Publications by authors named "Michaella J Levy"

9 Publications

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The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain.

Nat Commun 2021 03 4;12(1):1443. Epub 2021 Mar 4.

Stowers Institute for Medical Research, Kansas City, MO, USA.

Heterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.
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http://dx.doi.org/10.1038/s41467-021-21663-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933334PMC
March 2021

Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping.

Nature 2020 07 17;583(7817):638-643. Epub 2020 Jun 17.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

N-acetylcytidine (acC) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report acC-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of acC at single-nucleotide resolution. In human and yeast mRNAs, acC sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of acC across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. AcC is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of acC and its potential thermoadaptive role. Our studies quantitatively define the acC landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease.
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http://dx.doi.org/10.1038/s41586-020-2418-2DOI Listing
July 2020

A Systems Chemoproteomic Analysis of Acyl-CoA/Protein Interaction Networks.

Cell Chem Biol 2020 03 10;27(3):322-333.e5. Epub 2019 Dec 10.

Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA. Electronic address:

Acyl-coenzyme A (CoA)/protein interactions are essential for life. Despite this importance, their global scope and selectivity remains undefined. Here, we describe CATNIP (CoA/AcetylTraNsferase Interaction Profiling), a chemoproteomic platform for the high-throughput analysis of acyl-CoA/protein interactions in endogenous proteomes. First, we apply CATNIP to identify acetyl-CoA-binding proteins through unbiased clustering of competitive dose-response data. Next, we use this method to profile the selectivity of acyl-CoA/protein interactions, leading to the identification of specific acyl-CoA engagement signatures. Finally, we apply systems-level analyses to assess the features of protein networks that may interact with acyl-CoAs, and use a strategy for high-confidence proteomic annotation of acetyl-CoA-binding proteins to identify a site of non-enzymatic acylation in the NAT10 acetyltransferase domain that is likely driven by acyl-CoA binding. Overall, our studies illustrate how chemoproteomics and systems biology can be integrated to understand the roles of acyl-CoA metabolism in biology and disease.
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http://dx.doi.org/10.1016/j.chembiol.2019.11.011DOI Listing
March 2020

Probing the Sensitivity of the Orbitrap Lumos Mass Spectrometer Using a Standard Reference Protein in a Complex Background.

J Proteome Res 2018 10 13;17(10):3586-3592. Epub 2018 Sep 13.

Stowers Institute for Medical Research , Kansas City , Missouri 64110 , United States.

The use of mass spectrometry as a tool to detect proteins of biological interest has become a cornerstone of proteomics. The popularity of mass spectrometry-based methods has increased along with instrument improvements in detection and speed. The Orbitrap Fusion Lumos mass spectrometer has recently been shown to have better fragmentation and detection than its predecessors. Here, we determined the sensitivity of the Lumos using the NIST monoclonal antibody reference material at various concentrations to detect its peptides in a background of S. cerevisiae whole cell lysate, which was kept at a constant concentration. The data collected by data-dependent acquisition showed that the spiked protein could be detected at 10 pg by an average of 4 peptides in 250 ng of whole cell lysate when the instrument was operated by detecting the peptide masses in the Orbitrap and the fragment masses in the ion trap (FTIT mode). In contrast, when the peptides and fragments were both detected in the Orbitrap on either the Lumos or Q-Exactive Plus (FTFT mode), the lowest concentration of NIST monoclonal antibody detected was 50 pg. The Lumos can detect a single protein at a level 2500 times lower than the whole cell background and the combination of detecting ions in the Orbitrap and ion trap can improve the identification of low abundance proteins. Furthermore, the total number of proteins identified from decreasing starting amounts of whole cell extracts was determined. The Lumos, when operated in FTIT mode, was able to identify twice as many proteins compared to the Q-Exactive+ at 5 ng of whole cell lysate. Similar numbers of proteins were identified on both platforms at higher concentrations of starting material. Therefore, the Lumos mass spectrometer is especially useful for detecting proteins of low abundance in complex backgrounds or samples that have limited starting material.
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http://dx.doi.org/10.1021/acs.jproteome.8b00269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836688PMC
October 2018

Simple NMR methods for evaluating higher order structures of monoclonal antibody therapeutics with quinary structure.

J Pharm Biomed Anal 2016 Sep 7;128:398-407. Epub 2016 Jun 7.

Division of Pharmaceutical Analysis, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, US Food and Drug Administration, St. Louis, MO, 63110, United States.

Monoclonal antibody (mAb) drugs constitute the largest class of protein therapeutics currently on the market. Correctly folded protein higher order structure (HOS), including quinary structure, is crucial for mAb drug quality. The quinary structure is defined as the association of quaternary structures (e.g., oligomerized mAb). Here, several commonly available analytical methods, i.e., size-exclusion-chromatography (SEC) FPLC, multi-angle light scattering (MALS), circular dichroism (CD), NMR and multivariate analysis, were combined and modified to yield a complete profile of HOS and comparable metrics. Rituximab and infliximab were chosen for method evaluation because both IgG1 molecules are known to be homologous in sequence, superimposable in Fab crystal structure and identical in Fc structure. However, herein the two are identified to be significantly different in quinary structure in addition to minor secondary structure differences. All data collectively showed rituximab was mostly monomeric while infliximab was in mono-oligomer equilibrium driven by its Fab fragment. The quinary structure differences were qualitatively inferred from the less used but more reproducible dilution-injection-SEC-FPLC curve method. Quantitative principal component analysis (PCA) was performed on NMR spectra of either the intact or the in-situ enzymatic-digested mAb samples. The cleavage reactions happened directly in NMR tubes without further separation, which greatly enhanced NMR spectra quality and resulted in larger inter- and intra-lot variations based on PCA. The new in-situ enzymatic digestion method holds potential in identifying structural differences on larger therapeutic molecules using NMR.
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http://dx.doi.org/10.1016/j.jpba.2016.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066182PMC
September 2016

Marketplace Analysis of Conjugated Estrogens: Determining the Consistently Present Steroidal Content with LC-MS.

AAPS J 2015 Nov 5;17(6):1438-45. Epub 2015 Aug 5.

Division of Pharmaceutical Analysis, Office of Testing and Research, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 645 S. Newstead Avenue, St. Louis, Missouri, 63110, USA.

Conjugated estrogens purified from pregnant mares urine has been used as estrogen hormone replacement therapy since 1942. Previously, methods were proposed to identify and quantify the components of this complex mixture but ultimately were withdrawn due to incomplete characterization of the product and difficulties in transferring the method between laboratories. The aim of the current study is to develop a LC method that can reliably detect multiple steroidal components in conjugated estrogen tablets and measure their relative amount. The method developed was optimized for UHPLC columns, and the elution profile was analyzed using high-resolution mass spectrometry. A total of 60 steroidal components were identified using their exact m/z, product ion spectra of known, and predicted conjugated estrogen structures. These components were consistently present in 23 lots of Premarin tablets spanning two production years. The ten conjugated estrogens identified in the USP monograph and other additional estrogens reported elsewhere are among the 60 steroidal components reported here. The LC-MS method was tested in different laboratories using multiple samples, and the obtained results were reproducible among laboratories.
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http://dx.doi.org/10.1208/s12248-015-9805-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627451PMC
November 2015

Primary Sequence Confirmation of a Protein Therapeutic Using Top Down MS/MS and MS(3).

Anal Chem 2015 Jul 6;87(14):6995-9. Epub 2015 Jul 6.

U.S. Food and Drug Administration, Center for Drug Evaluation and Research, Office of Testing and Research, Division of Pharmaceutical Analysis, 645 S. Newstead Ave., St. Louis, Missouri 63110, United States.

Mass spectrometry has gained widespread acceptance for the characterization of protein therapeutics as a part of the regulatory approval process. Improvements in mass spectrometer sensitivity, resolution, and mass accuracy have enabled more detailed and confident analysis of larger biomolecules for confirming amino acid sequences, assessing sequence variants, and characterizing post translational modifications. This work demonstrates the suitability of a combined approach using intact MS and multistage top down MS/MS analyses for the characterization of a protein therapeutic drug. The protein therapeutic granulocyte-colony stimulating factor was analyzed using a Thermo Fusion Tribrid mass spectrometer using a multistage top down MS approach. Intact mass analysis identified the presence of two disulfide bonds based on exact mass shifts while a combined collision induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) MS/MS approach obtained 80% protein sequence coverage. Isolating MS/MS fragments for MS(3) analysis using HCD or CID increased the sequence coverage to 89%. 95% sequence coverage was obtained by reducing human granulocyte-colony stimulating factor (G-CSF) prior to MS/MS and MS(3) analysis to specifically target the residues between the disulfide bonds. The use of this combined intact MS and multistage top down MS approach allows for rapid and accurate determination of the primary sequence of a protein therapeutic drug product.
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http://dx.doi.org/10.1021/acs.analchem.5b01113DOI Listing
July 2015

Analytical techniques and bioactivity assays to compare the structure and function of filgrastim (granulocyte-colony stimulating factor) therapeutics from different manufacturers.

Anal Bioanal Chem 2014 Oct 22;406(26):6559-67. Epub 2013 Nov 22.

Division of Pharmaceutical Analysis, Office of Testing and Research, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, 645 S. Newstead Ave, St. Louis, MO, 63110, USA.

The FDA has approved more than 100 protein and peptide drugs with hundreds more in the pipeline (Lanthier et al. in Nat Rev Drug Discov 7(9):733-737, 2008). Many of these originator biologic products are now coming off patent and are being manufactured by alternate methods than the innovator as follow-on drugs. Because changes to the production method often lead to subtle differences (e.g., degradation products, different posttranslational modifications or impurities) in the therapeutic (Schiestl et al. in Nat Biotechnol 29(4):310-312, 2011), there is a critical need to define techniques to test and insure the quality of these drugs. In addition, the emergence of protein therapeutics manufactured by unapproved methodologies presents an ongoing and growing regulatory challenge. In this work, high-resolution mass spectrometry was used to determine the presence or absence of posttranslational modifications for one FDA-approved and three foreign-sourced, unapproved filgrastim products. Circular dichroism (CD) was used to compare the secondary structure and probe the temperature stability of these products. Native 2D (1)H,(15)N-heteronuclear singular quantum coherence (HSQC) NMR test was applied to these samples to compare the higher-order structure of the four products. Finally, a cell proliferation assay was performed on the filgrastims to compare their bioactivity, and stressed filgrastim was tested in the bioassay to better understand the effects of changes in protein structure on activity. The results showed that orthogonal approaches are capable of characterizing the physiochemical properties of this protein drug and assessing the impact of structural changes on filgrastim purity and potency.
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http://dx.doi.org/10.1007/s00216-013-7469-xDOI Listing
October 2014

Analysis of Fluorescent Proteins with a Nanoparticle Probe.

J Phys Chem Lett 2012 Feb;3(3):337-341

Department of Chemistry, Texas A&M University, College Station, Texas 77843-3255.

This letter presents the first application of high energy, single nanoparticle probes (e.g., 520 keV Au(400) 2nm NP) in the characterization of surfaces containing fluorescent proteins (e.g., GFP variants) by their co-emitted photon, electron and secondary ion signals. NP induced protein luminescence increases with the NP incident energy, is originated by the NP impact and is transferred to the protein fluorophor via electronic energy transfer. Multi-electron emission is observed per single NP impacts and their distributions are specific to the target morphology and composition. Fragment ions of protein sub-units consisting of 2-7 amino acid peptides are observed under individual NP impacts that can be correlated to the random protein orientation relative to the impact site (e.g., outer layer or "skin" of the protein).
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http://dx.doi.org/10.1021/jz201547xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3270945PMC
February 2012