| 1. |
- Albers, Michael Franz, 1987-
(författare)
-
Synthesis and investigation of bacterial effector molecules
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During infections, bacterial microorganisms initiate profound interactions with mammalian host cells. Usually defense mechanisms of the host destroy intruding bacteria in rapid manner. However, many bacterial pathogens have evolved in a way to avoid these mechanisms. By use of effector molecules, which can be small organic molecules or proteins with enzymatic activity, the host is manipulated on a molecular level. Effectors mediating post-translational modifications (PTMs) are employed by many pathogens to influence the biological activity of host proteins. In the presented thesis, two related PTMs are investigated in detail: Adenylylation, the covalent transfer of an adenosine monophosphate group from adenosine triphosphate onto proteins, and phosphocholination, the covalent transfer of a phosphocholine moiety onto proteins. Over the past years, enzymes mediating these modifications have been discovered in several pathogens, especially as a mechanism to influence the signaling of eukaryotic cells by adenylylating or phosphocholinating small GTPases. However, the development of reliable methods for the isolation and identification of adenylylated and phosphocholinated proteins remains a vehement challenge in this field of research. This thesis presents general procedures for the synthesis of peptides carrying adenylylated or phosphocholinated tyrosine, threonine and serine residues. From the resulting peptides, mono-selective polyclonal antibodies against adenylylated tyrosine and threonine have been raised. The antibodies were used as tools for proteomic research to isolate unknown substrates of adenylyl transferases from eukaryotic cells. Mass spectrometric fragmentation techniques have been investigated to ease the identification of adenylylated proteins. Furthermore, this work presents a new strategy to identify adenylylated proteins. Additionally, small effector molecules are involved in the regulation of infection mechanisms. In this work, the small molecule LAI-1 (Legionella autoinducer 1) from the pathogen Legionella pneumophila, the causative agent of the Legionnaire’s disease, was synthesised together with its amino-derivatives. LAI-1 showed are a clear pharmacological effect on the regulation of the life cycle of L. pneumophila, initiating transmissive traits like motility and virulence. Furthermore, LAI-1 was shown to have an effect on eukaryotic cells as well. Directed motility of the eukaryotic cells was significantly reduced and the cytoskeletal architecture was reorganised, probably by interfering with the small GTPase Cdc42.
|
|
| 2. |
- Du, Jiqing, et al.
(författare)
-
Rab1-AMPylation by Legionella DrrA is allosterically activated by Rab1
- 2021
-
Ingår i: Nature Communications. - : Nature Research. - 2041-1723. ; 12:1
-
Tidskriftsartikel (refereegranskat)abstract
- Legionella pneumophila infects eukaryotic cells by forming a replicative organelle – the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.
|
|
| 3. |
- Fauser, Joel, et al.
(författare)
-
Specificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD
- 2021
-
Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 12:1
-
Tidskriftsartikel (refereegranskat)abstract
- To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.
|
|
| 4. |
- Gulen, Burak, et al.
(författare)
-
Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture
- 2020
-
Ingår i: Nature Chemistry. - : Nature Publishing Group. - 1755-4330 .- 1755-4349. ; 12:8, s. 732-739
-
Tidskriftsartikel (refereegranskat)abstract
- Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme–nucleotide–substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.
|
|
| 5. |
- Hoepfner, Dorothea, et al.
(författare)
-
Monoclonal Anti-AMP Antibodies Are Sensitive and Valuable Tools for Detecting Patterns of AMPylation
- 2020
-
Ingår i: iScience. - Cambridge : Cell Press. - 2589-0042. ; 23:12
-
Tidskriftsartikel (refereegranskat)abstract
- AMPylation is a post-translational modification that modifies amino acid side chains with adenosine monophosphate (AMP). Recently, a role of AMPylation as a universal regulatory mechanism in infection and cellular homeostasis has emerged, driving the demand for universal tools to study this modification. Here, we describe three monoclonal anti-AMP antibodies (mAbs) from mouse that are capable of protein backbone-independent recognition of AMPylation, in denatured (western blot) as well as native (ELISA, IP) applications, thereby outperforming previously reported tools. These antibodies are highly sensitive and specific for AMP modifications, highlighting their potential as tools for new target identification, as well as for validation of known targets. Interestingly, applying the anti-AMP mAbs to various cancer cell lines reveals a previously undescribed broad and diverse AMPylation pattern. In conclusion, these anti-AMP mABs will further advance the current understanding of AMPylation and the spectrum of modified targets.
|
|
| 6. |
- Kaspers, Marietta S., et al.
(författare)
-
Dephosphocholination by Legionella effector Lem3 functions through remodelling of the switch II region of Rab1b
- 2023
-
Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
-
Tidskriftsartikel (refereegranskat)abstract
- Bacterial pathogens often make use of post-translational modifications to manipulate host cells. Legionella pneumophila, the causative agent of Legionnaires disease, secretes the enzyme AnkX that uses cytidine diphosphate-choline to post-translationally modify the human small G-Protein Rab1 with a phosphocholine moiety at Ser76. Later in the infection, the Legionella enzyme Lem3 acts as a dephosphocholinase, hydrolytically removing the phosphocholine. While the molecular mechanism for Rab1 phosphocholination by AnkX has recently been resolved, structural insights into the activity of Lem3 remained elusive. Here, we stabilise the transient Lem3:Rab1b complex by substrate mediated covalent capture. Through crystal structures of Lem3 in the apo form and in complex with Rab1b, we reveal Lem3's catalytic mechanism, showing that it acts on Rab1 by locally unfolding it. Since Lem3 shares high structural similarity with metal-dependent protein phosphatases, our Lem3:Rab1b complex structure also sheds light on how these phosphatases recognise protein substrates.
|
|
| 7. |
- Martinez, Nancy E., et al.
(författare)
-
Tetrahydroisoquinolines : New Inhibitors of Neutrophil Extracellular Trap (NET) Formation
- 2017
-
Ingår i: ChemBioChem. - : Wiley-VCH Verlagsgesellschaft. - 1439-4227 .- 1439-7633. ; 18:10, s. 888-893
-
Tidskriftsartikel (refereegranskat)abstract
- Neutrophils are short-lived leukocytes that migrate to sites of infection as part of the acute immune response, where they phagocytose, degranulate, and form neutrophil extracellular traps (NETs). During NET formation, the nuclear lobules of neutrophils disappear and the chromatin expands and, accessorized with neutrophilic granule proteins, is expelled. NETs can be pathogenic in, for example, sepsis, cancer, and autoimmune and cardiovascular diseases. Therefore, the identification of inhibitors of NET formation is of great interest. Screening of a focused library of natural-product-inspired compounds by using a previously validated phenotypic NET assay identified a group of tetrahydroisoquinolines as new NET formation inhibitors. This compound class opens up new avenues for the study of cellular death through NET formation (NETosis) at different stages, and might inspire new medicinal chemistry programs aimed at NET-dependent diseases.
|
|
| 8. |
- Aaboud, M, et al.
(författare)
-
Combined measurement of differential and total cross sections in the H → γγ and the H → ZZ⁎ → 4ℓ decay channels at s=13 TeV with the ATLAS detector
- 2018
-
Ingår i: Physics Letters, Section B: Nuclear, Elementary Particle and High-Energy Physics. - : Elsevier BV. - 0370-2693 .- 1873-2445. ; 786, s. 114-133
-
Tidskriftsartikel (refereegranskat)abstract
- A combined measurement of differential and inclusive total cross sections of Higgs boson production is performed using 36.1 fb−1 of 13 TeV proton–proton collision data produced by the LHC and recorded by the ATLAS detector in 2015 and 2016. Cross sections are obtained from measured H→γγ and H→ZZ⁎→4ℓ event yields, which are combined taking into account detector efficiencies, resolution, acceptances and branching fractions. The total Higgs boson production cross section is measured to be 57.0−5.9 +6.0 (stat.) −3.3 +4.0 (syst.) pb, in agreement with the Standard Model prediction. Differential cross-section measurements are presented for the Higgs boson transverse momentum distribution, Higgs boson rapidity, number of jets produced together with the Higgs boson, and the transverse momentum of the leading jet. The results from the two decay channels are found to be compatible, and their combination agrees with the Standard Model predictions. © 2018 The Author(s)
|
|
| 9. |
- Aaboud, M, et al.
(författare)
-
Measurement of colour flow using jet-pull observables in tt¯ events with the ATLAS experiment at √s=13TeV
- 2018
-
Ingår i: European Physical Journal C. - : Springer Science and Business Media LLC. - 1434-6044 .- 1434-6052. ; 78:10
-
Tidskriftsartikel (refereegranskat)abstract
- Previous studies have shown that weighted angular moments derived from jet constituents encode the colour connections between partons that seed the jets. This paper presents measurements of two such distributions, the jet-pull angle and jet-pull magnitude, both of which are derived from the jet-pull angular moment. The measurement is performed in tt¯ events with one leptonically decaying W boson and one hadronically decaying W boson, using 36.1fb-1 of pp collision data recorded by the ATLAS detector at s=13TeV delivered by the Large Hadron Collider. The observables are measured for two dijet systems, corresponding to the colour-connected daughters of the W boson and the two b-jets from the top-quark decays, which are not expected to be colour connected. To allow the comparison of the measured distributions to colour model predictions, the measured distributions are unfolded to particle level, after correcting for experimental effects introduced by the detector. While good agreement can be found for some combinations of predictions and observables, none of the predictions describes the data well across all observables. © 2018, CERN for the benefit of the ATLAS collaboration.
|
|
| 10. |
- Aaboud, M, et al.
(författare)
-
Measurement of fiducial and differential W+W- production cross-sections at √s=13 TeV with the ATLAS detector
- 2019
-
Ingår i: European Physical Journal C. - : Springer Science and Business Media LLC. - 1434-6044 .- 1434-6052. ; 79:10
-
Tidskriftsartikel (refereegranskat)abstract
- A measurement of fiducial and differential cross-sections for W+W- production in proton–proton collisions at s=13 TeV with the ATLAS experiment at the Large Hadron Collider using data corresponding to an integrated luminosity of 36.1 fb - 1 is presented. Events with one electron and one muon are selected, corresponding to the decay of the diboson system as WW→ e±νμ∓ν. To suppress top-quark background, events containing jets with a transverse momentum exceeding 35 GeV are not included in the measurement phase space. The fiducial cross-section, six differential distributions and the cross-section as a function of the jet-veto transverse momentum threshold are measured and compared with several theoretical predictions. Constraints on anomalous electroweak gauge boson self-interactions are also presented in the framework of a dimension-six effective field theory. © 2019, CERN for the benefit of the ATLAS collaboration.
|
|
