How the immune system readies for battleAlthough gene expression is tightly controlled at both the RNA and protein levels, the quantitative contribution of each step, especially during dynamic ...responses, remains largely unknown. Indeed, there has been much debate whether changes in RNA level contribute substantially to protein-level regulation. Jovanovic et al. built a genome-scale model of the temporal dynamics of differential protein expression during the stimulation of immunological dendritic cells (see the Perspective by Li and Biggin). Newly stimulated functions involved the up-regulation of specific RNAs and concomitant increases in the levels of the proteins they encode, whereas housekeeping functions were regulated posttranscriptionally at the protein level.Science, this issue 10.1126/science.1259038; see also p. 1066 Protein expression is regulated by the production and degradation of messenger RNAs (mRNAs) and proteins, but their specific relationships remain unknown. We combine measurements of protein production and degradation and mRNA dynamics so as to build a quantitative genomic model of the differential regulation of gene expression in lipopolysaccharide-stimulated mouse dendritic cells. Changes in mRNA abundance play a dominant role in determining most dynamic fold changes in protein levels. Conversely, the preexisting proteome of proteins performing basic cellular functions is remodeled primarily through changes in protein production or degradation, accounting for more than half of the absolute change in protein molecules in the cell. Thus, the proteome is regulated by transcriptional induction for newly activated cellular functions and by protein life-cycle changes for remodeling of preexisting functions.
Truncating mutations in the terminal exon of protein phosphatase Mg2+/Mn2+ 1D (PPM1D) have been identified in clonal hematopoiesis and myeloid neoplasms, with a striking enrichment in patients ...previously exposed to chemotherapy. In this study, we demonstrate that truncating PPM1D mutations confer a chemoresistance phenotype, resulting in the selective expansion of PPM1D-mutant hematopoietic cells in the presence of chemotherapy in vitro and in vivo. Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease mutational profiling of PPM1D in the presence of chemotherapy selected for the same exon 6 mutations identified in patient samples. These exon 6 mutations encode for a truncated protein that displays elevated expression and activity due to loss of a C-terminal degradation domain. Global phosphoproteomic profiling revealed altered phosphorylation of target proteins in the presence of the mutation, highlighting multiple pathways including the DNA damage response (DDR). In the presence of chemotherapy, PPM1D-mutant cells have an abrogated DDR resulting in altered cell cycle progression, decreased apoptosis, and reduced mitochondrial priming. We demonstrate that treatment with an allosteric, small molecule inhibitor of PPM1D reverts the phosphoproteomic, DDR, apoptotic, and mitochondrial priming changes observed in PPM1D-mutant cells. Finally, we show that the inhibitor preferentially kills PPM1D-mutant cells, sensitizes the cells to chemotherapy, and reverses the chemoresistance phenotype. These results provide an explanation for the enrichment of truncating PPM1D mutations in the blood of patients exposed to chemotherapy and in therapy-related myeloid neoplasms, and demonstrate that PPM1D can be a targeted in the prevention of clonal expansion of PPM1D-mutant cells and the treatment of PPM1D-mutant disease.
•Truncating PPM1D mutations confer chemotherapy resistance, leading to the selective expansion of PPM1D-mutant cells in vitro and in vivo.•PPM1D inhibitor treatment reverses the chemotherapy-resistance phenotype and selectively kills PPM1D-mutant cells.
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Here we present an optimized workflow for global proteome and phosphoproteome analysis of tissues or cell lines that uses isobaric tags (TMT (tandem mass tags)-10) for multiplexed analysis and ...relative quantification, and provides 3× higher throughput than iTRAQ (isobaric tags for absolute and relative quantification)-4-based methods with high intra- and inter-laboratory reproducibility. The workflow was systematically characterized and benchmarked across three independent laboratories using two distinct breast cancer subtypes from patient-derived xenograft models to enable assessment of proteome and phosphoproteome depth and quantitative reproducibility. Each plex consisted of ten samples, each being 300 μg of peptide derived from <50 mg of wet-weight tissue. Of the 10,000 proteins quantified per sample, we could distinguish 7,700 human proteins derived from tumor cells and 3100 mouse proteins derived from the surrounding stroma and blood. The maximum deviation across replicates and laboratories was <7%, and the inter-laboratory correlation for TMT ratio-based comparison of the two breast cancer subtypes was r > 0.88. The maximum deviation for the phosphoproteome coverage was <24% across laboratories, with an average of >37,000 quantified phosphosites per sample and differential quantification correlations of r > 0.72. The full procedure, including sample processing and data generation, can be completed within 10 d for ten tissue samples, and 100 samples can be analyzed in ~4 months using a single LC-MS/MS instrument. The high quality, depth, and reproducibility of the data obtained both within and across laboratories should enable new biological insights to be obtained from mass spectrometry-based proteomics analyses of cells and tissues together with proteogenomic data integration.
Hereditary autoinflammatory diseases are caused by gene mutations of the innate immune pathway, e.g. nucleotide receptor protein 3 (NLRP3). Here, we report a four-generation family with cold-induced ...urticarial rash, arthralgia, chills, headache and malaise associated with an autosomal-dominant inheritance. Genetic studies identify a substitution mutation in gene F12 (T859A, resulting in p.W268R) which encodes coagulation factor XII (FXII). Functional analysis reveals enhanced autocatalytic cleavage of the mutated protein and spontaneous FXII activation in patient plasma and in supernatant of transfected HEK293 cells expressing recombinant W268R-mutated proteins. Furthermore, we observe reduced plasma prekallikrein, cleaved high molecular weight kininogen and elevated plasma bradykinin. Neutrophils are identified as a local source of FXII. Interleukin-1β (IL-1β) is upregulated in lesional skin and mononuclear donor cells exposed to recombinant mutant proteins. Treatment with icatibant (bradykinin-B2-antagonist) or anakinra (interleukin-1-antagonist) reduces disease activity in patients. In conclusion, our findings provide a link between contact system activation and cytokine-mediated inflammation.
Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-di-glycine remnant (K-ε-GG) antibodies. Here, we describe a number of ...improvements to the K-ε-GG enrichment workflow, including optimized antibody and peptide input requirements, antibody cross-linking, and improved off-line fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of ∼20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input.
N6-methyladenosine (m6A) is a common modification of mRNA with potential roles in fine-tuning the RNA life cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of ...the methyltransferase complex, and show that three of them (WTAP, METTL14, and KIAA1429) are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps and their classification into WTAP-dependent and -independent sites. WTAP-dependent sites are located at internal positions in transcripts, topologically static across a variety of systems we surveyed, and inversely correlated with mRNA stability, consistent with a role in establishing “basal” degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data shed light on the proteomic and transcriptional underpinnings of this RNA modification.
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•METTL3, METTL14, WTAP, and KIAA1429 are required for mRNA methylation•Methylation maps, upon depletion of WTAP, reveal two classes of methylation•WTAP-dependent sites are mostly static and correlate with mRNA stability•Thousands of WTAP-independent sites are present at the first transcribed nucleotide
N6-methyladenosine (m6A) is a highly abundant modification of mRNA. Schwartz et al. identify and validate a network of proteins required for mRNA methylation in mammalian cells. They define two distinct classes of methylation sites. The majority of sites depend on the identified proteins, are located at internal positions in transcripts, and inversely correlate with mRNA stability. Sites independent of these proteins form at the first transcribed base as part of the cap structure, forming a previously unappreciated layer of transcriptome complexity.
Studying protein interactions
can reveal key molecular mechanisms of biological processes. Co-immunoprecipitation with mass spectrometry detects protein-protein interactions with high throughput. The ...nematode
is a powerful genetic model organism for
studies. Yet its rigid and complex tissues require optimization for biochemistry applications to ensure reproducibility. The authors optimized co-immunoprecipitation with mass spectrometry by combining a native co-immunoprecipitation procedure with single-pot, solid-phase enhanced sample preparation. The authors' results for the highly conserved chromatin regulator FACT subunits HMG-3 and HMG-4 demonstrated that single-pot, solid-phase enhanced sample preparation-integrated co-immunoprecipitation with mass spectrometry procedures for
samples are highly robust. Moreover, in an accompanying study about the chromodomain factor MRG-1 (MRG15 in humans), the authors demonstrated remarkably high reproducibility for ten replicate experiments.
Introduction of antibodies specific for acetylated lysine has significantly improved the detection of endogenous acetylation sites by mass spectrometry. Here, we describe a new, commercially ...available mixture of anti-lysine acetylation (Kac) antibodies and show its utility for in-depth profiling of the acetylome. Specifically, seven complementary monoclones with high specificity for Kac were combined into a final anti-Kac reagent which results in at least a twofold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline basic reversed phase separation, and use of state-of-the-art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods were applied to quantify acetylation sites in suberoylanilide hydroxamic acid-treated Jurkat cells. Over 10,000 Kac peptides from over 3000 Kac proteins were quantified from a single stable isotope labeling by amino acids in cell culture labeled sample using 7.5 mg of peptide input per state. This constitutes the deepest coverage of acetylation sites in quantitative experiments obtained to-date. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4, TMT6 and TMT10-plex reagents) for quantification. Greater than 6700 Kac peptides from over 2300 Kac proteins were quantified using 1 mg of tumor protein per iTRAQ 4-plex channel. The novel reagents and methods we describe here enable quantitative, global acetylome analyses with depth and sensitivity approaching that obtained for other well-studied post-translational modifications such as phosphorylation and ubiquitylation, and should have widespread application in biological and clinical studies employing mass spectrometry-based proteomics.
A fundamental goal of genomics is to identify the complete set of expressed proteins. Automated annotation strategies rely on assumptions about protein-coding sequences (CDSs), e.g., they are ...conserved, do not overlap, and exceed a minimum length. However, an increasing number of newly discovered proteins violate these rules. Here we present an experimental and analytical framework, based on ribosome profiling and linear regression, for systematic identification and quantification of translation. Application of this approach to lipopolysaccharide-stimulated mouse dendritic cells and HCMV-infected human fibroblasts identifies thousands of novel CDSs, including micropeptides and variants of known proteins, that bear the hallmarks of canonical translation and exhibit translation levels and dynamics comparable to that of annotated CDSs. Remarkably, many translation events are identified in both mouse and human cells even when the peptide sequence is not conserved. Our work thus reveals an unexpected complexity to mammalian translation suited to provide both conserved regulatory or protein-based functions.
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•ORF-RATER robustly identifies and quantifies translation from ribosome profiling data•ORF-RATER reveals thousands of novel micropeptides and variants of mammalian proteins•Hundreds of novel CDSs show evidence of protein-coding conservation among mammals•Many ORFs are translated in both mice and humans but lack protein-coding conservation
Fields et al. describe a ribosome profiling-based approach for empirical annotation of protein-coding regions of the genome. Of the thousands of previously unknown translated ORFs they identify in mouse and human, many are conserved or dynamically regulated. Surprisingly, a considerable subset is translated in both species despite weak sequence conservation.
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