Central resource facility for (quantitative) proteomics applications of RNA-virus infected cells

/in , /von

Research areas: Proteomics, PTM-analysis

Viral infection of target cells results in multiple alterations at the level of cellular signaling, transcription and translation to enable viral replication and to fight off cellular antiviral responses. Viral proteins interact with specific subsets of host cell proteins and are post-translationally modified by host cell enzymes. In turn, the virus infection affects expression rates of host cell proteins and their modification status either directly or indirectly. Changes at the level of the proteome precede or are a (direct) consequence of transcriptome changes. Additionally, both events can occur uncoupled. Thus, understanding proteome changes in relation to transcriptome alterations is instrumental to develop a holistic view on virus/cell interactions. The central aim of the Z03 project is to enable CRC1021 projects to investigate these aspects at a proteome-wide level quantitatively and with high resolution. To achieve this goal, the expertise from the group of M.Kracht in application of proteomics techniques to RNA virus biology will be combined with the expertise of U.Linne, who is heading a high-end mass spectrometry facility at the Faculty of Chemistry, Marburg. The facility is fully equipped with state of the art mass spectrometers including Orbitraps Velos Pro and XL (Thermo Scientific) and a Synapt G2Si, all connected to nanoHPLCs. Z03 will provide standardized work flows to study (i) protein-protein interactions, (ii) protein expression levels and (iii) post-translational modifications (PTM) of proteins. Additionally, mass spectrometric standard methods for quality control, amino acid sequence and mass determination of purified proteins will be provided to all members of CRC1021. These approaches can be used to study the interaction of viral components with host cells proteins or between host cell proteins including identification of proteins in samples derived from specific tagging strategies or the usage of cell-permeable crosslinkers  . Quantitative changes of expression across entire proteomes will be analyzed using stable isotope labelling strategies or label-free quantification methods. Phosphorylated, ubiquitylated or acetylated peptides will be enriched from denatured lysates by antibodies recognizing K-ε-G-G motifs or acetylated lysines or by immobilized metal ion affinity chromatography (IMAC) and will be identified by LC-MS/MS. A specific further aim of the Z03 project is to provide sophisticated analysis work flows for the resulting large data sets. This includes summary tables, detailed statistics and visualizations of modified and regulated residues mapped to peptides and genes as well as identification of consensus motifs and de novo motif searches for regulated subgroups of peptides. A work flow that has already been established in the R biostatistics environment by Dr. Axel Weber in the Kracht group (C02) will enable identification of statistically enriched components of KEGG or GO pathways, pathway mapping, protein network analyses and publication-ready visualizations using Cytoscape, STRING and several additional bioinformatics tools.

Project-related publications of the investigators:

  • Weiterer SS, Meier-Soelch J, Georgomanolis T, Mizi A, Beyerlein A, Weiser H, Brant L, Mayr-Buro C, Jurida L, Beuerlein K, Muller H, Weber A, Tenekeci U, Dittrich-Breiholz O, Bartkuhn M, Nist A, Stiewe T, van IWF, Riedlinger T, Schmitz ML, Papantonis A, Kracht M. 2020. Distinct IL-1alpha-responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner. EMBO J 39:e101533.
  • Aznaourova M, Janga H, Sefried S, Kaufmann A, Dorna J, Volkers SM, Georg P, Lechner M, Hoppe J, Dokel S, Schmerer N, Gruber AD, Linne U, Bauer S, Sander LE, Schmeck B, Schulte LN. 2020. Noncoding RNA MaIL1 is an integral component of the TLR4-TRIF pathway. Proc Natl Acad Sci U S A 117:9042-9053.
  • Weber A, Dam S, Saul VV, Kuznetsova I, Muller C, Fritz-Wolf K, Becker K, Linne U, Gu H, Stokes MP, Pleschka S, Kracht M, Schmitz ML. 2019. Phosphoproteome Analysis of Cells Infected with Adapted and Nonadapted Influenza A Virus Reveals Novel Pro- and Antiviral Signaling Networks. J Virol 93.
  • Franz-Badur S, Penner A, Strass S, von Horsten S, Linne U, Essen LO. 2019. Structural changes within the bifunctional cryptochrome/photolyase CraCRY upon blue light excitation. Sci Rep 9:9896.
  • Bruhl J, Trautwein J, Schafer A, Linne U, Bouazoune K. 2019. The DNA repair protein SHPRH is a nucleosome-stimulated ATPase and a nucleosome-E3 ubiquitin ligase. Epigenetics Chromatin 12:52.
  • Robledo M, Schluter JP, Loehr LO, Linne U, Albaum SP, Jimenez-Zurdo JI, Becker A. 2018. An sRNA and Cold Shock Protein Homolog-Based Feedforward Loop Post-transcriptionally Controls Cell Cycle Master Regulator CtrA. Front Microbiol 9:763.
  • Poppe M, Wittig S, Jurida L, Bartkuhn M, Wilhelm J, Muller H, Beuerlein K, Karl N, Bhuju S, Ziebuhr J, Schmitz ML, Kracht M. 2017. The NF-kappaB-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229E-infected cells. PLoS Pathog 13:e1006286.
  • Tenekeci U, Poppe M, Beuerlein K, Buro C, Muller H, Weiser H, Kettner-Buhrow D, Porada K, Newel D, Xu M, Chen ZJ, Busch J, Schmitz ML, Kracht M. 2016. K63-Ubiquitylation and TRAF6 Pathways Regulate Mammalian P-Body Formation and mRNA Decapping. Mol Cell 62:943-957.

Regulation of RNA metabolism, translation and protein degradation pathways in the host response to coronavirus infection

/in /von

Research areas: Signal Transduction, Molecular Virology

In this project, we aim to develop an integrative (and comprehensive) molecular view on the human coronavirus (CoV) host response and to achieve deep mechanistic insights into the (coordinated) changes affecting host cell gene expression during viral replication.  A specific overall goal is to define protein kinase modules that control transcriptional and post-transcriptional / translational levels of gene regulation. In the last funding period, by focusing on HCoV-229E, we systematically studied the host response to CoV at the signaling, chromatin and mRNA levels. We found that HCoV-229E causes an unconventional, attenuated activation of the NF-κB system, profoundly reshapes the genome-wide enhancer landscape in the nucleus of infected cells and strongly induces mRNAs encoding a set of transcription factors (TFs), inflammatory mediators and genes downstream of ER stress signaling involved in the unfolded protein response. However, genes upregulated at the mRNA level were often differentially expressed at the protein level. A combination of genetic and pharmacological loss of function experiments revealed a prominent activation of the PERK / IRE1α protein kinase systems by CoV and has led to the discovery of a potent antiviral effect of the ER stress trigger thapsigargin. Thapsigargin inhibits replication of HCoV-229E, MERS-CoV and SARS-CoV-2 in the lower nanomolar range and proteomic data suggest the involvement of ER-associated degradation (ERAD), autophagy and specific parts of the ubiquitin proteasome system in this effect. Together, these findings have resulted in multiple new insights into the CoV – host interactions, both at the level of individual molecules but also globally, that will be exploited in the third funding period. Part (A) of the work program defines four specific goals to identify (i) the chromatin-based mechanisms and genomic targets of the TFs ATF3, KLF6, ANKRD1 and c-JUN, (ii) the CoV-specific activation mechanisms, interactomes and functions of PERK and IRE1α, (iii) their roles in selective translation and (iv) the host cell factors required for CoV replication by unbiased genome-wide loss of function screens. Part (B) aims at identifying the molecular basis for the thapsigargin antiviral effects by focussing on (i) core ERAD components, (ii) factors involved in selective autophagy and (iii) candidate E1 and E2 enzymes, proteasome targeting factors (e.g. ubiquilins) and the role of the ubiquitin proteasome system in degrading viral components. In part (C) a final goal is to integrate the different levels of mechanistic, genome- and proteome-wide data into a (deep) holistic molecular view of the CoV host response also taking into account publically available data sets with the long-term aim to identify key nodes of signaling that distinguish the highly pathogenic from adapted human CoV and that are amenable to therapeutic intervention.

Project-related publications of the investigator:

  • Shaban MS, Muller C, Mayr-Buro C, Weiser H, Meier-Soelch J, Albert BV, Weber A, Linne U, Hain T, Babayev I, Karl N, Hofmann N, Becker S, Herold S, Schmitz ML, Ziebuhr J, Kracht M. 2021. Multi-level inhibition of coronavirus replication by chemical ER stress. Nat Commun 12:5536.
  • Meier-Soelch J, Mayr-Buro C, Juli J, Leib L, Linne U, Dreute J, Papantonis A, Schmitz ML, Kracht M. 2021. Monitoring the Levels of Cellular NF-kappaB Activation States. Cancers (Basel) 13.
  • Zarnack K, Balasubramanian S, Gantier MP, Kunetsky V, Kracht M, Schmitz ML, Strasser K. 2020. Dynamic mRNP Remodeling in Response to Internal and External Stimuli. Biomolecules 10.
  • Kracht M, Muller-Ladner U, Schmitz ML. 2020. Mutual regulation of metabolic processes and proinflammatory NF-kappaB signaling. J Allergy Clin Immunol 146:694-705.
  • Weber A, Dam S, Saul VV, Kuznetsova I, Muller C, Fritz-Wolf K, Becker K, Linne U, Gu H, Stokes MP, Pleschka S, Kracht M, Schmitz ML. 2019. Phosphoproteome Analysis of Cells Infected with Adapted and Nonadapted Influenza A Virus Reveals Novel Pro- and Antiviral Signaling Networks. J Virol 93.
  • Mayr-Buro C, Schlereth E, Beuerlein K, Tenekeci U, Meier-Soelch J, Schmitz ML, Kracht M. 2019. Single-Cell Analysis of Multiple Steps of Dynamic NF-kappaB Regulation in Interleukin-1alpha-Triggered Tumor Cells Using Proximity Ligation Assays. Cancers (Basel) 11.
  • Schmitz ML, Shaban MS, Albert BV, Gokcen A, Kracht M. 2018. The Crosstalk of Endoplasmic Reticulum (ER) Stress Pathways with NF-kappaB: Complex Mechanisms Relevant for Cancer, Inflammation and Infection. Biomedicines 6.
  • Meier-Soelch J, Jurida L, Weber A, Newel D, Kim J, Braun T, Schmitz ML, Kracht M. 2018. RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes. Front Immunol 9:775.
  • Poppe M, Wittig S, Jurida L, Bartkuhn M, Wilhelm J, Muller H, Beuerlein K, Karl N, Bhuju S, Ziebuhr J, Schmitz ML, Kracht M. 2017. The NF-kappaB-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229E-infected cells. PLoS Pathog 13:e1006286.