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Principal Investigator: Leesa Deterding
Organization: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
Fiscal Year: 2020
Award: $617,969
Funding agency: National Institute of Environmental Health Sciences
Mass spectrometry has been used to determine the extent of modification and the specific sites of modification on biomolecules. MS-based approaches have many advantages, including generally rapid analyses without radiolabeling. The MS analysis of a variety of proteins has been investigated using mass spectrometry. Products and digests have been analyzed by both positive and negative ion MALDI mass spectrometry and LC in combination with electrospray mass spectrometry. In addition, we are currently analyzing the use of the crosslinker BS3 with a variety of proteins.
1. Antibody Glycosylation Characterization. We are currently performing two projects related to glycosylation characterization. One project involves a recombinant SARS-CoV-2 spike glycoprotein which has recently been successfully expressed by the Structural Biology Core Laboratory at NIEHS. We have been asked to characterize the glycan structures and the degree of sequon occupancy on the glycoprotein. Our preliminary data indicate that all the sites on the recombinant protein are fully occupied and we are currently investigating the distribution of various glycoforms on each of 22 glycan sites by high resolution mass spectrometry. The other
project involves an extramural collaborative effort between the Division of Renal Diseases and Hypertension from the University of Minnesota at Minneapolis and our group at NIEHS. Abnormalities in O-glycosylation of circulating IgA1 are implicated in the pathogenesis of IgAN. This was initially demonstrated by the altered binding of lectins with specificity for O-linked glycans and confirmed later by mass spectrometry. We aim to identify potential sources of discrepancy between the two analytical methods.
2. Histone Proteins. We have been collaborating with the Archer laboratory in an effort to examine the role of H1 in controlling gene expression and protein levels when we knock out the H1 gene. Knockout cell lines of the histone protein H1.4 protein (as it was determined by MS previously that it was phosphorylated) was compared to a "wild-type" cell line. Data were acquired and are being analyzed to determine if other histone levels change and/or their PTMS change if the H1.4 isoform is knocked out.
3. Protein Crosslinking. Multiple projects are underway to characterize protein complexes by mass spectrometry in conjunction with chemical cross-linking. Most of these experiments have been conducted using BS3 as the cross-linking reagent followed by trypsin digestion and nanoLC-ESI-MS performed on a Q-Exactive Plus mass spectrometer. While there have been successful analyses on multiple projects, most recently we have determined the crosslinks in the protein Rix7 and a nonhomologous end joining complex. In an effort to understand the Rix7 structure, multiple approaches were used including chemical cross-linking and mass spectrometry as well as cryo-electron microscopy.
4. Characterization of RNA cleavage events. In collaboration with the Stanley lab at the NIEHS the MSRSG has characterized the site of RNA cleavage for multiple endoribonucleases including Las1 from yeast and NSP15 from SARS-CoV-2. This work also characterized the nature of the resulting 3 ends of the cleavage products.
5. Protein Stability as Measured by Limited Proteolysis. Bla g 1, the major cockroach allergen, forms a novel fold which encloses a large hydrophobic cavity that can bind lipids. Lipid-dependent immunoactivity has been observed for other allergens and understanding the structural basis of Bla g 1s interaction with lipids could help in the understanding of the molecular details of allergenicity. We found that fatty-acid cargoes significantly enhanced thermostability while inhibiting cleavage by cathepsin S. Both effects may relate to antigen processing, with potential downstream implications for immunogenicity. The MSRSG contributed by identifying the cathepsin S cleavage sites on Bla g 1 in the presence and absence of various lipids.
6. Phosphorylation of VRK1. Vaccinia-related kinase 1 (VRK1) is a Ser-Thr kinase and regulates numerous proteins. We found that VRK1 auto-phosphorylates at Ser376 and Thr386 in in vitro kinase assays. It was found that in cells, that Thr386 is phosphorylated in the low glucose (40 mg/dL) but not high glucose (140 mg/dL) media. This Thr386 phosphorylation correlates with an induced phosphorylation of p53 (Thr18) and C-Jun (Ser63) in the low glucose medium. Knock down of VRK1 attenuates phosphorylation of both C-Jun and p53 in the low glucose medium, while over-expression of VRK1 T386D (but not VRK1 WT or VRK1 T386A mutant) induces phosphorylation of p53 and C-Jun in the high glucose medium.
Terms: <2019 novel coronavirus><2019-nCoV><Acetylation><Affect><Allergens><Amino Acids><Antibodies><Assay><Attenuated><Bifunctional Reagents><Binding><Bioassay><Biologic Assays><Biological Assay><Bla g 1><Cathepsins><Cell Body><Cell Line><CellLine><Cells><Cellular biology><Chemicals><Collaborations><Complex><Cross-Linking Reagents><Crosslinker><Crosslinking Reagents><Cryo-electron Microscopy><Cryoelectron Microscopy><D-Glucose><Data><Dextrose><Digestion><Disease><Disorder><EXTMR><Electron Cryomicroscopy><Endoribonucleases><Enzyme Gene><Enzymes><Evaluation><Event><Extramural><Extramural Activities><Fatty Acids><Gene Expression><Gene Proteins><Genes><Glucose><Glycans><Glycoproteins><Histones><Hydrophobicity><Hypertensive Nephropathy><IgA1><In Vitro><Ions><Isoforms><JUN Family Gene><JUN Proto-oncogene Family><JUN gene><Kidney Diseases><Kinases><Knock-out><Knockout><Laboratories><Lectin><Life Cycle><Life Cycle Stages><Link><Lipid Binding><Lipids><MALD-MS><MALDI><MALDI-MS><Macromolecular Structure><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Measures><Mediating><Metabolic Glycosylation><Methylation><Minnesota><Modification><Molecular><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Molecular Structure><NHEJ><NIEHS><National Institute of Environmental Health Sciences><Nature><Nephropathy><Non-Homologous End Joining><Non-Polyadenylated RNA><Non-homologous DNA End Joining><Nonhomologous DNA End Joining><Nonhomologous End Joining><Normal Cell><P53><Pathogenesis><Peptides><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Polysaccharides><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Prevention><Protein Cleavage><Protein Gene Products><Protein Isoforms><Protein Modification><Protein Phosphorylation><Proteins><Proteolysis><Proteome><RNA><RNA Gene Products><RNA endonuclease><Radiolabeled><Recombinant Proteins><Recombinants><Renal Disease><Renal Hypertension><Resolution><Ribonucleic Acid><Role><SARS-CoV-2><SARS-CoV2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related coronavirus 2><Safety><Sampling><Severe acute respiratory syndrome coronavirus 2><Side><Site><Source><Specificity><Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization><Spectroscopy, Mass, Matrix-Assisted Laser Desorption-Ionization><Strains Cell Lines><Structure><System><TP53><TP53 gene><TRP53><Transphosphorylases><Tripcellim><Trypsin><Tumor Protein p53 Gene><Ubiquitilation><Ubiquitination><Ubiquitinoylation><Universities><Vaccinia><Work><Wuhan coronavirus><Yeasts><allergen Bla g 1><aminoacid><analytical method><antigen processing><base><c jun><c-jun Gene><cell biology><cell type><cockroach allergen><conformation><conformational state><cross-link><crosslink><cryo-EM><cryoEM><cultured cell line><experiment><experimental research><experimental study><functional group><glycosylation><hypertensive kidney><immunogenicity><insight><kidney disorder><knock-down><knockdown><life course><lipid bound><mass spectrometer><matrix assisted laser desorption ionization><mutant><novel><overexpress><overexpression><p53 Genes><protein complex><protein crosslink><renal disorder><social role><structural biology><thermolability><thermostability><ubiquination><ubiquitin conjugation>