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Heteroresistance (HR) in bacteria describes a subpopulational phenomenon of antibiotic resistant cells of a generally susceptible population. Here, we investigated the molecular mechanisms and phenotypic characteristics underlying HR to ceftazidime (CAZ) in a clinical Enterobacter cloacae complex strain (ECC). We identified a plasmid-borne gene duplication-amplification (GDA) event of a region harbouring an ampC gene encoding a β-lactamase bla DHA-1 as the key determinant of HR. Individual colonies exhibited variations in the copy number of the genes resulting in resistance level variation which correlated with growth onset (lag times) and growth rates in the presence of CAZ. GDA copy number heterogeneity occurred within single resistant colonies, demonstrating heterogeneity of GDA on the single-cell level. The interdependence between GDA, lag time and antibiotic treatment and the strong plasticity underlying HR underlines the high risk for misdetection of antimicrobial HR and subsequent treatment failure., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Cellular processes are remarkably effective across diverse temperature ranges, even with highly conserved proteins. In the context of the microtubule cytoskeleton, which is critically involved in a wide range of cellular activities, this is particularly striking, as tubulin is one of the most conserved proteins while microtubule dynamic instability is highly temperature sensitive. Here, we leverage the diversity of natural tubulin variants from three closely related frog species that live at different temperatures. We determine the microtubule structure across all three species at between 3.0 and 3.6 Å resolution by cryo-electron microscopy and find small differences at the β-tubulin lateral interactions. Using in vitro reconstitution assays and quantitative biochemistry, we show that tubulin's free energy scales inversely with temperature. The observed weakening of lateral contacts and the low apparent activation energy for tubulin incorporation provide an explanation for the overall stability and higher growth rates of microtubules in cold-adapted frog species. This study thus broadens our conceptual framework for understanding microtubule dynamics and provides insights into how conserved cellular processes are tailored to different ecological niches., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

CD8 + T-cell responses are meticulously orchestrated processes regulated by intercellular receptor:ligand interactions. These interactions critically control the dynamics of CD8 + T-cell populations that is crucial to overcome threats such as viral infections or cancer. Yet, the mechanisms governing these dynamics remain incompletely elucidated. Here, we identified a hitherto unknown T-cell referred function of the self-ligating surface receptor SLAMF7 (CD319) on CD8 + T cells during initiation of cytotoxic T-cell responses. According to its cytotoxicity related expression on T effector cells, we found that CD8 + T cells could utilize SLAMF7 to transduce environmental cues into cellular interactions and information exchange. Indeed, SLAMF7 facilitated a dose-dependent formation of stable homotypic contacts that ultimately resulted in stable cell-contacts, quorum populations and commitment to expansion and differentiation. Using pull-down assays and network analyses, we identified novel SLAMF7-binding intracellular signaling molecules including the CRK, CRKL, and Nck adaptors, which are involved in T-cell contact formation and may mediate SLAMF7 functions in sensing and adhesion. Hence, providing SLAMF7 signals during antigen recognition of CD8 + T cells enhanced their overall magnitude, particularly in responses towards low-affinity antigens, resulting in a significant boost in their proliferation and cytotoxic capacity. Overall, we have identified and characterized a potent initiator of the cytotoxic T lymphocyte response program and revealed advanced mechanisms to improve CD8 + T-cell response decisions against weak viral or tumor-associated antigens, thereby strengthening our defense against such adversaries., (© 2024. The Author(s).)

The question whether interference with the ubiquitous splicing machinery can lead to cell-type specific perturbation of cellular function is addressed here by T cell specific ablation of the general U5 snRNP assembly factor CD2BP2/U5-52K. This protein defines the family of nuclear GYF domain containing proteins that are ubiquitously expressed in eukaryotes with essential functions ascribed to early embryogenesis and organ function. Abrogating CD2BP2/U5-52K in T cells, allows us to delineate the consequences of splicing machinery interferences for T cell development and function. Increased T cell lymphopenia and T cell death are observed upon depletion of CD2BP2/U5-52K. A substantial increase in exon skipping coincides with the observed defect in the proliferation/differentiation balance in the absence of CD2BP2/U5-52K. Prominently, skipping of exon 7 in Mdm4 is observed, coinciding with upregulation of pro-apoptotic gene expression profiles upon CD2BP2/U5-52K depletion. Furthermore, we observe enhanced sensitivity of naïve T cells compared to memory T cells to changes in CD2BP2/U5-52K levels, indicating that depletion of this general splicing factor leads to modulation of T cell homeostasis. Given the recent structural characterization of the U5 snRNP and the crosslinking mass spectrometry data given here, design of inhibitors of the U5 snRNP conceivably offers new ways to manipulate T cell function in settings of disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bertazzon, Hurtado-Pico, Plaza-Sirvent, Schuster, Preußner, Kuropka, Liu, Kirsten, Schmitt, König, Álvaro-Benito, Abualrous, Albert, Kliche, Heyd, Schmitz and Freund.)

In recent years, proximity labeling has established itself as an unbiased and powerful approach to map the interactome of specific proteins. Although physiological expression of labeling enzymes is beneficial for the mapping of interactors, generation of the desired cell lines remains time-consuming and challenging. Using our established pipeline for rapid generation of C- and N-terminal CRISPR-Cas9 knock-ins (KIs) based on antibiotic selection, we were able to compare the performance of commonly used labeling enzymes when endogenously expressed. Endogenous tagging of the µ subunit of the adaptor protein (AP)-1 complex with TurboID allowed identification of known interactors and cargo proteins that simple overexpression of a labeling enzyme fusion protein could not reveal. We used the KI strategy to compare the interactome of the different AP complexes and clathrin and were able to assemble lists of potential interactors and cargo proteins that are specific for each sorting pathway. Our approach greatly simplifies the execution of proximity labeling experiments for proteins in their native cellular environment and allows going from CRISPR transfection to mass spectrometry analysis and interactome data in just over a month., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Saponin-mediated endosomal escape is a mechanism that increases the cytotoxicity of type I ribosome-inactivating proteins (type I RIPs). In order to actualize their cytotoxicity, type I RIPs must be released into the cytosol after endocytosis. Without release from the endosomes, type I RIPs are largely degraded and cannot exert their cytotoxic effects. Certain triterpene saponins are able to induce the endosomal escape of these type I RIPs, thus increasing their cytotoxicity. However, the molecular mechanism underlying the endosomal escape enhancement of type I RIPs by triterpene saponins has not been fully elucidated. In this report, we investigate the involvement of the basic amino acid residues of dianthin-30, a type I RIP isolated from the plant Dianthus caryophyllus L., in endosomal escape enhancement using alanine scanning. Therefore, we designed 19 alanine mutants of dianthin-30. Each mutant was combined with SO1861, a triterpene saponin isolated from the roots of Saponaria officinalis L., and subjected to a cytotoxicity screening in Neuro-2A cells. Cytotoxic screening revealed that dianthin-30 mutants with lysine substitutions did not impair the endosomal escape enhancement. There was one particular mutant dianthin, Arg24Ala, that exhibited significantly reduced synergistic cytotoxicity in three mammalian cell lines. However, this reduction was not based on an altered interaction with SO1861. It was, rather, due to the impaired endocytosis of dianthin Arg24Ala into the cells.

Introduction/objective: Suppression of the SOS response in combination with drugs damaging DNA has been proposed as a potential target to tackle antimicrobial resistance. The SOS response is the pathway used to repair bacterial DNA damage induced by antimicrobials such as quinolones. The extent of lexA -regulated protein expression and other associated systems under pressure of agents that damage bacterial DNA in clinical isolates remains unclear. The aim of this study was to assess the impact of this strategy consisting on suppression of the SOS response in combination with quinolones on the proteome profile of Escherichia coli clinical strains., Materials and Methods: Five clinical isolates of E. coli carrying different chromosomally- and/or plasmid-mediated quinolone resistance mechanisms with different phenotypes were selected, with E. coli ATCC 25922 as control strain. In addition, from each clinical isolate and control, a second strain was created, in which the SOS response was suppressed by deletion of the recA gene. Bacterial inocula from all 12 strains were then exposed to 1xMIC ciprofloxacin treatment (relative to the wild-type phenotype for each isogenic pair) for 1 h. Cell pellets were collected, and proteins were digested into peptides using trypsin. Protein identification and label-free quantification were done by liquid chromatography-mass spectrometry (LC-MS) in order to identify proteins that were differentially expressed upon deletion of recA in each strain. Data analysis and statistical analysis were performed using the MaxQuant and Perseus software., Results: The proteins with the lowest expression levels were: RecA (as control), AphA, CysP, DinG, DinI, GarL, PriS, PsuG, PsuK, RpsQ, UgpB and YebG; those with the highest expression levels were: Hpf, IbpB, TufB and RpmH. Most of these expression alterations were strain-dependent and involved DNA repair processes and nucleotide, protein and carbohydrate metabolism, and transport. In isolates with suppressed SOS response, the number of underexpressed proteins was higher than overexpressed proteins., Conclusion: High genomic and proteomic variability was observed among clinical isolates and was not associated with a specific resistant phenotype. This study provides an interesting approach to identify new potential targets to combat antimicrobial resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Recacha, Kuropka, Díaz-Díaz, García-Montaner, González-Tortuero, Docobo-Pérez, Rodríguez-Rojas and Rodríguez-Martínez.)

Introduction: Bloodwork is a widely used diagnostic tool in veterinary medicine, as diagnosis and therapeutic interventions often rely on blood biomarkers. However, biomarkers available in veterinary medicine often lack sensitivity or specificity. Mass spectrometry-based proteomics technology has been extensively used in the analysis of biological fluids. It offers excellent potential for a more comprehensive characterization of the plasma proteome in veterinary medicine., Methods: In this study, we aimed to identify and quantify plasma proteins in a cohort of healthy dogs and compare two techniques for depleting high-abundance plasma proteins to enable the detection of lower-abundance proteins via label-free quantification liquid chromatography-mass spectrometry. We utilized surplus lithium-heparin plasma from 30 healthy dogs, subdivided into five groups of pooled plasma from 6 randomly selected individuals each. Firstly, we used a commercial kit to deplete high-abundance plasma proteins. Secondly, we employed an in-house method to remove albumin using Blue-Sepharose., Results and Discussion: Among all the samples, some of the most abundant proteins identified were apolipoprotein A and B, albumin, alpha-2-macroglobulin, fibrinogen beta chain, fibronectin, complement C3, serotransferrin, and coagulation factor V. However, neither of the depletion techniques achieved significant depletion of highly abundant proteins. Despite this limitation, we could detect and quantify many clinically relevant proteins. Determining the healthy canine proteome is a crucial first step in establishing a reference proteome for canine plasma. After enrichment, this reference proteome can later be utilized to identify protein markers associated with different diseases, thereby contributing to the diagnosis and prognosis of various pathologies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Doulidis, Kuropka, Frizzo Ramos, Rodríguez-Rojas and Burgener.)

Protein-templated fragment ligation was established as a method for the rapid identification of high affinity ligands, and multicomponent reactions (MCR) such as the Ugi four-component reaction (Ugi 4CR) have been efficient in the synthesis of drug candidates. Thus, the combination of both strategies should provide a powerful approach to drug discovery. Here, we investigate protein-templated Ugi 4CR quantitatively using a fluorescence-based enzyme assay, HPLC-QTOF mass spectrometry (MS), and native protein MS with SARS-CoV-2 main protease as template. Ugi reactions were analyzed in aqueous buffer at varying pH and fragment concentration. Potent inhibitors of the protease were formed in presence of the protein via Ugi 4CR together with Ugi three-component reaction (Ugi 3CR) products. Binding of inhibitors to the protease was confirmed by native MS and resulted in the dimerization of the protein target. Formation of Ugi products was, however, more efficient in the non-templated reaction, apparently due to interactions of the protein with the isocyanide and imine fragments. Consequently, in-situ ligation screening of Ugi 4CR products was identified as a superior approach to the discovery of SARS-CoV-2 protease inhibitors., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Isogenic cells respond in a heterogeneous manner to interferon. Using a micropatterning approach combined with high-content imaging and spatial analyses, we characterized how the population context (position of a cell with respect to neighboring cells) of epithelial cells affects their response to interferons. We identified that cells at the edge of cellular colonies are more responsive than cells embedded within colonies. We determined that this spatial heterogeneity in interferon response resulted from the polarized basolateral interferon receptor distribution, making cells located in the center of cellular colonies less responsive to ectopic interferon stimulation. This was conserved across cell lines and primary cells originating from epithelial tissues. Importantly, cells embedded within cellular colonies were not protected from viral infection by apical interferon treatment, demonstrating that the population context-driven heterogeneous response to interferon influences the outcome of viral infection. Our data highlights that the behavior of isolated cells does not directly translate to their behavior in a population, placing the population context as one important factor influencing heterogeneity during interferon response in epithelial cells., (© 2024. The Author(s).)

Acute haemorrhagic diarrhoea is a common complaint in dogs. In addition to causes like intestinal parasites, dietary indiscretion, intestinal foreign bodies, canine parvovirus infection, or hypoadrenocorticism, acute haemorrhagic diarrhoea syndrome (AHDS) is an important and sometimes life-threatening differential diagnosis. There is some evidence supporting the link between Clostridium perfringens toxins and AHDS. These toxins may be partially responsible for the epithelial cell injury, but the pathogenesis of AHDS is still not fully understood. Recent studies have suggested that severe damage to the intestinal mucosa and associated barrier dysfunction can trigger chronic gastrointestinal illnesses. Besides bloodwork and classical markers for AHDS such as protein loss and intestinal bacterial dysbiosis, we focused mainly on the plasma-proteome to identify systemic pathological alterations during this disease and searched for potential biomarkers to improve the diagnosis. To accomplish the goals, we used liquid chromatography-mass spectrometry. We compared the proteomic profiles of 20 dogs with AHDS to 20 age-, breed-, and sex-matched control dogs. All dogs were examined, and several blood work parameters were determined and compared, including plasma biochemistry and cell counts. We identified and quantified (relative quantification) 207 plasmatic proteins, from which dozens showed significantly altered levels in AHDS. Serpina3, Lipopolysaccharide-binding protein, several Ig-like domain-containing proteins, Glyceraldehyde-3-phosphate dehydrogenase and Serum amyloid A were more abundant in plasma from AHDS affected dogs. In contrast, other proteins such as Paraoxonase, Selenoprotein, Amine oxidases, and Apolipoprotein C-IV were significantly less abundant. Many of the identified and quantified proteins are known to be associated with inflammation. Other proteins like Serpina3 and RPLP1 have a relevant role in oncogenesis. Some proteins and their roles have not yet been described in dogs with diarrhoea. Our study opens new avenues that could contribute to the understanding of the aetiology and pathophysiology of AHDS., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Huber et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Inflammatory bowel disease (IBD) is a chronic inflammatory condition that affects humans and several domestic animal species, including cats and dogs. In this study, we have analyzed duodenal organoids derived from canine IBD patients using quantitative proteomics. Our objective was to investigate whether these organoids show phenotypic traits of the disease compared with control organoids obtained from healthy donors. To this aim, IBD and control organoids were subjected to quantitative proteomics analysis via liquid chromatography-mass spectrometry. The obtained data revealed notable differences between the two groups. The IBD organoids exhibited several alterations at the levels of multiple proteins that are consistent with some known IBD alterations. The observed phenotype in the IBD organoids to some degree mirrors the corresponding intestinal condition, rendering them a compelling approach for investigating the disease and advancing drug exploration. Additionally, our study revealed similarities to some human IBD biomarkers, further emphasizing the translational and comparative value of dogs for future investigations related to the causes and treatment of IBD. Relevant proteins such as CALU, FLNA, MSN and HMGA2, which are related to intestinal diseases, were all upregulated in the IBD duodenal organoids. At the same time, other proteins such as intestinal keratins and the mucosal immunity PIGR were depleted in these IBD organoids. Based on these findings, we propose that these organoids could serve as a valuable tool for evaluating the efficacy of therapeutic interventions against canine IBD.

Staphylococcus aureus is a major pathogen, which has to defend against reactive oxygen and electrophilic species encountered during infections. Activated macrophages produce the immunometabolite itaconate as potent electrophile and antimicrobial upon pathogen infection. In this work, we used transcriptomics, metabolomics and shotgun redox proteomics to investigate the specific stress responses, metabolic changes and redox modifications caused by sublethal concentrations of itaconic acid in S. aureus. In the RNA-seq transcriptome, itaconic acid caused the induction of the GlnR, KdpDE, CidR, SigB, GraRS, PerR, CtsR and HrcA regulons and the urease-encoding operon, revealing an acid and oxidative stress response and impaired proteostasis. Neutralization using external urea as ammonium source improved the growth and decreased the expression of the glutamine synthetase-controlling GlnR regulon, indicating that S. aureus experienced ammonium starvation upon itaconic acid stress. In the extracellular metabolome, the amounts of acetate and formate were decreased, while secretion of pyruvate and the neutral product acetoin were strongly enhanced to avoid intracellular acidification. Exposure to itaconic acid affected the amino acid uptake and metabolism as revealed by the strong intracellular accumulation of lysine, threonine, histidine, aspartate, alanine, valine, leucine, isoleucine, cysteine and methionine. In the proteome, itaconic acid caused widespread S-bacillithiolation and S-itaconation of redox-sensitive antioxidant and metabolic enzymes, ribosomal proteins and translation factors in S. aureus, supporting its oxidative and electrophilic mode of action in S. aureus. In phenotype analyses, the catalase KatA, the low molecular weight thiol bacillithiol and the urease provided protection against itaconic acid-induced oxidative and acid stress in S. aureus. Altogether, our results revealed that under physiological infection conditions, such as in the acidic phagolysome, itaconic acid is a highly effective antimicrobial against multi-resistant S. aureus isolates, which acts as weak acid causing an acid, oxidative and electrophilic stress response, leading to S-bacillithiolation and itaconation., Competing Interests: Declaration of competing interest No competing financial interests exist., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Carbapenem resistance mediated by metallo-β-lactamases (MBL) such as New Delhi metallo-β-lactamase-1 (NDM-1) has become a major factor threatening the efficacy of essential β-lactam antibiotics. Starting from hit fragment dipicolinic acid (DPA), 8-hydroxy- and 8-sulfonamido-quinoline-2-carboxylic acids were developed as inhibitors of NDM-1 with highly improved inhibitory activity and binding affinity. The most active compounds formed reversibly inactive ternary protein-inhibitor complexes with two zinc ions as proven by native protein mass spectrometry and bio-layer interferometry. Modification of the NDM-1 structure with remarkable entropic gain was shown by isothermal titration calorimetry and NMR spectroscopy of isotopically labeled protein. The best compounds were potent inhibitors of NDM-1 and other representative MBL with no or little inhibition of human zinc-binding enzymes. These inhibitors significantly reduced the minimum inhibitory concentrations (MIC) of meropenem for multidrug-resistant bacteria recombinantly expressing bla NDM-1 as well as for several multidrug-resistant clinical strains at concentrations non-toxic to human cells.

Antibiotic resistance is a continuously increasing concern for public healthcare. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. The peptide antibiotic albicidin is such a promising candidate that, as a gyrase poison, shows bactericidal activity against a wide range of gram-positive and gram-negative bacteria. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against albicidin. RNA sequencing and proteomics data show that this novel mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of STM3175 (YgiV), a transcription regulator with a GyrI-like small molecule binding domain that traps albicidin with high affinity. X-ray crystallography and molecular docking reveal a new conserved motif in the binding groove of the GyrI-like domain that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in gram-negative bacteria, and our experiments confirm that STM3175 homologs can confer resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Saathoff et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Excessive discharge of quaternary ammonium disinfectants such as benzalkonium chloride (BAC) into aquatic systems can trigger several physiological responses in environmental microorganisms. In this study, we isolated a less-susceptible strain of Aeromonas hydrophila to BAC, designated as INISA09, from a wastewater treatment plant in Costa Rica. We characterized its phenotypic response upon exposure to three different concentrations of BAC and characterized mechanisms related to its resistance using genomic and proteomic approaches. The genome of the strain, mapped against 52 different sequenced A. hydrophila strains, consists of approximately 4.6 Mb with 4,273 genes. We found a massive genome rearrangement and thousands of missense mutations compared to the reference strain A. hydrophila ATCC 7966. We identified 15,762 missense mutations mainly associated with transport, antimicrobial resistance, and outer membrane proteins. In addition, a quantitative proteomic analysis revealed a significant upregulation of several efflux pumps and the downregulation of porins when the strain was exposed to three BAC concentrations. Other genes related to membrane fatty acid metabolism and redox metabolic reactions also showed an altered expression. Our findings indicate that the response of A. hydrophila INISA09 to BAC primarily occurs at the envelop level, which is the primary target of BAC. Our study elucidates the mechanisms of antimicrobial susceptibility in aquatic environments against a widely used disinfectant and will help better understand how bacteria can adapt to biocide pollution. To our knowledge, this is the first study addressing the resistance to BAC in an environmental A. hydrophila isolate. We propose that this bacterial species could also serve as a new model to study antimicrobial pollution in aquatic environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chacón, Kuropka, González-Tortuero, Schreiber, Rojas-Jiménez and Rodríguez-Rojas.)

Aims: The MarR/DUF24-family QsrR and YodB repressors control quinone detoxification pathways in Staphylococcus aureus and Bacillus subtilis . In S. aureus , the QsrR regulon also confers resistance to antimicrobial compounds with quinone-like elements, such as rifampicin, ciprofloxacin, and pyocyanin. Although QsrR was shown to be inhibited by thiol- S -alkylation of its conserved Cys4 residue by 1,4-benzoquinone, YodB senses quinones and diamide by the formation of reversible intermolecular disulfides. In this study, we aimed at further investigating the redox-regulation of QsrR and the role of its Cys4, Cys29, and Cys32 residues under quinone and oxidative stress in S. aureus . Results: The QsrR regulon was strongly induced by quinones and oxidants, such as diamide, allicin, hypochlorous acid (HOCl), and AGXX ® in S. aureus . Transcriptional induction of catE2 by quinones and oxidants required Cys4 and either Cys29' or Cys32' of QsrR for redox sensing in vivo . DNA-binding assays revealed that QsrR is reversibly inactivated by quinones and oxidants, depending on Cys4. Using mass spectrometry, QsrR was shown to sense diamide by an intermolecular thiol-disulfide switch, involving Cys4 and Cys29' of opposing subunits in vitro . In contrast, allicin caused S -thioallylation of all three Cys residues in QsrR, leading to its dissociation from the operator sequence. Further, the QsrR regulon confers resistance against quinones and oxidants, depending on Cys4 and either Cys29' or Cys32'. Conclusion and Innovation: QsrR was characterized as a two-Cys-type redox-sensing regulator, which senses the oxidative mode of quinones and strong oxidants, such as diamide, HOCl, and the antimicrobial compound allicin via different thiol switch mechanisms.

Regulation and functionality of species-specific alternative splicing has remained enigmatic to the present date. Calcium/calmodulin-dependent protein kinase IIβ (CaMKIIβ) is expressed in several splice variants and plays a key role in learning and memory. Here, we identify and characterize several primate-specific CAMK2B splice isoforms, which show altered kinetic properties and changes in substrate specificity. Furthermore, we demonstrate that primate-specific CAMK2B alternative splicing is achieved through branch point weakening during evolution. We show that reducing branch point and splice site strengths during evolution globally renders constitutive exons alternative, thus providing novel mechanistic insight into cis -directed species-specific alternative splicing regulation. Using CRISPR/Cas9, we introduce a weaker, human branch point sequence into the mouse genome, resulting in strongly altered Camk2b splicing in the brains of mutant mice. We observe a strong impairment of long-term potentiation in CA3-CA1 synapses of mutant mice, thus connecting branch point-controlled CAMK2B alternative splicing with a fundamental function in learning and memory., (© 2022 Franz et al.)

Nephridiophagids are unicellular fungi (Chytridiomycota), which infect the Malpighian tubules of insects. While most life cycle features are known, the effects of these endobionts on their hosts remain poorly understood. Here, we present results on the influence of an infection of the cockroach Blattella germanica with Nephridiophaga blattellae (Ni = Nephridiophaga-infected) on physical, physiological, and reproductive fitness parameters. Since the gut nematode Blatticola blattae is a further common parasite of B. germanica, we included double infected cockroaches (N + Ni = nematode plus Ni) in selected experiments. Ni individuals had lower fat reserves and showed reduced mobility. The lifespan of adult hosts was only slightly affected in these individuals but significantly shortened when both Nephridiophaga and nematodes were present. Ni as well as N + Ni females produced considerably less offspring than parasite-free (P-free) females. Immune parameters such as the number of hemocytes and phenoloxidase activity were barely changed by Nephridiophaga and/or nematode infections, while the ability to detoxify pesticides decreased. Quantitative proteomics from hemolymph of P-free, Ni, and N + Ni populations revealed clear differences in the expression profiles. For Ni animals, for example, the down-regulation of fatty acid synthases corroborates our finding of reduced fat reserves. Our study clearly shows that an infection with Nephridiophaga (and nematodes) leads to an overall reduced host fitness., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Type I ribosome-inactivating proteins (RIPs) are plant toxins that inhibit protein synthesis by exerting rRNA N -glycosylase activity (EC 3.2.2.22). Due to the lack of a cell-binding domain, type I RIPs are not target cell-specific. However once linked to antibodies, so called immunotoxins, they are promising candidates for targeted anti-cancer therapy. In this study, sapovaccarin-S1 and -S2, two newly identified type I RIP isoforms differing in only one amino acid, were isolated from the seeds of Saponaria vaccaria L. Sapovaccarin-S1 and -S2 were purified using ammonium sulfate precipitation and subsequent cation exchange chromatography. The determined molecular masses of 28,763 Da and 28,793 Da are in the mass range typical for type I RIPs and the identified amino acid sequences are homologous to known type I RIPs such as dianthin 30 and saporin-S6 (79% sequence identity each). Sapovaccarin-S1 and -S2 showed adenine-releasing activity and induced cell death in Huh-7 cells. In comparison to other type I RIPs, sapovaccarin-S1 and -S2 exhibited a higher thermostability as shown by nano-differential scanning calorimetry. These results suggest that sapovaccarin-S1 and -S2 would be optimal candidates for targeted anti-cancer therapy.

Background: Host-pathogen interactions can lead to dramatic changes in host feeding behaviour. One aspect of this includes self-medication, where infected individuals consume substances such as toxins or alter their macronutrient consumption to enhance immune competence. Another widely adopted animal response to infection is illness-induced anorexia, which is thought to assist host immunity directly or by limiting the nutritional resources available to pathogens. Here, we recorded macronutrient preferences of the global pest cockroach, Blatta orientalis to investigate how shifts in host macronutrient dietary preference and quantity of carbohydrate (C) and protein (P) interact with immunity following bacterial infection., Results: We find that B. orientalis avoids diets enriched for P under normal conditions, and that high P diets reduce cockroach survival in the long term. However, following bacterial challenge, cockroaches significantly reduced their overall nutrient intake, particularly of carbohydrates, and increased the relative ratio of protein (P:C) consumed. Surprisingly, these behavioural shifts had a limited effect on cockroach immunity and survival, with minor changes to immune protein abundance and antimicrobial activity between individuals placed on different diets, regardless of infection status., Conclusions: We show that cockroach feeding behaviour can be modulated by a pathogen, resulting in an illness-induced anorexia-like feeding response and a shift from a C-enriched to a more P:C equal diet. However, our results also indicate that such responses do not provide significant immune protection in B. orientalis, suggesting that the host's dietary shift might also result from random rather than directed behaviour. The lack of an apparent benefit of the shift in feeding behaviour highlights a possible reduced importance of diet in immune regulation in these invasive animals, although further investigations employing pathogens with alternative infection strategies are warranted., (© 2022. The Author(s).)

Species-specific diversities are particular features of mammalian chloride channel regulator, calcium activated (CLCA) genes. In contrast to four complex gene clusters in mammals, only two CLCA genes appear to exist in chickens. CLCA2 is conserved in both, while only the galline CLCA1 (gCLCA1) displays close genetic distance to mammalian clusters 1, 3 and 4. In this study, sequence analyses and biochemical characterizations revealed that gCLCA1 as a putative avian prototype shares common protein domains and processing features with all mammalian CLCA homologues. It has a transmembrane (TM) domain in the carboxy terminal region and its mRNA and protein were detected in the alimentary canal, where the protein was localized in the apical membrane of enterocytes, similar to CLCA4. Both mammals and birds seem to have at least one TM domain containing CLCA protein with complex glycosylation in the apical membrane of enterocytes. However, some characteristic features of mammalian CLCA1 and 3 including entire protein secretion and expression in cell types other than enterocytes seem to be dispensable for chicken. Phylogenetic analyses including twelve bird species revealed that avian CLCA1 and mammalian CLCA3 form clades separate from a major branch containing mammalian CLCA1 and 4. Overall, our data suggest that gCLCA1 and mammalian CLCA clusters 1, 3 and 4 stem from a common ancestor which underwent complex gene diversification in mammals but not in birds., Competing Interests: The authors have declared that no competing interests exist.

Applications of key technologies in biomedical research, such as qRT-PCR or LC-MS-based proteomics, are generating large biological (-omics) datasets which are useful for the identification and quantification of biomarkers in any research area of interest. Genome, transcriptome and proteome databases are already available for a number of model organisms including vertebrates and invertebrates. However, there is insufficient information available for protein sequences of certain invertebrates, such as the great pond snail Lymnaea stagnalis, a model organism that has been used highly successfully in elucidating evolutionarily conserved mechanisms of memory function and dysfunction. Here, we used a bioinformatics approach to designing and benchmarking a comprehensive central nervous system (CNS) proteomics database (LymCNS-PDB) for the identification of proteins from the CNS of Lymnaea by LC-MS-based proteomics. LymCNS-PDB was created by using the Trinity TransDecoder bioinformatics tool to translate amino acid sequences from mRNA transcript assemblies obtained from a published Lymnaea transcriptomics database. The blast-style MMSeq2 software was used to match all translated sequences to UniProtKB sequences for molluscan proteins, including those from Lymnaea and other molluscs. LymCNS-PDB contains 9628 identified matched proteins that were benchmarked by performing LC-MS-based proteomics analysis with proteins isolated from the Lymnaea CNS. MS/MS analysis using the LymCNS-PDB database led to the identification of 3810 proteins. Only 982 proteins were identified by using a non-specific molluscan database. LymCNS-PDB provides a valuable tool that will enable us to perform quantitative proteomics analysis of protein interactomes involved in several CNS functions in Lymnaea, including learning and memory and age-related memory decline., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and their recycling after splicing require numerous assembly/recycling factors whose modes of action are often poorly understood. The intrinsically disordered TSSC4 protein has been identified as a nuclear-localized U5 snRNP and U4/U6-U5 tri-snRNP assembly/recycling factor, but how TSSC4's intrinsic disorder supports TSSC4 functions remains unknown. Using diverse interaction assays and cryogenic electron microscopy-based structural analysis, we show that TSSC4 employs four conserved, non-contiguous regions to bind the PRPF8 Jab1/MPN domain and the SNRNP200 helicase at functionally important sites. It thereby inhibits SNRNP200 helicase activity, spatially aligns the proteins, coordinates formation of a U5 sub-module and transiently blocks premature interaction of SNRNP200 with at least three other spliceosomal factors. Guided by the structure, we designed a TSSC4 variant that lacks stable binding to the PRPF8 Jab1/MPN domain or SNRNP200 in vitro. Comparative immunoprecipitation/mass spectrometry from HEK293 nuclear extract revealed distinct interaction profiles of wild type TSSC4 and the variant deficient in PRPF8/SNRNP200 binding with snRNP proteins, other spliceosomal proteins as well as snRNP assembly/recycling factors and chaperones. Our findings elucidate molecular strategies employed by an intrinsically disordered protein to promote snRNP assembly, and suggest multiple TSSC4-dependent stages during snRNP assembly/recycling., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recent advances in imaging technology have highlighted that scaffold proteins and receptors are arranged in subsynaptic nanodomains. The synaptic membrane-associated guanylate kinase (MAGUK) scaffold protein membrane protein palmitoylated 2 (MPP2) is a component of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-associated protein complexes and also binds to the synaptic cell adhesion molecule SynCAM 1. Using superresolution imaging, we show that-like SynCAM 1-MPP2 is situated at the periphery of the postsynaptic density (PSD). In order to explore MPP2-associated protein complexes, we used a quantitative comparative proteomics approach and identified multiple γ-aminobutyric acid (GABA)A receptor subunits among novel synaptic MPP2 interactors. In line with a scaffold function for MPP2 in the assembly and/or modulation of intact GABAA receptors, manipulating MPP2 expression had effects on inhibitory synaptic transmission. We further show that GABAA receptors are found together with MPP2 in a subset of dendritic spines and thus highlight MPP2 as a scaffold that serves as an adaptor molecule, linking peripheral synaptic elements critical for inhibitory regulation to central structures at the PSD of glutamatergic synapses., Competing Interests: The authors have declared that no competing interests exist.

The intrinsically unstructured C9ORF78 protein was detected in spliceosomes but its role in splicing is presently unclear. We find that C9ORF78 tightly interacts with the spliceosome remodeling factor, BRR2, in vitro. Affinity purification/mass spectrometry and RNA UV-crosslinking analyses identify additional C9ORF78 interactors in spliceosomes. Cryogenic electron microscopy structures reveal how C9ORF78 and the spliceosomal B complex protein, FBP21, wrap around the C-terminal helicase cassette of BRR2 in a mutually exclusive manner. Knock-down of C9ORF78 leads to alternative NAGNAG 3'-splice site usage and exon skipping, the latter dependent on BRR2. Inspection of spliceosome structures shows that C9ORF78 could contact several detected spliceosome interactors when bound to BRR2, including the suggested 3'-splice site regulating helicase, PRPF22. Together, our data establish C9ORF78 as a late-stage splicing regulatory protein that takes advantage of a multi-factor trafficking site on BRR2, providing one explanation for suggested roles of BRR2 during splicing catalysis and alternative splicing., (© 2022. The Author(s).)

Cytoskeletal proteins are essential for parasite proliferation, growth, and transmission, and therefore have the potential to serve as drug targets. 1-5 While microtubules and their molecular building block αβ-tubulin are established drug targets in a variety of cancers, 6 , 7 we still lack sufficient knowledge of the biochemistry of parasite tubulins to exploit the structural divergence between parasite and human tubulins. For example, it remains to be determined whether compounds of interest can specifically target parasite microtubules without affecting the host cell cytoskeleton. Such mechanistic insights have been limited by the lack of functional parasite tubulin. In this study, we report the purification and characterization of tubulin from Plasmodium falciparum, the causative agent of malaria. We show that the highly purified tubulin is fully functional, as it efficiently assembles into microtubules with specific parameters of dynamic instability. There is a high degree of amino-acid conservation between human and P. falciparum α- and β-tubulin, sharing approximately 83.7% and 88.5% identity, respectively. However, Plasmodium tubulin is more similar to plant than to mammalian tubulin, raising the possibility of identifying compounds that would selectively disrupt parasite microtubules without affecting the host cell cytoskeleton. As a proof of principle, we describe two compounds that exhibit selective toxicity toward parasite tubulin. Thus, the ability to specifically disrupt protozoan microtubule growth without affecting human microtubules provides an exciting opportunity for the development of novel antimalarials., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Evolution of bacterial tolerance to antimicrobials precedes evolution of resistance and may result in cross-tolerance, cross-resistance, or collateral sensitivity to other antibiotics. Transient exposure of gut bacteria to glyphosate, the world's most widely used herbicide, has been linked to the activation of the stress response and changes in susceptibility to antibiotics. In this study, we investigated whether chronic exposure to a glyphosate-based herbicide (GBH) results in resistance, a constitutive activation of the tolerance and stress responses, and cross-tolerance or cross-resistance to antibiotics. Of the 10 farm animal-derived clinical isolates of Salmonella enterica subjected to experimental evolution in increasing concentrations of GBH, three isolates showed stable resistance with mutations associated with the glyphosate target gene aroA and no fitness costs. Global quantitative proteomics analysis demonstrated activation of the cellular tolerance and stress response during the transient exposure to GBH but not constitutively in the resistant mutants. Resistant mutants displayed no cross-resistance or cross-tolerance to antibiotics. These results suggest that while transient exposure to GBH triggers cellular tolerance response in Salmonella enterica , this response does not become genetically fixed after selection for resistance to GBH and does not result in increased cross-tolerance or cross-resistance to clinically important antibiotics under our experimental conditions. IMPORTANCE Glyphosate-based herbicides (GBH) are among the world's most popular, with traces commonly found in food, feed, and the environment. Such high ubiquity means that the herbicide may come into contact with various microorganisms, on which it acts as an antimicrobial, and it may select for resistance and cross-resistance to clinically important antibiotics. It is therefore important to estimate whether the widespread use of pesticides may be an underappreciated source of antibiotic-resistant microorganisms that may compromise efficiency of antibiotic treatments in humans and animals., (Copyright © 2020 Pöppe et al.)

Cationic antimicrobial peptides (AMPs) are active immune effectors of multicellular organisms and are also considered as new antimicrobial drug candidates. One of the problems encountered when developing AMPs as drugs is the difficulty of reaching sufficient killing concentrations under physiological conditions. Here, using pexiganan, a cationic peptide derived from a host defense peptide of the African clawed frog and the first AMP developed into an antibacterial drug, we studied whether sub-lethal effects of AMPs can be harnessed to devise treatment combinations. We studied the pexiganan stress response of Staphylococcus aureus at sub-lethal concentrations using quantitative proteomics. Several proteins involved in nucleotide metabolism were elevated, suggesting a metabolic demand. We then show that Staphylococcus aureus is highly susceptible to antimetabolite nucleoside analogs when exposed to pexiganan, even at sub-inhibitory concentrations. These findings could be used to enhance pexiganan potency while decreasing the risk of resistance emergence, and our findings can likely be extended to other antimicrobial peptides., (Copyright © 2020 Rodríguez-Rojas, Nath, El Shazely, Santi, Kim, Weise, Kuropka and Rolff.)

At presynaptic active zones, arrays of large conserved scaffold proteins mediate fast and temporally precise release of synaptic vesicles (SVs). SV release sites could be identified by clusters of Munc13, which allow SVs to dock in defined nanoscale relation to Ca2+ channels. We here show in Drosophila that RIM-binding protein (RIM-BP) connects release sites physically and functionally to the ELKS family Bruchpilot (BRP)-based scaffold engaged in SV recruitment. The RIM-BP N-terminal domain, while dispensable for SV release site organization, was crucial for proper nanoscale patterning of the BRP scaffold and needed for SV recruitment of SVs under strong stimulation. Structural analysis further showed that the RIM-BP fibronectin domains form a "hinge" in the protein center, while the C-terminal SH3 domain tandem binds RIM, Munc13, and Ca2+ channels release machinery collectively. RIM-BPs' conserved domain architecture seemingly provides a relay to guide SVs from membrane far scaffolds into membrane close release sites., (© 2020 Petzoldt et al.)

The RNA helicase bad response to refrigeration 2 homolog (BRR2) is required for the activation of the spliceosome before the first catalytic step of RNA splicing. BRR2 represents a distinct subgroup of Ski2-like nucleic acid helicases whose members comprise tandem helicase cassettes. Only the N-terminal cassette of BRR2 is an active ATPase and can unwind substrate RNAs. The C-terminal cassette represents a pseudoenzyme that can stimulate RNA-related activities of the N-terminal cassette. However, the molecular mechanisms by which the C-terminal cassette modulates the activities of the N-terminal unit remain elusive. Here, we show that N- and C-terminal cassettes adopt vastly different relative orientations in a crystal structure of BRR2 in complex with an activating domain of the spliceosomal Prp8 protein at 2.4 Å resolution compared with the crystal structure of BRR2 alone. Likewise, inspection of BRR2 structures within spliceosomal complexes revealed that the cassettes occupy different relative positions and engage in different intercassette contacts during different splicing stages. Engineered disulfide bridges that locked the cassettes in two different relative orientations had opposite effects on the RNA-unwinding activity of the N-terminal cassette, with one configuration enhancing and the other configuration inhibiting RNA unwinding compared with the unconstrained protein. Moreover, we found that differences in relative positioning of the cassettes strongly influence RNA-stimulated ATP hydrolysis by the N-terminal cassette. Our results indicate that the inactive C-terminal cassette of BRR2 can both positively and negatively affect the activity of the N-terminal helicase unit from a distance., (© 2020 Vester et al.)

Rationale: Pathological biomechanical signaling induces vascular anomalies including cerebral cavernous malformations (CCM), which are caused by a clonal loss of CCM1/KRIT1 (Krev interaction trapped protein 1), CCM2/MGC4607, or CCM3/PDCD10. Why patients typically experience lesions only in lowly perfused venous capillaries of the cerebrovasculature is completely unknown., Objective: In contrast, animal models with a complete loss of CCM proteins lack a functional heart and blood flow and exhibit vascular anomalies within major blood vessels as well. This finding raises the possibility that hemodynamics may play a role in the context of this vascular pathology., Methods and Results: Here, we used a genetic approach to restore cardiac function and blood flow in a zebrafish model of CCM1. We find that blood flow prevents cardiovascular anomalies including a hyperplastic expansion within a large Ccm1-deficient vascular bed, the lateral dorsal aorta., Conclusions: This study identifies blood flow as an important physiological factor that is protective in the cause of this devastating vascular pathology.

The scaffolding protein intersectin 1 plays important roles in clathrin-mediated endocytosis and in the replenishment of release-ready synaptic vesicles (SV). Two splice variants of intersectin's SH3A domain are expressed in the brain, and association of the neuron-specific variant with synapsin I has been shown to enable sustained neurotransmission and to be regulated by an adjacent C-terminal motif. Here, we demonstrate that the ubiquitously expressed short SH3A variant of intersectin 1 interacts with an N-terminal intramolecular sequence that operates synergistically with the C-terminal motif. NMR spectroscopic investigations show that the five-amino acid insertion into the β strand 2 of the neuronal SH3A variant introduces conformational plasticity incompatible with binding of the N-terminal sequence. The difference in the autoregulatory mechanism of the domain's variants differentially affects its synaptic binding partners, thereby establishing alternative splicing in conjunction with autoinhibitory motif variation as a mechanism to regulate protein interaction networks., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Protein scaffolds at presynaptic active zone membranes control information transfer at synapses. For scaffold biogenesis and maintenance, scaffold components must be safely transported along axons. A spectrum of kinases has been suggested to control transport of scaffold components, but direct kinase-substrate relationships and operational principles steering phosphorylation-dependent active zone protein transport are presently unknown. Here, we show that extensive phosphorylation of a 150-residue unstructured region at the N-terminus of the highly elongated Bruchpilot (BRP) active zone protein is crucial for ordered active zone precursor transport in Drosophila Point mutations that block SRPK79D kinase-mediated phosphorylation of the BRP N-terminus interfered with axonal transport, leading to BRP-positive axonal aggregates that also contain additional active zone scaffold proteins. Axonal aggregates formed only in the presence of non-phosphorylatable BRP isoforms containing the SRPK79D-targeted N-terminal stretch. We assume that specific active zone proteins are pre-assembled in transport packages and are thus co-transported as functional scaffold building blocks. Our results suggest that transient post-translational modification of a discrete unstructured domain of the master scaffold component BRP blocks oligomerization of these building blocks during their long-range transport., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

PSD-95 MAGUK family scaffold proteins are multi-domain organisers of synaptic transmission that contain three PDZ domains followed by an SH3-GK domain tandem. This domain architecture allows coordinated assembly of protein complexes composed of neurotransmitter receptors, synaptic adhesion molecules and downstream signalling effectors. Here we show that binding of monomeric CRIPT-derived PDZ 3 ligands to the third PDZ domain of PSD-95 induces functional changes in the intramolecular SH3-GK domain assembly that influence subsequent homotypic and heterotypic complex formation. We identify PSD-95 interactors that differentially bind to the SH3-GK domain tandem depending on its conformational state. Among these interactors, we further establish the heterotrimeric G protein subunit Gnb5 as a PSD-95 complex partner at dendritic spines of rat hippocampal neurons. The PSD-95 GK domain binds to Gnb5, and this interaction is triggered by CRIPT-derived PDZ 3 ligands binding to the third PDZ domain of PSD-95, unraveling a hierarchical binding mechanism of PSD-95 complex formation., Competing Interests: NR, BK, SK, MW, CF, SS No competing interests declared, (© 2019, Rademacher et al.)

Intestinal release of dietary triglycerides via chylomicrons is the major contributor to elevated postprandial triglyceride levels. Dietary lipids can be transiently stored in cytosolic lipid droplets (LDs) located in intestinal enterocytes for later release. ADP ribosylation factor-related protein 1 (ARFRP1) participates in processes of LD growth in adipocytes and in lipidation of lipoproteins in liver and intestine. This study aims to explore the impact of ARFRP1 on LD organization and its interplay with chylomicron-mediated triglyceride release in intestinal-like Caco-2 cells. Suppression of Arfrp1 reduced release of intracellularly derived triglycerides (0.69-fold) and increased the abundance of transitional endoplasmic reticulum ATPase TERA/VCP, fatty acid synthase-associated factor 2 (FAF2) and perilipin 2 (Plin2) at the LD surface. Furthermore, TERA/VCP and FAF2 co-occurred more frequently with ATGL at LDs, suggesting a reduced adipocyte triglyceride lipase (ATGL)-mediated lipolysis. Accordingly, inhibition of lipolysis reduced lipid release from intracellular storage pools by the same magnitude as Arfrp1 depletion. Thus, the lack of Arfrp1 increases the abundance of lipolysis-modulating enzymes TERA/VCP, FAF2 and Plin2 at LDs, which might decrease lipolysis and reduce availability of fatty acids for triglyceride synthesis and their release via chylomicrons., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim: We analyzed the protein corona of thermoresponsive, poly(N-isopropylacrylamide)- or poly(N-isopropylmethacrylamide)-based nanogels., Materials & Methods: Traces of protein corona detected after incubation in human serum were characterized by proteomics and dynamic light scattering in undiluted serum., Results: Apolipoprotein B-100 and albumin were the main components of the protein coronae. For dendritic polyglycerol-poly(N-isopropylacrylamide) nanogels at 37°C, an increase in adsorbed immunoglobulin light chains was detected, followed by partially reversible nanogel aggregation. All nanogels in their hydrophilic state are colloidally stable in serum and bear a dysopsonin-rich protein corona., Conclusion: We observed strong changes in NG stability upon slight alterations in the composition of the protein coronae according to nanogel solvation state. Nanogels in their hydrophilic state possess safe protein coronae.

The major histocompatibility complex of class II (MHCII) immunopeptidome represents the repertoire of antigenic peptides with the potential to activate CD4 + T cells. An understanding of how the relative abundance of specific antigenic epitopes affects the outcome of T cell responses is an important aspect of adaptive immunity and offers a venue to more rationally tailor T cell activation in the context of disease. Recent advances in mass spectrometric instrumentation, computational power, labeling strategies, and software analysis have enabled an increasing number of stratified studies on HLA ligandomes, in the context of both basic and translational research. A key challenge in the case of MHCII immunopeptidomes, often determined for different samples at distinct conditions, is to derive quantitative information on consensus epitopes from antigenic peptides of variable lengths. Here, we present the design and benchmarking of a new algorithm [peptide landscape antigenic epitope alignment utility (PLAtEAU)] allowing the identification and label-free quantification (LFQ) of shared consensus epitopes arising from series of nested peptides. The algorithm simplifies the complexity of the dataset while allowing the identification of nested peptides within relatively short segments of protein sequences. Moreover, we apply this algorithm to the comparison of the ligandomes of cell lines with two different expression levels of the peptide-exchange catalyst HLA-DM. Direct comparison of LFQ intensities determined at the peptide level is inconclusive, as most of the peptides are not significantly enriched due to poor sampling. Applying the PLAtEAU algorithm for grouping of the peptides into consensus epitopes shows that more than half of the total number of epitopes is preferentially and significantly enriched for each condition. This simplification and deconvolution of the complex and ambiguous peptide-level dataset highlights the value of the PLAtEAU algorithm in facilitating robust and accessible quantitative analysis of immunopeptidomes across cellular contexts. In silico analysis of the peptides enriched for each HLA-DM expression conditions suggests a higher affinity of the pool of peptides isolated from the high DM expression samples. Interestingly, our analysis reveals that while for certain autoimmune-relevant epitopes their presentation increases upon DM expression others are clearly edited out from the peptidome.

ATAD2 (ANCCA) is an epigenetic regulator and transcriptional cofactor, whose overexpression has been linked to the progress of various cancer types. Here, we report a DNA-encoded library screen leading to the discovery of BAY-850, a potent and isoform selective inhibitor that specifically induces ATAD2 bromodomain dimerization and prevents interactions with acetylated histones in vitro, as well as with chromatin in cells. These features qualify BAY-850 as a chemical probe to explore ATAD2 biology.

Fragment-based screening presents a promising alternative to high-throughput screening and has gained great attention in recent years. So far, only a few studies have discussed mass spectrometry as a screening technology for fragments. Herein, we report the application of native electrospray ionization mass spectrometry (MS) for screening defined sets of fragments against four different target proteins. Fragments were selected from a primary screening conducted with a thermal shift assay (TSA) and represented different binding categories. Our data indicated that, beside specific complex formation, many fragments show extensive multiple binding and also charge-state shifts. Both of these factors complicate automated data analysis and decrease the attractiveness of native MS as a primary screening tool for fragments. A comparison of the hits identified by native MS and TSA showed good agreement for two of the proteins. Furthermore, we discuss general challenges, including the determination of an optimal fragment concentration and the question of how to rank fragment hits according to their affinity. In conclusion, we consider native MS to be a highly valuable tool for the validation and deeper investigation of promising fragment hits rather than a method for primary screening., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)