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Abstract Despite their lack of a defined 3D structure, intrinsically disordered regions (IDRs) of proteins play important biological roles. Many IDRs contain short linear motifs (SLiMs) that mediate protein-protein interactions (PPIs), which can be regulated by post-translational modifications like phosphorylation. 20% of pathogenic missense mutations are found in IDRs, and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms. Here, we employ peptide-based interaction proteomics to investigate 36 disease-associated mutations affecting phosphorylation sites. Our results unveil significant differences in interactomes between phosphorylated and non-phosphorylated peptides, often due to disrupted phosphorylation-dependent SLiMs. We focused on a mutation of a serine phosphorylation site in the transcription factor GATAD1, which causes dilated cardiomyopathy. We find that this phosphorylation site mediates interaction with 14-3-3 family proteins. Follow-up experiments reveal the structural basis of this interaction and suggest that 14-3-3 binding affects GATAD1 nucleocytoplasmic transport by masking a nuclear localisation signal. Our results demonstrate that pathogenic mutations of human phosphorylation sites can significantly impact protein-protein interactions, offering insights into potential molecular mechanisms underlying pathogenesis.

The production of ribosomes is a precisely orchestrated energy consuming cellular process of highest priority. Here, the authors use cryo-EM to show that bacterial ribosomal subunits, self-assembled from their purified RNA and protein components, mature along parallel pathways.

Alveolar rhabdomyosarcoma is a clinically challenging disease due to the lack of druggable targets. Here the authors show preclinical evidence for ATR inhibitors as a therapeutic option for alveolar rhabdomyosarcoma.

RNA binding protein HDLBP (or Vigilin) localizes in the endoplasmic reticulum (ER) membrane. Here the authors show that HDLBP contributes to translation of ER-targeted mRNAs.

Alternative lengthening of telomeres (ALT) is associated with a poor outcome in neuroblastoma. Here, the authors find that ALT is associated with mutated ATRX and/or reduced protein abundance, frequent telomeric repeat loci and heterochromatic telomeric chromatin.

Herpesviruses code for conserved protein kinases (CHPKs) that exert several regulatory functions by interacting with cellular factors. Here, the authors use affinity purification mass spectrometry (AP–MS) to identify differential interaction partners of CHPKs from seven different human herpesviruses, finding Cyclin A and associated factors as a specific signature of β-herpesvirus kinases.

Avian influenza A virus (IAV) strains replicate poorly in mammalian hosts, but mechanisms underlying species restriction are incompletely understood. Here, Bogdanow et al. show that avian and mammalian adapted IAV strains have evolved different RNA structure features for regulation of M segment RNA splicing.

Very high risk neuroblastoma is characterised by increased MAPK signalling, and targeting MAPK signalling is a promising therapeutic strategy. We used a deeply characterised panel of neuroblastoma cell lines and found that the sensitivity to MEK inhibitors varied drastically between these cell lines. By generating quantitative perturbation data and mathematical modelling, we determined potential resistance mechanisms. We found that negative feedbacks within MAPK signalling and via the IGF receptor mediate re-activation of MAPK signalling upon treatment in resistant cell lines. By using cell-line specific models, we predict that combinations of MEK inhibitors with RAF or IGFR inhibitors can overcome resistance, and tested these predictions experimentally. In addition, phospho-proteomic profiling confirmed the cell-specific feedback effects and synergy of MEK and IGFR targeted treatment. Our study shows that a quantitative understanding of signalling and feedback mechanisms facilitated by models can help to develop and optimise therapeutic strategies. Our findings should be considered for the planning of future clinical trials introducing MEKi in the treatment of neuroblastoma. Author summary Only few targeted therapies are currently available to treat high-risk neuroblastoma. To address this issue we characterized the drug response of high risk neuroblastoma cell lines and correlated it with genomic and transcriptomic data. Particularly for MEK inhibition, we saw that our panel could be nicely separated into two groups of resistant and sensitive cell lines. Genomic and transcriptomic markers alone did not help to discriminate between responders and non-responders. We used signalling perturbation data to build cell line specific signalling models. Our models suggest that negative feedbacks within MAPK signalling lead to a stronger reactivation of MEK in MEKi resistant cell lines after MEK inhibition. Model analysis suggested that combining MEK inhibition with IGF1R or RAF inhibition could be an effective treatment and we characterised this combination using phosphoproteomics by mass-spectrometry and growth assays. Our study confirms the importance of quantitative understanding of signalling and may help plan future clinical trials involving MEK inhibition for the treatment of neuroblastoma.

We describe MIP-1 and MIP-2, novel paralogous C. elegans germ granule components that interact with the intrinsically disordered MEG-3 protein. These proteins promote P granule condensation, form granules independently of MEG-3 in the postembryonic germ line, and balance each other in regulating P granule growth and localization. MIP-1 and MIP-2 each contain two LOTUS domains and intrinsically disordered regions and form homo- and heterodimers. They bind and anchor the Vasa homolog GLH-1 within P granules and are jointly required for coalescence of MEG-3, GLH-1, and PGL proteins. Animals lacking MIP-1 and MIP-2 show temperature-sensitive embryonic lethality, sterility, and mortal germ lines. Germline phenotypes include defects in stem cell self-renewal, meiotic progression, and gamete differentiation. We propose that these proteins serve as scaffolds and organizing centers for ribonucleoprotein networks within P granules that help recruit and balance essential RNA processing machinery to regulate key developmental transitions in the germ line.

RNA binding proteins are important regulators of RNA function. Here the authors describe a method for isolation of RNA-protein complexes that does not rely on a specific RNA sequence or motif, and demonstrate the approach by providing the global RNA-bound proteomes of human HEK293 cells and Salmonella Typhimurium.

RNA-binding proteins (RBPs) are key mediators of posttranscriptional gene expression control. However, the links between cell signaling on the one hand and RBP function on the other are understudied. While thousands of posttranslational modification (PTM) sites on RBPs have been identified, their functional roles are only poorly characterized. RNA-interactome capture (RIC) and cross-linking and immunoprecipitation (CLIP) are attractive methods that provide information about RBP-RNA interactions on a genome-wide scale. Both approaches rely on the in situ UV cross-linking of RBPs and RNAs, biochemical enrichment and analysis by RNA-sequencing (CLIP) or mass spectrometry (RIC). In principle, RIC- and CLIP-like methods could be used to globally quantify RBP-RNA interactions in response to perturbations. However, several biases have to be taken into account to avoid misinterpretation of the results obtained. Here, we focus on RIC-like methods and discuss four key aspects relevant for quantitative interpretation: (1) the RNA isolation efficiency, (2) the inefficient and highly variable UV cross-linking, (3) the baseline RNA occupancy of RBPs, and (4) indirect factors affecting RBP-RNA interaction. We highlight these points by presenting selected examples of PTMs that might induce differential quantification in RIC-like experiments without necessarily affecting RNA-binding. We conclude that quantifying RBP-RNA interactions via RIC or CLIP-like methods should not be regarded as an end in itself but rather as starting points for deeper analysis.

Summary: Detailed knowledge of the molecular biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is crucial for understanding of viral replication, host responses, and disease progression. Here, we report gene expression profiles of three SARS-CoV- and SARS-CoV-2-infected human cell lines. SARS-CoV-2 elicited an approximately two-fold higher stimulation of the innate immune response compared to SARS-CoV in the human epithelial cell line Calu-3, including induction of miRNA-155. Single-cell RNA sequencing of infected cells showed that genes induced by virus infections were broadly upregulated, whereas interferon beta/lambda genes, a pro-inflammatory cytokines such as IL-6, were expressed only in small subsets of infected cells. Temporal analysis suggested that transcriptional activities of interferon regulatory factors precede those of nuclear factor κB. Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 activity resulted in a reduction of viral replication and pro-inflammatory cytokine expression in primary human airway epithelial cells.

Subcellular localization of RNAs and proteins is important for polarized cells such as neurons. Here the authors differentiate mouse embryonic stem cells into neurons, and analyze the local transcriptome, proteome, and translated transcriptome in their cell bodies and neurites, providing a unique resource for future studies on neuronal polarity.

[This corrects the article DOI: 10.1371/journal.pgen.1004103.].

Summary: Chlamydia trachomatis (Ctr) causes a range of infectious diseases and is epidemiologically associated with cervical and ovarian cancers. To obtain a panoramic view of Ctr-induced signaling, we performed global phosphoproteomic and transcriptomic analyses. We identified numerous Ctr phosphoproteins and Ctr-regulated host phosphoproteins. Bioinformatics analysis revealed that these proteins were predominantly related to transcription regulation, cellular growth, proliferation, and cytoskeleton organization. In silico kinase substrate motif analysis revealed that MAPK and CDK were the most overrepresented upstream kinases for upregulated phosphosites. Several of the regulated host phosphoproteins were transcription factors, including ETS1 and ERF, that are downstream targets of MAPK. Functional analysis of phosphoproteome and transcriptome data confirmed their involvement in epithelial-to-mesenchymal transition (EMT), a phenotype that was validated in infected cells, along with the essential role of ERK1/2, ETS1, and ERF for Ctr replication. Our data reveal the extent of Ctr-induced signaling and provide insights into its pro-carcinogenic potential. : Zadora et al. performed an integrated global phosphoproteomic and transcriptomic analysis, revealing a comprehensive map of Chlamydia-induced host cell signaling and identifying transcription factors ETS1 and ERF, which drive epithelial-to-mesenchymal transition. These insights provide mechanistic clues to Chlamydia pathogenesis and serve as an important resource for future studies. Keywords: Chlamydia trachomatis, signaling, human papillomavirus, transcription factors, cervical cancer, ovarian cancer, human primary cells

Protein kinase A (PKA) is a key mediator of cyclic AMP signalling. Here, Eccles et al. show that ARHGAP36 antagonizes PKA by acting as a kinase inhibitor and targeting the catalytic subunit for endolysosomal degradation, thus reducing sensitivity of cells to cAMP and promoting Hedgehog signalling.

The AAA+ ATPase p97 is an essential hexameric protein with multiple protein interaction partners and cellular functions. Here, the authors use interaction mapping to examine partner proteins of this large complex, and assess the effects of these proteins on the disassembly of the p97 complex.

The naked mole-rat is a subterranean rodent lacking several pain behaviors found in humans, rats, and mice. For example, nerve growth factor (NGF), an important mediator of pain sensitization, fails to produce thermal hyperalgesia in naked mole-rats. The sensitization of capsaicin-sensitive TRPV1 ion channels is necessary for NGF-induced hyperalgesia, but naked mole-rats have fully functional TRPV1 channels. We show that exposing isolated naked mole-rat nociceptors to NGF does not sensitize TRPV1. However, the naked mole-rat NGF receptor TrkA displays a reduced ability to engage signal transduction pathways that sensitize TRPV1. Between one- and three-amino-acid substitutions in the kinase domain of the naked mole-rat TrkA are sufficient to render the receptor hypofunctional, and this is associated with the absence of heat hyperalgesia. Our data suggest that evolution has selected for a TrkA variant that abolishes a robust nociceptive behavior in this species but is still compatible with species fitness.

Insertion of reporter cassettes into the Lgr5 locus has enabled the characterization of mouse intestinal stem cells (ISCs). However, low cell surface abundance of LGR5 protein and lack of high-affinity anti-LGR5 antibodies represent a roadblock to efficiently isolate human colonic stem cells (hCoSCs). We set out to identify stem cell markers that would allow for purification of hCoSCs. In an unbiased approach, membrane-enriched protein fractions derived from in vitro human colonic organoids were analyzed by quantitative mass spectrometry. Protein tyrosine pseudokinase PTK7 specified a cell population within human colonic organoids characterized by highest self-renewal and re-seeding capacity. Antibodies recognizing the extracellular domain of PTK7 allowed us to isolate and expand hCoSCs directly from patient-derived mucosa samples. Human PTK7+ cells display features of canonical Lgr5+ ISCs and include a fraction of cells that undergo differentiation toward enteroendocrine lineage that resemble crypt label retaining cells (LRCs).

Eukaryotic gene expression requires the coordinated action of transcription factors, chromatin remodelling complexes and RNA polymerase. The conserved nuclear protein Akirin plays a central role in immune gene expression in insects and mammals, linking the SWI/SNF chromatin-remodelling complex with the transcription factor NFκB. Although nematodes lack NFκB, Akirin is also indispensable for the expression of defence genes in the epidermis of Caenorhabditis elegans following natural fungal infection. Through a combination of reverse genetics and biochemistry, we discovered that in C. elegans Akirin has conserved its role of bridging chromatin-remodellers and transcription factors, but that the identity of its functional partners is different since it forms a physical complex with NuRD proteins and the POU-class transcription factor CEH-18. In addition to providing a substantial step forward in our understanding of innate immune gene regulation in C. elegans, our results give insight into the molecular evolution of lineage-specific signalling pathways.

Abstract Translational regulation is mediated through the interaction between diffusible trans‐factors and cis‐elements residing within mRNA transcripts. In contrast to extensively studied transcriptional regulation, cis‐regulation on translation remains underexplored. Using deep sequencing‐based transcriptome and polysome profiling, we globally profiled allele‐specific translational efficiency for the first time in an F1 hybrid mouse. Out of 7,156 genes with reliable quantification of both alleles, we found 1,008 (14.1%) exhibiting significant allelic divergence in translational efficiency. Systematic analysis of sequence features of the genes with biased allelic translation revealed that local RNA secondary structure surrounding the start codon and proximal out‐of‐frame upstream AUGs could affect translational efficiency. Finally, we observed that the cis‐effect was quantitatively comparable between transcriptional and translational regulation. Such effects in the two regulatory processes were more frequently compensatory, suggesting that the regulation at the two levels could be coordinated in maintaining robustness of protein expression.

Several proteins have been linked to neurodegenerative disorders (NDDs), but their molecular function is not completely understood. Here, we used quantitative interaction proteomics to identify binding partners of Amyloid beta precursor protein (APP) and Presenilin-1 (PSEN1) for Alzheimer’s disease (AD), Huntingtin (HTT) for Huntington’s disease, Parkin (PARK2) for Parkinson’s disease, and Ataxin-1 (ATXN1) for spinocerebellar ataxia type 1. Our network reveals common signatures of protein degradation and misfolding and recapitulates known biology. Toxicity modifier screens and comparison to genome-wide association studies show that interaction partners are significantly linked to disease phenotypes in vivo. Direct comparison of wild-type proteins and disease-associated variants identified binders involved in pathogenesis, highlighting the value of differential interactome mapping. Finally, we show that the mitochondrial protein LRPPRC interacts preferentially with an early-onset AD variant of APP. This interaction appears to induce mitochondrial dysfunction, which is an early phenotype of AD.

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.

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.

Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. 'Cut and paste' DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB) and piggyBac (PB) that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1), a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for PB.

We studied the dynamics of the proteome of influenza virus A/PR/8/34 (H1N1) infected Madin-Darby canine kidney cells up to 12 hours post infection by mass spectrometry based quantitative proteomics using the approach of stable isotope labeling by amino acids in cell culture (SILAC). We identified 1311 cell proteins and, apart from the proton channel M2, all major virus proteins. Based on their abundance two groups of virus proteins could be distinguished being in line with the function of the proteins in genesis and formation of new virions. Further, the data indicate a correlation between the amount of proteins synthesized and their previously determined copy number inside the viral particle. We employed bioinformatic approaches such as functional clustering, gene ontology, and pathway (KEGG) enrichment tests to uncover co-regulated cellular protein sets, assigned the individual subsets to their biological function, and determined their interrelation within the progression of viral infection. For the first time we are able to describe dynamic changes of the cellular and, of note, the viral proteome in a time dependent manner simultaneously. Through cluster analysis, time dependent patterns of protein abundances revealed highly dynamic up- and/or down-regulation processes. Taken together our study provides strong evidence that virus infection has a major impact on the cell status at the protein level.

microRNAs (miRNAs) repress target genes by destabilizing mRNAs and/or by inhibiting translation. The best known factor for target recognition is the so called seed--a short continuous region of Watson-Crick base pairing between nucleotides 2-7 of the miRNA and complementary sequences in 3' untranslated regions of target mRNAs. The miR-34 family consists of three conserved members with important tumor suppressor functions linked to the p53 pathway. The family members share the same seed, raising the question if they also have the same targets. Here, we analyse the effect of miR-34a and miR-34c on protein synthesis by pSILAC. Despite significant overlap, we observe that the impact of both family members on protein synthesis differs. The ability to identify specific targets of a family member is complicated by the occurrence of * strand mediated repression. Transfection of miR-34 chimeras indicates that the 3'end of the miRNA might be responsible for differential regulation in case of targets without a perfect seed site. Pathway analysis of regulated proteins indicates overlapping functions related to cell cycle and the p53 pathway and preferential targeting of several anti-apoptotic proteins by miR-34a. We used luciferase assays to confirm that Vcl and Fkbp8, an important anti-apoptotic protein, are specifically repressed by miR-34a. In summary, we find that miR-34a and miR-34c down-regulate distinct subsets of targets which might mediate different cellular outcomes. Our data provides a rich resource of miR-34 targets that might be relevant for clinical trials that want to implement the miR-34 family in cancer therapy.

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.

Mitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, which shapes the oxidative phosphorylation complexes essential for cellular energy metabolism. Despite the importance of mitochondrial translation (MT) control, it is challenging to identify and quantify the mitochondrial-encoded proteins because of their hydrophobic nature and low abundance. Here, we introduce a mass spectrometry-based proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture. Our method provides the highest protein identification rate with the shortest measurement time among currently available methods, enabling us to quantify 12 of the 13 mitochondrialencoded proteins. We applied this method to uncover the global picture of (post-)translational regulation of both mitochondrial- and nuclear-encoded subunits of oxidative phosphorylation complexes. We found that inhibition of MT led to degradation of orphan nuclear-encoded subunits that are considered to form subcomplexes with the mitochondrialencoded subunits. This method should be readily applicable to study MT programs in many contexts, including oxidative stress and mitochondrial disease. [ABSTRACT FROM AUTHOR]