Dynamic ADP-Ribosylome, Phosphoproteome, and Interactome in LPS-Activated Macrophages

Abstract

We have used mass spectrometry (MS) to characterize protein signaling in lipopolysaccharide (LPS)-stimulated macrophages from human blood, human THP1 cells, mouse bone marrow, and mouse Raw264.7 cells. Protein ADP-ribosylation was truncated down to phosphoribose, allowing for enrichment and identification of the resulting phosphoribosylated peptides alongside phosphopeptides. Size exclusion chromatography-MS (SEC-MS) was used to separate proteoforms by size; protein complexes were then identified by weighted correlation network analysis (WGCNA) based on their correlated movement into or out of SEC fractions following stimulation, presenting an analysis method for SEC-MS that does not rely on established databases. We highlight two modules of interest: one linked to the apoptosis signal-regulating kinase (ASK) signalosome and the other containing poly(ADP-ribose) polymerase 9 (PARP9). Finally, PARP inhibition was used to perturb the characterized systems, demonstrating the importance of ADP-ribosylation for the global interactome. All post-translational modification (PTM) and interactome data have been aggregated into a meta-database of 6729 proteins, with ADP-ribosylation characterized on 2905 proteins and phosphorylation characterized on 2669 proteins. This databasetitled MAPCD, for Macrophage ADP-ribosylation, Phosphorylation, and Complex Dynamicsserves as an invaluable resource for studying crosstalk between the ADP-ribosylome, phosphoproteome, and interactome.

Spatiotemporal proteomic profiling of the pro-inflammatory response to lipopolysaccharide in the THP-1 human leukaemia cell line

“…For example, we see colocalisation of (i) CDC42 and TRIP10 (CDC42-interacting protein 4) 75 , (ii) the endocytic molecules HGS and TSG101 76 and (iii) the mitochondrial NLRX1 and FASTKD5 77 . Proteins known to co-localise upon LPS stimulation were also shown together in hyperLOPIT space, including IKBKG (Nemo) with IKBKB 78 . The LPS-responsive interactors PARP9 and DTX3L 41 were both upregulated in abundance by 12 h-LPS and co-localised in hyperLOPIT space.…”

Intracellular mono-ADP-ribosyltransferases at the host–virus interphase

“…While mass spectrometry studies define increasing numbers of substrates and ADP-ribosylation sites (see e.g. [ 76 – 82 ]), information that specifies enzyme–substrate pairs is still rare. Interestingly, a recent study suggests that ADP-ribosylation increases upon treatment with IFN as well as poly(I:C), a vRNA mimic [ 83 ].…”

Global characterization of macrophage polarization mechanisms and identification of M2-type polarization inhibitors

“…This is, in part, explained by the lack of comparative, global quantitative time-course proteomic and phosphoproteomic analyses of the changes that occur during the induction phase of M1- versus M2-type macrophage polarization. Previous phosphoproteomic studies were limited in scale and temporal resolution and only assessed LPS-induced, but not IL-4-induced, macrophage polarization and mainly examined only the very early phase of M1-type polarization ( Daniels et al, 2020 ; Sharma et al, 2010 ; Sjoelund et al, 2014 ; Weintz et al, 2010 ). Similarly, prior proteomic studies of fully polarized macrophages have been limited in depth and scale ( Court et al, 2017 ; Meijer et al, 2015 ; Wiktorowicz et al, 2019 ).…”