA (mtDNA) polymerase gamma and interferes with SP-BER by covalently trapping polymerase gamma during attempted dL excision. However, repair of dL was detected in human mitochondrial extracts, and we show that this repair is via long-patch BER (LP-BER) dependent on flap endonuclease 1 (FEN1), not previously known to be present in mitochondria. FEN1 was retained in protease-treated mitochondria and detected in mitochondrial nucleoids that contain known mitochondrial replication and transcription proteins. Results of immunofluorescence and subcellular fractionation studies were also consistent with the presence of FEN1 in the mitochondria of intact cells. Immunodepletion experiments showed that the LP-BER activity of mitochondrial extracts was strongly diminished in parallel with the removal of FEN1, although some activity remained, suggesting the presence of an additional flap-removing enzyme. Biological evidence for a FEN1 role in repairing mitochondrial oxidative DNA damage was provided by RNA interference experiments, with the extent of damage greater and the recovery slower in FEN1-depleted cells than in control cells. The mitochondrial LP-BER pathway likely plays important roles in repairing dL lesions and other oxidative lesions and perhaps in normal mtDNA replication.
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http://dx.doi.org/10.1128/MCB.00457-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519700PMC
August 2008

The layered structure of human mitochondrial DNA nucleoids.

J Biol Chem 2008 Feb 6;283(6):3665-3675. Epub 2007 Dec 6.

Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651.

Mitochondrial DNA (mtDNA) occurs in cells in nucleoids containing several copies of the genome. Previous studies have identified proteins associated with these large DNA structures when they are biochemically purified by sedimentation and immunoaffinity chromatography. In this study, formaldehyde cross-linking was performed to determine which nucleoid proteins are in close contact with the mtDNA. A set of core nucleoid proteins is found in both native and cross-linked nucleoids, including 13 proteins with known roles in mtDNA transactions. Several other metabolic proteins and chaperones identified in native nucleoids, including ATAD3, were not observed to cross-link to mtDNA. Additional immunofluorescence and protease susceptibility studies showed that an N-terminal domain of ATAD3 previously proposed to bind to the mtDNA D-loop is directed away from the mitochondrial matrix, so it is unlikely to interact with mtDNA in vivo. These results are discussed in relation to a model for a layered structure of mtDNA nucleoids in which replication and transcription occur in the central core, whereas translation and complex assembly may occur in the peripheral region.
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http://dx.doi.org/10.1074/jbc.M708444200DOI Listing
February 2008

A quantitative proteomic analysis of mitochondrial participation in p19 cell neuronal differentiation.

J Proteome Res 2008 Jan 23;7(1):328-38. Epub 2007 Nov 23.

Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651, USA.

A quantitative proteomic analysis of changes in protein expression accompanying the differentiation of P19 mouse embryonal carcinoma cells into neuron-like cells using isobaric tag technology coupled with LC-MS/MS revealed protein changes reflecting withdrawal from the cell cycle accompanied by a dynamic reorganization of the cytoskeleton and an up-regulation of mitochondrial biogenesis. Further study of quantitative changes in abundance of individual proteins in a purified mitochondrial fraction showed that most mitochondrial proteins increased significantly in abundance. A set of chaperone proteins did not participate in this increase, suggesting that neuron-like cells are relatively deficient in mitochondrial chaperones. We developed a procedure to account for differences in recovery of mitochondrial proteins during purification of organelles from distinct cell or tissue sources. Proteomic data supported by RT-PCR analysis suggests that enhanced mitochondrial biogenesis during neuronal differentiation may reflect a large increase in expression of PGC-1alpha combined with down-regulation of its negative regulator, p160 Mybbp1a.
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http://dx.doi.org/10.1021/pr070300gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2547413PMC
January 2008

The EM structure of human DNA polymerase gamma reveals a localized contact between the catalytic and accessory subunits.

EMBO J 2007 Oct 30;26(19):4283-91. Epub 2007 Aug 30.

Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA.

We used electron microscopy to examine the structure of human DNA pol gamma, the heterotrimeric mtDNA replicase implicated in certain mitochondrial diseases and aging models. Separate analysis of negatively stained preparations of the catalytic subunit, pol gammaA, and of the holoenzyme including a dimeric accessory factor, pol gammaB(2), permitted unambiguous identification of the position of the accessory factor within the holoenzyme. The model explains protection of a partial chymotryptic cleavage site after residue L(549) of pol gammaA upon binding of the accessory subunit. This interaction region is near residue 467 of pol gammaA, where a disease-related mutation has been reported to impair binding of the B subunit. One pol gammaB subunit dominates contacts with the catalytic subunit, while the second B subunit is largely exposed to solvent. A model for pol gamma is discussed that considers the effects of known mutations in the accessory subunit and the interaction of the enzyme with DNA.
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http://dx.doi.org/10.1038/sj.emboj.7601843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2230839PMC
October 2007

Proteolytic activation of monocyte chemoattractant protein-1 by plasmin underlies excitotoxic neurodegeneration in mice.

J Neurosci 2007 Feb;27(7):1738-45

Department of Pharmacological Sciences and Program in Molecular and Cellular Pharmacology, Stony Brook University, Stony Brook, New York 11794-8651, USA.

Exposure of neurons to high concentrations of excitatory neurotransmitters causes them to undergo excitotoxic death via multiple synergistic injury mechanisms. One of these mechanisms involves actions undertaken locally by microglia, the CNS-resident macrophages. Mice deficient in the serine protease plasmin exhibit decreased microglial migration to the site of excitatory neurotransmitter release and are resistant to excitotoxic neurodegeneration. Microglial chemotaxis can be signaled by the chemokine monocyte chemoattractant protein-1 (MCP-1)/CCL2 (CC chemokine ligand 2). We show here that mice genetically deficient for MCP-1 phenocopy plasminogen deficiency by displaying decreased microglial recruitment and resisting excitotoxic neurodegeneration. Connecting these pathways, we demonstrate that MCP-1 undergoes a proteolytic processing step mediated by plasmin. The processing, which consists of removal of the C terminus of MCP-1, enhances the potency of MCP-1 in in vitro migration assays. Finally, we show that infusion of the cleaved form of MCP-1 into the CNS restores microglial recruitment and excitotoxicity in plasminogen-deficient mice. These findings identify MCP-1 as a key downstream effector in the excitotoxic pathway triggered by plasmin and identify plasmin as an extracellular chemokine activator. Finally, our results provide a mechanism that explains the resistance of plasminogen-deficient mice to excitotoxicity.
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http://dx.doi.org/10.1523/JNEUROSCI.4987-06.2007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6673734PMC
February 2007

Human mitochondrial DNA nucleoids are linked to protein folding machinery and metabolic enzymes at the mitochondrial inner membrane.

J Biol Chem 2006 Sep 6;281(35):25791-802. Epub 2006 Jul 6.

Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651, USA.

Mitochondrial DNA (mtDNA) is packaged into bacterial nucleoid-like structures, each containing several mtDNA molecules. The distribution of nucleoids during mitochondrial fission and fusion events and during cytokinesis is important to the segregation of mitochondrial genomes in heteroplasmic cells bearing a mixture of wild-type and mutant mtDNA molecules. We report fractionation of HeLa cell mtDNA nucleoids into two subsets of complexes that differ in their sedimentation velocity and their association with cytoskeletal proteins. Pulse labeling studies indicated that newly replicated mtDNA molecules are evenly represented in the rapidly and slowly sedimenting fractions. Slowly sedimenting nucleoids were immunoaffinity purified using antibodies to either of two abundant mtDNA-binding proteins, TFAM or mtSSB. These two different immunoaffinity procedures yielded very similar sets of proteins, with 21 proteins in common, including most of the proteins previously shown to play roles in mtDNA replication and transcription. In addition to previously identified mitochondrial proteins, multiple peptides were observed for one novel DNA metabolic protein, the DEAH-box helicase DHX30. Antibodies raised against a recombinant fragment of this protein confirmed the mitochondrial localization of a specific isoform of DHX30.
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http://dx.doi.org/10.1074/jbc.M604501200DOI Listing
September 2006

MiGenes: a searchable interspecies database of mitochondrial proteins curated using gene ontology annotation.

Bioinformatics 2006 Feb 20;22(4):485-92. Epub 2005 Dec 20.

Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA.

Motivation: There has been an explosion of interest in the role of mitochondria in programmed cell death and other fundamental pathological processes underlying the development of human diseases. Nevertheless, the inventory of mitochondrial proteins encoded in the nuclear genome remains incomplete, providing an impediment to mitochondrial research at the interface with systems biology. We created the MiGenes database to further define the scope of the mitochondrial proteome in humans and model organisms including mice, rats, flies and worms as well as budding and fission yeasts. MiGenes is intended to stimulate mitochondrial research using model organisms.

Summary: MiGenes is a large-scale relational database that is automatically updated to keep pace with advances in mitochondrial proteomics and is curated to assure that the designation of proteins as mitochondrial reflects gene ontology (GO) annotations supported by high-quality evidence codes. A set of postulates is proposed to help define which proteins are authentic components of mitochondria. MiGenes incorporates >1160 new GO annotations to human, mouse and rat protein records, 370 of which represent the first GO annotation reflecting a mitochondrial localization. MiGenes employs a flexible search interface that permits batchwise accession number searches to support high-throughput proteomic studies. A web interface is provided to permit members of the mitochondrial research community to suggest modifications in protein annotations or mitochondrial status.
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http://dx.doi.org/10.1093/bioinformatics/btk009DOI Listing
February 2006

Functional human mitochondrial DNA polymerase gamma forms a heterotrimer.

J Biol Chem 2006 Jan 1;281(1):374-82. Epub 2005 Nov 1.

Department of Pharmacological Sciences and Center for Structural Biology, State University of New York, Stony Brook, New York 11794-8651, USA.

Mitochondrial DNA polymerase gamma (pol gamma) is responsible for replication and repair of mtDNA and is mutated in individuals with genetic disorders such as chronic external ophthalmoplegia and Alpers syndrome. pol gamma is also an adventitious target for toxic side effects of several antiviral compounds, and mutation of its proofreading exonuclease leads to accelerated aging in mouse models. We have used a variety of physical and functional approaches to study the interaction of the human pol gamma catalytic subunit with both the wild-type accessory factor, pol gammaB, and a deletion derivative that is unable to dimerize and consequently is impaired in its ability to stimulate processive DNA synthesis. Our studies clearly showed that the functional human holoenzyme contains two subunits of the processivity factor and one catalytic subunit, thereby forming a heterotrimer. The structure of pol gamma seems to be variable, ranging from a single catalytic subunit in yeast to a heterodimer in Drosophila and a heterotrimer in mammals.
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http://dx.doi.org/10.1074/jbc.M509730200DOI Listing
January 2006

The influence of the DNA polymerase gamma accessory subunit on base excision repair by the catalytic subunit.

DNA Repair (Amst) 2006 Jan 30;5(1):121-8. Epub 2005 Sep 30.

Department of Pharmacological Sciences, State University of New York at Stony Brook, Basic Health Science, Stony Brook, NY 11794-8651, USA.

Mammalian DNA polymerase gamma, the sole polymerase responsible for replication and repair of mitochondrial DNA, contains a large catalytic subunit and a smaller accessory subunit, pol gammaB. In addition to the polymerase domain, the large subunit contains a 3'-5' editing exonuclease domain as well as a dRP lyase activity that can remove a 5'-deoxyribosephosphate (dRP) group during base excision repair. We show that the accessory subunit enhances the ability of the catalytic subunit to function in base excision repair mainly by stimulating two subreactions in the repair process. Pol gammaB appeared to specifically enhance the rate at which pol gamma was able to locate damage in high molecular weight DNA. One pol gammaB point mutant known to have impaired ability to bind duplex DNA stimulated repair poorly, suggesting that duplex DNA binding through pol gammaB may help the catalytic subunit locate sites of DNA damage. In addition, the small subunit significantly stimulated the dRP lyase activity of pol gammaA, although it did not increase the rate at which the dRP group dissociated from the enzyme. The ability of DNA pol gamma to process a high load of damaged DNA may be compromised by the slow release of the dRP group.
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http://dx.doi.org/10.1016/j.dnarep.2005.08.014DOI Listing
January 2006

Protein components of mitochondrial DNA nucleoids in higher eukaryotes.

Mol Cell Proteomics 2003 Nov 26;2(11):1205-16. Epub 2003 Sep 26.

Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794, USA.

Mitochondrial DNA (mtDNA) is not packaged in nucleosomal particles, but has been reported to associate with the mitochondrial inner membrane. Gentle lysis of Xenopus oocyte mitochondria with nonionic detergent liberates a nucleoprotein complex containing mtDNA associated with a previously characterized DNA binding partner, mitochondrial transcription factor A (mtTFA), as well as a series of inner membrane proteins identified by sequencing. More extensive detergent treatment stripped most of these proteins from the DNA, leaving a limited number of proteins in a nucleoid core. Sequencing of the major proteins retained in association with mtDNA revealed the expected mtDNA binding proteins, mtTFA and mitochondrial single-stranded DNA binding protein (mtSSB), as well as four proteins not previously reported to associate with mtDNA. These include adenine nucleotide translocator 1, the lipoyl-containing E2 subunits of pyruvate dehydrogenase and branched chain alpha-ketoacid dehydrogenase and prohibitin 2. The association of several of these proteins with mtTFA-containing mtDNA nucleoids was confirmed by immunoprecipitation.
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http://dx.doi.org/10.1074/mcp.M300035-MCP200DOI Listing
November 2003

The mitochondrial DNA replication bubble has not burst.

Trends Biochem Sci 2003 Jul;28(7):357-60

Department of Pharmacology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA.

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http://dx.doi.org/10.1016/S0968-0004(03)00132-4DOI Listing
July 2003