Highlights d The release of conventionally secreted cytokines is reduced in necroptotic cells d Receptor shedding occurs during TNF-induced necroptosis and apoptosis d Lysosomal components are released via lysosomal exocytosis during necroptosis

Multinucleated giant cells (MGCs) are implicated in many diseases including schistosomiasis, sarcoidosis and arthritis. MGC generation is energy intensive to enforce membrane fusion and cytoplasmic expansion. Using receptor activator of nuclear factor kappa-Β ligand (RANKL) induced osteoclastogenesis to model MGC formation, here we report RANKL cellular programming requires extracellular arginine. Systemic arginine restriction improves outcome in multiple murine arthritis models and its removal induces preosteoclast metabolic quiescence, associated with impaired tricarboxylic acid (TCA) cycle function and metabolite induction. Effects of arginine deprivation on osteoclastogenesis are independent of mTORC1 activity or global transcriptional and translational inhibition. Arginine scarcity also dampens generation of IL-4 induced MGCs. Strikingly, in extracellular arginine absence, both cell types display flexibility as their formation can be restored with select arginine precursors. These data establish how environmental amino acids control the metabolic fate of polykaryons and suggest metabolic ways to manipulate MGC-associated pathologies and bone remodelling.

Highlights d Mass spectrometry resource for inflammasome-activated protein release in macrophages d Inhibition of inflammasome assembly and protein release by ER-Golgi disruption d Dissection of protein release by gasdermin, organelle damage, and extracellular vesicles d Identification of released constitutive and induced alarmins

Secreted proteins such as cytokines, interleukins, growth factors, and hormones have pleiotropic functions and facilitate intercellular communication in organisms. Quantification of these proteins conventionally relies on antibody-based methods, i.e., enzyme-linked immunosorbent assays (ELISA), whose large-scale use is limited by availability, specificity, and affordability.Here, we describe an experimental and bioinformatics workflow to comprehensively quantify cellular protein secretion by mass spectrometry. Secreted proteins are collected in vitro or ex vivo, digested with proteases and the resulting peptide mixtures are analyzed in single liquid chromatography-mass spectrometry (LC-MS/MS) runs. Label-free quantification and bioinformatics analysis is conducted in the MaxQuant and Perseus computational environment. Our workflow allows the quantification of thousands of secreted proteins spanning a concentration range of four orders of magnitude and permits the systems-level characterization of secretory programs as well as the discovery of proteins with unexpected extracellular functions.

Cells signal through rearrangements of protein communities governed by covalent modifications and reversible interactions of distinct sets of proteins. A method that identifies those posttranscriptional modifications regulating signaling complex composition and functional phenotypes in one experimental setup would facilitate an efficient identification of novel molecular signaling checkpoints. Here, we devised modifications, interactions and phenotypes by affinity purification mass spectrometry (MIP-APMS), comprising the streamlined cloning and transduction of tagged proteins into functionalized reporter cells as well as affinity chromatography, followed by MS-based quantification. We report the time-resolved interplay of more than 50 previously undescribed modification and hundreds of protein-protein interactions of 19 immune protein complexes in monocytes. Validation of interdependencies between covalent, reversible, and functional protein complex regulations by knockout or site-specific mutation revealed ISGylation and phosphorylation of TRAF2 as well as ARHGEF18 interaction in Toll-like receptor 2 signaling. Moreover, we identify distinct mechanisms of action for small molecule inhibitors of p38 (MAPK14). Our method provides a fast and cost-effective pipeline for the molecular interrogation of protein communities in diverse biological systems and primary cells.