Marieluise Kirchner

GABA Blocks Pathological but Not Acute TRPV1 Pain Signals

Sensitization of the capsaicin receptor TRPV1 is central to the initiation of pathological forms of pain, and multiple signaling cascades are known to enhance TRPV1 activity under inflammatory conditions. How might detrimental escalation of TRPV1 activity be counteracted? Using a genetic-proteomic approach, we identify the GABAB1 receptor subunit as bona fide inhibitor of TRPV1 sensitization in the context of diverse inflammatory settings. We find that the endogenous GABAB agonist, GABA, is released from nociceptive nerve terminals, suggesting an autocrine feedback mechanism limiting TRPV1 sensitization. The effect of GABAB on TRPV1 is independent of canonical G protein signaling and rather relies on close juxtaposition of the GABAB1 receptor subunit and TRPV1. Activating the GABAB1 receptor subunit does not attenuate normal functioning of the capsaicin receptor but exclusively reverts its sensitized state. Thus, harnessing this mechanism for anti-pain therapy may prevent adverse effects associated with currently available TRPV1 blockers.

Conservation of mRNA and Protein Expression during Development of C. elegans

Spatiotemporal control of gene expression is crucial for development and subject to evolutionary changes. Although proteins are the final product of most genes, the developmental proteome of an animal has not yet been comprehensively defined, and the correlation between mRNA and protein abundance during development is largely unknown. Here, we globally measured and compared protein and mRNA expression changes during the life cycle of the nematodes C. elegans and C. briggsae, separated by ~30 million years of evolution. We observed that developmental mRNA and protein changes were highly conserved to a surprisingly similar degree but were poorly correlated within a species, suggesting important and widespread posttranscriptional regulation. Posttranscriptional control was particularly well conserved if mRNA fold changes were buffered on the protein level, indicating a predominant repressive function. Finally, among divergently expressed genes, we identified insulin signaling, a pathway involved in lifespan determination, as a putative target of adaptive evolution.

Global characterization of the oocyte-to-embryo transition in Caenorhabditis elegans uncovers a novel mRNA clearance mechanism

The oocyte‐to‐embryo transition (OET) is thought to be mainly driven by post‐transcriptional gene regulation. However, expression of both RNAs and proteins during the OET has not been comprehensively assayed. Furthermore, specific molecular mechanisms that regulate gene expression during OET are largely unknown. Here, we quantify and analyze transcriptome‐wide, expression of mRNAs and thousands of proteins in Caenorhabditis elegans oocytes, 1‐cell, and 2‐cell embryos. This represents a first comprehensive gene expression atlas during the OET in animals. We discovered a first wave of degradation in which thousands of mRNAs are cleared shortly after fertilization. Sequence analysis revealed a statistically highly significant presence of a polyC motif in the 3′ untranslated regions of most of these degraded mRNAs. Transgenic reporter assays demonstrated that this polyC motif is required and sufficient for mRNA degradation after fertilization. We show that orthologs of human polyC‐binding protein specifically bind this motif. Our data suggest a mechanism in which the polyC motif and binding partners direct degradation of maternal mRNAs. Our data also indicate that endogenous siRNAs but not miRNAs promote mRNA clearance during the OET.

Drep-2 is a novel synaptic protein important for learning and memory

CIDE-N domains mediate interactions between the DNase Dff40/CAD and its inhibitor Dff45/ICAD. In this study, we report that the CIDE-N protein Drep-2 is a novel synaptic protein important for learning and behavioral adaptation. Drep-2 was found at synapses throughout the Drosophila brain and was strongly enriched at mushroom body input synapses. It was required within Kenyon cells for normal olfactory short- and intermediate-term memory. Drep-2 colocalized with metabotropic glutamate receptors (mGluRs). Chronic pharmacological stimulation of mGluRs compensated for drep-2 learning deficits, and drep-2 and mGluR learning phenotypes behaved non-additively, suggesting that Drep 2 might be involved in effective mGluR signaling. In fact, Drosophila fragile X protein mutants, shown to benefit from attenuation of mGluR signaling, profited from the elimination of drep-2. Thus, Drep-2 is a novel regulatory synaptic factor, probably intersecting with metabotropic signaling and translational regulation. DOI: http://dx.doi.org/10.7554/eLife.03895.001

CLCN2 chloride channel mutations in familial hyperaldosteronism type II

Primary aldosteronism, a common cause of severe hypertension1, features constitutive production of the adrenal steroid aldosterone. We analyzed a multiplex family with familial hyperaldosteronism type II (FH-II)2 and 80 additional probands with unsolved early-onset primary aldosteronism. Eight probands had novel heterozygous variants in CLCN2, including two de novo mutations and four independent occurrences of a mutation encoding an identical p.Arg172Gln substitution; all relatives with early-onset primary aldosteronism carried the CLCN2 variant found in the proband. CLCN2 encodes a voltage-gated chloride channel expressed in adrenal glomerulosa that opens at hyperpolarized membrane potentials. Channel opening depolarizes glomerulosa cells and induces expression of aldosterone synthase, the rate-limiting enzyme for aldosterone biosynthesis. Mutant channels show gain of function, with higher open probabilities at the glomerulosa resting potential. These findings for the first time demonstrate a role of anion channels in glomerulosa membrane potential determination, aldosterone production and hypertension. They establish the cause of a substantial fraction of early-onset primary aldosteronism.Whole-exome sequencing identifies mutations in CLCN2 in individuals with familial hyperaldosteronism type II or early-onset primary aldosteronism. These gain-of-function mutations cause chloride channel opening and glomerulosa cell depolarization, showing a role for anion channels in aldosterone production.

In Vivo Quantitative Proteome Profiling: Planning and Evaluation of SILAC Experiments

Mass spectrometry-based quantitative proteomics can identify and quantify thousands of proteins in complex biological samples. Improved instrumentation, quantification strategies and data analysis tools now enable protein analysis on a genome-wide scale. Particularly, quantification based on stable isotope labeling with amino acids (SILAC) has emerged as a robust, reliable and simple method for accurate large-scale protein quantification. The spectrum of applications ranges from bacteria and eukaryotic cell culture systems to multicellular organisms. Here, we provide a step-by-step protocol on how to plan and perform large-scale quantitative proteome analysis using SILAC, from sample preparation to final data analysis.

In Vivo Conditions to Identify Prkci Phosphorylation Targets Using the Analog-Sensitive Kinase Method in Zebrafish

Protein kinase C iota is required for various cell biological processes including epithelial tissue polarity and organ morphogenesis. To gain mechanistic insight into different roles of this kinase, it is essential to identify specific substrate proteins in their cellular context. The analog-sensitive kinase method provides a powerful tool for the identification of kinase substrates under in vivo conditions. However, it has remained a major challenge to establish screens based on this method in multicellular model organisms. Here, we report the methodology for in vivo conditions using the analog-sensitive kinase method in a genetically-tractable vertebrate model organism, the zebrafish. With this approach, kinase substrates can uniquely be labeled in the developing zebrafish embryo using bulky ATPγS analogs which results in the thiophosphorylation of substrates. The labeling of kinase substrates with a thiophosphoester epitope differs from phosphoesters that are generated by all other kinases and allows for an enrichment of thiophosphopeptides by immunoaffinity purification. This study provides the foundation for using the analog-sensitive kinase method in the context of complex vertebrate development, physiology, or disease.

Conservation of miRNA-mediated silencing mechanisms across 600 million years of animal evolution

Our current knowledge about the mechanisms of miRNA silencing is restricted to few lineages such as vertebrates, arthropods, nematodes and land plants. miRNA-mediated silencing in bilaterian animals is dependent on the proteins of the GW182 family. Here, we dissect the function of GW182 protein in the cnidarian Nematostella, separated by 600 million years from other Metazoa. Using cultured human cells, we show that Nematostella GW182 recruits the CCR4-NOT deadenylation complexes via its tryptophan-containing motifs, thereby inhibiting translation and promoting mRNA decay. Further, similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with Argonaute proteins, and functions downstream to repress mRNA. Thus, our work suggests that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria.

Picky: a simple online PRM and SRM method designer for targeted proteomics

To the Editor: Targeted proteomics methods such as selected reaction monitoring (SRM) and parallel reaction monitoring (PRM) are increasingly popular because they allow sensitive and rapid analysis of preselected proteins1–3. However, the development of targeted assays is tedious and typically requires selection, synthesis and mass spectrometric analysis of candidate peptides. SRMAtlas and ProteomeTools provide fragmentation spectra of synthetic peptides covering the entire human proteome4,5, but it is difficult to extract relevant data for specific proteins. Also, the development of scheduled acquisition methods (i.e., for the analysis of specific peptides in defined elution time windows) requires adjustments to specific chromatographic conditions. The number of peptides to be targeted in parallel often exceeds the speed of the mass spectrometer, which forces researchers to ask which peptides can be omitted without sacrificing too much information. Available method design tools do not generate optimized scheduled acquisition methods (Supplementary Fig. 1). Here, we present Picky (https://picky.mdc-berlin.de), a fast and user-friendly online design tool for PRM and SRM assays (Fig. 1a, Supplementary Methods). Users simply provide identifiers for human (or mouse) proteins of interest. Picky then selects corresponding tryptic peptides and their experimentally observed retention times (RTs) from the ProteomeTools data set for targeted analysis. Picky uses a scheduling algorithm that adapts to different HPLC gradients (Supplementary Fig. 2), which allows users to upload a list of experimentally observed peptide RTs acquired on their HPLC system. Picky uses these data to rescale the experimentally observed RTs from ProteomeTools and thus to predict their RTs under the chromatographic conditions used. More than 80% of RTs are correctly predicted within an elution time window of ±3 min, which is a considerable improvement compared with predictions based on hydrophobicity scores (Supplementary Figs. 3–5). Alternatively, users can directly provide experimentally observed RTs of target peptides (Supplementary Methods). The acquisition list is further optimized if the number of peptides monitored in parallel exceeds a user-defined threshold. In this case, the lowest-scoring peptide from the protein with the greatest number of targeted peptides is removed in an iterative manner (Supplementary Fig. 2). Hence, Picky selects the best set of peptides covering the targeted proteins under the given chromatographic constraints. For SRM, Picky selects transitions based on the most intense fragment ions observed. Options such as isotope labels, fragmentation types and protein-abundance-specific SRM dwell times (Supplementary Fig. 6) can be freely adjusted by the user. The tool exports an inclusion list, which can be imported into the acquisition software of different mass spectrometers. Picky also displays annotated fragmentation spectra and exports the corresponding spectral library. This library can be imported into Skyline6 to validate the acquired SRM/PRM data. To assess the performance of PRM assays designed by Picky, we spiked different amounts of standard human proteins into 1.4 μg of yeast extract (Supplementary Methods). We provided Picky with (i) identifiers of target human proteins and (ii) an RT calibration file obtained through measurement of the yeast digest alone. On the basis of this input, Picky designed an optimized PRM method in less than a minute. We then used this method to analyze the reference samples by PRM and by standard data-dependent acquisition (DDA) for comparison. PRM markedly outperformed DDA at higher dilutions of the spiked-in proteins (Fig. 1b). We also targeted the same number of randomly selected human proteins and did not observe a single false-positive hit. Thus, Picky enables the detection of human proteins with high sensitivity and specificity. SRM/PRM data are typically validated by monitoring of the chromatographic coelution of multiple transitions for a given peptide6. In our experiment, this yielded convincing profiles for high amounts of

Protein Interaction Screen on Peptide Matrix (PRISMA) reveals interaction footprints and the PTM-dependent interactome of intrinsically disordered C/EBPβ

CCAAT enhancer binding protein beta (C/EBPβ) is a pioneer transcription factor that specifies cell differentiation. C/EBPβ is intrinsically unstructured, a molecular feature common to many proteins involved in signal processing and epigenetics. The structure of C/EBPβ differs depending on alternative translation start site usage and multiple post-translational modifications (PTM). Mutation of distinct PTM sites in C/EBPβ alters designated protein interactions and cell differentiation, suggesting a C/EBPβ PTM indexing code determines epigenetic outcomes. Herein, we systematically explored the interactome of C/EBPβ using an array of spot-synthesised C/EBPβ-derived linear tiling peptides with and without PTM, combined with mass spectrometric proteomic analysis of protein interactions. We identified interaction footprints of ~1300 proteins in nuclear cell extracts, many with chromatin modifying, remodelling and RNA processing functions. The results suggest C/EBPβ acts as a multi-tasking molecular switchboard, integrating signal-dependent modifications and structural plasticity to orchestrate interactions with numerous protein complexes directing cell fate and function. Highlights Peptide array based interaction proteomics map SLiM and PTM dependent C/EBPβ interactome Novel links between C/EBPβ, RNA processing, transcription elongation, MLL, NuRD were revealed C/EBPβ structure organizes modular hub function for gene regulatory machinery PRISMA is suitable to resolve protein interactions and networks based on intrinsically disordered proteins