Research area: 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:
- 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(13). pii: e00528-19. doi: 10.1128
- 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. 2019. Distinct IL-1alpha-responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner. EMBO J 39(1):e101533. doi: 10.15252.
- 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. Frontiers in Immunology 9:775. doi: 10.3389.
- 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(2). pii: E58. doi: 10.3390.
- 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(3):e1006286. doi: 10.1371.
- Brühl J, Trautwein J, Schäfer 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(1):52. doi:10.1186.
- Robledo M, Schlüter JP, Loehr LO, Linne U, Albaum SP, Jiménez-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. doi:10.3389.
- Nickel AI, Wäber NB, Gößringer M, Lechner M, Linne U, Toth U, Rossmanith W, Hartmann RK 2017. Minimal and RNA-free RNase P in Aquifex aeolicus. Proc Natl Acad Sci U S A. 114(42):11121–11126. doi:10.1073.
- Hajipour MJ, Ghasemi F, Aghaverdi H, Raoufi M, Linne U, Atyabi F, Nabipour I, Azhdarzadeh M, Derakhshankhah H, Lotfabadi A, Bargahi A, Alekhamis Z, Aghaie A, Hashemi E, Tafakhori A, Aghamollaii V, Mashhadi MM, Sheibani S, Vani H, Mahmoudi M 2017. Sensing of Alzheimer's Disease and Multiple Sclerosis Using Nano-Bio Interfaces. J Alzheimers Dis. 59(4):1187–1202. doi:10.3233.
- Bertram K, Valcu CM, Weitnauer M, Linne U, Görlach A. 2015. NOX1 supports the metabolic remodeling of HepG2 cells. PLoS One. 10(3):e0122002. doi:10.1371.