Search

Nucleic acids Research 49(6), 3507-3523 (2021). doi:10.1093/nar/gkab116, Published by Oxford Univ. Press, Oxford

Stem cells such as mesenchymal stem cells (MSCs) enhance neurological recovery in preclinical stroke models by secreting extracellular vesicles (EVs). Since previous reports have focused on the application of MSC-EVs only, the role of the most suitable host cell for EV enrichment and preclinical stroke treatment remains elusive. The present study aimed to evaluate the therapeutic potential of EVs derived from neural progenitor cells (NPCs) following experimental stroke. Using the PEG technique, EVs were enriched and characterized by electron microscopy, proteomics, rt-PCR, nanosight tracking analysis, and Western blotting. Different dosages of NPC-EVs displaying a characteristic profile in size, shape, cargo protein, and non-coding RNA contents were incubated in the presence of cerebral organoids exposed to oxygen-glucose deprivation (OGD), significantly reducing cell injury when compared with control organoids. Systemic administration of NPC-EVs in male C57BL6 mice following experimental ischemia enhanced neurological recovery and neuroregeneration for as long as 3 months. Interestingly, the therapeutic impact of such NPC-EVs was found to be not inferior to MSC-EVs. Flow cytometric analyses of blood and brain samples 7 days post-stroke demonstrated increased blood concentrations of B and T lymphocytes after NPC-EV delivery, without affecting cerebral cell counts. Likewise, a biodistribution analysis after systemic delivery of NPC-EVs revealed the majority of NPC-EVs to be found in extracranial organs such as the liver and the lung. This proof-of-concept study supports the idea of EVs being a general concept of stem cell–induced neuroprotection under stroke conditions, where EVs contribute to reverting the peripheral post-stroke immunosuppression. Electronic supplementary material The online version of this article (10.1007/s12975-020-00814-z) contains supplementary material, which is available to authorized users.

Sleep is an essential state that allows for recuperation and survival processes. Disturbing sleep triggers stress responses that promote protective gene expression. Sleep and its deprivation grossly impact gene expression, but little is known about how normal or disturbed sleep control gene expression. Central to the induction of sleep are sleep-active neurons, which inhibit wakefulness and promote survival. Sleep and sleep-active neurons are highly conserved. In Caenorhabditis elegans, the sleep-active RIS neuron is crucial for sleep and survival. Here, we show that RIS depolarization promotes the protective gene expression response that occurs during developmental arrest. This response includes the activation of FOXO/DAF-16 and expression of DAF-16 target genes such as HSP-12.6, a small heat-shock protein that is required for starvation survival. Disturbing sleep by mechanical stimulation increases RIS depolarization. RIS activation in turn activates DAF-16 and other genes required for survival. Hence, during normal sleep, RIS depolarization promotes protective gene expression. When sleep is disturbed, protective gene expression gets further increased by raised RIS depolarization. We thus link sleep-active neuron depolarization to protective gene expression changes and suggest that the cellular stress response following sleep deprivation could be understood as a safeguarding process that is caused by the overactivation of sleep-active neurons.

The mechanical properties of biological cells are determined by the cytoskeleton, a composite biopolymer network consisting of microtubules, actin filaments and intermediate filaments (IFs). By differential expression of cytoskeletal proteins, modulation of the network architecture and interactions between the filaments, cell mechanics may be adapted to varying requirements on the cell. Here, we focus on the intermediate filament protein vimentin and introduce post-translational modifications as an additional, much faster mechanism for mechanical modulation. We study the impact of phosphorylation on filament mechanics by recording force-strain curves using optical traps. Partial phosphorylation softens the filaments. We show that binding of the protein 14–3–3 to phosphorylated vimentin IFs further enhances this effect and speculate that in the cell 14–3–3 may serve to preserve the softening and thereby the altered cell mechanics. We explain our observation by the additional charges introduced during phosphorylation.

Protein–DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein–DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, we present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by ultraviolet (UV) light. Our approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 we show that our technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei we determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that our workflow is not restricted to reconstituted materials. As our approach can distinguish between protein–RNA and DNA interactions in one single experiment, we project that it will be possible to obtain insights into chromatin and its regulation in the future., Cross-linking mass spectrometry (XLMS) allows mapping of protein-protein and protein-RNA interactions, but the analysis of protein-DNA complexes remains challenging. Here, the authors develop a UV light-based XLMS workflow to determine protein-DNA interfaces in reconstituted chromatin and isolated nuclei.

Dendritic spines, the postsynaptic compartments of excitatory neurotransmission, have different shapes classified from 'stubby' to 'mushroom-like'. Whereas mushroom spines are essential for adult brain function, stubby spines disappear during brain maturation. It is still unclear whether and how they differ in protein composition. To address this, we combined electron microscopy and quantitative biochemistry with super-resolution microscopy to annotate more than 47,000 spines for more than 100 synaptic targets. Surprisingly, mushroom and stubby spines have similar average protein copy numbers and topologies. However, an analysis of the correlation of each protein to the postsynaptic density mass, used as a marker of synaptic strength, showed substantially more significant results for the mushroom spines. Secretion and trafficking proteins correlated particularly poorly to the strength of stubby spines. This suggests that stubby spines are less likely to adequately respond to dynamic changes in synaptic transmission than mushroom spines, which possibly explains their loss during brain maturation.

The costimulatory receptor, CD28, synergizes with the T cell antigen receptor (TCR) to promote IL-2 production, cell survival and proliferation. Despite their profound synergy, the obligatory interdependence of the signaling pathways initiated by these two receptors is not well understood. Upon TCR stimulation, Gads, a Grb2-family adaptor, bridges the interaction of two additional adaptors, LAT and SLP-76, to form a TCR-induced effector signaling complex. SLP-76 binds the Tec-family tyrosine kinase, Itk, which phosphorylates SLP-76 at Y173 and PLC-γ1 at Y783. Here we identified Gads Y45 as an additional TCR-inducible, Itk-mediated phosphorylation site. Y45 is found within the N-terminal SH3 domain of Gads, an evolutionarily conserved domain with no known binding partners or signaling function. Gads Y45 phosphorylation depended on the interaction of Gads with SLP-76 and on the preferentially-paired binding of Gads to phospho-LAT. Three Itk-related features, Gads Y45, SLP-76 Y173, and a proline-rich Itk SH3-binding motif on SLP-76, were selectively required for activation of the CD28 RE/AP transcriptional element from the IL-2 promoter, but were not required to activate NFAT. This study illuminates a new regulatory module, in which Itk-targeted phosphorylation sites on two adaptor proteins, SLP-76 and Gads, control the transcriptional response to TCR/CD28 costimulation, thus enforcing the obligatory interdependence of the TCR and CD28 signaling pathways.

SummaryDipeptidyl peptidase 9 (DPP9) is a serine protease cleaving N-terminal dipeptides preferentially post-proline with (patho)physiological roles in the immune system and cancer. Only few DPP9 substrates are known. Here we identify an association of human DPP9 with the tumour suppressor BRCA2, a key player in repair of DNA double-strand breaks that promotes the formation of RAD51 filaments. This interaction is triggered by DNA-damage and requires access to the DPP9 active-site. We present crystallographic structures documenting the N-terminal Met1-Pro2 of a BRCA21-40 peptide captured in the DPP9 active-site. Mechanistically, DPP9 targets BRCA2 for degradation by the N-degron pathway, and promotes RAD51 foci formation. Both processes are phenocopied by BRCA2 N-terminal truncation mutants, indicating that DPP9 regulates both stability and the cellular stoichiometric interactome of BRCA2. Consistently, DPP9-deprived cells are hypersensitive to DNA-damage. Together, we identify DPP9 as a regulator of BRCA2, providing a possible explanation for DPP9 involvement in cancer development.

Dissecting the degradation of the Alzheimer’s disease–related protein Tau by the 20S proteasome at single-residue resolution., Intrinsically disordered proteins (IDPs) can be degraded in a ubiquitin-independent process by the 20S proteasome. Decline in 20S activity characterizes neurodegenerative diseases. Here, we examine 20S degradation of IDP tau, a protein that aggregates into insoluble deposits in Alzheimer’s disease. We show that cleavage of tau by the 20S proteasome is most efficient within the aggregation-prone repeat region of tau and generates both short, aggregation-deficient peptides and two long fragments containing residues 1 to 251 and 1 to 218. Phosphorylation of tau by the non-proline–directed Ca2+/calmodulin-dependent protein kinase II inhibits degradation by the 20S proteasome. Phosphorylation of tau by GSK3β, a major proline-directed tau kinase, modulates tau degradation kinetics in a residue-specific manner. The study provides detailed insights into the degradation products of tau generated by the 20S proteasome, the residue specificity of degradation, single-residue degradation kinetics, and their regulation by posttranslational modification.

Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain associates with Pol II subunit RPB12 and the phosphorylated C-terminal region of DSIF subunit SPT5. RTF1 also forms four α-helices that extend from the Plus3 domain along the Pol II protrusion and RPB10 to the polymerase funnel. The C-terminal ‘fastener’ helix retains PAF and is followed by a ‘latch’ that reaches the end of the bridge helix, a flexible element of the Pol II active site. RTF1 strongly stimulates Pol II elongation, and this requires the latch, possibly suggesting that RTF1 activates transcription allosterically by influencing Pol II translocation. Cryo-EM elucidation of a fully reconstituted Pol II–DSF–PAF1–SPT6 elongation complex defines the position of PAF1 subunit RTF1 and reveals contacts with the Pol II bridge helix that may allosterically stimulate transcription elongation.

Candida yeasts causing human infections are spread across the yeast phylum with Candida glabrata being related to Saccharomyces cerevisiae, Candida krusei grouping to Pichia spp., and Candida albicans, Candida parapsilosis and Candida tropicalis belonging to the CTG-clade. The latter lineage contains yeasts with an altered genetic code translating CUG codons as serine using a serine-tRNA with a mutated anticodon. It has been suggested that the CTG-clade CUG codons are mistranslated to a small extent as leucine due to mischarging of the serine-tRNA(CAG). The mistranslation was suggested to result in variable surface proteins explaining fast host adaptation and pathogenicity. Here, we re-assessed this potential mistranslation by high-resolution mass spectrometry-based proteogenomics of multiple CTG-clade yeasts, various C. albicans strains, isolated from colonized and from infected human body sites, and C. albicans grown in yeast and hyphal forms. Our in vivo data do not support CUG codon mistranslation by leucine. Instead, (i) CUG codons are mistranslated only to the extent of ribosomal mistranslation with no preference for specific amino acids, (ii) CUG codons are as unambiguous (or ambiguous) as the related CUU leucine and UCC serine codons, (iii) tRNA anticodon loop variation across the CTG-clade yeasts does not result in any difference of the mistranslation level, and (iv) CUG codon unambiguity is independent of C. albicans’ strain pathogenicity or growth form.

Cross-linking mass spectrometry (XL-MS) is steadily expanding its range of applications from purified protein complexes to more complex samples like organelles and even entire cells. One main challenge using non-cleavable cross-linkers is the so-called n2 problem: With linearly increasing database size, the search space for the identification of two covalently linked peptides per spectrum increases quadratically. Here, we report an alternative search strategy that focuses on only those peptides, which were demonstrated to cross-link under the applied experimental conditions. The performance of a parallel XL-MS experiment using a thiol-cleavable cross-linker enabled the identification of peptides that carried a cleaved cross-link moiety after reduction and hence were involved in cross-linking reactions. Based on these identifications, a peptide database was generated and used for the database search of the actual cross-linking experiment with a non-cleavable cross-linker. This peptide-focused approach was tested on protein complexes with a reported structural model and obtained results corresponded well to a conventional database search. An application of the strategy on in vivo cross-linked Bacillus subtilis and Bacillus cereus cells revealed a five- to tenfold reduction in search time and led to significantly more identifications with the latter species than a search against the entire proteome. Significance Instead of considering all theoretically cross-linkable peptides in a proteome, identification and pre-filtering for a subset of cross-link peptide candidates allows for a dramatically decreased search space. Hence, there is less potential for the random accumulation of false positives ultimately leading to a higher sensitivity in the XL-MS experiment. Using the peptide-focused approach, a cross-linking database search can be conducted in a fraction of time while yielding a similar or higher number of identifications, thereby enabling the cross-linking analysis of samples of mammalian proteome complexity.

Site-specific regulation of protein N-glycosylation is essential in human cells. However, accurate quantification of glycosylation sites and their individual glycan moieties in a cell-wide manner is still technically challenging. Here, we introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising fast protein aggregation capture (PAC)-based sample preparation, optimized multi-notch MS3 LC-MS acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that allows for confident, global identification and quantification of intact glycopeptides in complex biological samples. PAC greatly reduces the overall samplehandling time without compromising sensitivity. Glyco-SPS-MS3 combines high-resolution MS2 and MS3 scans, resulting in enhanced reporter signals of isobaric mass tags, improved detection of N-glycopeptide fragments, and significantly lowered interference in multiplexed quantification. GlycoBinder enables streamlined processing of Glyco-SPS-MS3 data, followed by a two-step database search which increases the identification rates of intact glycopeptides by up to 22% when compared with one-step database search strategies. SugarQuant was applied to identify and quantify more than 5,000 unique glycoforms in Burkitt’s lymphoma cells, and determined complex site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence.

Proteasomes are the main producers of antigenic peptides presented to CD8+ T cells. They can cut proteins and release their fragments or recombine non-contiguous fragments thereby generating novel sequences, i.e. spliced peptides. Understanding which are the driving forces and the sequence preferences of both reactions can streamline target discovery in immunotherapies against cancer, infection and autoimmunity. Here, we present a large database of spliced and non-spliced peptides generated by proteasomes in vitro, which is available as simple CSV file and as a MySQL database. To generate the database, we performed in vitro digestions of 55 unique synthetic polypeptide substrates with different proteasome isoforms and experimental conditions. We measured the samples using three mass spectrometers, filtered and validated putative peptides, identified 22,333 peptide product sequences (15,028 spliced and 7,305 non-spliced product sequences). Our database and datasets have been deposited to the Mendeley (doi:10.17632/nr7cs764rc.1) and PRIDE (PXD016782) repositories. We anticipate that this unique database can be a valuable source for predictors of proteasome-catalyzed peptide hydrolysis and splicing, with various future translational applications., Measurement(s)peptideTechnology Type(s)mass spectrometryFactor Type(s)spliced/non-spliced • instrument • synthetic polypeptide • proteasome isoform • time of reactionSample Characteristic - OrganismHomo sapiens Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12205274

Summary Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 A core resolution and 4.5–5.7 A at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome’s architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome’s catalytically active RNP (ribonucleoprotein) conformation. It reveals the spatial organization of the metazoan-specific proteins PPWD1, WDR70, FRG1, and CIR1 in human C complexes, indicating they stabilize functionally important protein domains and RNA structures rearranged/repositioned during the Bact to C transition. Structural comparisons with human Bact, C∗, and P complexes reveal an intricate cascade of RNP rearrangements during splicing catalysis, with intermediate RNP conformations not found in yeast, and additionally elucidate the structural basis for the sequential recruitment of metazoan-specific spliceosomal proteins.

Diabetic nephropathy (DN) is the main reason for end-stage renal disease. Microalbuminuria as the non-invasive available diagnosis marker lacks specificity and gives high false positive rates. To identify and validate biomarkers for DN, we used in the present study urine samples from four patient groups: diabetes without nephropathy, diabetes with microalbuminuria, diabetes with macroalbuminuria and proteinuria without diabetes. For the longitudinal validation, we recruited 563 diabetic patients and collected 1363 urine samples with the clinical data during a follow-up of 6 years. Comparative urinary proteomics identified four proteins Apolipoprotein A-I (APOA1), Beta-2-microglobulin (B2M), E-cadherin (CDH1) and Lithostathine-1-alpha (REG1A), which differentiated with high statistical strength (p &lt, 0.05) between DN patients and the other groups. Label-free mass spectrometric quantification of the candidates confirmed the discriminatory value of E-cadherin and Lithostathine-1-alpha (p &lt, 0.05). Immunological validation highlighted E-cadherin as the only marker able to differentiate significantly between the different DN stages with an area under the curve (AUC) of 0.85 (95%-CI: [0.72, 0.97]). The analysis of the samples from the longitudinal study confirmed the prognostic value of E-cadherin, the critical increase in urinary E-cadherin level was measured 20 &plusmn, 12.5 months before the onset of microalbuminuria and correlated significantly (p &lt, 0.05) with the glomerular filtration rate measured by estimated glomerular filtration rate (eGFR).

Background Dystroglycanopathies are a group of inherited disorders characterized by vast clinical and genetic heterogeneity and caused by abnormal functioning of the ECM receptor dystroglycan (Dg). Remarkably, among many cases of diagnosed dystroglycanopathies, only a small fraction can be linked directly to mutations in Dg or its regulatory enzymes, implying the involvement of other, not-yet-characterized, Dg-regulating factors. To advance disease diagnostics and develop new treatment strategies, new approaches to find dystroglycanopathy-related factors should be considered. The Dg complex is highly evolutionarily conserved; therefore, model genetic organisms provide excellent systems to address this challenge. In particular, Drosophila is amenable to experiments not feasible in any other system, allowing original insights about the functional interactors of the Dg complex. Methods To identify new players contributing to dystroglycanopathies, we used Drosophila as a genetic muscular dystrophy model. Using mass spectrometry, we searched for muscle-specific Dg interactors. Next, in silico analyses allowed us to determine their association with diseases and pathological conditions in humans. Using immunohistochemical, biochemical, and genetic interaction approaches followed by the detailed analysis of the muscle tissue architecture, we verified Dg interaction with some of the discovered factors. Analyses of mouse muscles and myocytes were used to test if interactions are conserved in vertebrates. Results The muscle-specific Dg complexome revealed novel components that influence the efficiency of Dg function in the muscles. We identified the closest human homologs for Dg-interacting partners, determined their significant enrichment in disease-associations, and verified some of the newly identified Dg interactions. We found that Dg associates with two components of the mechanosignaling Hippo pathway: the WW domain-containing proteins Kibra and Yorkie. Importantly, this conserved interaction manages adult muscle size and integrity. Conclusions The results presented in this study provide a new list of muscle-specific Dg interactors, further analysis of which could aid not only in the diagnosis of muscular dystrophies, but also in the development of new therapeutics. To regulate muscle fitness during aging and disease, Dg associates with Kibra and Yorkie and acts as a transmembrane Hippo signaling receptor that transmits extracellular information to intracellular signaling cascades, regulating muscle gene expression.

Cell 180(6), 1130 - 1143.e20 (2020). doi:10.1016/j.cell.2020.02.034, Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a �� subunit of the 2.6 megadalton ��6-��6 S. cerevisiae FAS, which is shown by high-resolution structures to stabilize a rotated FAS conformation and rearrange ACP domains from equatorial to axial positions. The �� subunit spans the length of the FAS inner cavity, impeding reductase activities of FAS, regulating NADPH turnover by kinetic hysteresis at the ketoreductase, and suppressing off-pathway reactions at the enoylreductase. The �� subunit delineates the functional compartment within FAS. As a scaffold, it may be exploited to incorporate natural and designed enzymatic activities that are not present in natural FAS., Published by Elsevier, New York, NY

At the heart of protein ubiquitination cascades, ubiquitin-conjugating enzymes (E2s) form reactive ubiquitin-thioester intermediates to enable efficient transfer of ubiquitin to cellular substrates. The precise regulation of E2s is thus crucial for cellular homeostasis, and their deregulation is frequently associated with tumorigenesis. In addition to driving substrate ubiquitination together with ubiquitin ligases (E3s), many E2s can also autoubiquitinate, thereby promoting their own proteasomal turnover. To investigate the mechanisms that balance these disparate activities, we dissected the regulatory dynamics of UBE2S, a human APC/C-associated E2 that ensures the faithful ubiquitination of cell cycle regulators during mitosis. We uncovered a dimeric state of UBE2S that confers autoinhibition by blocking a catalytically critical ubiquitin binding site. Dimerization is stimulated by the lysine-rich carboxyl-terminal extension of UBE2S that is also required for the recruitment of this E2 to the APC/C and is autoubiquitinated as substrate abundance becomes limiting. Consistent with this mechanism, we found that dimerization-deficient UBE2S turned over more rapidly in cells and did not promote mitotic slippage during prolonged drug-induced mitotic arrest. We propose that dimerization attenuates the autoubiquitination-induced turnover of UBE2S when the APC/C is not fully active. More broadly, our data illustrate how the use of mutually exclusive macromolecular interfaces enables modulation of both the activities and the abundance of E2s in cells to facilitate precise ubiquitin signaling.

While many aspects of archaeal cell biology remain relatively unexplored, systems biology approaches like mass spectrometry (MS) based proteomics offer an opportunity for rapid advances. Unfortunately, the enormous amount of MS data generated often remains incompletely analyzed due to a lack of sophisticated bioinformatic tools and field-specific biological expertise for data interpretation. Here we present the initiation of the Archaeal Proteome Project (ArcPP), a community-based effort to comprehensively analyze archaeal proteomes. Starting with the model archaeon Haloferax volcanii, we reanalyze MS datasets from various strains and culture conditions. Optimized peptide spectrum matching, with strict control of false discovery rates, facilitates identifying > 72% of the reference proteome, with a median protein sequence coverage of 51%. These analyses, together with expert knowledge in diverse aspects of cell biology, provide meaningful insights into processes such as N-terminal protein maturation, N-glycosylation, and metabolism. Altogether, ArcPP serves as an invaluable blueprint for comprehensive prokaryotic proteomics., While archaeal proteomics advanced rapidly, a comprehensive proteome database for archaea is lacking. Therefore, the authors here launch the Archaeal Proteome Project, a community-effort providing insights into archaeal cell biology via the combined reanalysis of Haloferax volcanii proteomics data.

The cardiac membrane protein phospholamban (PLN) is targeted by protein kinase A (PKA) at Ser16 and by Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr17 β-Adrenergic stimulation and PKA-dependent phosphorylation of Ser16 acutely stimulate the sarcoplasmic reticulum calcium pump (SERCA) by relieving its inhibition by PLN. CaMKII-dependent phosphorylation may lead to longer-lasting SERCA stimulation and may sustain maladaptive Ca2+ handling. Here, we demonstrated that phosphorylation at either Ser16 or Thr17 converted PLN into a target for the phosphoadaptor protein 14-3-3 with different affinities. 14-3-3 proteins were localized within nanometers of PLN and endogenous 14-3-3 coimmunoprecipitated with pentameric PLN from cardiac membranes. Molecular dynamics simulations predicted different molecular contacts for peptides phosphorylated at Ser16 or Thr17 with the binding groove of 14-3-3, resulting in varied binding affinities. 14-3-3 binding protected either PLN phosphosite from dephosphorylation. β-Adrenergic stimulation of isolated adult cardiomyocytes resulted in the membrane recruitment of endogenous 14-3-3. The exogenous addition of 14-3-3 to β-adrenergic-stimulated cardiomyocytes led to prolonged SERCA activation, presumably because 14-3-3 protected PLN pentamers from dephosphorylation. Phosphorylation of Ser16 was disrupted by the cardiomyopathy-associated ∆Arg14 mutation, implying that phosphorylation of Thr17 by CaMKII may become crucial for 14-3-3 recruitment to ∆Arg14 PLN. Consistent with PLN acting as a dynamic hub in the control of Ca2+ handling, our results identify 14-3-3 binding to PLN as a contractility-augmenting mechanism.

Acute myeloid leukemia (AML) is an aggressive blood cancer with a poor prognosis. We report a comprehensive proteogenomic analysis of bone marrow biopsies from 252 uniformly treated AML patients to elucidate the molecular pathophysiology of AML in order to inform future diagnostic and therapeutic approaches. In addition to in-depth quantitative proteomics, our analysis includes cytogenetic profiling and DNA/RNA sequencing. We identify five proteomic AML subtypes, each reflecting specific biological features spanning genomic boundaries. Two of these proteomic subtypes correlate with patient outcome, but none is exclusively associated with specific genomic aberrations. Remarkably, one subtype (Mito-AML), which is captured only in the proteome, is characterized by high expression of mitochondrial proteins and confers poor outcome, with reduced remission rate and shorter overall survival on treatment with intensive induction chemotherapy. Functional analyses reveal that Mito-AML is metabolically wired toward stronger complex I-dependent respiration and is more responsive to treatment with the BCL2 inhibitor venetoclax.

13 pags, 6 figs, The molecular chaperone Hsp90 is critical for the maintenance of cellular homeostasis and represents a promising drug target. Despite increasing knowledge on the structure of Hsp90, the molecular basis of substrate recognition and pro-folding by Hsp90/co-chaperone complexes remains unknown. Here, we report the solution structures of human full-length Hsp90 in complex with the PPIase FKBP51, as well as the 280 kDa Hsp90/FKBP51 complex bound to the Alzheimer's disease-related protein Tau. We reveal that the FKBP51/Hsp90 complex, which synergizes to promote toxic Tau oligomers in vivo, is highly dynamic and stabilizes the extended conformation of the Hsp90 dimer resulting in decreased Hsp90 ATPase activity. Within the ternary Hsp90/FKBP51/Tau complex, Hsp90 serves as a scaffold that traps the PPIase and nucleates multiple conformations of Tau's proline-rich region next to the PPIase catalytic pocket in a phosphorylation-dependent manner. Our study defines a conceptual model for dynamic Hsp90/co-chaperone/client recognition., This work was supported by the National Institutes of Mental Health Grant R01 MH103848. J.O. was supported by a Marie Curie Intra-European fellowship (project number 626526) and B.J.C. by a Fulbright scholarship. M.Z. was supported by the European Community’s Seventh Framework Programme (FP7/ 2007-2013) under BioStruct-X (grant agreement 283570), and the advanced grant “‘87679 – LLPS-NMR” of the European Research Council. H.U. and M.Z. were supported by the German Science Foundation (Collaborative Research Center 860; projects A10 and B2). L.J.B. was supported by the NIH/NINDS R01 NS073899 Grant.

Multi-omic approaches are promising a broader view on cellular processes and a deeper understanding of biological systems. with strongly improved high-throughput methods the amounts of data generated have become huge, and their handling challenging. Area Covered: New bioinformatic tools and pipelines for the integration of data from different omics disciplines continue to emerge, and will support scientists to reliably interpret data in the context of biological processes. comprehensive data integration strategies will fundamentally improve systems biology and systems medicine. to present recent developments of integrative omics, the göttingen proteomics forum (gpf) organized its 6th symposium on the 23rd of november 2017, as part of a series of regular gpf symposia. more than 140 scientists attended the event that highlighted the challenges and opportunities but also the caveats of integrating data from different omics disciplines. Expert commentary: The continuous exponential growth in omics data require similar development in software solutions for handling this challenge. Integrative omics tools offer the chance to handle this challenge but profound investigations and coordinated efforts are required to boost this field.

Cryo-electron microscopy (cryo-EM) can solve structures of highly dynamic macromolecular complexes. To characterize less well defined regions in cryo-EM images, cross-linking coupled with mass spectrometry (CX-MS) provides valuable information on the arrangement of domains and amino acids. CX-MS involves covalent linkage of protein residues close to each other and identifying these connections by mass spectrometry. Here, we summarise the advances of CX-MS and its integration with cryo-EM for structural reconstruction.

Utilization of energy-rich carbon sources such as glucose is fundamental to the evolutionary success of bacteria. Glucose can be catabolized via glycolysis for feeding the intermediary metabolism. The methylglyoxal synthase MgsA produces methylglyoxal from the glycolytic intermediate dihydroxyacetone phosphate. Methylglyoxal is toxic, requiring stringent regulation of MgsA activity. In the Gram-positive bacterium Bacillus subtilis, an interaction with the phosphoprotein Crh controls MgsA activity. In the absence of preferred carbon sources, Crh is present in the nonphosphorylated state and binds to and thereby inhibits MgsA. To better understand the mechanism of regulation of MgsA, here we performed biochemical and structural analyses of B. subtilis MgsA and of its interaction with Crh. Our results indicated that MgsA forms a hexamer (i.e. a trimer of dimers) in the crystal structure, whereas it seems to exist in an equilibrium between a dimer and hexamer in solution. In the hexamer, two alternative dimers could be distinguished, but only one appeared to prevail in solution. Further analysis strongly suggested that the hexamer is the biologically active form. In vitro cross-linking studies revealed that Crh interacts with the N-terminal helices of MgsA and that the Crh–MgsA binding inactivates MgsA by distorting and thereby blocking its active site. In summary, our results indicate that dimeric and hexameric MgsA species exist in an equilibrium in solution, that the hexameric species is the active form, and that binding to Crh deforms and blocks the active site in MgsA.

Several noncoding RNAs potentially involved in nitrogen (N)-regulation have been detected in Methanosarcina mazei, however, targets have been identified only for one of them. Here, we report on the function of sRNA41 , highly expressed under N-sufficiency. Comprising 120 nucleotides, sRNA41 shows high sequence and structural conservation within draft genomes of numerous Methanosarcina species. In silico target prediction revealed several potential targets, including genes of two homologous operons encoding for acetyl-CoA-decarbonylase/synthase complexes (ACDS) representing highly probable target candidates. A highly conserved single stranded region of sRNA41 was predicted to mask six independent ribosome binding sites of these two polycistronic mRNAs and was verified in vitro by microscale thermophoresis. Proteome analysis of the respective sRNA41 -deletion mutant showed increased protein expression of both ACDS complexes in the absence of sRNA41 , whereas no effect on transcript levels was detected, arguing for sRNA41 -mediated post-transcriptional fine-tuning of ACDS expression. We hypothesize that the physiological advantage of downregulating sRNA41 under N-limiting conditions is the resulting increase of ACDS protein levels. This provides sufficient amounts of amino acids for nitrogenase synthesis as well as reducing equivalents and energy for N2 -fixation, thus linking the carbon and N-metabolism.

β-adrenergic receptor (β-AR) stimulation represents a major mechanism of modulating cardiac output. In spite of its fundamental importance, its molecular basis on the level of cell signalling has not been characterised in detail yet. We employed mass spectrometry-based proteome and phosphoproteome analysis using SuperSILAC (spike-in stable isotope labelling by amino acids in cell culture) standardization to generate a comprehensive map of acute phosphoproteome changes in mice upon administration of isoprenaline (ISO), a synthetic β-AR agonist that targets both β1-AR and β2-AR subtypes. Our data describe 8597 quantitated phosphopeptides corresponding to 10,164 known and novel phospho-events from 2975 proteins. In total, 197 of these phospho-events showed significantly altered phosphorylation, indicating an intricate signalling network activated in response to β-AR stimulation. In addition, we unexpectedly detected significant cardiac expression and ISO-induced fragmentation of junctophilin-1, a junctophilin isoform hitherto only thought to be expressed in skeletal muscle. Data are available via ProteomeXchange with identifier PXD025569.

Introduction Cumin (Cuminum cyminum), a popular spice has been widely used in traditional medicine to cure various ailments. Despite the existence of scientific literature about its pharmacological properties, no successful proteome profiling has yet been attempted. Objective To optimise extraction of cumin proteins and analyse its profile by shotgun proteomics, using one-dimensional electrophoresis coupled with nano-ESI-LC–MS/MS. Methodology As a first step, we have compared three extraction protocols for total proteins extraction from cumin. Extracted proteins were separated on one-dimensional gel and analysed by state-of-the-art linear ion trap (LTQ)-Orbitrap Velose and Q Exactive HF mass spectrometer. Results Evaluation of extraction method revealed significant differences in protein yield and proteome composition between the three extracts. LC–MS/MS allowed identification of several proteins with functional significance in various biological processes. Conclusion This study provides identification of a large number of proteins and offers a molecular basis for future research on potential pharmacologically active cumin proteins. Copyright © 2017 John Wiley & Sons, Ltd.

Determining the structures of, and gaining insight into, the function of large protein complexes at the molecular or atomic level has become a key part of modern structural biology. Electron cryo-microscopy (cryo-EM) can solve structures of highly dynamic macromolecular complexes that are not feasible with other structural techniques like X-ray of crystallized proteins (protein complexes) or nuclear magnetic resonance (NMR) spectroscopy of proteins (protein complexes) in solution. To resolve the regions that are less well defined in cryo-EM images, cross-linking coupled with mass spectrometry (CX-MS) provides valuable information on the proximity between amino-acid residues as distance constraints for homology or de novo modelling. The CX-MS strategy involves covalent linkage, with chemical cross-linkers, of residues close to each other in three-dimensional space and identifying these connections by mass spectrometry. In this article, we summarise the advances of CX-MS and its integration with cryo-EM for structural reconstruction. We further evaluate a number of important examples of structure determination that followed this combinatorial strategy.

Tissue engineering offers promising perspectives in the therapy of osteoarthritis. In the context of cell-based therapy, chondrogenic progenitor cells (CPCs) may be used to regenerate defects in cartilage tissue. An in-depth characterization of the secretome of CPCs is a prerequisite to this approach. In this study, a method was developed for the qualitative and quantitative analysis of the secretome of undifferentiated and differentiated CPCs. Secreted proteins from cells grown in two-dimensional as well as three-dimensional alginate cultures were extracted and analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Quantitation was achieved by internal standardization using stable isotope-labeled amino acids in cell culture (SILAC). Qualitative analysis of CPC secretomes revealed ECM-components, signal proteins and growth factors most of which were also found in healthy cartilage. A quantitative comparison revealed significantly upregulated proteins with regenerative potential during differentiation, while proteins involved in catabolic metabolism were significantly downregulated. The development of methods for qualitative and quantitative analysis of the secretome of CPCs by mass spectrometry provides a foundation for the investigation of progenitor or stem cells from other sources.

Assembly of the SNARE proteins syntaxin1, SNAP25, and synaptobrevin into a SNARE complex is essential for exocytosis in neurons. For efficient assembly, SNAREs interact with additional proteins but neither the nature of the intermediates nor the sequence of protein assembly is known. Here, we have characterized a ternary complex between syntaxin1, SNAP25, and the SM protein Munc18‐1 as a possible acceptor complex for the R‐SNARE synaptobrevin. The ternary complex binds synaptobrevin with fast kinetics, resulting in the rapid formation of a fully zippered SNARE complex to which Munc18‐1 remains tethered by the N‐terminal domain of syntaxin1. Intriguingly, only one of the synaptobrevin truncation mutants (Syb1‐65) was able to bind to the syntaxin1:SNAP25:Munc18‐1 complex, suggesting either a cooperative zippering mechanism that proceeds bidirectionally or the progressive R‐SNARE binding via an SM template. Moreover, the complex is resistant to disassembly by NSF. Based on these findings, we consider the ternary complex as a strong candidate for a physiological intermediate in SNARE assembly. ![][1] The ternary complex syntaxin1, SNAP25a and Munc18‐1 acts as an intermediate in the SNARE assembly pathway by functioning as an acceptor for synaptobrevin/VAMP‐2. [1]: /embed/graphic-1.gif

A hallmark of protein N-glycosylation is extensive heterogeneity associated with each glycosylation site. In human cells, the constituent glycoforms differ mostly in numerous ways of extensions from an invariable trimannosyl core and terminal modifications. The efficient identification of these glycoforms at the glycopeptide level by mass spectrometry (MS) requires a precursor sampling technique that is not dictated by signal intensity or by preset targets during MS2 data acquisition. We show here that the recently developed data-independent acquisition (DIA) approach is best suited to this demanding task. It allows postacquisition extraction of glycopeptide-specific fragment-ion chromatograms to be aligned with that of precursor MS1 ion by nanoLC elution time. For any target glycoprotein, judicious selection of the most favorable MS1/MS2 transitions can first be determined from prior analysis of a purified surrogate standard that carries similar site-specific glycosylation but may differ in its exact range of glycoforms. Since the MS2 transitions to be used for extracting DIA data is common to that glycosylation site and not dictated by a specific MS1 value, our workflow applies equally well to the identification of both targeted and unexpected glycoforms. Using a case example, we show that, in targeted mode, it identified more site-specific glycoforms than the more commonly used data-dependent acquisition method when the amount of the target glycoprotein was limited in a sample of high complexity. In discovery mode, it allows detection, with supporting MS2 evidence, of under-sampled glycoforms and of those that failed to be identified by searching against a predefined glycan library owing to unanticipated modifications.

Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C*). The architecture of the catalytic U2–U6 ribonucleoprotein (RNP) core of the human C* spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated from the catalytic centre by approximately 20 A, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains. RNA helicase PRP22 is located about 100 A from the catalytic centre, suggesting that it destabilizes the spliced mRNA after step two from a distance. Comparison of the structure of the yeast C and human C* complexes reveals numerous RNP rearrangements that are likely to be facilitated by PRP16, including a large-scale movement of the U2 small nuclear RNP. The cryo-EM structure of the splicing intermediate known as the C* complex from human. Recent years have seen substantial progress in understanding the structure of various intermediates of the splicing process. Two groups, led by Reinhard Luhrmann and Kiyoshi Nagai, now describe the cryo-electron microscopy structures (from human and yeast cells, respectively) of the splicing intermediate known as the C* complex. The notable feature observed in this complex, relative to the preceding catalytic intermediate (the C complex), is a remodelling that positions the branch-site adenosine and the branched intron out of the catalytic core, opening up space for the 3′ exon to dock in preparation for exon ligation.

Epithelial tissues are fundamental for the establishment and maintenance of different body compartments in multicellular animals. To achieve this specific task epithelial sheets secrete an apical extracellular matrix for tissue strength and protection and they organize a transepithelial barrier function, which is mediated by tight junctions in vertebrates or septate junctions in invertebrates. Here, we show that the bicistronic gene wurmchen is functionally expressed in epithelial tissues. CRISPR/Cas9-mediated mutations in both coding sequences reveal two essential polypeptides, Wurmchen1 and Wurmchen2, which are both necessary for normal epithelial tissue development. Wurmchen1 represents a genuine septate junction core component. It is required during embryogenesis for septate junction organization, the establishment of a transepithelial barrier function, distinct cellular transport processes and tracheal system morphogenesis. Wurmchen2 is localized in the apical membrane region of epithelial tissues and in a central core of the tracheal lumen during embryogenesis. It is essential during the later larval development.

The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing1. Stable addition of U2 during early spliceosome formation requires the DEAD-box ATPase PRP52–7. Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem–loop (BSL)8, but whether the human U2 snRNA folds in a similar manner is unknown. The U2 SF3B1 protein, a common mutational target in haematopoietic cancers9, contains a HEAT domain (SF3B1HEAT) with an open conformation in isolated SF3b10, but a closed conformation in spliceosomes11, which is required for stable interaction between U2 and the branch site. Here we report a 3D cryo-electron microscopy structure of the human 17S U2 snRNP at a core resolution of 4.1 A and combine it with protein crosslinking data to determine the molecular architecture of this snRNP. Our structure reveals that SF3B1HEAT interacts with PRP5 and TAT-SF1, and maintains its open conformation in U2 snRNP, and that U2 snRNA forms a BSL that is sandwiched between PRP5, TAT-SF1 and SF3B1HEAT. Thus, substantial remodelling of the BSL and displacement of BSL-interacting proteins must occur to allow formation of the U2–branch-site helix. Our studies provide a structural explanation of why TAT-SF1 must be displaced before the stable addition of U2 to the spliceosome, and identify RNP rearrangements facilitated by PRP5 that are required for stable interaction between U2 and the branch site. The cryo-EM structure of human U2 small nuclear ribonucleoprotein (snRNP) offers insights into what rearrangements are required for this snRNP to be stably incorporated into the spliceosome, and the role that the DEAD-box ATPase PRP5 may have in these rearrangements.

Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain associates with Pol II subunit RPB12 and the phosphorylated C-terminal region of DSIF subunit SPT5. RTF1 also forms four α-helices that extend from the Plus3 domain along the Pol II protrusion and RPB10 to the polymerase funnel. The C-terminal 'fastener' helix retains PAF and is followed by a 'latch' that reaches the end of the bridge helix, a flexible element of the Pol II active site. RTF1 strongly stimulates Pol II elongation, and this requires the latch, possibly suggesting that RTF1 activates transcription allosterically by influencing Pol II translocation.

The mammalian Staufen proteins (Stau1 and Stau2) mediate degradation of mRNA containing complex secondary structures in their 3’-untranslated region (UTR) through a pathway known as Staufen-mediated mRNA decay (SMD). This pathway also involves the RNA helicase UPF1, which is best known for its role in the nonsense-mediated mRNA decay (NMD) pathway. Here we present a biochemical reconstitution of the recruitment and activation of UPF1 in context of the SMD pathway. We demonstrate the involvement of UPF2, a core NMD factor and a known activator of UPF1, in SMD. UPF2 acts as an adaptor between Stau1 and UPF1, stimulates the catalytic activity of UPF1 and plays a central role in the formation of an SMD-competent mRNP. Our study elucidates the molecular mechanisms of SMD and points towards extensive cross-talk between UPF1-mediated mRNA decay pathways in cells., The Staufen proteins recognize secondary structures in 3’-untranslated regions in mRNA transcripts and induce degradation of these mRNAs with the help of the RNA helicase UPF1. Here the authors report that the nonsense-mediated mRNA decay factor UPF2 mediates the interaction between Stau1 and UPF1 in Staufen-mediated mRNA decay.

Proteins are continually produced and degraded, to avoid the accumulation of old or damaged molecules and to maintain the efficiency of physiological processes. Despite its importance, protein turnover has been difficult to measure in vivo. Previous approaches to evaluating turnover in vivo have required custom labeling approaches, involved complex mass spectrometry (MS) analyses, or used comparative strategies that do not allow direct quantitative measurements. Here, we describe a robust protocol for quantitative proteome turnover analysis in mice that is based on a commercially available diet for stable isotope labeling of amino acids in mammals (SILAM). We start by discussing fundamental concepts of protein turnover, including different methodological approaches. We then cover in detail the practical aspects of metabolic labeling and explain both the experimental and computational steps that must be taken to obtain accurate in vivo results. Finally, we present a simple experimental workflow that enables measurement of precise turnover rates in a time frame of ~4-5 weeks, including the labeling time. We also provide all the scripts needed for the interpretation of the MS results and for comparing turnover across different conditions. Overall, the workflow presented here comprises several improvements in the determination of protein lifetimes with respect to other available methods, including a minimally invasive labeling strategy and a robust interpretation of MS results, thus enhancing reproducibility across laboratories.

SummaryMolecular switches are essential modules in signaling networks and transcriptional reprogramming. Here, we describe a role for small ubiquitin-related-modifier SUMO as a molecular switch in epidermal growth factor receptor (EGFR) signaling. Using quantitative mass spectrometry, we compared the endogenous SUMO-proteomes of Hela cells before and after EGF-stimulation. Thereby, we identified a small group of transcriptional co-regulators including IRF2BP1, IRF2BP2 and IRF2BPL as novel players in EGFR signaling. Comparison of cells expressing wildtype or SUMOylation deficient IRF2BP1 indicated that transient deSUMOylation of IRF2BP1 is important for appropriate expression of immediate early genes including Dual specificity phosphatase 1 (DUSP1, MKP-1), an important feedback regulator of EGFR signaling. We find that IRF2BP1 is a SUMO-dependent repressor, whose transient deSUMOylation on the DUSP1 promotor allows - and whose timely reSUMOylation restricts - DUSP1 expression. Our work thus provides a paradigm how comparative SUMO proteome analyses serve to reveal novel regulators in signal transduction and transcription.

Axonal degeneration is a key and early pathological feature in traumatic and neurodegenerative disorders of the CNS. Following a focal lesion to axons, extended axonal disintegration by acute axonal degeneration (AAD) occurs within several hours. During AAD, the accumulation of autophagic proteins including Unc-51 like autophagy activating kinase 1 (ULK1) has been demonstrated, but its role is incompletely understood. Here, we study the effect of ULK1 inhibition in different models of lesion-induced axonal degeneration in vitro and in vivo. Overexpression of a dominant negative of ULK1 (ULK1.DN) in primary rat cortical neurons attenuates axotomy-induced AAD in vitro. Both ULK1.DN and the ULK1 inhibitor SBI-0206965 protect against AAD after rat optic nerve crush in vivo. ULK1.DN additionally attenuates long-term axonal degeneration after rat spinal cord injury in vivo. Mechanistically, ULK1.DN decreases autophagy and leads to an mTOR-mediated increase in translational proteins. Consistently, treatment with SBI-0206965 results in enhanced mTOR activation. ULK1.DN additionally modulates the differential splicing of the degeneration-associated genesKif1bandDdit3. These findings uncover ULK1 as an important mediator of axonal degeneration in vitro and in vivo, and elucidate its function in splicing, defining it as a putative therapeutic target.

Gene transcription by RNA polymerase II is regulated by activator proteins that recruit the coactivator complexes SAGA (Spt–Ada–Gcn5–acetyltransferase)1,2 and transcription factor IID (TFIID)2–4. SAGA is required for all regulated transcription5 and is conserved among eukaryotes6. SAGA contains four modules7–9: the activator-binding Tra1 module, the core module, the histone acetyltransferase (HAT) module and the histone deubiquitination (DUB) module. Previous studies provided partial structures10–14, but the structure of the central core module is unknown. Here we present the cryo-electron microscopy structure of SAGA from the yeast Saccharomyces cerevisiae and resolve the core module at 3.3 A resolution. The core module consists of subunits Taf5, Sgf73 and Spt20, and a histone octamer-like fold. The octamer-like fold comprises the heterodimers Taf6–Taf9, Taf10–Spt7 and Taf12–Ada1, and two histone-fold domains in Spt3. Spt3 and the adjacent subunit Spt8 interact with the TATA box-binding protein (TBP)2,7,15–17. The octamer-like fold and its TBP-interacting region are similar in TFIID, whereas Taf5 and the Taf6 HEAT domain adopt distinct conformations. Taf12 and Spt20 form flexible connections to the Tra1 module, whereas Sgf73 tethers the DUB module. Binding of a nucleosome to SAGA displaces the HAT and DUB modules from the core-module surface, allowing the DUB module to bind one face of an ubiquitinated nucleosome. Structural studies on the yeast transcription coactivator complex SAGA (Spt–Ada–Gcn5–acetyltransferase) provide insights into the mechanism of initiation of regulated transcription by this multiprotein complex, which is conserved among eukaryotes.

Alzheimer’s disease is the most prevalent neurodegenerative disorder leading to progressive cognitive decline. Despite decades of research, understanding AD progression at the molecular level, especially at its early stages, remains elusive. Here, we identified several presymptomatic AD markers by investigating brain proteome changes over the course of neurodegeneration in a transgenic mouse model of AD (3×Tg-AD). We show that one of these markers, heme-binding protein 1 (Hebp1), is elevated in the brains of both 3×Tg-AD mice and patients affected by rapidly-progressing forms of AD. Hebp1, predominantly expressed in neurons, interacts with the mitochondrial contact site complex (MICOS) and exhibits a perimitochondrial localization. Strikingly, wildtype, but not Hebp1-deficient, neurons showed elevated cytotoxicity in response to heme-induced apoptosis. Increased survivability in Hebp1-deficient neurons is conferred by blocking the activation of the mitochondrial-associated caspase signaling pathway. Taken together, our data highlight a role of Hebp1 in progressive neuronal loss during AD progression.

Mitochondrial membrane proteins with internal targeting signals are inserted into the inner membrane by the carrier translocase (TIM22 complex). For this, precursors have to be initially directed from the TOM complex in the outer mitochondrial membrane across the intermembrane space toward the TIM22 complex. How these two translocation processes are topologically coordinated is still unresolved. Using proteomic approaches, we find that the human TIM22 complex associates with the mitochondrial contact site and cristae organizing system (MICOS) complex. This association does not appear to be conserved in yeast, whereby the yeast MICOS complex instead interacts with the presequence translocase. Using a yeast mic10Δ strain and a HEK293T MIC10 knockout cell line, we characterize the role of MICOS for protein import into the mitochondrial inner membrane and matrix. We find that a physiological cristae organization promotes efficient import via the presequence pathway in yeast, while in human mitochondria, the MICOS complex is dispensable for protein import along the presequence pathway. However, in human mitochondria, the MICOS complex is required for the efficient import of carrier proteins into the mitochondrial inner membrane. Our analyses suggest that in human mitochondria, positioning of the carrier translocase at the crista junction, and potentially in vicinity to the TOM complex, is required for efficient transport into the inner membrane. peerReviewed

Summary Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryo-EM structures of core and complete vRNAP enzymes from Vaccinia virus at 2.8 A resolution. The vRNAP core enzyme resembles eukaryotic RNA polymerase II (Pol II) but also reveals many virus-specific features, including the transcription factor Rap94. The complete enzyme additionally contains the transcription factor VETF, the mRNA processing factors VTF/CE and NPH-I, the viral core protein E11, and host tRNAGln. This complex can carry out the entire early transcription cycle. The structures show that Rap94 partially resembles the Pol II initiation factor TFIIB, that the vRNAP subunit Rpo30 resembles the Pol II elongation factor TFIIS, and that NPH-I resembles chromatin remodeling enzymes. Together with the accompanying paper ( Hillen et al., 2019 ), these results provide the basis for unraveling the mechanisms of poxvirus transcription and RNA processing.

© 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group. Carrier state viral infection constitutes an equilibrium state in which a limited fraction of a cellular population is infected while the remaining cells are transiently resistant to infection. This type of infection has been characterized for several bacteriophages but not, to date, for archaeal viruses. Here we demonstrate that the rudivirus SIRV3 can produce a host-dependent carrier state infection in the model crenarchaeon Sulfolobus. SIRV3 only infected a fraction of a Sulfolobus islandicus REY15A culture over several days during which host growth was unimpaired and no chromosomal DNA degradation was observed. CRISPR spacer acquisition from SIRV3 DNA was induced by coinfecting with the monocaudavirus SMV1 and it was coincident with increased transcript levels from subtype I-A adaptation and interference cas genes. However, this response did not significantly affect the carrier state infection of SIRV3 and both viruses were maintained in the culture over 12 days during which SIRV3 anti-CRISPR genes were shown to be expressed. Transcriptome and proteome analyses demonstrated that most SIRV3 genes were expressed at varying levels over time whereas SMV1 gene expression was generally low. The study yields insights into the basis for the stable infection of SIRV3 and the resistance to the different host CRISPR-Cas interference mechanisms. It also provides a rationale for the commonly observed coinfection of archaeal cells by different viruses in natural environments.

Transcriptome and proteome profiling in isogenic CRISPR/Cas9 engineered human colon organoids reveals qualitatively distinct Wnt responses in APC-deficient adenoma and normal stem/progenitor cells. The data represent a comprehensive resource for molecular markers that are linked with colon cancer prognosis., Constitutive Wnt activation upon loss of Adenoma polyposis coli (APC) acts as main driver of colorectal cancer (CRC). Targeting Wnt signaling has proven difficult because the pathway is crucial for homeostasis and stem cell renewal. To distinguish oncogenic from physiological Wnt activity, we have performed transcriptome and proteome profiling in isogenic human colon organoids. Culture in the presence or absence of exogenous ligand allowed us to discriminate receptor-mediated signaling from the effects of CRISPR/Cas9-induced APC loss. We could catalog two nonoverlapping molecular signatures that were stable at distinct levels of stimulation. Newly identified markers for normal stem/progenitor cells and adenomas were validated by immunohistochemistry and flow cytometry. We found that oncogenic Wnt signals are associated with good prognosis in tumors of the consensus molecular subtype 2 (CMS2). In contrast, receptor-mediated signaling was linked to CMS4 tumors and poor prognosis. Together, our data represent a valuable resource for biomarkers that allow more precise stratification of Wnt responses in CRC., Graphical Abstract

Cell fate decisions are controlled by complex signal transduction processes that transmit information via posttranslational protein modifications such as phosphorylation. In lymphoma, as in other cancer types, these signaling networks are often dysregulated and thus contribute to malignant transformation and tumor maintenance. For example, B-cell antigen receptor signals are rewired in certain lymphoma types, such as diffuse large B-cell lymphomas, to promote cell growth and survival of the malignant cell clones. Hence, global elucidation of such intricate signaling networks is important for an improved understanding of the biology of these tumors and the identification of target proteins for therapeutic purposes.We describe here a mass spectrometry-based phosphoproteomic approach for characterization of intracellular signaling events and their dynamics. This integrated phosphoproteomic technology combines phosphopeptide enrichment and fractionation with liquid-chromatography-coupled mass spectrometry for the site-specific mapping and quantification of thousands of phosphorylation events in a given cell type. Such global signaling analyses provide valuable insights into oncogenic signaling networks and can inform drug development efforts.