| 1. |
- Adolfsson, Dan E., 1989-, et al.
(author)
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Enhanement of amyloid fibril binding by ring expansion of thiazolino fused 2-pyridone peptidomimetics
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Other publication (other academic/artistic)abstract
- Thiazolino fused 2-pyridones undergo thiazoline ring opening by reaction with 2-nitrobenzyl bromide through thi- oether attack, and base promoted fragmentation of the resulting sulfonium ions. Subsequent deprotonation of the benzylic carbon and intramolecular 1,4-addition leads to ring closure, generating dihydrothiazine fused 2-pyridones by net ring expansion of the thiazoline ring. Application of the ring expansion procedure to the pyridine and pyrimidine fused thiazolino 2-pyridones provided compounds with enhanced fibril binding activity.
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| 2. |
- Adolfsson, Dan E., 1989-, et al.
(author)
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Intramolecular Povarov Reactions for the Synthesis of Chromenopyridine fused 2-Pyridone Polyheterocycles Binding to α-Synuclein and Amyloid-β fibrils
- 2020
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In: Journal of Organic Chemistry. - : American Chemical Society (ACS). - 0022-3263 .- 1520-6904. ; 85:21, s. 14174-14189
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Journal article (other academic/artistic)abstract
- A BF3×OEt2 catalyzed intramolecular Povarov reaction was used to synthesize a library of 15 chromenopyridine fused thiazolino-2-pyridone peptidomimetics. The reaction works with a range of O-alkylated salicylaldehydes and amino functionalized thiazolino-2-pyridones, to generate polyheterocycles with diverse substitution. The synthesized compounds were screened for their ability to bind α-synuclein and amyloid β fibrils in vitro. Analogs substituted with a nitro group bind to mature amyloid fibrils, and the activity moreover depends on the positioning of this functional group.
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| 3. |
- Alam, Athar, et al.
(author)
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Dissociation between the critical role of ClpB of Francisella tularensis for the heat shock response and the DnaK interaction and its important role for efficient type VI secretion and bacterial virulence
- 2020
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In: PLoS Pathogens. - : Public Library of Science. - 1553-7366 .- 1553-7374. ; 16:4, s. 1-27
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Journal article (peer-reviewed)abstract
- Author summary Type VI secretion systems (T6SSs) are essential virulence determinants of many Gram-negative pathogens, including Francisella tularensis. This highly virulent bacterium encodes an atypical T6SS lacking ClpV, the ATPase crucial for prototypic T6SS sheath disassembly. It, however, possesses ClpB, a protein critical for heat shock survival via its interaction with DnaK. Since ClpB possesses ATPase activity, it has been hypothesized to provide a compensatory function for the absence of ClpV, a hypothesis supported by the recent findings from us and others. Here, we investigated how F. tularensis ClpB controls T6S. In silico modelling of the ClpB-DnaK complex identified key interactions that were experimentally verified. For example, mutating one of the DnaK-interacting residues rendered the bacterium exquisitely susceptible to heat shock, but had no effect on T6S and virulence. In contrast, removing the N-terminal of ClpB only had a slight effect on the heat shock response, but strongly compromised both T6S and virulence. Intriguingly, the Escherichia coli ClpB could fully complement the function of F. tularensis ClpB. The data demonstrate that the two critical roles of ClpB, mediating heat shock survival and effective T6S, are dissociated and that the N-terminal is crucial for T6S and virulence. Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for its virulence. The chaperone ClpB, a member of the Hsp100/Clp family, is involved in Francisella T6SS disassembly and type VI secretion (T6S) is impaired in its absence. We asked if the role of ClpB for T6S was related to its prototypical role for the disaggregation activity. The latter is dependent on its interaction with the DnaK/Hsp70 chaperone system. Key residues of the ClpB-DnaK interaction were identified by molecular dynamic simulation and verified by targeted mutagenesis. Using such targeted mutants, it was found that the F. novicida ClpB-DnaK interaction was dispensable for T6S, intracellular replication, and virulence in a mouse model, although essential for handling of heat shock. Moreover, by mutagenesis of key amino acids of the Walker A, Walker B, and Arginine finger motifs of each of the two Nucleotide-Binding Domains, their critical roles for heat shock, T6S, intracellular replication, and virulence were identified. In contrast, the N-terminus was dispensable for heat shock, but required for T6S, intracellular replication, and virulence. Complementation of the Delta clpB mutant with a chimeric F. novicida ClpB expressing the N-terminal of Escherichia coli, led to reconstitution of the wild-type phenotype. Collectively, the data demonstrate that the ClpB-DnaK interaction does not contribute to T6S, whereas the N-terminal and NBD domains displayed critical roles for T6S and virulence.
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| 4. |
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| 5. |
- Bharate, Jaideep B., et al.
(author)
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K2S2O8-mediated coupling of 6-amino-7-aminomethyl-thiazolino-pyridones with aldehydes to construct amyloid affecting pyrimidine-fused thiazolino-2-pyridones
- 2021
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In: Organic and biomolecular chemistry. - : The Royal Society of Chemistry. - 1477-0520 .- 1477-0539. ; 19:44, s. 9758-9772
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Journal article (peer-reviewed)abstract
- We herein present the synthesis of diversely functionalized pyrimidine fused thiazolino-2-pyridones via K2S2O8-mediated oxidative coupling of 6-amino-7-(aminomethyl)-thiazolino-2-pyridones with aldehydes. The developed protocol is mild, has wide substrate scope, and does not require transition metal catalyst or base. Some of the synthesized compounds have an ability to inhibit the formation of Amyloid-β fibrils associated with Alzheimer's disease, while others bind to mature amyloid-β and α-synuclein fibrils.
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| 6. |
- Cairns, Andrew G., et al.
(author)
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Increased Brain Exposure of an Alpha-Synuclein Fibrillization Modulator by Utilization of an Activated Ester Prodrug Strategy
- 2018
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In: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 9:11, s. 2542-2547
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Journal article (peer-reviewed)abstract
- Previous work in our laboratories has identified a series of peptidomimetic 2-pyridone molecules as modulators of alpha-synuclein (α-syn) fibrillization in vitro. As a first step toward developing molecules from this scaffold as positron emission tomography imaging agents, we were interested in evaluating their blood-brain barrier permeability in nonhuman primates (NHP) in vivo. For this purpose, 2-pyridone 12 was prepared and found to accelerate α-syn fibrillization in vitro. Acid 12, and its acetoxymethyl ester analogue 14, were then radiolabeled with 11C (t1/2 = 20.4 min) at high radiochemical purity (>99%) and high specific radioactivity (>37 GBq/μmol). Following intravenous injection of each compound in NHP, a 4-fold higher radioactivity in brain was observed for [11C]14 compared to [11C]12 (0.8 vs 0.2 SUV, respectively). [11C]14 was rapidly eliminated from plasma, with [11C]12 as the major metabolic product observed by radio-HPLC. The presented prodrug approach paves the way for future development of 2-pyridones as imaging biomarkers for in vivo imaging of α-synuclein deposits in brain.
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| 7. |
- Chorell, Erik, et al.
(author)
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Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts.
- 2015
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 10:10, s. e0140194-
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Journal article (peer-reviewed)abstract
- Amyloid formation is historically associated with cytotoxicity, but many organisms produce functional amyloid fibers (e.g., curli) as a normal part of cell biology. Two E. coli genes in the curli operon encode the chaperone-like proteins CsgC and CsgE that both can reduce in vitro amyloid formation by CsgA. CsgC was also found to arrest amyloid formation of the human amyloidogenic protein α-synuclein, which is involved in Parkinson's disease. Here, we report that the inhibitory effects of CsgC arise due to transient interactions that promote the formation of spherical α-synuclein oligomers. We find that CsgE also modulates α-synuclein amyloid formation through transient contacts but, in contrast to CsgC, CsgE accelerates α-synuclein amyloid formation. Our results demonstrate the significance of transient protein interactions in amyloid regulation and emphasize that the same protein may inhibit one type of amyloid while accelerating another.
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| 8. |
- Clifton, Luke A., et al.
(author)
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Creation of distinctive Bax-lipid complexes at mitochondrial membrane surfaces drives pore formation to initiate apoptosis
- 2023
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In: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 9:22
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Journal article (peer-reviewed)abstract
- Apotosis is an essential process tightly regulated by the Bcl-2 protein family where proapoptotic Bax triggers cell death by perforating the mitochondrial outer membrane. Although intensively studied, the molecular mechanism by which these proteins create apoptotic pores remains elusive. Here, we show that Bax creates pores by extracting lipids from outer mitochondrial membrane mimics by formation of Bax/lipid clusters that are deposited on the membrane surface. Time-resolved neutron reflectometry and Fourier transform infrared spectroscopy revealed two kinetically distinct phases in the pore formation process, both of whichwere critically dependent on cardiolipin levels. The initially fast adsorption of Bax on the mitochondrial membrane surface is followed by a slower formation of pores and Bax-lipid clusters on the membrane surface. Our findings provide a robust molecular understanding of mitochondrial membrane perforation by cell-killing Bax protein and illuminate the initial phases of programmed cellular death.
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| 9. |
- Clifton, Luke A., et al.
(author)
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Insight into Bcl-2 proteins' functioning at mitochondrial membrane level
- 2023
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In: Biophysical Journal. - : Elsevier. - 0006-3495 .- 1542-0086. ; 122:3S1, s. 232a-232a
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Journal article (peer-reviewed)abstract
- Programmed cell death (apoptosis) is essential in life. In its intrinsic apoptotic pathway opposing members of the B-cell lymphoma 2 (Bcl-2) protein family control the permeability of the mitochondrial outer membrane (MOM) and the release of apoptotic factors such as cytochrome c. Any misregulation of this process can cause disorders most prominently cancer, where often upregulation of cell protecting (anti-apoptotic) Bcl-2 members such as the Bcl-2 membrane protein itself plays a notorious role by blocking MOM perforation by - often drug induced - apoptotic proteins such as Bax which would cause cancer cell death normally. Here, we apply neutron reflectometry (NR) on supported lipid bilayers which mimic MOM environment and solid state/liquid state NMR spectroscopy to unravel the molecular basis driving opposing proteins to interact with each other at the MOM; a mechanism which is not really understood yet due to lack of high-resolution structural insight. Based on our central hypothesis that Bcl-2 drives its cell-protecting function at a membrane-embedded location as revealed by NR (1), we focus i) to determine the structure of human Bcl-2 protein in its membrane setting by combining solution and solid-state NMR; ii) use NR to study the kinetics and lipid/protein pore assemblied upon binding of Bax to mitochondrial membranes and its membrane destroying activities there; and iii) unravel the nature of direct interaction between Bcl-2 and Bax to neutralize each other. Knowledge generated here, will be indispensable in understanding the regulative function of the Bcl-2 family at mitochondrial membranes.
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| 10. |
- Dingeldein, Artur Peter Günther, et al.
(author)
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Bax to the future : A novel, high-yielding approach for purification and expression of full-length Bax protein for structural studies
- 2019
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In: Protein Expression and Purification. - : Elsevier. - 1046-5928 .- 1096-0279. ; 158, s. 20-26
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Journal article (peer-reviewed)abstract
- Mitochondria-mediated apoptosis (programmed cell death) involves a sophisticated signaling and regulatory network that is regulated by the Bcl-2 protein family. Members of this family have either pro- or anti-apoptotic functions. An important pro-apoptotic member of this family is the cytosolic Bax. This protein is crucial for the onset of apoptosis by perforating the mitochondrial outer membrane (MOM). This process can be seen as point of no return, since disintegration of the MOM leads to the release of apotogenic factors such as cytochrome c into the cytosol triggering the activation of caspases and subsequent apoptotic steps. Bax is able to interact with the MOM with both its termini, making it inherently difficult to express in E. coli. In this study, we present a novel approach to express and purify full-length Bax with significantly increased yields, when compared to the commonly applied strategy. Using a double fusion approach with an N-terminal GST-tag and a C-terminal Intein-CBD-tag, we were able to render both Bax termini inactive and prevent disruptive interactions from occurring during gene expression. By deploying an Intein-CBD-tag at the C-terminus we were further able to avoid the introduction of any artificial residues, hence ensuring the native like activity of the membrane-penetrating C-terminus of Bax. Further, by engineering a His6-tag to the C-terminus of the CBD-tag we greatly improved the robustness of the purification procedure. We report yields for pure, full-length Bax protein that are increased by an order of magnitude, when compared to commonly used Bax expression protocols.
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