Mass spectrometry-based proteomics coupled to liquid chromatography has matured into an automatized, high-throughput technology, producing data on the scale of multiple gigabytes per instrument per ...day. Consequently, an automated quality control (QC) and quality analysis (QA) capable of detecting measurement bias, verifying consistency, and avoiding propagation of error is paramount for instrument operators and scientists in charge of downstream analysis. We have developed an R-based QC pipeline called Proteomics Quality Control (PTXQC) for bottom-up LC–MS data generated by the MaxQuant software pipeline. PTXQC creates a QC report containing a comprehensive and powerful set of QC metrics, augmented with automated scoring functions. The automated scores are collated to create an overview heatmap at the beginning of the report, giving valuable guidance also to nonspecialists. Our software supports a wide range of experimental designs, including stable isotope labeling by amino acids in cell culture (SILAC), tandem mass tags (TMT), and label-free data. Furthermore, we introduce new metrics to score MaxQuant’s Match-between-runs (MBR) functionality by which peptide identifications can be transferred across Raw files based on accurate retention time and m/z. Last but not least, PTXQC is easy to install and use and represents the first QC software capable of processing MaxQuant result tables. PTXQC is freely available at https://github.com/cbielow/PTXQC.
In response to swelling, mammalian cells release chloride and organic osmolytes through volume-regulated anion channels (VRACs). VRACs are heteromers of LRRC8A and other LRRC8 isoforms (LRRC8B to ...LRRC8E), which are co-expressed in HEK293 and most other cells. The spectrum of VRAC substrates and its dependence on particular LRRC8 isoforms remains largely unknown. We show that, besides the osmolytes taurine and
-inositol, LRRC8 channels transport the neurotransmitters glutamate, aspartate and γ-aminobutyric acid (GABA) and the co-activator D-serine. HEK293 cells engineered to express defined subsets of LRRC8 isoforms were used to elucidate the subunit-dependence of transport. Whereas LRRC8D was crucial for the translocation of overall neutral compounds like
-inositol, taurine and GABA, and sustained the transport of positively charged lysine, flux of negatively charged aspartate was equally well supported by LRRC8E. Disruption of LRRC8B or LRRC8C failed to decrease the transport rates of all investigated substrates, but their inclusion into LRRC8 heteromers influenced the substrate preference of VRAC. This suggested that individual VRACs can contain three or more different LRRC8 subunits, a conclusion confirmed by sequential co-immunoprecipitations. Our work suggests a composition-dependent role of VRACs in extracellular signal transduction.
Loss of pluripotency is a gradual event whose initiating factors are largely unknown. Here we report the earliest metabolic changes induced during the first hours of differentiation. High-resolution ...NMR identified 44 metabolites and a distinct metabolic transition occurring during early differentiation. Metabolic and transcriptional analyses showed that pluripotent cells produced acetyl-CoA through glycolysis and rapidly lost this function during differentiation. Importantly, modulation of glycolysis blocked histone deacetylation and differentiation in human and mouse embryonic stem cells. Acetate, a precursor of acetyl-CoA, delayed differentiation and blocked early histone deacetylation in a dose-dependent manner. Inhibitors upstream of acetyl-CoA caused differentiation of pluripotent cells, while those downstream delayed differentiation. Our results show a metabolic switch causing a loss of histone acetylation and pluripotent state during the first hours of differentiation. Our data highlight the important role metabolism plays in pluripotency and suggest that a glycolytic switch controlling histone acetylation can release stem cells from pluripotency.
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•The metabolic profile of embryonic stem cell changes within hours of differentiation•Glycolytic production of acetyl-CoA promotes histone acetylation during pluripotency•Glycolysis inhibition leads to deacetylation and differentiation of pluripotent cells•Pharmacologic modulation of acetyl-CoA regulates pluripotency
Moussaieff et al. show that the initial induction of human and mouse pluripotent stem cell differentiation is accompanied by a reduction of glycolysis-generated acetyl-CoA, which affects histone acetylation. Inhibition of glycolysis or acetyl-CoA led to differentiation of embryonic stem cells. See also associated Preview by Daley.
Growing empirical evidence suggests that nutrition and bacterial metabolites might impact the systemic immune response in the context of disease and autoimmunity. We report that long-chain fatty ...acids (LCFAs) enhanced differentiation and proliferation of T helper 1 (Th1) and/or Th17 cells and impaired their intestinal sequestration via p38-MAPK pathway. Alternatively, dietary short-chain FAs (SCFAs) expanded gut T regulatory (Treg) cells by suppression of the JNK1 and p38 pathway. We used experimental autoimmune encephalomyelitis (EAE) as a model of T cell-mediated autoimmunity to show that LCFAs consistently decreased SCFAs in the gut and exacerbated disease by expanding pathogenic Th1 and/or Th17 cell populations in the small intestine. Treatment with SCFAs ameliorated EAE and reduced axonal damage via long-lasting imprinting on lamina-propria-derived Treg cells. These data demonstrate a direct dietary impact on intestinal-specific, and subsequently central nervous system-specific, Th cell responses in autoimmunity, and thus might have therapeutic implications for autoimmune diseases such as multiple sclerosis.
•Dietary fatty acids have profound influence on T cell differentiation in the gut•Middle- and long-chain fatty acids (LCFAs) support Th1 and Th17 cell differentiation•Short-chain fatty acids (SCFAs) lead to increased Treg cell differentiation•LCFAs worsen disease in an animal model of MS; SCFAs exert the opposite effect
Haghikia and colleagues show that dietary fatty acids (FAs) influence T cell differentiation in the gut, with short FAs leading to increased Treg cell differentiation and long FAs supporting Th1 and/or Th17 cell differentiation. These FAs differentially affect EAE severity, demonstrating a direct dietary impact on central nervous system autoimmunity.
RNA helicases are important regulators of gene expression that act by remodeling RNA secondary structures and RNA-protein interactions. Here, we demonstrate that MOV10 has an ATP-dependent 5′ to 3′ ...in vitro RNA unwinding activity and determine the RNA-binding sites of MOV10 and its helicase mutants using PAR-CLIP. We find that MOV10 predominantly binds to 3′ UTRs upstream of regions predicted to form local secondary structures and provide evidence that MOV10 helicase mutants are impaired in their ability to translocate 5′ to 3′ on their mRNA targets. MOV10 interacts with UPF1, the key component of the nonsense-mediated mRNA decay pathway. PAR-CLIP of UPF1 reveals that MOV10 and UPF1 bind to RNA in close proximity. Knockdown of MOV10 resulted in increased mRNA half-lives of MOV10-bound as well as UPF1-regulated transcripts, suggesting that MOV10 functions in UPF1-mediated mRNA degradation as an RNA clearance factor to resolve structures and displace proteins from 3′ UTRs.
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•MOV10 has in vitro 5′ to 3′ directional RNA unwinding activity•MOV10 displays widespread binding to 3′ untranslated regions (3′ UTRs) of mRNAs•PAR-CLIP of helicase-deficient MOV10 indicates impaired translocation along 3′ UTRs•MOV10 interacts with UPF1 and contributes to degradation of UPF1-regulated transcripts
RNA helicases regulate gene expression by remodeling RNA secondary structures and RNA-protein interactions. Gregersen et al. show that the RNA helicase MOV10 binds to 3′ UTRs and translocates 5′ to 3′ on its mRNA targets. MOV10 interacts with UPF1 and functions in UPF1-mediated mRNA degradation as mRNPase clearance factor.
Activated oncogenes and anticancer chemotherapy induce cellular senescence, a terminal growth arrest of viable cells characterized by S-phase entry-blocking histone 3 lysine 9 trimethylation ...(H3K9me3). Although therapy-induced senescence (TIS) improves long-term outcomes, potentially harmful properties of senescent tumour cells make their quantitative elimination a therapeutic priority. Here we use the Eµ-myc transgenic mouse lymphoma model in which TIS depends on the H3K9 histone methyltransferase Suv39h1 to show the mechanism and therapeutic exploitation of senescence-related metabolic reprogramming in vitro and in vivo. After senescence-inducing chemotherapy, TIS-competent lymphomas but not TIS-incompetent Suv39h1(-) lymphomas show increased glucose utilization and much higher ATP production. We demonstrate that this is linked to massive proteotoxic stress, which is a consequence of the senescence-associated secretory phenotype (SASP) described previously. SASP-producing TIS cells exhibited endoplasmic reticulum stress, an unfolded protein response (UPR), and increased ubiquitination, thereby targeting toxic proteins for autophagy in an acutely energy-consuming fashion. Accordingly, TIS lymphomas, unlike senescence models that lack a strong SASP response, were more sensitive to blocking glucose utilization or autophagy, which led to their selective elimination through caspase-12- and caspase-3-mediated endoplasmic-reticulum-related apoptosis. Consequently, pharmacological targeting of these metabolic demands on TIS induction in vivo prompted tumour regression and improved treatment outcomes further. These findings unveil the hypercatabolic nature of TIS that is therapeutically exploitable by synthetic lethal metabolic targeting.
There is increasing evidence that transcripts or transcript regions annotated as non-coding can harbor functional short open reading frames (sORFs). Loss-of-function experiments have identified ...essential developmental or physiological roles for a few of the encoded peptides (micropeptides), but genome-wide experimental or computational identification of functional sORFs remains challenging.
Here, we expand our previously developed method and present results of an integrated computational pipeline for the identification of conserved sORFs in human, mouse, zebrafish, fruit fly, and the nematode C. elegans. Isolating specific conservation signatures indicative of purifying selection on amino acid (rather than nucleotide) sequence, we identify about 2,000 novel small ORFs located in the untranslated regions of canonical mRNAs or on transcripts annotated as non-coding. Predicted sORFs show stronger conservation signatures than those identified in previous studies and are sometimes conserved over large evolutionary distances. The encoded peptides have little homology to known proteins and are enriched in disordered regions and short linear interaction motifs. Published ribosome profiling data indicate translation of more than 100 novel sORFs, and mass spectrometry data provide evidence for more than 70 novel candidates.
Taken together, we identify hundreds of previously unknown conserved sORFs in major model organisms. Our computational analyses and integration with experimental data show that these sORFs are expressed, often translated, and sometimes widely conserved, in some cases even between vertebrates and invertebrates. We thus provide an integrated resource of putatively functional micropeptides for functional validation in vivo.
Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master ...regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.
Abiotic stresses adversely affect plant growth and development. The hormone abscisic acid (ABA) plays a central role in the response and adaptation to environmental constraints. However, apart from ...the well established role of ABA in regulating gene expression programmes, little is known about its function in plant stress metabolism.
Using an integrative multiparallel approach of metabolome and transcriptome analyses, we studied the dynamic response of the model glyophyte Arabidopsis thaliana to ABA and high salt conditions. Our work shows that salt stress induces complex re-adjustment of carbohydrate metabolism and that ABA triggers the initial steps of carbon mobilisation.
These findings open new perspectives on how high salinity and ABA impact on central carbohydrate metabolism and highlight the power of iterative combinatorial approaches of non-targeted and hypothesis-driven experiments in stress biology.
Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive ...nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide binding sites of GLD-1, a conserved, germline-specific translational repressor in
C. elegans. We identified 439 reproducible target mRNAs and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knockdown, protein but not mRNA expression of the 439 targets was specifically upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites near the start codon of target genes. These sites are functional in vitro and likely confer strong repression in vivo. We propose that GLD-1 interacts with the translation machinery near the start codon, a so-far-unknown mode of gene regulation in eukaryotes.
► iPAR-CLIP identifies binding sites of RNA binding proteins (RBPs) in
C. elegans ► iPAR-CLIP identified hundreds of direct targets of the germline-specific RBP GLD-1 ► Effects of GLD-1 knockdown on protein expression of targets confirm functionality ► Conserved 5′ UTR sites near start codons are bound by GLD-1 and functional in vitro
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