Publications by authors named "Bernd Wollscheid"

100 Publications

Elucidation of host-virus surfaceome interactions using spatial proteotyping.

Adv Virus Res 2021 12;109:105-134. Epub 2021 Apr 12.

Department of Health Sciences and Technology (D-HEST), ETH Zurich, Institute of Translational Medicine (ITM), Zurich, Switzerland; Swiss Institute of Bioinformatics (SIB), Lausanne, Switzerland. Electronic address:

The cellular surfaceome and its residing extracellularly exposed proteins are involved in a multitude of molecular signaling processes across the viral infection cycle. Successful viral propagation, including viral entry, immune evasion, virion release and viral spread rely on dynamic molecular interactions with the surfaceome. Decoding of these viral-host surfaceome interactions using advanced technologies enabled the discovery of fundamental new functional insights into cellular and viral biology. In this review, we highlight recently developed experimental strategies, with a focus on spatial proteotyping technologies, aiding in the rational design of theranostic strategies to combat viral infections.
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http://dx.doi.org/10.1016/bs.aivir.2021.03.002DOI Listing
April 2021

PCprophet: a framework for protein complex prediction and differential analysis using proteomic data.

Nat Methods 2021 May 15;18(5):520-527. Epub 2021 Apr 15.

Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland.

Despite the availability of methods for analyzing protein complexes, systematic analysis of complexes under multiple conditions remains challenging. Approaches based on biochemical fractionation of intact, native complexes and correlation of protein profiles have shown promise. However, most approaches for interpreting cofractionation datasets to yield complex composition and rearrangements between samples depend considerably on protein-protein interaction inference. We introduce PCprophet, a toolkit built on size exclusion chromatography-sequential window acquisition of all theoretical mass spectrometry (SEC-SWATH-MS) data to predict protein complexes and characterize their changes across experimental conditions. We demonstrate improved performance of PCprophet over state-of-the-art approaches and introduce a Bayesian approach to analyze altered protein-protein interactions across conditions. We provide both command-line and graphical interfaces to support the application of PCprophet to any cofractionation MS dataset, independent of separation or quantitative liquid chromatography-MS workflow, for the detection and quantitative tracking of protein complexes and their physiological dynamics.
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http://dx.doi.org/10.1038/s41592-021-01107-5DOI Listing
May 2021

Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen.

Nat Commun 2021 03 16;12(1):1693. Epub 2021 Mar 16.

Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland.

Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in a mass spectrometer and a custom data analysis pipeline. The method is significantly faster, cheaper, technically less demanding, easy to multiplex and produces accurate protease fingerprints. Using the blood cascade proteases as a case study, we obtain protease substrate profiles that can be used to map specificity, cleavage entropy and allosteric effects and to design protease probes. The data further show that protease substrate predictions enable the selection of potential physiological substrates for targeted validation in biochemical assays.
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http://dx.doi.org/10.1038/s41467-021-21754-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7966775PMC
March 2021

CD20 as a gatekeeper of the resting state of human B cells.

Proc Natl Acad Sci U S A 2021 Feb;118(7)

Biology III, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany;

CD20 is a B cell-specific membrane protein and represents an attractive target for therapeutic antibodies. Despite widespread usage of anti-CD20 antibodies for B cell depletion therapies, the biological function of their target remains unclear. Here, we demonstrate that CD20 controls the nanoscale organization of receptors on the surface of resting B lymphocytes. CRISPR/Cas9-mediated ablation of CD20 in resting B cells resulted in relocalization and interaction of the IgM-class B cell antigen receptor with the coreceptor CD19. This receptor rearrangement led to a transient activation of B cells, accompanied by the internalization of many B cell surface marker proteins. Reexpression of CD20 restored the expression of the B cell surface proteins and the resting state of Ramos B cells. Similarly, treatment of Ramos or naive human B cells with the anti-CD20 antibody rituximab induced nanoscale receptor rearrangements and transient B cell activation in vitro and in vivo. A departure from the resting B cell state followed by the loss of B cell identity of CD20-deficient Ramos B cells was accompanied by a PAX5 to BLIMP-1 transcriptional switch, metabolic reprogramming toward oxidative phosphorylation, and a shift toward plasma cell development. Thus, anti-CD20 engagement or the loss of CD20 disrupts membrane organization, profoundly altering the fate of human B cells.
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http://dx.doi.org/10.1073/pnas.2021342118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896350PMC
February 2021

Alterations in Are Associated with Cisplatin Resistance through Inhibition of Apoptosis in Malignant Pleural Mesothelioma.

Clin Cancer Res 2021 Apr 5;27(8):2277-2291. Epub 2021 Feb 5.

Institute of Pathology and Molecular Pathology, University Hospital Zurich, Zurich, Switzerland.

Purpose: The clinical standard treatment for patients with malignant pleural mesothelioma (MPM) includes a cisplatin-based chemotherapy, leading to reduction of tumor size in only a minority of patients. Predicting response to chemotherapy in patients with MPM by using a genetic marker would, therefore, enable patient stratification.

Experimental Design: In this retrospective biomarker study, eligible patients had resectable MPM, measurable disease, and available primary MPM tissue. All patients underwent first-line treatment with cisplatin and pemetrexed, followed by surgery. Thorough molecular analysis was performed (whole-exome and targeted deep sequencing, and copy-number analyses), and also mechanistic data (viability assays, Western blots, and immunoprecipitation) using mesothelioma cell lines with and without siRNA-mediated BRCA1-associated protein 1 (BAP1) knockdown were provided.

Results: In a training cohort of patients with MPM ( = 28), mutations or deletions of each predicted resistance to chemotherapy in patients with primary MPM. The negative predictive value of loss in patients with MPM was confirmed by amplicon sequencing and copy-number array technology in an independent test cohort ( = 39). Preliminary mechanistic studies using siRNA-based knockdown of BAP1 in MPM cell culture models along with immunoprecipitation assays confirmed chemoresistance , possibly through inhibition of apoptosis and transcriptional regulation of the BAP1/HCF1/E2F1 axis.

Conclusions: Alterations in in MPM were a negative predictor for response to chemotherapy and could possibly be used as a companion biomarker for treatment decision.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-4037DOI Listing
April 2021

The Tumor Profiler Study: integrated, multi-omic, functional tumor profiling for clinical decision support.

Cancer Cell 2021 Mar 21;39(3):288-293. Epub 2021 Jan 21.

Roche Pharmaceutical Research and Early Development, Roche Innovation Center Zurich, Wagistrasse 10, 8952 Schlieren, Switzerland.

The application and integration of molecular profiling technologies create novel opportunities for personalized medicine. Here, we introduce the Tumor Profiler Study, an observational trial combining a prospective diagnostic approach to assess the relevance of in-depth tumor profiling to support clinical decision-making with an exploratory approach to improve the biological understanding of the disease.
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http://dx.doi.org/10.1016/j.ccell.2021.01.004DOI Listing
March 2021

Enzymatic Dissociation Induces Transcriptional and Proteotype Bias in Brain Cell Populations.

Int J Mol Sci 2020 Oct 26;21(21). Epub 2020 Oct 26.

Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, CH-8057 Zurich, Switzerland.

Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37 °C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4 °C. These findings emphasize the risk of introducing technical biases and biological artifacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.
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http://dx.doi.org/10.3390/ijms21217944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663484PMC
October 2020

Standardization and harmonization of distributed multi-center proteotype analysis supporting precision medicine studies.

Nat Commun 2020 10 16;11(1):5248. Epub 2020 Oct 16.

School of Life and Environmental Science, The University of Sydney, NSW, 2006, Sydney, Australia.

Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwide is necessary to gain statistically significant clinical insights for the benefit of patients. Here we conceived and standardized a proteotype data generation and analysis workflow enabling distributed data generation and evaluated the quantitative data generated across laboratories of the international Cancer Moonshot consortium. Using harmonized mass spectrometry (MS) instrument platforms and standardized data acquisition procedures, we demonstrate robust, sensitive, and reproducible data generation across eleven international sites on seven consecutive days in a 24/7 operation mode. The data presented from the high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) workflow shows that coordinated proteotype data acquisition is feasible from clinical specimens using such standardized strategies. This work paves the way for the distributed multi-omic digitization of large clinical specimen cohorts across multiple sites as a prerequisite for turning molecular precision medicine into reality.
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http://dx.doi.org/10.1038/s41467-020-18904-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568553PMC
October 2020

Surfaceome dynamics reveal proteostasis-independent reorganization of neuronal surface proteins during development and synaptic plasticity.

Nat Commun 2020 10 5;11(1):4990. Epub 2020 Oct 5.

Neuroscience Center Zurich, Zurich, Switzerland.

Neurons are highly compartmentalized cells with tightly controlled subcellular protein organization. While brain transcriptome, connectome and global proteome maps are being generated, system-wide analysis of temporal protein dynamics at the subcellular level are currently lacking. Here, we perform a temporally-resolved surfaceome analysis of primary neuron cultures and reveal dynamic surface protein clusters that reflect the functional requirements during distinct stages of neuronal development. Direct comparison of surface and total protein pools during development and homeostatic synaptic scaling demonstrates system-wide proteostasis-independent remodeling of the neuronal surface, illustrating widespread regulation on the level of surface trafficking. Finally, quantitative analysis of the neuronal surface during chemical long-term potentiation (cLTP) reveals fast externalization of diverse classes of surface proteins beyond the AMPA receptor, providing avenues to investigate the requirement of exocytosis for LTP. Our resource (neurosurfaceome.ethz.ch) highlights the importance of subcellular resolution for systems-level understanding of cellular processes.
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http://dx.doi.org/10.1038/s41467-020-18494-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536423PMC
October 2020

MassIVE.quant: a community resource of quantitative mass spectrometry-based proteomics datasets.

Nat Methods 2020 10 14;17(10):981-984. Epub 2020 Sep 14.

Khoury College of Computer Sciences, Northeastern University, Boston, MA, USA.

MassIVE.quant is a repository infrastructure and data resource for reproducible quantitative mass spectrometry-based proteomics, which is compatible with all mass spectrometry data acquisition types and computational analysis tools. A branch structure enables MassIVE.quant to systematically store raw experimental data, metadata of the experimental design, scripts of the quantitative analysis workflow, intermediate input and output files, as well as alternative reanalyses of the same dataset.
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http://dx.doi.org/10.1038/s41592-020-0955-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541731PMC
October 2020

Cell-Derived Vesicles as TRPC1 Channel Delivery Systems for the Recovery of Cellular Respiratory and Proliferative Capacities.

Adv Biosyst 2020 11 2;4(11):e2000146. Epub 2020 Sep 2.

Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, MD6, 14 Medical Drive, Singapore, 117599, Singapore.

Pulsed electromagnetic fields (PEMFs) are capable of specifically activating a TRPC1-mitochondrial axis underlying cell expansion and mitohormetic survival adaptations. This study characterizes cell-derived vesicles (CDVs) generated from C2C12 murine myoblasts and shows that they are equipped with the sufficient molecular machinery to confer mitochondrial respiratory capacity and associated proliferative responses upon their fusion with recipient cells. CDVs derived from wild type C2C12 myoblasts include the cation-permeable transient receptor potential (TRP) channels, TRPC1 and TRPA1, and directly respond to PEMF exposure with TRPC1-mediated calcium entry. By contrast, CDVs derived from C2C12 muscle cells in which TRPC1 has been genetically knocked-down using CRISPR/Cas9 genome editing, do not. Wild type C2C12-derived CDVs are also capable of restoring PEMF-induced proliferative and mitochondrial activation in two C2C12-derived TRPC1 knockdown clonal cell lines in accordance to their endogenous degree of TRPC1 suppression. C2C12 wild type CDVs respond to menthol with calcium entry and accumulation, likewise verifying TRPA1 functional gating and further corroborating compartmental integrity. Proteomic and lipidomic analyses confirm the surface membrane origin of the CDVs providing an initial indication of the minimal cellular machinery required to recover mitochondrial function. CDVs hence possess the potential of restoring respiratory and proliferative capacities to senescent cells and tissues.
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http://dx.doi.org/10.1002/adbi.202000146DOI Listing
November 2020

The hematopoietic stem cell marker VNN2 is associated with chemoresistance in pediatric B-cell precursor ALL.

Blood Adv 2020 09;4(17):4052-4064

Department of Stem Cell Transplantation, University Children's Hospital Zurich, Zurich, Switzerland; and.

Most relapses of acute lymphoblastic leukemia (ALL) occur in patients with a medium risk (MR) for relapse on the Associazione Italiana di Ematologia e Oncologia Pediatrica and Berlin-Frankfurt-Münster (AIEOP-BFM) ALL protocol, based on persistence of minimal residual disease (MRD). New insights into biological features that are associated with MRD are needed. Here, we identify the glycosylphosphatidylinositol-anchored cell surface protein vanin-2 (VNN2; GPI-80) by charting the cell surface proteome of MRD very high-risk (HR) B-cell precursor (BCP) ALL using a chemoproteomics strategy. The correlation between VNN2 transcript and surface protein expression enabled a retrospective analysis (ALL-BFM 2000; N = 770 cases) using quantitative polymerase chain reaction to confirm the association of VNN2 with MRD and independent prediction of worse outcome. Using flow cytometry, we detected VNN2 expression in 2 waves, in human adult bone marrow stem and progenitor cells and in the mature myeloid compartment, in line with proposed roles for fetal hematopoietic stem cells and inflammation. Prospective validation by flow cytometry in the ongoing clinical trial (AIEOP-BFM 2009) identified 10% (103/1069) of VNN2+ BCP ALL patients at first diagnosis, primarily in the MRD MR (48/103, 47%) and HR (37/103, 36%) groups, across various cytogenetic subtypes. We also detected frequent mutations in epigenetic regulators in VNN2+ ALLs, including histone H3 methyltransferases MLL2, SETD2, and EZH2 and demethylase KDM6A. Inactivation of the VNN2 gene did not impair leukemia repopulation capacity in xenografts. Taken together, VNN2 marks a cellular state of increased resistance to chemotherapy that warrants further investigations. Therefore, this marker should be included in diagnostic flow cytometry panels.
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http://dx.doi.org/10.1182/bloodadvances.2019000938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7479947PMC
September 2020

ESRRG and PERM1 Govern Mitochondrial Conversion in Brite/Beige Adipocyte Formation.

Front Endocrinol (Lausanne) 2020 12;11:387. Epub 2020 Jun 12.

Institute of Food, Nutrition and Health, Department of Health Sciences and Technology (D-HEST), ETH Zürich, Zurich, Switzerland.

When exposed to cold temperatures, mice increase their thermogenic capacity by an expansion of brown adipose tissue mass and the formation of brite/beige adipocytes in white adipose tissue depots. However, the process of the transcriptional changes underlying the conversion of a phenotypic white to brite/beige adipocytes is only poorly understood. By analyzing transcriptome profiles of inguinal adipocytes during cold exposure and in mouse models with a different propensity to form brite/beige adipocytes, we identified ESRRG and PERM1 as modulators of this process. The production of heat by mitochondrial uncoupled respiration is a key feature of brite/beige compared to white adipocytes and we show here that both candidates are involved in PGC1α transcriptional network to positively regulate mitochondrial capacity. Moreover, we show that an increased expression of ESRRG or PERM1 supports the formation of brown or brite/beige adipocytes and . These results reveal that ESRRG and PERM1 are early induced in and important regulators of brite/beige adipocyte formation.
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http://dx.doi.org/10.3389/fendo.2020.00387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7304443PMC
June 2020

An adverse outcome pathway-based approach to assess steatotic mixture effects of hepatotoxic pesticides in vitro.

Food Chem Toxicol 2020 May 19;139:111283. Epub 2020 Mar 19.

German Federal Institute for Risk Assessment, Dept. Food Safety, Berlin, Germany.

Exposure to complex chemical mixtures requires a tiered strategy for efficient mixture risk assessment. As a part of the EuroMix project we developed an adverse outcome pathway (AOP)-based assay toolbox to investigate the combined effects of the liver steatosis-inducing compounds imazalil, thiacloprid, and clothianidin in human HepaRG hepatocarcinoma cells. Compound-specific relative potency factors were determined using a benchmark dose approach. Equipotent mixtures were tested for nuclear receptor activation, gene and protein expression, and triglyceride accumulation, according to the molecular initiating events and key events proposed in the steatosis AOP. All three compounds affected the activity of nuclear receptors, but not key genes/proteins as proposed. Triglyceride accumulation was observed with three different methods. Mixture effects were in agreement with the assumption of dose additivity for all the combinations and endpoints tested. Compound-specific RPFs remained similar over the different endpoints studied downstream the AOP. Therefore, it might be possible to reduce testing to a smaller battery of key tests. The results demonstrate the suitability of our in vitro assay toolbox, integrated within an AOP framework and combined with the RPF approach, for the analysis of steatotic effects of chemical mixtures. However, mRNA results suggest that the steatosis AOP still needs improvement.
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http://dx.doi.org/10.1016/j.fct.2020.111283DOI Listing
May 2020

A Proteogenomic Resource Enabling Integrated Analysis of Genotype-Proteotype-Phenotype Relationships.

J Proteome Res 2020 04 6;19(4):1647-1662. Epub 2020 Mar 6.

Department of Health Sciences and Technology (D-HEST), ETH Zürich, 8092 Zürich, Switzerland.

is an opportunistic foodborne pathogen responsible for listeriosis, a potentially fatal foodborne disease. Many different strains and serotypes exist, but a proteogenomic resource that bridges the gap in our molecular understanding of the relationships between the genotypes and phenotypes via proteotypes is still missing. Here, we devised a next-generation proteogenomics strategy that enables the community to rapidly proteotype strains and relate this information back to the genotype. Based on sequencing and assembly of the two most commonly used model strains, EGD-e and ScottA, we established two comprehensive proteogenomic databases. A genome comparison established core- and strain-specific genes potentially responsible for virulence differences. Next, we established a DIA/SWATH-based proteotyping strategy, including a new and robust sample preparation workflow, that enables the reproducible, sensitive, and relative quantitative measurement of proteotypes. This reusable and publicly available DIA/SWATH library covers 70% of open reading frames of and represents the most extensive spectral library for proteotype analysis to date. We used these two new resources to investigate the proteotype in states mimicking the upper gastrointestinal passage. Exposure of to bile salts at 37 °C, which simulates conditions encountered in the duodenum, showed significant proteotype perturbations including an increase of FlaA, the structural protein of flagella. Given that is known to lose its flagella above 30 °C, this was an unexpected finding. The formation of flagella, which might have implications on infectivity, was validated by parallel reaction monitoring and light and scanning electron microscopy. transcript levels did not change significantly upon exposure to bile salts at 37 °C, suggesting regulation at the post-transcriptional level. Together, these analyses provide a comprehensive proteogenomic resource and toolbox for the community enabling the analysis of genotype-proteotype-phenotype relationships.
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http://dx.doi.org/10.1021/acs.jproteome.9b00842DOI Listing
April 2020

Classification of mouse B cell types using surfaceome proteotype maps.

Nat Commun 2019 12 16;10(1):5734. Epub 2019 Dec 16.

Biomedical Proteomics Platform, Department of Health Sciences and Technology, ETH Zurich, 8093, Zurich, Switzerland.

System-wide quantification of the cell surface proteotype and identification of extracellular glycosylation sites is challenging when samples are limited. Here, we miniaturize and automate the previously described Cell Surface Capture (CSC) technology, increasing sensitivity, reproducibility and throughput. We use this technology, which we call autoCSC, to create population-specific surfaceome maps of developing mouse B cells and use targeted flow cytometry to uncover developmental cell subpopulations.
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http://dx.doi.org/10.1038/s41467-019-13418-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6915781PMC
December 2019

Structure-function relationships of HDL in diabetes and coronary heart disease.

JCI Insight 2020 01 16;5(1). Epub 2020 Jan 16.

Institute of Clinical Chemistry, University of Zurich and University Hospital of Zurich, Zurich, Switzerland.

High-density lipoproteins (HDL) contain hundreds of lipid species and proteins and exert many potentially vasoprotective and antidiabetogenic activities on cells. To resolve structure-function-disease relationships of HDL, we characterized HDL of 51 healthy subjects and 98 patients with diabetes (T2DM), coronary heart disease (CHD), or both for protein and lipid composition, as well as functionality in 5 cell types. The integration of 40 clinical characteristics, 34 nuclear magnetic resonance (NMR) features, 182 proteins, 227 lipid species, and 12 functional read-outs by high-dimensional statistical modeling revealed, first, that CHD and T2DM are associated with different changes of HDL in size distribution, protein and lipid composition, and function. Second, different cellular functions of HDL are weakly correlated with each other and determined by different structural components. Cholesterol efflux capacity (CEC) was no proxy of other functions. Third, 3 potentially novel determinants of HDL function were identified and validated by the use of artificially reconstituted HDL, namely the sphingadienine-based sphingomyelin SM 42:3 and glycosylphosphatidylinositol-phospholipase D1 for the ability of HDL to inhibit starvation-induced apoptosis of human aortic endothelial cells and apolipoprotein F for the ability of HDL to promote maximal respiration of brown adipocytes.
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http://dx.doi.org/10.1172/jci.insight.131491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7030825PMC
January 2020

Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

Nature 2019 12 23;576(7787):452-458. Epub 2019 Oct 23.

Polyphor AG, Allschwil, Switzerland.

There is an urgent need for new antibiotics against Gram-negative pathogens that are resistant to carbapenem and third-generation cephalosporins, against which antibiotics of last resort have lost most of their efficacy. Here we describe a class of synthetic antibiotics inspired by scaffolds derived from natural products. These chimeric antibiotics contain a β-hairpin peptide macrocycle linked to the macrocycle found in the polymyxin and colistin family of natural products. They are bactericidal and have a mechanism of action that involves binding to both lipopolysaccharide and the main component (BamA) of the β-barrel folding complex (BAM) that is required for the folding and insertion of β-barrel proteins into the outer membrane of Gram-negative bacteria. Extensively optimized derivatives show potent activity against multidrug-resistant pathogens, including all of the Gram-negative members of the ESKAPE pathogens. These derivatives also show favourable drug properties and overcome colistin resistance, both in vitro and in vivo. The lead candidate is currently in preclinical toxicology studies that-if successful-will allow progress into clinical studies that have the potential to address life-threatening infections by the Gram-negative pathogens, and thus to resolve a considerable unmet medical need.
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http://dx.doi.org/10.1038/s41586-019-1665-6DOI Listing
December 2019

Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlB-mediated invasion.

PLoS Pathog 2019 10 7;15(10):e1008032. Epub 2019 Oct 7.

Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland.

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen.
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http://dx.doi.org/10.1371/journal.ppat.1008032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779246PMC
October 2019

In vitro quantification of botulinum neurotoxin type A1 using immobilized nerve cell-mimicking nanoreactors in a microfluidic platform.

Analyst 2019 Sep;144(19):5755-5765

Institute for Food, Nutrition and Health, ETH Zürich, Zurich, Switzerland.

The bacterial toxin botulinum neurotoxin A (BoNT/A) is not only an extremely toxic substance but also a potent pharmaceutical compound that is used in a wide spectrum of neurological disorders and cosmetic applications. The quantification of the toxin is extremely challenging due to its extraordinary high physiological potency and is further complicated by the toxin's three key functionalities that are necessary for its activity: receptor binding, internalization-translocation, and catalytic activity. So far, the industrial standard to measure the active toxin has been the mouse bioassay (MBA) that is considered today as outdated due to ethical issues. Therefore, recent introductions of cell-based assays were highly anticipated; their impact however remains limited due to their labor-intensive implementation. This report describes a new in vitro approach that combines a nanosensor based on the use of nerve cell-mimicking nanoreactors (NMN) with microfluidic technology. The nanosensor was able to measure all three key functionalities, and therefore suitable to quantify the amount of physiologically active BoNT/A. The integration of such a sensor in a microfluidic device allowed the detection and quantification of BoNT/A amounts in a much shorter time than the MBA (<10 h vs. 2-4 days). Lastly, the system was also able to reliably quantify physiologically active BoNT/A within a simple final pharmaceutical formulation. This complete in vitro testing system and its unique combination of a highly sensitive nanosensor and microfluidic technology represent a significant ethical advancement over in vivo measures and a possible alternative to cell-based in vitro detection methods.
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http://dx.doi.org/10.1039/c9an00817aDOI Listing
September 2019

Antibiotic Discovery with Synthetic Fermentation: Library Assembly, Phenotypic Screening, and Mechanism of Action of β-Peptides Targeting Penicillin-Binding Proteins.

ACS Chem Biol 2019 05 24;14(5):1030-1040. Epub 2019 Apr 24.

Laboratorium für Organische Chemie, Department of Chemistry and Applied Biosciences , ETH-Zürich , 8093 Zürich , Switzerland.

In analogy to biosynthetic pathways leading to bioactive natural products, synthetic fermentation generates mixtures of molecules from simple building blocks under aqueous, biocompatible conditions, allowing the resulting cultures to be directly screened for biological activity. In this work, a novel β-peptide antibiotic was successfully identified using the synthetic fermentation platform. Phenotypic screening was carried out in an initially random fashion, allowing simple identification of active cultures. Subsequent deconvolution, focused screening, and structure-activity relationship studies led to the identification of a potent antimicrobial peptide, showing strong selectivity for our model system Bacillus subtilis over human HEK293 cells. To determine the antibacterial mechanism of action, a peptide probe bearing a photoaffinity tag was readily synthesized through the use of appropriate synthetic fermentation building blocks and utilized for target identification using a quantitative mass spectrometry-based strategy. The chemoproteomic approach led to the identification of a number of bacterial membrane proteins as prospective targets. These findings were validated through binding affinity studies with penicillin-binding protein 4 using microscale thermophoresis, with the bioactive peptide showing a dissociation constant ( K) in the nanomolar range. Through these efforts, we provide a proof of concept for the synthetic fermentation approach presented here as a new strategy for the phenotypic discovery of novel bioactive compounds.
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http://dx.doi.org/10.1021/acschembio.9b00227DOI Listing
May 2019

Listeriolysin O-dependent host surfaceome remodeling modulates Listeria monocytogenes invasion.

Pathog Dis 2018 11 1;76(8). Epub 2018 Nov 1.

Institut Pasteur, Unité des Interactions Bactéries Cellules, Paris F-75015, France.

Listeria monocytogenes is a pathogenic bacterium that invades epithelial cells by activating host signaling cascades, which promote bacterial engulfment within a phagosome. The pore-forming toxin listeriolysin O (LLO), which is required for bacteria phagosomal escape, has also been associated with the activation of several signaling pathways when secreted by extracellular bacteria, including Ca2+ influx and promotion of L. monocytogenes entry. Quantitative host surfaceome analysis revealed significant quantitative remodeling of a defined set of cell surface glycoproteins upon LLO treatment, including a subset previously identified to play a role in the L. monocytogenes infection process. Our data further shows that the lysosomal-associated membrane proteins LAMP-1 and LAMP-2 are translocated to the cellular surface and those LLO-induced Ca2+ fluxes are required to trigger the surface relocalization of LAMP-1. Finally, we identify late endosomes/lysosomes as the major donor compartments of LAMP-1 upon LLO treatment and by perturbing their function, we suggest that these organelles participate in L. monocytogenes invasion.
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http://dx.doi.org/10.1093/femspd/fty082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282100PMC
November 2018

Thanatin targets the intermembrane protein complex required for lipopolysaccharide transport in .

Sci Adv 2018 11 14;4(11):eaau2634. Epub 2018 Nov 14.

Chemistry Department, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

With the increasing resistance of many Gram-negative bacteria to existing classes of antibiotics, identifying new paradigms in antimicrobial discovery is an important research priority. Of special interest are the proteins required for the biogenesis of the asymmetric Gram-negative bacterial outer membrane (OM). Seven Lpt proteins (LptA to LptG) associate in most Gram-negative bacteria to form a macromolecular complex spanning the entire envelope, which transports lipopolysaccharide (LPS) molecules from their site of assembly at the inner membrane to the cell surface, powered by adenosine 5'-triphosphate hydrolysis in the cytoplasm. The periplasmic protein LptA comprises the protein bridge across the periplasm, which connects LptBFGC at the inner membrane to LptD/E anchored in the OM. We show here that the naturally occurring, insect-derived antimicrobial peptide thanatin targets LptA and LptD in the network of periplasmic protein-protein interactions required to assemble the Lpt complex, leading to the inhibition of LPS transport and OM biogenesis in .
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http://dx.doi.org/10.1126/sciadv.aau2634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235536PMC
November 2018

The in silico human surfaceome.

Proc Natl Acad Sci U S A 2018 11 29;115(46):E10988-E10997. Epub 2018 Oct 29.

Institute of Molecular Systems Biology at the Department of Biology, ETH Zurich, 8093 Zurich, Switzerland;

Cell-surface proteins are of great biomedical importance, as demonstrated by the fact that 66% of approved human drugs listed in the DrugBank database target a cell-surface protein. Despite this biomedical relevance, there has been no comprehensive assessment of the human surfaceome, and only a fraction of the predicted 5,000 human transmembrane proteins have been shown to be located at the plasma membrane. To enable analysis of the human surfaceome, we developed the surfaceome predictor SURFY, based on machine learning. As a training set, we used experimentally verified high-confidence cell-surface proteins from the Cell Surface Protein Atlas (CSPA) and trained a random forest classifier on 131 features per protein and, specifically, per topological domain. SURFY was used to predict a human surfaceome of 2,886 proteins with an accuracy of 93.5%, which shows excellent overlap with known cell-surface protein classes (i.e., receptors). In deposited mRNA data, we found that between 543 and 1,100 surfaceome genes were expressed in cancer cell lines and maximally 1,700 surfaceome genes were expressed in embryonic stem cells and derivative lines. Thus, the surfaceome diversity depends on cell type and appears to be more dynamic than the nonsurface proteome. To make the predicted surfaceome readily accessible to the research community, we provide visualization tools for intuitive interrogation (wlab.ethz.ch/surfaceome). The in silico surfaceome enables the filtering of data generated by multiomics screens and supports the elucidation of the surfaceome nanoscale organization.
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http://dx.doi.org/10.1073/pnas.1808790115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6243280PMC
November 2018

Surfaceome nanoscale organization and extracellular interaction networks.

Curr Opin Chem Biol 2019 02 9;48:26-33. Epub 2018 Oct 9.

Institute of Molecular Systems Biology & Department of Health Sciences and Technology, ETH Zürich, Zürich, Switzerland. Electronic address:

The reductionist view of 'one target-one drug' has fueled the development of therapeutic agents to treat human disease. However, many compounds that have efficacy in vitro are inactive in complex in vivo systems. It has become clear that a molecular understanding of signaling networks is needed to address disease phenotypes in the human body. Protein signaling networks function at the molecular level through information transfer via protein-protein interactions. Cell surface exposed proteins, termed the surfaceome, are the gatekeepers between the intra- and extracellular signaling networks, translating extracellular cues into intracellular responses and vice versa. As 66% of drugs in the DrugBank target the surfaceome, these proteins are a key source for potential diagnostic and therapeutic agents. In this review article, we will discuss current knowledge about the spatial organization and molecular interactions of the surfaceome and provide a perspective on the technologies available for studying the extracellular surfaceome interaction network.
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http://dx.doi.org/10.1016/j.cbpa.2018.09.020DOI Listing
February 2019

Adverse Outcome Pathway-Driven Analysis of Liver Steatosis in Vitro: A Case Study with Cyproconazole.

Chem Res Toxicol 2018 08 11;31(8):784-798. Epub 2018 Jul 11.

Department Food Safety , German Federal Institute for Risk Assessment , 10589 Berlin , Germany.

Adverse outcome pathways (AOPs) describe causal relationships between molecular perturbation and adverse cellular effects and are being increasingly adopted for linking in vitro mechanistic toxicology to in vivo data from regulatory toxicity studies. In this work, a case study was performed by developing a bioassay toolbox to assess key events in the recently proposed AOP for chemically induced liver steatosis. The toolbox is comprised of in vitro assays to measure nuclear receptor activation, gene and protein expression, lipid accumulation, mitochondrial respiration, and formation of fatty liver cells. Assay evaluation was performed in human HepaRG hepatocarcinoma cells exposed to the model compound cyproconazole, a fungicide inducing steatosis in rodents. Cyproconazole dose-dependently activated RARα and PXR, two molecular initiating events in the steatosis AOP. Moreover, cyproconazole provoked a disruption of mitochondrial functions and induced triglyceride accumulation and the formation of fatty liver cells as described in the AOP. Gene and protein expression analysis, however, showed expression changes different from those proposed in the AOP, thus suggesting that the current version of the AOP might not fully reflect the complex mechanisms linking nuclear receptor activation and liver steatosis. Our study shows that cyproconazole induces steatosis in human liver cells in vitro and demonstrates the utility of systems-based approaches in the mechanistic assessment of molecular and cellular key events in an AOP. AOP-driven in vitro testing as demonstrated can further improve existing AOPs, provide insight regarding molecular mechanisms of toxicity, and inform predictive risk assessment.
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http://dx.doi.org/10.1021/acs.chemrestox.8b00112DOI Listing
August 2018

Leukocyte Differentiation by Histidine-Rich Glycoprotein/Stanniocalcin-2 Complex Regulates Murine Glioma Growth through Modulation of Antitumor Immunity.

Mol Cancer Ther 2018 09 26;17(9):1961-1972. Epub 2018 Jun 26.

Uppsala University, Department of Immunology, Genetics and Pathology, Science for Life Laboratory, The Rudbeck Laboratory, Uppsala, Sweden.

The plasma-protein histidine-rich glycoprotein (HRG) is implicated in phenotypic switching of tumor-associated macrophages, regulating cytokine production and phagocytotic activity, thereby promoting vessel normalization and antitumor immune responses. To assess the therapeutic effect of HRG gene delivery on CNS tumors, we used adenovirus-encoded HRG to treat mouse intracranial GL261 glioma. Delivery of Ad5-HRG to the tumor site resulted in a significant reduction in glioma growth, associated with increased vessel perfusion and increased CD45 leukocyte and CD8 T-cell accumulation in the tumor. Antibody-mediated neutralization of colony-stimulating factor-1 suppressed the effects of HRG on CD45 and CD8 infiltration. Using a novel protein interaction-decoding technology, TRICEPS-based ligand receptor capture (LRC), we identified Stanniocalcin-2 (STC2) as an interacting partner of HRG on the surface of inflammatory cells and colocalization of HRG and STC2 in gliomas. HRG reduced the suppressive effects of STC2 on monocyte CD14 differentiation and STC2-regulated immune response pathways. In consequence, Ad5-HRG-treated gliomas displayed decreased numbers of IL35 Treg cells, providing a mechanistic rationale for the reduction in GL261 growth in response to Ad5-HRG delivery. We conclude that HRG suppresses glioma growth by modulating tumor inflammation through monocyte infiltration and differentiation. Moreover, HRG acts to balance the regulatory effects of its partner, STC2, on inflammation and innate and/or acquired immunity. HRG gene delivery therefore offers a potential therapeutic strategy to control antitumor immunity. .
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0097DOI Listing
September 2018

HATRIC-based identification of receptors for orphan ligands.

Nat Commun 2018 04 17;9(1):1519. Epub 2018 Apr 17.

Department of Health Sciences and Technology & Institute of Molecular Systems Biology & BioMedical Proteomics Platform (BMPP), ETH Zurich, Zurich, Switzerland.

Cellular responses depend on the interactions of extracellular ligands, such as nutrients, growth factors, or drugs, with specific cell-surface receptors. The sensitivity of these interactions to non-physiological conditions, however, makes them challenging to study using in vitro assays. Here we present HATRIC-based ligand receptor capture (HATRIC-LRC), a chemoproteomic technology that successfully identifies target receptors for orphan ligands on living cells ranging from small molecules to intact viruses. HATRIC-LRC combines a click chemistry-based, protein-centric workflow with a water-soluble catalyst to capture ligand-receptor interactions at physiological pH from as few as 1 million cells. We show HATRIC-LRC utility for general antibody target validation within the native nanoscale organization of the surfaceome, as well as receptor identification for a small molecule ligand. HATRIC-LRC further enables the identification of complex extracellular interactomes, such as the host receptor panel for influenza A virus (IAV), the causative agent of the common flu.
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http://dx.doi.org/10.1038/s41467-018-03936-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904110PMC
April 2018