PTMs in Disease and Infection
Understanding the molecular processes of life requires an understanding of proteins: Almost every biological process involves these biomolecules. It is therefore not surprising that detailed studies of proteins at the molecular level have provided fundamental insights into human biochemistry and cell biology.
Our group is interested in studying the biochemistry and structure of proteins under the influence of interaction partners. In particular, the contribution of posttranslational modifications to the regulation of protein activity is a central research topic in our laboratory.
In this respect, it is interesting to consider the influence of pathogenic bacteria on the function and activity of human proteins: During evolution, these pathogens have developed fascinating mechanisms to manipulate infected human cells by manipulating the activity of key proteins, e.g. by applying posttranslational modifications. The study of the molecular basis of such manipulation processes enables us to identify and describe strategies of pathogens.
PTMs in the context of disease and infection
In the course of evolution, several bacterial pathogens have developed sophisticated strategies to escape their elimination by the human immune system. For this purpose, they release a multitude of bacterial proteins during an infection process, which interfere with essential intracellular processes in the host cell and thus guarantee the survival of the intruder. Our goal is to understand the underlying mechanisms of manipulation of human proteins using selected bacterial factors in molecular detail. For this purpose, we isolate the desired bacterial proteins in high purity, so that we can subsequently study their biochemical and functional properties using biophysical and structural biology methods.
A current focus of our work is the investigation of post-translational modifications (PTMs) that occur in the context of bacterial pathogens. Post-translational modifications are chemical changes in proteins caused by enzymes. These transformations significantly influence the activity and functionality of the modified proteins. Therefore, many pathogens release enzymes that selectively and specifically modify central factors of human cells to give the pathogen an advantage.
Of particular interest for us among the post-translational modifications is the so-called AMPylation of human proteins. Many bacterial pathogens inject enzymes into host cells that use the generally available adenosine triphosphate (ATP) to link target proteins with an adenosine monophosphate (AMP). We now know that the AMP-transmitting enzymes are present in many bacterial pathogens, but their target proteins cannot be predicted.
Therefore, a central topic of our work is the development of methods to identify AMP-modified proteins. For this purpose, we use a spectrum of biochemical, chemical, mass spectrometric and immunological methods, which allow the targeted enrichment and analysis of AMPylated molecules. But also other PTMs (phosphocholination, phosphorylation, proteolysis) are subject of our research.
In addition, we want to understand the biochemical, functional and structural consequences of PTMs in the context of bacterial infections in molecular detail. The introduction and analysis of PTMs (e.g. AMPylations) are technically challenging and require extensive knowledge of the biochemistry and function of the respective proteins and enzymes. A core expertise of our group is therefore the generation of proteins, the introduction of PTMs, and the comprehensive characterization of these molecules. Our research approach enables us to identify targets of bacteria and to study their possible cellular consequences.
Technical Expertise
- Expression and production of purified proteins
- Biophysical characterization of proteins
- Development of enzymatic test methods
Methods available in the institute
- Molecular biology for pro- and eukaryotic systems
- Recombinant protein expression
- Production of pure proteins by chromatographic methods on a multi-milligram scale (affinity and size exclusion chromatography, chromatography systems, proteolytic digestion)
- Mass spectrometric analysis of recombinant proteins
- Biophysical characterization of proteins and protein interactions (fluorescence spectrometry, fluorescence anisotropy, fluorescence titration, isothermal titration calorimetry, thermophoresis, biolayer interferometry)
- Protein crystallization and structure determination by X-ray crystallography.
- Interaction analysis of proteins using yeast 2-hybrid approaches, analytical size exclusion chromatography, affinity studies
- Immunological detection techniques (e.g. Western blotting)
- Establishment of enzyme kinetics (based on fluorescence methods, mass spectrometry, quantifying Western blot and chromatographic methods)
- Eukaryotic cell culture (Hela, CHO, THP1) in combination with fluorescence microscopy
- Establishment of new detection and enrichment methods for post-translational modifications (generation and application of specific antibodies, application of new chemical concepts)
- Stabilization, preparation and characterization of low affinity protein complexes
Publications
2024
Pronucleotide Probes Reveal a Diverging Specificity for AMPylation vs UMPylation of Human and Bacterial Nucleotide Transferases
Mostert D, Bubeneck W, Rauh T, Kielkowski P, Itzen A, Jung K, Sieber S
BIOCHEMISTRY-US. 2024;63(5):651-659.
2023
The Alarmone Diadenosine Tetraphosphate as a Cosubstrate for Protein AMPylation
Frese M, Saumer P, Yuan Y, Herzog D, Höpfner D, Itzen A, Marx A
ANGEW CHEM INT EDIT. 2023;62(8):e202213279.
The DNA-binding induced (de)AMPylation activity of a Coxiella burnetii Fic enzyme targets Histone H3
Höpfner D, Cichy A, Pogenberg V, Krisp C, Mezouar S, Bach N, Grotheer J, Zarza S, Martinez E, Bonazzi M, Feige M, Sieber S, Schlüter H, Itzen A
COMMUN BIOL. 2023;6(1):1124.
Dephosphocholination by Legionella effector Lem3 functions through remodelling of the switch II region of Rab1b
Kaspers M, Pogenberg V, Pett C, Ernst S, Ecker F, Ochtrop P, Groll M, Hedberg C, Itzen A
NAT COMMUN. 2023;14(1):2245.
2022
Revisiting AMPylation through the lens of Fic enzymes
Gulen B, Itzen A
TRENDS MICROBIOL. 2022;30(4):350-363.
2021
Rab1-AMPylation by Legionella DrrA is allosterically activated by Rab1
Du J, Wrisberg M, Gulen B, Stahl M, Pett C, Hedberg C, Lang K, Schneider S, Itzen A
NAT COMMUN. 2021;12(1):.
Specificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD
Fauser J, Gulen B, Pogenberg V, Pett C, Pourjafar-Dehkordi D, Krisp C, Höpfner D, König G, Schlüter H, Feige M, Zacharias M, Hedberg C, Itzen A
NAT COMMUN. 2021;12(1):.
Current Advances in Covalent Stabilization of Macromolecular Complexes for Structural Biology
Fauser J, Itzen A, Gulen B
BIOCONJUGATE CHEM. 2021;32(5):879-890.
Erratum: Monoclonal Anti-AMP Antibodies Are Sensitive and Valuable Tools for Detecting Patterns of AMPylation
Höpfner D, Fauser J, Kaspers M, Pett C, Hedberg C, Itzen A
ISCIENCE. 2021;24(7):102731.
SopD from Salmonella specifically inactivates Rab8
Savitskiy S, Itzen A
BBA-PROTEINS PROTEOM. 2021;1869(8):140661.
Proteolysis of Rab32 by Salmonella GtgE induces an inactive GTPase conformation
Savitskiy S, Wachtel R, Pourjafar-Dehkordi D, Kang H, Trauschke V, Lamb D, Sattler M, Zacharias M, Itzen A
ISCIENCE. 2021;24(1):.
2020
Conformational control of small GTPases by AMPylation
Barthelmes K, Ramcke E, Kang H, Sattler M, Itzen A
P NATL ACAD SCI USA. 2020;117(11):5772-5781.
The trimer to monomer transition of Tumor Necrosis Factor-Alpha is a dynamic process that is significantly altered by therapeutic antibodies
Daub H, Traxler L, Ismajli F, Groitl B, Itzen A, Rant U
SCI REP-UK. 2020;10(1):9265.
Legionella effector AnkX displaces the switch II region for Rab1b phosphocholination
Ernst S, Ecker F, Kaspers M, Ochtrop P, Hedberg C, Groll M, Itzen A
SCI ADV. 2020;6(20):eaaz8041.
Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture
Gulen B, Rosselin M, Fauser J, Albers M, Pett C, Krisp C, Pogenberg V, Schlüter H, Hedberg C, Itzen A
NAT CHEM. 2020;12(8):732-739.
Monoclonal Anti-AMP Antibodies Are Sensitive and Valuable Tools for Detecting Patterns of AMPylation
Höpfner D, Fauser J, Kaspers M, Pett C, Hedberg C, Itzen A
ISCIENCE. 2020;23(12):.
Validation of the Slow Off-Kinetics of Sirtuin-Rearranging Ligands (SirReals) by Means of Label-Free Electrically Switchable Nanolever Technology
Schiedel M, Daub H, Itzen A, Jung M
CHEMBIOCHEM. 2020;21(8):1161-1166.
Divergent Evolution of Legionella RCC1 Repeat Effectors Defines the Range of Ran GTPase Cycle Targets
Swart A, Steiner B, Gomez-Valero L, Schütz S, Hannemann M, Janning P, Irminger M, Rothmeier E, Buchrieser C, Itzen A, Panse V, Hilbi H
MBIO. 2020;11(2):.
PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2 mediated phosphorylation at Threonine72
Vieweg S, Mulholland K, Brauning B, Kacharia N, Lai Y, Toth R, Singh P, Volpi I, Sattler M, Groll M, Itzen A, Muqit M
BIOCHEM J. 2020;477(9):1651-1668.
2019
Phosphorylation of Ser111 in Rab8a Modulates Rabin8-Dependent Activation by Perturbation of Side Chain Interaction Networks
Pourjafar-Dehkordi D, Vieweg S, Itzen A, Zacharias M
BIOCHEMISTRY-US. 2019;58(33):3546-3554.
Nucleotide exchange factor Rab3GEP requires DENN and non-DENN elements for activation and targeting of Rab27a
Sanzà P, Evans R, Briggs D, Cantero M, Montoliu L, Patel S, Sviderskaya E, Itzen A, Figueiredo A, Seabra M, Hume A
J CELL SCI. 2019;132(9):.
2018
The protease GtgE from Salmonella exclusively targets inactive Rab GTPases
Wachtel R, Bräuning B, Mader S, Ecker F, Kaila V, Groll M, Itzen A
NAT COMMUN. 2018;9(1):44.
2017
Proximity-Triggered Covalent Stabilization of Low-Affinity Protein Complexes In Vitro and In Vivo
Cigler M, Müller T, Horn-Ghetko D, von Wrisberg M, Fottner M, Goody R, Itzen A, Lang K
ANGEW CHEM INT EDIT. 2017;56(49):15737-15741.
2016
A pull-down procedure for the identification of unknown GEFs for small GTPases
Koch D, Rai A, Ali I, Bleimling N, Friese T, Brockmeyer A, Janning P, Goud B, Itzen A, Goody R
Small GTPases. 2016;7(2):93-106.
Adenylylation of Tyr77 stabilizes Rab1b GTPase in an active state: A molecular dynamics simulation analysis
Luitz M, Bomblies R, Ramcke E, Itzen A, Zacharias M
SCI REP-UK. 2016;6:19896.
bMERB domains are bivalent Rab8 family effectors evolved by gene duplication
Rai A, Oprisko A, Campos J, Fu Y, Friese T, Itzen A, Goody R, Gazdag E
ELIFE. 2016;5:.
2015
Molecular perspectives on protein adenylylation
Hedberg C, Itzen A
ACS CHEM BIOL. 2015;10(1):12-21.
Covalent Protein Labeling by Enzymatic Phosphocholination
Heller K, Ochtrop P, Albers M, Zauner F, Itzen A, Hedberg C
ANGEW CHEM INT EDIT. 2015;54(35):10327-30.
Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1
Lai Y, Kondapalli C, Lehneck R, Procter J, Dill B, Woodroof H, Gourlay R, Peggie M, Macartney T, Corti O, Corvol J, Campbell D, Itzen A, Trost M, Muqit M
EMBO J. 2015;34(22):2840-61.
Locking GTPases covalently in their functional states
Wiegandt D, Vieweg S, Hofmann F, Koch D, Li F, Wu Y, Itzen A, Goody R
NAT COMMUN. 2015;6:7773.
2014
Exploring adenylylation and phosphocholination as post-translational modifications
Albers M, Itzen A, Hedberg C
CHEMBIOCHEM. 2014;15(1):19-26.
The Legionella longbeachae Icm/Dot substrate SidC selectively binds phosphatidylinositol 4-phosphate with nanomolar affinity and promotes pathogen vacuole-endoplasmic reticulum interactions
Dolinsky S, Haneburger I, Cichy A, Hannemann M, Itzen A, Hilbi H
INFECT IMMUN. 2014;82(10):4021-33.
Reaction mechanism of adenylyltransferase DrrA from Legionella pneumophila elucidated by time-resolved fourier transform infrared spectroscopy
Gavriljuk K, Schartner J, Itzen A, Goody R, Gerwert K, Kötting C
J AM CHEM SOC. 2014;136(26):9338-45.
The structure of the N-terminal domain of the Legionella protein SidC
Gazdag E, Schöbel S, Shkumatov A, Goody R, Itzen A
J STRUCT BIOL. 2014;186(1):188-94.
Diversity and plasticity in Rab GTPase nucleotide release mechanism has consequences for Rab activation and inactivation
Langemeyer L, Nunes Bastos R, Cai Y, Itzen A, Reinisch K, Barr F
ELIFE. 2014;3:e01623.
The role of the hypervariable C-terminal domain in Rab GTPases membrane targeting
Li F, Yi L, Zhao L, Itzen A, Goody R, Wu Y
P NATL ACAD SCI USA. 2014;111(7):2572-7.
Direct targeting of Rab-GTPase-effector interactions
Spiegel J, Cromm P, Itzen A, Goody R, Grossmann T, Waldmann H
ANGEW CHEM INT EDIT. 2014;53(9):2498-503.
α-Synuclein interacts with the switch region of Rab8a in a Ser129 phosphorylation-dependent manner
Yin G, Lopes da Fonseca T, Eisbach S, Anduaga A, Breda C, Orcellet M, Szegő É, Guerreiro P, Lázaro D, Braus G, Fernandez C, Griesinger C, Becker S, Goody R, Itzen A, Giorgini F, Outeiro T, Zweckstetter M
NEUROBIOL DIS. 2014;70:149-61.
2013
RabGEFs are a major determinant for specific Rab membrane targeting
Blümer J, Rey J, Dehmelt L, Mazel T, Wu Y, Bastiaens P, Goody R, Itzen A
J CELL BIOL. 2013;200(3):287-300.
Membrane extraction of Rab proteins by GDP dissociation inhibitor characterized using attenuated total reflection infrared spectroscopy
Gavriljuk K, Itzen A, Goody R, Gerwert K, Kötting C
P NATL ACAD SCI USA. 2013;110(33):13380-5.
Protein-DNA arrays as tools for detection of protein-protein interactions by mass spectrometry
Gogolin L, Schroeder H, Itzen A, Goody R, Niemeyer C, Becker C
CHEMBIOCHEM. 2013;14(1):92-9.
Modulation of small GTPases by Legionella
Goody R, Itzen A
CURR TOP MICROBIOL. 2013;376:117-33.
Intermediates in the guanine nucleotide exchange reaction of Rab8 protein catalyzed by guanine nucleotide exchange factors Rabin8 and GRAB
Guo Z, Hou X, Goody R, Itzen A
J BIOL CHEM. 2013;288(45):32466-74.
Mechanism of Rab1b deactivation by the Legionella pneumophila GAP LepB
Mihai Gazdag E, Streller A, Haneburger I, Hilbi H, Vetter I, Goody R, Itzen A
EMBO REP. 2013;14(2):199-205.
Activation of Ran GTPase by a Legionella effector promotes microtubule polymerization, pathogen vacuole motility and infection
Rothmeier E, Pfaffinger G, Hoffmann C, Harrison C, Grabmayr H, Repnik U, Hannemann M, Wölke S, Bausch A, Griffiths G, Müller-Taubenberger A, Itzen A, Hilbi H
PLOS PATHOG. 2013;9(9):e1003598.
2012
Specific localization of Rabs at intracellular membranes
Blümer J, Wu Y, Goody R, Itzen A
BIOCHEM SOC T. 2012;40(6):1421-5.
Catalytic mechanism of a mammalian Rab·RabGAP complex in atomic detail
Gavriljuk K, Gazdag E, Itzen A, Kötting C, Goody R, Gerwert K
P NATL ACAD SCI USA. 2012;109(52):21348-53.
Reversible phosphocholination of Rab proteins by Legionella pneumophila effector proteins
Goody P, Heller K, Oesterlin L, Itzen A, Goody R
EMBO J. 2012;31(7):1774-84.
Crystal structure of the Rab binding domain of OCRL1 in complex with Rab8 and functional implications of the OCRL1/Rab8 module for Lowe syndrome
Hagemann N, Hou X, Goody R, Itzen A, Erdmann K
Small GTPases. 2012;3(2):107-10.
Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor
Oesterlin L, Goody R, Itzen A
P NATL ACAD SCI USA. 2012;109(15):5621-6.
Characterization of enzymes from Legionella pneumophila involved in reversible adenylylation of Rab1 protein
Shkumatov A, Oesterlin L, Schoebel S, Goody P, Goody R, Itzen A
J BIOL CHEM. 2012;287(42):35036-46.
2011
The versatile Legionella effector protein DrrA
Goody R, Schoebel S, Oesterlin L, Blümer J, Peters H, Blankenfeldt W, Itzen A
Commun Integr Biol. 2011;4(1):72-4.
A structural basis for Lowe syndrome caused by mutations in the Rab-binding domain of OCRL1
Hou X, Hagemann N, Schoebel S, Blankenfeldt W, Goody R, Erdmann K, Itzen A
EMBO J. 2011;30(8):1659-70.
Adenylylation: renaissance of a forgotten post-translational modification
Itzen A, Blankenfeldt W, Goody R
TRENDS BIOCHEM SCI. 2011;36(4):221-8.
Covalent coercion by Legionella pneumophila
Itzen A, Goody R
CELL HOST MICROBE. 2011;10(2):89-91.
GTPases involved in vesicular trafficking: structures and mechanisms
Itzen A, Goody R
SEMIN CELL DEV BIOL. 2011;22(1):48-56.
Rab GTPase-Myo5B complexes control membrane recycling and epithelial polarization
Roland J, Bryant D, Datta A, Itzen A, Mostov K, Goldenring J
P NATL ACAD SCI USA. 2011;108(7):2789-94.
Protein LidA from Legionella is a Rab GTPase supereffector
Schoebel S, Cichy A, Goody R, Itzen A
P NATL ACAD SCI USA. 2011;108(44):17945-50.
Efficient synthesis and applications of peptides containing adenylylated tyrosine residues
Smit C, Blümer J, Eerland M, Albers M, Goody R, Itzen A, Hedberg C
ANGEW CHEM INT EDIT. 2011;50(39):9200-4.
Identification and characterisation of novel Mss4-binding Rab GTPases
Wixler V, Wixler L, Altenfeld A, Ludwig S, Goody R, Itzen A
BIOL CHEM. 2011;392(3):239-48.
One-pot dual-labeling of a protein by two chemoselective reactions
Yi L, Sun H, Itzen A, Triola G, Waldmann H, Goody R, Wu Y
ANGEW CHEM INT EDIT. 2011;50(36):8287-90.
Atomic resolution structure of EhpR: phenazine resistance in Enterobacter agglomerans Eh1087 follows principles of bleomycin/mitomycin C resistance in other bacteria
Yu S, Vit A, Devenish S, Mahanty H, Itzen A, Goody R, Blankenfeldt W
BMC STRUCT BIOL. 2011;11:33.
2010
The Legionella effector protein DrrA AMPylates the membrane traffic regulator Rab1b
Peters H, Blümer J, Blankenfeldt W, Goody R, Itzen A
SCIENCE. 2010;329(5994):946-9.
High-affinity binding of phosphatidylinositol 4-phosphate by Legionella pneumophila DrrA
Schoebel S, Blankenfeldt W, Goody R, Itzen A
EMBO REP. 2010;11(8):598-604.
2009
Chaperone-assisted production of active human Rab8A GTPase in Escherichia coli
Bleimling N, Alexandrov K, Goody R, Itzen A
PROTEIN EXPRES PURIF. 2009;65(2):190-5.
RabGDI displacement by DrrA from Legionella is a consequence of its guanine nucleotide exchange activity
Schoebel S, Oesterlin L, Blankenfeldt W, Goody R, Itzen A
MOL CELL. 2009;36(6):1060-72.
2008
Key determinants of Rab specificity
Itzen A, Goody R
STRUCTURE. 2008;16(10):1437-9.
2007
Sec2 is a highly efficient exchange factor for the Rab protein Sec4
Itzen A, Rak A, Goody R
J MOL BIOL. 2007;365(5):1359-67.
2006
Purification, crystallization and preliminary X-ray crystallographic analysis of mammalian MSS4-Rab8 GTPase protein complex
Itzen A, Bleimling N, Ignatev A, Pylypenko O, Rak A
ACTA CRYSTALLOGR F. 2006;62(Pt 2):113-6.
Nucleotide exchange via local protein unfolding--structure of Rab8 in complex with MSS4
Itzen A, Pylypenko O, Goody R, Alexandrov K, Rak A
EMBO J. 2006;25(7):1445-55.
Letzte Aktualisierung aus dem FIS: 21.12.2024 - 03:13 Uhr