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Principal Investigator: Lisa Jenkins
Organization: DIVISION OF BASIC SCIENCES - NCI
Fiscal Year: 2024
Award: $1,295,034
Funding agency: National Cancer Institute
Overall, the expertise of the Mass Spectrometry Resource is being used to further the research of multiple groups within the NIH. In FY24, we collaborated in more than 65 different projects from 40 different investigators, with over 4000 sample runs. Among these are projects to characterize the post-translational modifications of target proteins, including sites of phosphorylation, acetylation, and methylation, to better understand signal transduction, protein regulation, and the effects of small molecule inhibitors. The resource is also being used to identify protein interactors of both proteins and nucleic acids. Mass spectrometry is additionally being used extensively for large-scale quantitative proteomics projects, using both isotopic labeling and label-free approaches. Structural mass spectrometry applications, such as crosslinking and limited proteolysis methods, are used to investigate protein conformation. Finally, the resource is using inductively-coupled plasma mass spectrometry (ICP-MS) to quantify the level of metals in biological samples. In the past year, thirteen collaborative studies have been published; several other projects are nearing completion or manuscripts are under review. Highlights of some of these are described below. Three studies were published in which mass spectrometry-based characterization of interactomes was crucial for better understanding of the functions of the molecule of interest. The first project focused on the interactors of the mRNA of forkhead box A1 (FOXA1), a pioneer transcription factor that functions as an oncogene in prostate and breast cancer but may have potential tumor suppressor functions in colorectal cancer. Genome-wide RNA stability assays identified FOXA1 as an unstable mRNA in CRC cells, with the 3' UTR responsible for instability of the FOXA1 transcript. RNA pulldowns and mass spectrometry identified Staufen1 (STAU1) as a potential regulator of FOXA1 mRNA. STAU1 knockdown resulted in increased FOXA1 mRNA and protein expression due to increased mRNA stability. This work, performed in collaboration with the lab of Dr. Ashish Lal, Genetics Branch, was published in Molecular and Cellular Biology. Next, with Dr. Sergio Ruiz, Laboratory of Genome Integrity, we performed mass spectrometry analysis of the interactome of DUXBL, a member of the double homeobox protein (DUX) family embryonic transcription factors, to better understand the mechanism by which DUXBL regulates the two-cell-associated transcriptional program. We identified consistent interaction of DUXBL with TRIM24 and TRIM33, related members of the members of the TRIM superfamily involved in gene silencing. Further experiments demonstrated that DUXBL colocalizes with both proteins in nuclear foci upon DUX expression. The findings were published in Nature Genetics. The third study continued a long-standing collaboration with the lab of Dr. Yves Pommier, Developmental Therapeutics Branch, studying the functions of topoisomerase proteins. In the current work, published in Nature Communications, mass spectrometry was used to identify any deubiquitinating enzymes that might co-immunoprecipitate with TOP3B. The Pommier group showed increased ubiquitination of TOP3B in the absence of TDRD3 and increased stability of TOP3B in the presence of TDRD3. Hypothesizing that TDRD3 recruits a deubiquitinase to interact with TOP3B and stabilize it, we characterized the interactors of TOP3B in HEK293 cells. In addition to TDRD3, several deubiquitinating enzymes were also identified. Among these, USP9X was a top hit and further experiments demonstrated that its inactivation destabilizes TOP3B. In a separate project with the Pommier lab, global quantitative proteomics were used to examine the molecular effect of combination strategies of the oral ATR inhibitor tuvusertib (M1774) with the DNA-damaging agent SN-38. We found that single treatment with SN-38 significantly increased the levels of proteins related to replication stress and reduced protein involved in S-phase. Addition of low-dose of M1774 to the SN-38-treated cells increased proteins associated with active replication, replication fork protection complex, and cell cycle progression to G2-M-phase. However, non-toxic low doses of M1774 alone did not change the proteins that were increased in combination. The findings were published in Molecular Cancer Therapeutics. Although many of the collaborative projects make use of proteomic workflows, we also participate in projects performing targeted quantitation of small molecules. Three papers were published reporting quantitative results. In collaboration with the labs of Dr. Michael Gottesman and Dr. Pedro Batista, both Laboratory of Cell Biology, quantitative mass spectrometry was used to measure potential methyltransferase activity of the enzyme TMT1A (METTL7A) on the histone deacetylase inhibitor romidepsin. TMT1A was identified as increased in a screen to identify proteins that give rise to romidepsin resistance. As the paralog of TMT1A was previously shown to methylate thiol groups on hydrogen sulfide and captopril and romidepsin has a thiol as the zinc-binding moiety, experiments were performed to test whether TMT1A could inactivate romidepsin via methylation of the thiol group. Our mass spectrometry results demonstrated that romidepsin was methylated was increased in the resistant lines and in cells overexpressing TMT1A. In a follow-up study, we demonstrated that this activity is conserved in the mouse, rat, chicken, and zebrafish homologs. The results of the first study were published in Molecular Cancer Therapeutics and the follow-up in Chemico-Biological Interactions. Finally, the Mass Spectrometry Resource has collaborated on two projects related to development of new software packages for analysis of mass spectrometry data. The first, performed in collaboration with Dr. Doug Lowy, Laboratory of Cellular Oncology, described a cProSite, a web-based tool for analysis of proteomic, phosphoproteomic, and genomic data sets in the CPTAC database. We performed phosphosite mapping on ECT2 to validate findings from the cProSite tool; we found, as predicted, that several phosphorylation sites were increased in cells synchronized at the G2/M phase and were decreased upon treatment with a CDK1 inhibitor. The paper on the tool was published in the Journal of Biotechnology and Biomedicine. The second tool is an algorithm for rapid searching of mass spectrometry data, focusing on identification of microorganisms by the proteins present. Mass spectrometry analysis using tandem mass tags for sample multiplexing combined with the enhanced search algorithm resulted in an identification sensitivity of 93-97% and a specificity of 100% at the species level. Furthermore, by multiplexing, the time for analysis of a single sample could be decreased significantly. Overall, this represents an important step forward to having a fast, accurate, and broad spectrum method for the identification of microorganisms in the clinical setting. This study was performed in collaboration with the group of Dr. Yi-Kuo Yu, National Center for Biotechnology Information, National Library of Medicine, and was published in the Journal of the American Society for Mass Spectrometry. In addition to the above projects, mass spectrometry has been used to investigate the mechanism of action of SAMT-247, a thioester molecule developed to prevent and treat HIV infection. Working with Drs. Genoveffa Franchini, Vaccine Branch, and Daniel Appella, NIDDK, mass spectrometry experiments have demonstrated that SAMT-247 covalently modifies cysteine and lysine residues in HIV Gag, resulting in protein aggregation and loss of function. Additionally, new work has identified a host protein that can also be targeted by SAMT-247, resulting in decreased HIV infection of cells.
Terms: <3' Untranslated Regions><3'UTR><AIDS Virus><Acetylation><Acquired Immune Deficiency Syndrome Virus><Acquired Immunodeficiency Syndrome Virus><Active Follow-up><Algorithms><American><Assay><Basal Transcription Factor><Basal transcription factor genes><Basic Research><Basic Science><Binding><Bioassay><Biological><Biological Assay><Biotech><Biotechnology><Brachydanio rerio><Breast Cancer><CCR><CDC2><CDC2 Protein Kinase><CDC2 gene><CDK1><Cancer Genes><Cancer-Promoting Gene><Cancers><Captopril><Cell Body><Cell Communication and Signaling><Cell Cycle Controller CDC2 Gene><Cell Cycle Controller cdc2><Cell Cycle Progression><Cell Division Control Protein 2 Homolog><Cell Division Cycle 2><Cell Division Cycle 2 Protein><Cell Signaling><Cells><Cellular biology><Chickens><Clinical><Co-Immunoprecipitations><Collaborations><Colorectal Cancer><Common Rat Strains><Communication><Communities><Complex><Computer software><Cyclin-Dependent Kinase 1><Cysteine><DNA Damage><DNA Injury><DNA replication fork><Danio rerio><Data><Data Analyses><Data Analysis><Data Bases><Databases><Deubiquitinating Enzyme><Development><Developmental Therapeutics><Developmental Therapeutics Program><Developmental Therapy><Dose><EC 2.1.1><Embryo><Embryonic><Experimental Designs><Follow-Up Studies><Followup Studies><Gallus domesticus><Gallus gallus><Gallus gallus domesticus><Gene Inactivation><Gene Silencing><Gene Transcription><GeneHomolog><General Transcription Factor Gene><General Transcription Factors><Genetic><Genetic Transcription><HDAC Agent><HDAC inhibitor><HIV><HIV Infections><HTLV-III Infections><HTLV-III-LAV Infections><Half-Cystine><Histone Deacetylase Inhibitor><Histone deacetylase inhibition><Homeo Domain Proteins><Homeobox Family Protein><Homeobox Proteins><Homeodomain Family Protein><Homeodomain Proteins><Homeoproteins><Homeotic Proteins><Homolog><Homologous Gene><Homologue><Human Immunodeficiency Viruses><Human T-Lymphotropic Virus Type III Infections><Hydrogen Sulfide><ICP-MS><Inductively Coupled Plasma Mass Spectrometry><Intracellular Communication and Signaling><Investigators><Isotope Labeling><Journals><L-Cysteine><L-Lysine><LAV-HTLV-III><Label><Laboratories><Lymphadenopathy-Associated Virus><Lysine><M Phase><Magazine><Malignant Breast Neoplasm><Malignant Neoplasms><Malignant Tumor><Malignant Tumor of the Prostate><Malignant neoplasm of prostate><Malignant prostatic tumor><Manuscripts><Maps><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Measures><Mercaptans><Mercapto Compounds><Messenger RNA><Metals><Methods><Methylation><Methyltransferase><Mice><Mice Mammals><Mitosis><Mitosis Stage><Molecular><Molecular Interaction><Molecular and Cellular Biology><Murine><Mus><NIDDK><NIH><NLM><National Institute of Diabetes and Digestive and Kidney Diseases><National Institutes of Health><National Library of Medicine><National Medical Library><Nature><Non-Polyadenylated RNA><Nuclear><Nucleic Acids><Oncogenes><Oral><Outcome><Paper><Phosphorylation Site><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Preparation><Prostate CA><Prostate Cancer><Prostate malignancy><Prostatic Cancer><Protein Cleavage><Protein Conformation><Protein Modification><Proteins><Proteolysis><Proteomics><Publishing><RNA><RNA Expression><RNA Gene Products><RNA Stability><Rat><Rats Mammals><Rattus><Regulation><Reporting><Research><Research Personnel><Research Resources><Researchers><Resistance><Resources><Ribonucleic Acid><Running><S Period><S phase><SN-38><Sampling><Signal Transduction><Signal Transduction Systems><Signaling><Site-Directed Mutagenesis><Site-Specific Mutagenesis><Societies><Software><Specificity><Sulfhydryl Compounds><Synthesis Period><Synthesis Phase><T-Cell Antigen Receptor-Interacting Molecule><T-Cell Receptor-Associated Transmembrane Adaptor 1><TRAT1><TRC-Interacting Molecule><TRIM><TRIM Gene><Targeted DNA Modification><Targeted Modification><Techniques><Technology><Testing><Therapeutic><Thiols><Time><Topoisomerase><Transcript><Transcription><Transcription Factor Proto-Oncogene><Transcription factor genes><Transforming Genes><Translational Research><Translational Science><Tumor Suppressor Proteins><Ubiquitilation><Ubiquitination><Ubiquitinoylation><United States National Institutes of Health><United States National Library of Medicine><Vaccines><Virus-HIV><Work><Zebra Danio><Zebra Fish><Zebrafish><Zinc><Zn element><active followup><biologic><biological signal transduction><cdc2 gene product><cdc2+ Protein><cdk1 Kinase><cell biology><cellular oncology><crosslink><data base><data interpretation><de-ubiquitinase><de-ubiquitinating enzyme><design><designing><developmental><enzyme activity><experiment><experimental research><experimental study><experiments><follow up><follow-up><followed up><followup><genome integrity><genome scale><genome-wide><genomewide><genomic data><genomic data-set><genomic dataset><genomic integrity><inhibitor><insoluble aggregate><interest><knock-down><knockdown><loss of function><mRNA><mRNA Expression><mRNA Stability><malignancy><malignant breast tumor><member><methylase><microorganism><neoplasm/cancer><overexpress><overexpression><p34 Protein Kinase><p34 Protein Kinase Gene><p34(CDC2) Gene><p34CDC2><paralog><paralogous gene><phospho-proteomics><phosphoproteomics><preparations><prevent><preventing><programs><protein aggregate><protein aggregation><protein expression><recruit><replication fork><replication stress><resistant><small molecular inhibitor><small molecule><small molecule inhibitor><sulfhydryl group><thioester><tool><transcription factor><transcriptional silencing><translation research><translational investigation><transmethylase><tumor suppressor><ubiquination><ubiquitin conjugation><ubiquitin isopeptidase><ubiquitin-specific isopeptidase><web tool><web-based tool>