Protein Modifications Involved in Cell Signaling

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

1. Quantification of the modification dynamics in TLR signaling 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 they are each activated by one or more PAMP ligands. TLRs are all 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). Among the most important genes to be regulated by TLR 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, in particular in the area of the development of therapies for the treatment of chronic inflammatory diseases. A clearer understanding of the TLR 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 are studying the changes in phosphorylation-dependent signaling in cells where the TLR signaling pathway components (MyD88, TRIF, IRAK family proteins, CD14) are knocked down or knocked out. We currently use the phosphorylation datasets as well as datasets quantifying other PTMs (ADP-ribosylation, ubiquitination, nucleic acid complexing) as constraints for a computational model of the TLR signaling network (project AI001085-07). The candidate proteins whose phosphorylation and ADP-ribosylation (2, 3) changed significantly during the investigated time course are being further examined in biological experiments. We characterized the changes in phosphorylation of specific sites of MARCKS upon LPS stimulation and we are now exploring the biological significance of these sites. We have performed site-directed mutagenesis of the individual sites and the mutant MARCKS has been expressed in the cells where we knocked out the wild-type protein using CRISPR technology and characterized by mass spectrometry. B). We have 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 have validated the data using ELISA-based assays of cytokine production and targeted proteomics. We have performed data correlation with the transcriptome (in collaboration with Dr. Fraser). We identified differences in signaling between individual TLRs and revealed specifics of pathway regulation at the protein level (4, 5). The data will provide more stringent constraints for the TLR signaling model. 2. We are studying the dynamics of the MyD88-associated protein complex (myddosome) following the stimulation of mouse macrophages with pathogen-derived molecules. Our results indicate that MyD88 exists in macrophages in a complex with inhibitory molecules which are released after LPS stimulation, allowing the proteins activating the inflammatory response to interact with MyD88 and initiate the inflammatory signaling cascade. 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 are currently exploring the interactomes of macrophages exposed to different pathogens (6, 7). 3. We performed a quantitative analysis of the proteome of the cells from the terminal ileum (chosen as 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-related protein expression and in specific metabolic pathways. The correlation of the proteome and transcriptome data revealed several differentially regulated pathways and significant transcriptome-proteome discordance in the adaptation of the host to the microbiota. This discovery leads to a conclusion that transcript level analysis is not sufficient to predict protein levels and their influence on the function of many specific cellular pathways, so only the combination of the quantitative data at different levels will lead to the complete understanding of the complex relationships between the host and the microbiota (8). Our further 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 (9). 4. In collaboration with Dr. Ernst (University of Maryland) we are examining the effects of different LPS structures on bacterial pathogenesis, focusing on macrophage signaling and the proteome, phosphoproteome and cytokine secretion changes. We have identified LPS-structure-dependent differences in the TLR signaling and inflammasome signaling (10, 11, 12, 13) and now study the effects of synthetic LPS-mimics on the TLR-signaling network components. 5. In collaboration with Dr. Leelahavanichkul from the Chulalongkorn University in Thailand we have been performing proteome, secretome and phosphoproteome studies of the endotoxin-induced exhaustion and sepsis (14, 15, 16, 17, 18). We are following up with studies of LPS-tolerance in the context of acyloxacyl hydrolase (AOAH) deficiency (collaboration with Dr. Robert Munford, NIAID). 6. Our new collaborative efforts focus on infection and disease. With Dr. Zelazny (NIH CC) we study the effects of clarithromycin on Mycobacterium abscessus. With Dr. Machner (NICHD) we uncovered the role of N-Ras in the Legionella pneumophila infection and its effect on the macrophage phosphoproteome. With Dr. Rochman (Cincinnati Childrens) we have uncovered the role or the minichromosome complex in eosinophilic esophagitis in the proteomic screen of esophageal biopsies (19). 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. Daniels CM, Nuccio A, Kaplan PR, Nita-Lazar A (2020). Methods Mol Biol 2184, 145-160. 4. Koppenol-Raab M, et al. (2017) Mol Cell Proteomics 16(4 suppl 1):S172-S186. 5. Koppenol-Raab M., and Nita-Lazar A. (2017) Methods Mol Biol. 1636:301-312 6. Gillen J, Nita-Lazar A (2019). Front Physiol 10, 425. 7. Gillen J, et al. (2020). Expert Rev Proteomics 17, 341-354. 8. Manes, N.P., et al. (2017). mSystems. 2(5). pii: e00107-17. 9. Li, Z. et al (2022) J. Exp. Med. 219 (7). 10. Khan MM, et al. (2019). 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