Overall, the expertise of the Mass Spectrometry Unit is being widely used to further the research of multiple groups within the NIH. In FY2018, the unit collaborated in 54 different projects, with more than 3000 samples processed and analyzed. These projects are being performed in collaboration with 46 different investigators. Among these are projects to characterize the post-translational modifications of target proteins, including sites of phosphorylation, ubiquitination, 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, including identification of those that change following post-translational modification. 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 being 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, including copper, iron, and platinum. In the past year, six collaborative studies have been published; several other projects are nearing completion or manuscripts are under review. The first study, a collaboration with Dr. Ashish Lal, Genetics Branch, focused on the biological function of the long non-coding RNA PURPL. PURPL is an intergenic lncRNA that was identified by RNA sequencing from multiple colorectal cancer lines as being regulated by p53. Further experiments demonstrated that in the context of DNA damage, PURPL functions as a pro-survival factor and suppresses p53 protein level. To better understand how PURPL exerts these activities, mass spectrometry was used to identify the protein interactors of PURPL. Among the protein interactors we identified, MYBBP1A, a protein known to stabilize p53, was strongly enriched in the PURPL pull-downs. MYBBP1A is a predominantly nucleolar protein that associates with RNA and directly binds to p53 in the nucleoplasm, resulting in p53 activation and stabilization. Further cell-based experiments revealed that MYBBP1A associates with PURPL through association with HuR, a second protein identified by mass spectrometry to be strongly enriched in the PURPL pull-downs. These studies identify an auto-regulatory feedback loop between p53 and PURPL and was published in Cell Reports. We worked with Dr. Jonathan Ashwell, Laboratory of Immune Cell Biology, on two projects related to T-cell receptor (TCR) signaling. In the first study, reported in PLOS Biology, mass spectrometry analysis identified a novel site of phosphorylation on nuclear factor of activated T cells 1 (NFAT1), a transcription factor required for induction of T-cell cytokine production and effector function after TCR activation. We found that T cell p38, activated by an alternative pathway independent of the mitogen-activated protein kinase (MAPK) cascade, phosphorylated NFAT1 on S79. Whereas p38 activated via the MAPK cascade phosphorylated NFAT1 and NFAT2 resulting in cytosolic retention and inhibition, alternatively activated p38 phosphorylation of NFAT1 results in calcineurin binding and migration to the nucleus. Therefore, the stress-induced classic p38 pathway leads to inhibitory effects on NFAT1 and NFAT2, whereas alternatively activated p38 activates NFAT1 and NFAT2. In the second study, we showed by mass spectrometry that alternatively activated p38 phosphorylates its upstream tyrosine kinase ZAP-70. T cell p38 MAPK, which is directly phosphorylated and activated by ZAP-70 downstream of the TCR, in turn phosphorylates Thr-293 of ZAP-70. Mutant T cells expressing ZAP-70 with an alanine substitution at this residue had enhanced TCR proximal signaling and increased effector responses. Lack of ZAP-70 T293 phosphorylation increased association of ZAP-70 with the TCR and prolonged the existence of TCR signaling microclusters. These results identify a tight negative feedback loop in which ZAP-70-activated p38 reciprocally phosphorylates ZAP-70 and destabilizes the signaling complex. The results of this work were published in the Proceedings of the National Academy of Sciences of the USA. We worked with Dr. Yves Pommier, Developmental Therapeutics Branch, on two studies centered on proteins involved in DNA repair. First, we used mass spectrometry to confirm the presence of a novel short form of Tyrosyl-DNA phosphodiesterase 2 (TDP2), which repairs abortive topoisomerase II cleavage complexes. TDP2S is expressed from an alternative transcription start site and contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol. We found that both TDP2 isoforms are present and active in the mitochondria, and their knockout renders cells hypersensitive to mitochondrial-targeted doxorubicin. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we found that TDP2S protects against mtDox. Finally, lack of TDP2 in the mitochondria reduces mitochondrial transcription levels. Thus, in addition to identifying a novel TDP2S isoform, this research, published in EMBO Reports, demonstrates the presence and importance of both TDP2 isoforms in the mitochondria. The second project, published in Molecular Cell, focused on the function of SLFN11, a protein that is a dominant determinant of response to widely used anticancer drugs that induce replication stress. We found that in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci. Using mass spectrometry analysis, we found that this binding occurs through SLFN11 interaction with RPA1 and the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block, and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites. Finally, we used mass spectrometry to elucidate the mechanism by which human mutant p53 (mutp53) cancer cells reprogram macrophages to a tumor supportive and anti-inflammatory state. Mass spectrometry analysis of the secretome of M2 macrophages co-cultured with mutp53 or WT p53 tumor cells, or without any co-culture, showed that when macrophages were exposed to tumor cells carrying mutp53, increased secretion of several pro-tumorigenic factors, such as matrix metallopeptidase 9, vascular non-inflammatory molecule 1, and transforming growth factor beta-induced (TGFBI), was observed. Further analysis revealed that colon cancer cells harboring gain-of-function mutp53 selectively shed miR-1246-enriched exosomes. Uptake of these exosomes by neighboring macrophages triggers their reprogramming into a cancer-promoting state. These findings, associated with poor survival in colon cancer patients, strongly support a microenvironmental GOF role for mutp53 in actively engaging the immune system to promote cancer progression and metastasis. This study, performed in collaboration with Dr. Curtis Harris, Laboratory of Human Carcinogenesis, was published in Nature Communications.

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Kriebel, Paul W; Majumdar, Ritankar; Jenkins, Lisa M et al. (2018) Extracellular vesicles direct migration by synthesizing and releasing chemotactic signals. J Cell Biol 217:2891-2910
Murai, Junko; Tang, Sai-Wen; Leo, Elisabetta et al. (2018) SLFN11 Blocks Stressed Replication Forks Independently of ATR. Mol Cell 69:371-384.e6
Chaudhary, Ritu; Gryder, Berkley; Woods, Wendy S et al. (2017) Prosurvival long noncoding RNA PINCR regulates a subset of p53 targets in human colorectal cancer cells by binding to Matrin 3. Elife 6:
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Appella, Ettore; Jenkins, Lisa M Miller; Guengerich, F Peter (2015) Introduction to thematic series: protein interactions, structures, and networks. J Biol Chem 290:26393-4

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