Protein Modifications Involved in Cell Signaling

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

1. Quantification of the protein modifications and state dynamics in TLR signaling pathways and inflammasome pathways. The TLRs are a family of pathogen recognition receptors that alert the host to the presence of pathogens by recognizing molecular signatures, termed pathogen-associated molecular patterns (PAMPs). These sensors act as the first step in the induction of protective innate and adaptive immune responses. There are 11 human TLR homologues and each is activated by one or more ligands. TLRs are transmembrane proteins and their signaling is mediated by association of their internal domains with intracellular components. Classically, the TLR signaling cascade involves the myeloid differentiation primary response gene 88 (MyD88), interleukin-1 receptor-activated kinase (IRAK), and tumor-necrosis factor receptor-associated factor 6 (TRAF6), leading to the activation of Nuclear Factor kappaB (NF-kB). Intracellularly, canonical inflammasome activation is governed by a two-signal system where TLR or inflammatory cytokine receptor stimulation provides an initial transcriptional priming signal, while a second signal acts as an intracellular stress trigger leading to caspase cascade activation and release of proinflammatory IL-1 family cytokines. Among the most important genes to be regulated by TLR and inflammasome signaling are those encoding cytokines. Given the key role of cytokines in the orchestration of the inflammatory response, mechanisms of modulating their production garnered substantial interest, especially for the development of therapies for the treatment of chronic inflammatory diseases. A clearer understanding of the TLR pathway and inflammasome pathway leading to the cytokine production is required for a successful pharmacological intervention. A) We investigated differences in the phosphoprotein signaling cascades triggered by TLR2, TLR4, and TLR7 ligands in a murine macrophage cell line. We performed a global, quantitative, early post-stimulation kinetic analysis of the global mouse macrophage phosphoproteome using high-resolution mass spectrometry (1). We study the changes in phosphorylation-dependent signaling in cells where the TLR signaling pathway components are knocked down or knocked out. We use the phosphorylation datasets as well as datasets quantifying other PTMs (ADP-ribosylation (2), ubiquitination, nucleic acid complexing) as constraints for a computational model of the TLR signaling network (project AI001085-07). We further examine functions of candidate proteins whose modifications change significantly during the investigated time course. This year, we have performed deep profiling of the phosphoproteome changes using data-independent analysis by mass spectrometry, which resulted in a comprehensive coverage of the phosphosites and their changes in response to LPS, with a novel discovery of hundreds of LPS-dependent phosphotyrosine events. B). We characterized the changes in phosphorylation of specific sites on MARCKS upon LPS stimulation and we are now exploring their influence on the function of MARCKS. We performed site-directed mutagenesis of the individual sites and the mutant MARCKS was expressed in the cells where we knocked out the wild-type protein using CRISPR technology and characterized by mass spectrometry. We discovered previously uncharacterized role of MARCKS in changing the macrophage metabolism by regulating mitochondrial function (3) and obtained evidence on the opposing roles of MARCKS in inflammatory and interferon signaling pathways. C). We conducted parallel studies of the proteome and secretome changes using the same cells and ligands as for the phosphoproteome analysis but collecting data after longer periods of time to allow for changes in protein expression and secretion (4). We validated the data using ELISA-based assays of cytokine production and targeted proteomics. We performed data correlation with the transcriptome (with Dr. Fraser). We identified differences in signaling between individual TLRs and revealed specifics of pathway regulation at the protein level (4). The data will provide more stringent constraints for the TLR signaling model. We continue this work expanding to other types of human myeloid lineage cells (neutrophils) and human monocytes and well as T-cells (in collaboration with Jeff Zhu) 2. We study the dynamics of the MyD88-associated protein complex (myddosome) post-stimulation of mouse macrophages with PAMPs. Our results indicate that MyD88 exists in macrophages in a complex with inhibitory molecules released after LPS stimulation, allowing the proteins activating the inflammatory response to interact with MyD88 and initiate the inflammatory signaling. We showed that the dynamics of the myddosome is proteolysis-dependent. We performed quantitative studies of changes in the myddosome and phosphorylation of the myddosome components in cells stimulated with different PAMPs and we have detected compositional and temporal differences in the signaling networks. We explored the interactomes of macrophages exposed to different pathogens. Now, we focus on MyD88 interactions with specific proteins we observed, on the protein-protein and protein-RNA (5) interactions. We have recently started to expand the work on protein complexes in macrophages using limited proteolysis mass spectrometry (LiP-MS) with tandem mass tag (TMT) labeling and thermal proteome profiling. We identified cytoplasmic proteins whose surface exposure to proteinase K changes in response to LPS treatment, indicating changes in the complex formation. Using modified LOPIT method, we have been mapping the protein subcellular localization changes upon macrophage exposure to PAMPS (LPS and flagellin) which affect complex formation dynamics. 3. We performed quantitative analysis of the proteome of the cells from the terminal ileum (a site of intense host-microbe interactions) of germ-free and normal mice (collaboration with Drs. Shulzhenko and Morgun). The data showed changes in the immune processes- and metabolism-related protein expression. The correlation of the proteome and transcriptome data revealed differentially regulated pathways and significant transcriptome-proteome discordance in the adaptation of the host to the microbiota. We concluded that only the combination of the quantitative data at different omics levels will lead to the complete understanding of the complex relationships between the host and the microbiota (6). Our studies of the differences between the germ-free and normal mice dependent on the dietary changes led to the discovery of the role of Mmp12+ macrophages in white adipose tissue (7). We are now working to further elucidate the mechanisms of action of this macrophage population exposed to a specific component of the gut microbiome, Oscillibacter spp. 4. In collaboration with Dr. Ernst (UMD) we study the effects of different LPS structures on bacterial pathogenesis, focusing on macrophage signaling and the proteome, phosphoproteome and cytokine secretion changes. We identified LPS-structure-dependent differences in the TLR signaling and inflammasome signaling (8, 9, 10, 11) and now study the effects of synthetic LPS-mimics on the TLR-signaling and inflammasome signaling. 5. We have long been working on proteome, secretome and phosphoproteome studies of the endotoxin-induced exhaustion and sepsis (12, 13, 14, 15, 16). This year, we follow up with studies of LPS-tolerance in the context of acyloxacyl hydrolase (AOAH) deficiency (with Dr. Munford, NIAID). 6. Our collaborative efforts focus on infection and disease. With Dr. Machner (NICHD) we uncovered the role of N-Ras in the Legionella pneumophila infection and its effect on the macrophage phosphoproteome (17) and currently work on the unbiquitinome. With Dr. Gough (NIAID) we study the phosphoproteome changes in macrophages exposed to Roseomonas and its mutant strains. With Dr. Grigg (NIAID) we explore the roles of specific Toxoplasma proteins in the infection process. With Dr. Chatterjee (UMD) we study different aspects of the S. aureus infection mechanisms and antibiotic resistance. With Dr. Torabi-Parizi we have been studying interactions leading to the activation of different immune cell populations upon human IV-endotoxin challenge. With Dr. Thompson (Howard University) we examine the PTMs and protein expression changes in E.coli strains grown under defined conditions. With Dr. Adoro (NCI) we work on the cGAS regulation in autoinflammatory myopathy. References: 1. Sjoelund V, Smelkinson M, and Nita-Lazar A. (2014) J Proteome Res. 2014 Nov 7;13(11):5185-97 2. Daniels CM, et al. (2020) J Proteome Res. 19 (9) 3. Issara-Amphorn, J. et al (2023) Sci Rep. 13(1):19562 4. Koppenol-Raab M, et al. (2017) Mol Cell Proteomics 16(4 suppl 1):S172-S186 5. Rathore, D. et al (2024) J. Proteome Res. 1 3(8):3280-329 6. Manes, N.P., et al. (2017). mSystems. 2(5). pii: e00107-17 7. Li, Z. et al (2022) J. Exp. Med. 219 (7) 8. Khan MM, et al. (2019). Pathog Dis 77 9. Khan MM, et al. (2019). ACS Infect Dis 5, 493-505 10. Khan MM. et al. (2018). J Mol Biol 430, 2641-2660 11. Ernst O. et al. (2021). mSystems 6 (4) 12. Ondee T, et al. (2019). Int J Mol Sci 20 13. Ondee T. et al. (2019) Cells. Sep 11;8(9):1064 14. Gillen J, Ondee T, et al (2021). Biomolecules 11 15. Phuengmaung P. et al. (2023) Int J Mol Sci 24 (10) 16. Saisorn W. et al. (2023) Int J Mol Sci 24 (12) 17. Lehman S. S. et al. Cell Reports 43(4):114033