The innate immune system is the organism's first line of defense against pathogens. Pattern recognition receptors (PRRs) are responsible for sensing the presence of pathogen-associated molecules. The prototypic PRRs, the membrane-bound receptors of the Toll-like receptor (TLR) family, recognize pathogen-associated molecular patterns (PAMPs) and initiate an innate immune response through signaling pathways that depend on the adaptor molecules MyD88 and TRIF. Deciphering the differences in the complex signaling events that lead to pathogen recognition and initiation of the correct response remains challenging. Here we report the discovery of temporal changes in the protein signaling components involved in innate immunity. Using an integrated strategy combining unbiased proteomics, transcriptomics and macrophage stimulations with three different PAMPs, we identified differences in signaling between individual TLRs and revealed specifics of pathway regulation at the protein level.

Osteoclasts are monocyte-derived multinuclear cells that directly attach to and resorb bone. Sphingosine-1-phosphate (S1P) 1 regulates bone resorption by functioning as both a chemoattractant and chemorepellent of osteoclast precursors through two G-protein coupled receptors that antagonize each other in an S1P-concentration-dependent manner. To quantitatively explore the behavior of this chemosensing pathway, we applied targeted proteomics, transcriptomics, and rule-based pathway modeling using the Simmune toolset. RAW264.7 cells (a mouse monocyte/macrophage cell line) were used as model osteoclast precursors, RNA-seq was used to identify expressed target proteins, and selected reaction monitoring (SRM) mass spectrometry using internal peptide standards was used to perform absolute abundance measurements of pathway proteins. The resulting transcript and protein abundance values were strongly correlated. Measured protein abundance values, used as simulation input parameters, led to in silico pathway behavior matching in vitro measurements. Moreover, once model parameters were established, even simulated responses toward stimuli that were not used for parameterization were consistent with experimental findings. These findings demonstrate the feasibility and value of combining targeted mass spectrometry with pathway modeling for advancing biological insight. Molecular & Cellular Proteomics 14: 10.1074/mcp.M115.048918, 2661-2681, 2015.Chemotaxis is defined as directed movement of a cell (or of an organism) resulting from stimulation by a chemokine or other chemotactic chemical. Eukaryotic cells employ intricate intracellular pathways to sense concentration differences of chemoattractants at their surface and move along such gradients using multiple synchronized cellular processes, including cell protrusion and adhesion at the leading end, de-adhesion at the trailing end, and mechanical force generation at both the leading and trailing ends (1-4).Activation of chemotactic receptors through chemoattractant concentration gradients results in nonuniform intracellular signaling responses that depend on numerous feedback mechanisms (5-7). Downstream, F-actin synthesis at the leading end causes the formation of cell protrusions (for example, filopodia, lamellipodia, and lamellae) that, in concert with actomyosin contraction, generates force at both the leading and trailing ends (8 -10). Chemotactic traction is produced by cell protrusions interacting with a confined environment and/or by adhesions that bind to the extracellular matrix and/or to cell adhesion molecules (11).Chemotaxis plays a major role in a wide range of physiological and pathophysiological processes. Sphingosine-1-phosphate (S1P), a phosphosphingolipid, mediates chemotaxis of many circulating cell types, including osteoclast precursors (OPs) (12)(13)(14). Osteoclasts are monocyte-derived multinuclear cells that directly attach to the bone matrix and resorb bone. They are solely responsible for bone resorption, and their misregulated activity has been imp...

The tail-anchored protein Fis1 is implicated as a passive tether in yeast mitochondrial fission. We probed the functional role of Fis1 Glu-78, whose elevated side chain pK a suggests participation in protein interactions. Fis1 binds partners Mdv1 or Dnm1 tightly, but mutation E78A weakens Fis1 interaction with Mdv1, alters mitochondrial morphology, and abolishes fission in a growth assay. In fis1⌬ rescue experiments, Fis1-E78A causes a novel localization pattern in which Dnm1 uniformly coats the mitochondria. By contrast, Fis1-E78A at lower expression levels recruits Dnm1 into mitochondrial punctate structures but fails to support normal fission. Thus, Fis1 makes multiple interactions that support Dnm1 puncta formation and may be essential after this step, supporting a revised model for assembly of the mitochondrial fission machinery. The insights gained by mutating a residue with a perturbed pK a suggest that side chain pK a values inferred from routine NMR sample pH optimization could provide useful leads for functional investigations.