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Article ; Online: Oncometabolite D-2-Hydroxyglutarate enhances gene silencing through inhibition of specific H3K36 histone demethylases

Ryan Janke / Anthony T Iavarone / Jasper Rine

eLife, Vol

2017 Volume 6

Abstract: Certain mutations affecting central metabolism cause accumulation of the oncometabolite D-2-hydroxyglutarate which promotes progression of certain tumors. High levels of D-2-hydroxyglutarate inhibit the TET family of DNA demethylases and Jumonji family ... ...

Abstract	Certain mutations affecting central metabolism cause accumulation of the oncometabolite D-2-hydroxyglutarate which promotes progression of certain tumors. High levels of D-2-hydroxyglutarate inhibit the TET family of DNA demethylases and Jumonji family of histone demethylases and cause epigenetic changes that lead to altered gene expression. The link between inhibition of DNA demethylation and changes in expression is strong in some cancers, but not in others. To determine whether D-2-hydroxyglutarate can affect gene expression through inhibiting histone demethylases, orthologous mutations to those known to cause accumulation of D-2-hydroxyglutarate in tumors were generated in Saccharomyces cerevisiae, which has histone demethylases but not DNA methylases or demethylases. Accumulation of D-2-hydroxyglutarate caused inhibition of several histone demethylases. Inhibition of two of the demethylases that act specifically on histone H3K36me2,3 led to enhanced gene silencing. These observations pinpointed a new mechanism by which this oncometabolite can alter gene expression, perhaps repressing critical inhibitors of proliferation.
Keywords	Heterochromatin ; D-2-Hydroxyglutarate ; Isocitrate Dehydrogenase ; Oncometabolite ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
Subject code	570
Language	English
Publishing date	2017-03-01T00:00:00Z
Publisher	eLife Sciences Publications Ltd
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System.

Tina Y Liu / Anthony T Iavarone / Jennifer A Doudna

PLoS ONE, Vol 12, Iss 1, p e

2017 Volume 0170552

Abstract: CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit ... ...

Abstract	CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus) Type III-A Csm complex (TthCsm) with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA) by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5' tag) of the crRNA and the 3' flanking region of the ssRNA. Mutation of the histidine-aspartate (HD) nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA) during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography.
Keywords	Medicine ; R ; Science ; Q
Subject code	612
Language	English
Publishing date	2017-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Correction

Tina Y Liu / Anthony T Iavarone / Jennifer A Doudna

PLoS ONE, Vol 12, Iss 4, p e

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System.

2017 Volume 0175612

Abstract: This corrects the article DOI:10.1371/journal.pone.0170552.]. ...

Abstract	[This corrects the article DOI:10.1371/journal.pone.0170552.].
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2017-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Mapping the H-NOX/HK Binding Interface in Vibrio cholerae by Hydrogen/Deuterium Exchange Mass Spectrometry

Guo, Yirui / Anthony T. Iavarone / Matthew M. Cooper / Michael A. Marletta

Biochemistry. 2018 Feb. 19, v. 57, no. 11

2018

Abstract: Heme-nitric oxide/oxygen binding (H-NOX) proteins are a group of hemoproteins that bind diatomic gas ligands such as nitric oxide (NO) and oxygen (O2). H-NOX proteins typically regulate histidine kinases (HK) located within the same operon. It has been ... ...

Abstract	Heme-nitric oxide/oxygen binding (H-NOX) proteins are a group of hemoproteins that bind diatomic gas ligands such as nitric oxide (NO) and oxygen (O2). H-NOX proteins typically regulate histidine kinases (HK) located within the same operon. It has been reported that NO-bound H-NOXs inhibit cognate histidine kinase autophosphorylation in bacterial H-NOX/HK complexes; however, a detailed mechanism of NO-mediated regulation of the H-NOX/HK activity remains unknown. In this study, the binding interface of Vibrio cholerae (Vc) H-NOX/HK complex was characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS) and further validated by mutagenesis, leading to a new model for NO-dependent kinase inhibition. A conformational change in Vc H-NOX introduced by NO generates a new kinase-binding interface, thus locking the kinase in an inhibitory conformation.
Keywords	deuterium ; enzyme inhibition ; histidine kinase ; ligands ; mass spectrometry ; models ; mutagenesis ; nitric oxide ; operon ; oxygen ; protein phosphorylation ; proteins ; Vibrio cholerae
Language	English
Dates of publication	2018-0219
Size	p. 1779-1789.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/acs.biochem.8b00027
Database	NAL-Catalogue (AGRICOLA)

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Article: Characterization of Chlamydomonas reinhardtii Core Histones by Top-Down Mass Spectrometry Reveals Unique Algae-Specific Variants and Post-Translational Modifications

Khan, Aliyya / Anthony T. Iavarone / Carlo K. Eikani / Hana Khan / James J. Pesavento

Journal of proteome research. 2018 Jan. 05, v. 17, no. 1

2018

Abstract: The unicellular microalga Chlamydomonas reinhardtii has played an instrumental role in the development of many new fields (bioproducts, biofuels, etc.) as well as the advancement of basic science (photosynthetic apparati, flagellar function, etc.). ... ...

Abstract	The unicellular microalga Chlamydomonas reinhardtii has played an instrumental role in the development of many new fields (bioproducts, biofuels, etc.) as well as the advancement of basic science (photosynthetic apparati, flagellar function, etc.). Chlamydomonas’ versatility ultimately derives from the genes encoded in its genome and the way that the expression of these genes is regulated, which is largely influenced by a family of DNA binding proteins called histones. We characterize C. reinhardtii core histones, both variants and their post-translational modifications, by chromatographic separation, followed by top-down mass spectrometry (TDMS). Because TDMS has not been previously used to study Chlamydomonas proteins, we show rampant artifactual protein oxidation using established nuclei purification and histone extraction methods. After addressing oxidation, both histones H3 and H4 are found to each have a single polypeptide sequence that is minimally acetylated and methylated. Surprisingly, we uncover a novel monomethylation at lysine 79 on histone H4 present on all observed molecules. Histone H2B and H2A are found to have two and three variants, respectively, and both are minimally modified. This study provides an updated assessment of the core histone proteins in the green alga C. reinhardtii by top-down mass spectrometry and lays the foundation for further investigation of these essential proteins.
Keywords	biofuels ; Chlamydomonas reinhardtii ; chromatography ; DNA-binding proteins ; genes ; histones ; lysine ; mass spectrometry ; methylation ; microalgae ; oxidation ; photosynthesis ; polypeptides ; post-translational modification ; proteome
Language	English
Dates of publication	2018-0105
Size	p. 23-32.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	2078618-9
ISSN	1535-3907 ; 1535-3893
ISSN (online)	1535-3907
ISSN	1535-3893
DOI	10.1021/acs.jproteome.7b00780
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Promiscuous Diffusible Signal Factor Production and Responsiveness of the Xylella fastidiosa Rpf System

Michael Ionescu / Kenji Yokota / Elena Antonova / Angelica Garcia / Ellen Beaulieu / Terry Hayes / Anthony T. Iavarone / Steven E. Lindow

mBio, Vol 7, Iss 4, p e01054-

2016 Volume 16

Abstract: Cell density-dependent regulation of gene expression in Xylella fastidiosa that is crucial to its switching between plant hosts and insect vectors is dependent on RpfF and its production of 2-enoic acids known as diffusible signal factor (DSF). We show ... ...

Abstract	Cell density-dependent regulation of gene expression in Xylella fastidiosa that is crucial to its switching between plant hosts and insect vectors is dependent on RpfF and its production of 2-enoic acids known as diffusible signal factor (DSF). We show that X. fastidiosa produces a particularly large variety of similar, relatively long-chain-length 2-enoic acids that are active in modulating gene expression. Both X. fastidiosa itself and a Pantoea agglomerans surrogate host harboring X. fastidiosa RpfF (XfRpfF) is capable of producing a variety of both saturated and unsaturated free fatty acids. However, only 2-cis unsaturated acids were found to be biologically active in X. fastidiosa. X. fastidiosa produces, and is particularly responsive to, a novel DSF species, 2-cis-hexadecanoic acid that we term XfDSF2. It is also responsive to other, even longer 2-enoic acids to which other taxa such as Xanthomonas campestris are unresponsive. The 2-enoic acids that are produced by X. fastidiosa are strongly affected by the cellular growth environment, with XfDSF2 not detected in culture media in which 2-tetradecenoic acid (XfDSF1) had previously been found. X. fastidiosa is responsive to much lower concentrations of XfDSF2 than XfDSF1. Apparently competitive interactions can occur between various saturated and unsaturated fatty acids that block the function of those agonistic 2-enoic fatty acids. By altering the particular 2-enoic acids produced and the relative balance of free enoic and saturated fatty acids, X. fastidiosa might modulate the extent of DSF-mediated quorum sensing.
Keywords	Science ; Q ; Microbiology ; QR1-502
Subject code	571
Language	English
Publishing date	2016-07-01T00:00:00Z
Publisher	American Society for Microbiology
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Targeted Lipid Profiling Discovers Plasma Biomarkers of Acute Brain Injury.

Sunil A Sheth / Anthony T Iavarone / David S Liebeskind / Seok Joon Won / Raymond A Swanson

PLoS ONE, Vol 10, Iss 6, p e

2015 Volume 0129735

Abstract: Prior efforts to identify a blood biomarker of brain injury have relied almost exclusively on proteins; however their low levels at early time points and poor correlation with injury severity have been limiting. Lipids, on the other hand, are the most ... ...

Abstract	Prior efforts to identify a blood biomarker of brain injury have relied almost exclusively on proteins; however their low levels at early time points and poor correlation with injury severity have been limiting. Lipids, on the other hand, are the most abundant molecules in the brain and readily cross the blood-brain barrier. We previously showed that certain sphingolipid (SL) species are highly specific to the brain. Here we examined the feasibility of using SLs as biomarkers for acute brain injury. A rat model of traumatic brain injury (TBI) and a mouse model of stroke were used to identify candidate SL species though our mass-spectrometry based lipid profiling approach. Plasma samples collected after TBI in the rat showed large increases in many circulating SLs following injury, and larger lesions produced proportionately larger increases. Plasma samples collected 24 hours after stroke in mice similarly revealed a large increase in many SLs. We constructed an SL score (sum of the two SL species showing the largest relative increases in the mouse stroke model) and then evaluated the diagnostic value of this score on a small sample of patients (n = 14) who presented with acute stroke symptoms. Patients with true stroke had significantly higher SL scores than patients found to have non-stroke causes of their symptoms. The SL score correlated with the volume of ischemic brain tissue. These results demonstrate the feasibility of using lipid biomarkers to diagnose brain injury. Future studies will be needed to further characterize the diagnostic utility of this approach and to transition to an assay method applicable to clinical settings.
Keywords	Medicine ; R ; Science ; Q
Subject code	616
Language	English
Publishing date	2015-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Hydrogen–Deuterium Exchange of Lipoxygenase Uncovers a Relationship between Distal, Solvent Exposed Protein Motions and the Thermal Activation Barrier for Catalytic Proton-Coupled Electron Tunneling

Adam R. Offenbacher / Shenshen Hu / Erin M. Poss / Cody A. M. Carr / Alexander D. Scouras / Daniil M. Prigozhin / Anthony T. Iavarone / Ali Palla / Tom Alber / James S. Fraser / Judith P. Klinman

ACS Central Science, Vol 3, Iss 6, Pp 570-

2017 Volume 579

Keywords	Chemistry ; QD1-999
Language	English
Publishing date	2017-06-01T00:00:00Z
Publisher	American Chemical Society
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation

Jinek, Martin / Anthony T. Iavarone / Carolin Anders / David W. Taylor / Emine Kaya / Emmanuelle Charpentier / Enbo Ma / Eva Nogales / Fuguo Jiang / Jennifer A. Doudna / Kaihong Zhou / Matias Kaplan / Michael Hauer / Samuel H. Sternberg / Steven Lin

Science. 2014 Mar. 14, v. 343, no. 6176

2014

Abstract: Cas9 Solved Clustered regularly interspaced short palindromic repeats (CRISPR)âassociated (Cas) loci allow prokaryotes to identify and destroy invading DNA. Not only important to bacteria, the universal value of Cas endonuclease specificity has also ... ...

Abstract	Cas9 Solved Clustered regularly interspaced short palindromic repeats (CRISPR)âassociated (Cas) loci allow prokaryotes to identify and destroy invading DNA. Not only important to bacteria, the universal value of Cas endonuclease specificity has also resulted in Cas9 being exploited as a tool for genome editing. Jinek et al. (10.1126/science.1247997, published online 6 February) determined the 2.6 and 2.2 angstrom resolution crystal structures of two Cas9 enzymes to reveal a common structural core with distinct peripheral elaborations. The enzymes are autoinhibited, undergo large conformational changes on binding RNA, and have channels lined with basic residues that are candidates for an RNA-DNA binding groove. Based on these and other insights from the structures, this work provides important revelations both for the CRISPR mechanism and for genome editing.
Keywords	bacteria ; crystal structure ; DNA ; enzymes ; genetic engineering ; loci ; prokaryotic cells ; RNA
Language	English
Dates of publication	2014-0314
Size	p. 1247997.
Publishing place	American Association for the Advancement of Science
Document type	Article
ZDB-ID	128410-1
ISSN	1095-9203 ; 0036-8075
ISSN (online)	1095-9203
ISSN	0036-8075
DOI	10.1126/science.1247997
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Macro-to-micro structural proteomics

Monica Totir / Nathaniel Echols / Max Nanao / Christine L Gee / Alisa Moskaleva / Scott Gradia / Anthony T Iavarone / James M Berger / Andrew P May / Chloe Zubieta / Tom Alber

PLoS ONE, Vol 7, Iss 2, p e

native source proteins for high-throughput crystallization.

2012 Volume 32498

Abstract: Structural biology and structural genomics projects routinely rely on recombinantly expressed proteins, but many proteins and complexes are difficult to obtain by this approach. We investigated native source proteins for high-throughput protein ... ...

Abstract	Structural biology and structural genomics projects routinely rely on recombinantly expressed proteins, but many proteins and complexes are difficult to obtain by this approach. We investigated native source proteins for high-throughput protein crystallography applications. The Escherichia coli proteome was fractionated, purified, crystallized, and structurally characterized. Macro-scale fermentation and fractionation were used to subdivide the soluble proteome into 408 unique fractions of which 295 fractions yielded crystals in microfluidic crystallization chips. Of the 295 crystals, 152 were selected for optimization, diffraction screening, and data collection. Twenty-three structures were determined, four of which were novel. This study demonstrates the utility of native source proteins for high-throughput crystallography.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2012-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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