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Rapid temporal dynamics of transcription, protein synthesis, and secretion during macrophage activation.

ABSTRACT: Macrophages provide the first line of host defense with their capacity to react to an array of cytokines and bacterial components requiring tight regulation of protein expression and secretion to invoke a properly tuned innate immune response. To capture the dynamics of this system, we introduce a novel method combining pulsed stable isotope labeling with amino acids in cell culture (SILAC) with pulse labeling using the methionine analog azidohomoalanine that allows the enrichment of newly synthesized proteins via click-chemistry followed by their identification and quantification by mass spectrometry. We show that this permits the analysis of proteome changes on a rapid time scale, as evidenced by the detection of 4852 newly synthesized proteins after only a 20-min SILAC pulse. We have applied this methodology to study proteome response during macrophage activation in a time-course manner. We have combined this with full proteome, transcriptome, and secretome analyses, producing an integrative analysis of the first 3 h of lipopolysaccharide-induced macrophage activation. We observed the rapid induction of multiple processes well known to TLR4 signaling, as well as anti-inflammatory proteins and proteins not previously associated with immune response. By correlating transcriptional, translational, and secretory events, we derived novel mechanistic principles of processes specifically induced by lipopolysaccharides, including ectodomain shedding and proteolytic processing of transmembrane and extracellular proteins and protein secretion independent of transcription. In conclusion, we demonstrate that the combination of pulsed azidohomoalanine and pulsed SILAC permits the detailed characterization of proteomic events on a rapid time scale. We anticipate that this approach will be very useful in probing the immediate effects of cellular stimuli and will provide mechanistic insight into cellular perturbation in multiple biological systems. The data have been deposited in ProteomeXchange with the identifier PXD000600.

SUBMITTER: Eichelbaum K

PROVIDER: S-EPMC3945909 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Rapid temporal dynamics of transcription, protein synthesis, and secretion during macrophage activation.

Eichelbaum Katrin K Krijgsveld Jeroen J

Molecular & cellular proteomics : MCP 20140106 3

Macrophages provide the first line of host defense with their capacity to react to an array of cytokines and bacterial components requiring tight regulation of protein expression and secretion to invoke a properly tuned innate immune response. To capture the dynamics of this system, we introduce a novel method combining pulsed stable isotope labeling with amino acids in cell culture (SILAC) with pulse labeling using the methionine analog azidohomoalanine that allows the enrichment of newly synth ...[more]

PMID: 24396086

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Project description:Macrophages provide the first line of host defense by their capacity to react to an array of cytokines and bacterial components, requiring tight regulation of protein expression and secretion to invoke a properly tuned innate immune response. To capture the dynamics of this system, we introduce a novel method combining pulsed SILAC labeling with pulse-labeling using the methionine-analogue azidohomoalanine (AHA), allowing the enrichment of newly synthesized proteins via click-chemistry followed by their identification and quantification by mass spectrometry. We show that this permits the analysis of proteome changes at a rapid time scale evidenced by the detection of 4852 newly synthesized proteins after only a 20-minute SILAC pulse. We have applied this methodology to study proteome response during macrophage activation in a time-course manner. We have combined this with full proteome and secretome analyses producing an integrative analysis of the first 3 hours of lipopolysaccharides (LPS)-induced macrophage activation. Data analysis: The mass spectrometric raw data were processed using MaxQuant (version 1.2.2.5) (1) and MS/MS spectra were searched using the Andromeda search engine (2) against mouse proteins in UniProt (53623 entries) (3), to which 265 frequently-observed contaminants as well as reversed sequences of all entries had been added. Enzyme specificity was set to trypsin/P and a maximum of two missed cleavages were allowed. Cysteine carbamidomethylation was used as fixed modification and methionine oxidation, protein N-terminal acetylation, as well as replacement of methionine by AHA (in case of AHA treatment experiments) were used as variable modifications. The minimal peptide length was set to 6 amino acids. Initial maximal allowed mass tolerance was set to 20 ppm for peptide masses, followed by 6 ppm in the main search and 0.5 Dalton for fragment ion masses. False discovery rates for peptide and protein identification were set to 1%. At least one unique peptide was required for protein identification. The protein identification was reported as an indistinguishable “protein group” if no unique peptide sequence to a single database entry was identified. For protein quantification a minimum of two ratio counts was set and the “requantify” and “match between runs” function enabled. For pSILAC samples a protein group was kept for the further analysis if the number of identified peptide species carrying an medium-heavy or heavy label divided by the total number of peptide species detected in the complete experimental setup was higher than 0.2. For all other samples proteins assigned to contaminants or reverse sequences were removed. Average protein ratios were reported, if they were quantified in two replicates each based on at least two ratio counts.

Project description:BackgroundMacrophages show versatile functions in innate immunity, infectious diseases, and progression of cancers and cardiovascular diseases. These versatile functions of macrophages are conducted by different macrophage phenotypes classified as classically activated macrophages and alternatively activated macrophages due to different stimuli in the complex in vivo cytokine environment. Dissecting the regulation of macrophage activations will have a significant impact on disease progression and therapeutic strategy. Mathematical modeling of macrophage activation can improve the understanding of this biological process through quantitative analysis and provide guidance to facilitate future experimental design. However, few results have been reported for a complete model of macrophage activation patterns.ResultsWe globally searched and reviewed literature for macrophage activation from PubMed databases and screened the published experimental results. Temporal in vitro macrophage cytokine expression profiles from published results were selected to establish Boolean network models for macrophage activation patterns in response to three different stimuli. A combination of modeling methods including clustering, binarization, linear programming (LP), Boolean function determination, and semi-tensor product was applied to establish Boolean networks to quantify three macrophage activation patterns. The structure of the networks was confirmed based on protein-protein-interaction databases, pathway databases, and published experimental results. Computational predictions of the network evolution were compared against real experimental results to validate the effectiveness of the Boolean network models.ConclusionThree macrophage activation core evolution maps were established based on the Boolean networks using Matlab. Cytokine signatures of macrophage activation patterns were identified, providing a possible determination of macrophage activations using extracellular cytokine measurements.

Project description:UNLABELLED:Understanding cellular responses to environmental stimuli requires not only the knowledge of specific regulatory components but also the quantitative characterization of the magnitude and timing of regulatory events. The two-component system is one of the major prokaryotic signaling schemes and is the focus of extensive interest in quantitative modeling and investigation of signaling dynamics. Here we report how the binding affinity of the PhoB two-component response regulator (RR) to target promoters impacts the level and timing of expression of PhoB-regulated genes. Information content has often been used to assess the degree of conservation for transcription factor (TF)-binding sites. We show that increasing the information content of PhoB-binding sites in designed phoA promoters increased the binding affinity and that the binding affinity and concentration of phosphorylated PhoB (PhoB~P) together dictate the level and timing of expression of phoA promoter variants. For various PhoB-regulated promoters with distinct promoter architectures, expression levels appear not to be correlated with TF-binding affinities, in contrast to the intuitive and oversimplified assumption that promoters with higher affinity for a TF tend to have higher expression levels. However, the expression timing of the core set of PhoB-regulated genes correlates well with the binding affinity of PhoB~P to individual promoters and the temporal hierarchy of gene expression appears to be related to the function of gene products during the phosphate starvation response. Modulation of the information content and binding affinity of TF-binding sites may be a common strategy for temporal programming of the expression profile of RR-regulated genes. IMPORTANCE:A single TF often orchestrates the expression of multiple genes in response to environmental stimuli. It is not clear how different TF-binding sites within the regulon dictate the expression profile. Our studies of Escherichia coli PhoB, a response regulator that controls expression of a core set of phosphate assimilation genes in response to phosphate starvation, showed that expression levels of PhoB-regulated genes are under sophisticated control and do not follow a simple correlation with the binding affinity of PhoB~P to individual promoters. However, the expression timing correlates with the PhoB-binding affinity and gene functions. Genes involved in direct Pi uptake contain high-affinity sites and are transcribed earlier than genes involved in phosphorus scavenging. This illustrates an elaborate mechanism of temporally programmed gene expression, even for nondevelopmental pathways.

Dataset Information

Rapid temporal dynamics of transcription, protein synthesis, and secretion during macrophage activation.

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Rapid temporal dynamics of transcription, protein synthesis, and secretion during macrophage activation.

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