| 1. |
- Aspholm, Emelie E., et al.
(författare)
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Keeping α-synuclein at bay: A more active role of molecular chaperones in preventing mitochondrial interactions and transition to pathological states?
- 2020
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Ingår i: Life. - : MDPI AG. - 2075-1729. ; 10:11, s. 1-19
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Tidskriftsartikel (refereegranskat)abstract
- The property of molecular chaperones to dissolve protein aggregates of Parkinson-related α-synuclein has been known for some time. Recent findings point to an even more active role of molecular chaperones preventing the transformation of α-synuclein into pathological states subsequently leading to the formation of Lewy bodies, intracellular inclusions containing protein aggregates as well as broken organelles found in the brains of Parkinson’s patients. In parallel, a short motif around Tyr39 was identified as being crucial for the aggregation of α-synuclein. Interestingly, this region is also one of the main segments in contact with a diverse pool of molecular chaperones. Further, it could be shown that the inhibition of the chaperone:α-synuclein interaction leads to a binding of α-synuclein to mitochondria, which could also be shown to lead to mitochondrial membrane disruption as well as the possible proteolytic processing of α-synuclein by mitochondrial proteases. Here, we will review the current knowledge on the role of molecular chaperones in the regulation of physiological functions as well as the direct consequences of impairing these interactions—i.e., leading to enhanced mitochondrial interaction and consequential mitochondrial breakage, which might mark the initial stages of the structural transition of α-synuclein towards its pathological states. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
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| 2. |
- Burmann, Björn Marcus, 1979, et al.
(författare)
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Regulation of alpha-synuclein by chaperones in mammalian cells
- 2020
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 577:7788, s. 127-32
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Tidskriftsartikel (refereegranskat)abstract
- Neurodegeneration in patients with Parkinson's disease is correlated with the occurrence of Lewy bodies-intracellular inclusions that contain aggregates of the intrinsically disordered protein alpha-synuclein(1). The aggregation propensity of alpha-synuclein in cells is modulated by specific factors that include post-translational modifications(2,3), Abelson-kinase-mediated phosphorylation(4,5) and interactions with intracellular machineries such as molecular chaperones, although the underlying mechanisms are unclear(6-8). Here we systematically characterize the interaction of molecular chaperones with alpha-synuclein in vitro as well as in cells at the atomic level. We find that six highly divergent molecular chaperones commonly recognize a canonical motif in alpha-synuclein, consisting of the N terminus and a segment around Tyr39, and hinder the aggregation of alpha-synuclein. NMR experiments(9) in cells show that the same transient interaction pattern is preserved inside living mammalian cells. Specific inhibition of the interactions between alpha-synuclein and the chaperone HSC70 and members of the HSP90 family, including HSP90 beta, results in transient membrane binding and triggers a remarkable re-localization of alpha-synuclein to the mitochondria and concomitant formation of aggregates. Phosphorylation of alpha-synuclein at Tyr39 directly impairs the interaction of alpha-synuclein with chaperones, thus providing a functional explanation for the role of Abelson kinase in Parkinson's disease. Our results establish a master regulatory mechanism of alpha-synuclein function and aggregation in mammalian cells, extending the functional repertoire of molecular chaperones and highlighting new perspectives for therapeutic interventions for Parkinson's disease.
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| 3. |
- Kazimierczuk, Krzysztof, et al.
(författare)
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Resolution enhancement in NMR spectra by deconvolution with compressed sensing reconstruction
- 2020
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Ingår i: Chemical Communications. - : Royal Society of Chemistry (RSC). - 1359-7345 .- 1364-548X. ; 56, s. 14585-14588
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Tidskriftsartikel (refereegranskat)abstract
- NMR spectroscopy is one of the basic tools for molecular structure elucidation. Unfortunately, the resolution of the spectra is often limited by inter-nuclear couplings. The existing workarounds often alleviate the problem by trading it for another deficiency, such as spectral artefacts or difficult sample preparation and, thus, are rarely used. We suggest an approach using the coupling deconvolution in the framework of compressed sensing (CS) spectra processing that leads to a major increase in resolution, sensitivity, and overall quality of NUS reconstruction. A new mathematical description of the decoupling by deconvolution explains the effects of thermal noise and reveals a relation with the underlying assumption of the CS. The gain in resolution and sensitivity for challenging molecular systems is demonstrated for the key HNCA experiment used for protein backbone assignment applied to two large proteins: intrinsically disordered 441-residue Tau and a 509-residue globular bacteriophytochrome fragment. The approach will be valuable in a multitude of chemistry applications, where NMR experiments are compromised by the homonuclear scalar coupling. This journal is
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| 4. |
- Kurzawa-Akanbi, M., et al.
(författare)
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Altered ceramide metabolism is a feature in the extracellular vesicle-mediated spread of alpha-synuclein in Lewy body disorders
- 2021
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Ingår i: Acta Neuropathologica. - : Springer Science and Business Media LLC. - 0001-6322 .- 1432-0533. ; 142, s. 961-984
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in glucocerebrosidase (GBA) are the most prevalent genetic risk factor for Lewy body disorders (LBD)-collectively Parkinson's disease, Parkinson's disease dementia and dementia with Lewy bodies. Despite this genetic association, it remains unclear how GBA mutations increase susceptibility to develop LBD. We investigated relationships between LBD-specific glucocerebrosidase deficits, GBA-related pathways, and alpha-synuclein levels in brain tissue from LBD and controls, with and without GBA mutations. We show that LBD is characterised by altered sphingolipid metabolism with prominent elevation of ceramide species, regardless of GBA mutations. Since extracellular vesicles (EV) could be involved in LBD pathogenesis by spreading disease-linked lipids and proteins, we investigated EV derived from post-mortem cerebrospinal fluid (CSF) and brain tissue from GBA mutation carriers and non-carriers. EV purified from LBD CSF and frontal cortex were heavily loaded with ceramides and neurodegeneration-linked proteins including alpha-synuclein and tau. Our in vitro studies demonstrate that LBD EV constitute a "pathological package" capable of inducing aggregation of wild-type alpha-synuclein, mediated through a combination of alpha-synuclein-ceramide interaction and the presence of pathological forms of alpha-synuclein. Together, our findings indicate that abnormalities in ceramide metabolism are a feature of LBD, constituting a promising source of biomarkers, and that GBA mutations likely accelerate the pathological process occurring in sporadic LBD through endolysosomal deficiency.
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| 5. |
- Lantero Rodriguez, Juan, et al.
(författare)
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Tau protein profiling in tauopathies: a human brain study
- 2024
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Ingår i: MOLECULAR NEURODEGENERATION. - 1750-1326. ; 19:1
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Tidskriftsartikel (refereegranskat)abstract
- Abnormal accumulation of misfolded and hyperphosphorylated tau protein in brain is the defining feature of several neurodegenerative diseases called tauopathies, including Alzheimer's disease (AD). In AD, this pathological change is reflected by highly specific cerebrospinal fluid (CSF) tau biomarkers, including both phosphorylated and non-phosphorylated variants. Interestingly, despite tau pathology being at the core of all tauopathies, CSF tau biomarkers remain unchanged in certain tauopathies, e.g., progressive supranuclear palsy (PSP), Pick's disease (PiD), and corticobasal neurodegeneration (CBD). To better understand commonalities and differences between tauopathies, we report a multiplex assay combining immunoprecipitation and high-resolution mass spectrometry capable of detecting and quantifying peptides from different tau protein isoforms as well as non-phosphorylated and phosphorylated peptides, including those carrying multiple phosphorylations. We investigated the tau proteoforms in soluble and insoluble fractions of brain tissue from subjects with autopsy-confirmed tauopathies, including sporadic AD (n = 10), PSP (n = 11), PiD (n = 10), and CBD (n = 10), and controls (n = 10). Our results demonstrate that non-phosphorylated tau profiles differ across tauopathies, generally showing high abundance of microtubule-binding region (MTBR)-containing peptides in insoluble protein fractions compared with controls; the AD group showed 12-72 times higher levels of MTBR-containing aggregates. Quantification of tau isoforms showed the 3R being more abundant in PiD and the 4R isoform being more abundant in CBD and PSP in the insoluble fraction. Twenty-three different phosphorylated peptides were quantified. Most phosphorylated peptides were measurable in all investigated tauopathies. All phosphorylated peptides were significantly increased in AD insoluble fraction. However, doubly and triply phosphorylated peptides were significantly increased in AD even in the soluble fraction. Results were replicated using a validation cohort comprising AD (n = 10), CBD (n = 10), and controls (n = 10). Our study demonstrates that abnormal levels of phosphorylation and aggregation do indeed occur in non-AD tauopathies, however, both appear pronouncedly increased in AD, becoming a distinctive characteristic of AD pathology.
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| 6. |
- Lesovoy, D. M., et al.
(författare)
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Unambiguous Tracking of Protein Phosphorylation by Fast High-Resolution FOSY NMR**
- 2021
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Ingår i: Angewandte Chemie-International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 60:44, s. 23540-23544
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Tidskriftsartikel (refereegranskat)abstract
- Dysregulation of post-translational modifications (PTMs) like phosphorylation is often involved in disease. NMR may elucidate exact loci and time courses of PTMs at atomic resolution and near-physiological conditions but requires signal assignment to individual atoms. Conventional NMR methods for this base on tedious global signal assignment that may often fail, as for large intrinsically disordered proteins (IDPs). We present a sensitive, robust alternative to rapidly obtain only the local assignment near affected signals, based on FOcused SpectroscopY (FOSY) experiments using selective polarisation transfer (SPT). We prove its efficiency by identifying two phosphorylation sites of glycogen synthase kinase 3 beta (GSK3 beta) in human Tau40, an IDP of 441 residues, where the extreme spectral dispersion in FOSY revealed unprimed phosphorylation also of Ser409. FOSY may broadly benefit NMR studies of PTMs and other hotspots in IDPs, including sites involved in molecular interactions.
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| 7. |
- Lidman, Jens, 1994, et al.
(författare)
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Structure and dynamics of the mitochondrial DNA-compaction factor Abf2 from S. cerevisiae
- 2023
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Ingår i: Journal of Structural Biology. - 1047-8477. ; 215:3
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondria are essential organelles that produce most of the energy via the oxidative phosphorylation (OXPHOS) system in all eukaryotic cells. Several essential subunits of the OXPHOS system are encoded by the mitochondrial genome (mtDNA) despite its small size. Defects in mtDNA maintenance and expression can lead to severe OXPHOS deficiencies and are amongst the most common causes of mitochondrial disease. The mtDNA is packaged as nucleoprotein structures, referred to as nucleoids. The conserved mitochondrial proteins, ARSbinding factor 2 (Abf2) in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and mitochondrial transcription factor A (TFAM) in mammals, are nucleoid associated proteins (NAPs) acting as condensing factors needed for packaging and maintenance of the mtDNA. Interestingly, gene knockout of Abf2 leads, in yeast, to the loss of mtDNA and respiratory growth, providing evidence for its crucial role. On a structural level, the condensing factors usually contain two DNA binding domains called high-mobility group boxes (HMG boxes). The co-operating mechanical activities of these domains are crucial in establishing the nucleoid architecture by bending the DNA strands. Here we used advanced solution NMR spectroscopy methods to characterize the dynamical properties of Abf2 together with its interaction with DNA. We find that the two HMG-domains react notably different to external cues like temperature and salt, indicating distinct functional properties. Biophysical characterizations show the pronounced difference of these domains upon DNA-binding, suggesting a refined picture of the Abf2 functional cycle.
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| 8. |
- Matečko-Burmann, Irena, et al.
(författare)
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Recording In-Cell NMR-Spectra in Living Mammalian Cells
- 2020
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Ingår i: Methods in molecular biology (Clifton, N.J.). - New York, NY : Springer US. - 1940-6029. ; 2141, s. 857-871
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Tidskriftsartikel (refereegranskat)abstract
- At the foundation of many cellular processes as well as a large number of diseases is the (mis)folding of important intrinsically disordered proteins (IDPs). Despite tremendous scientific efforts, the factors driving their structural changes within the cellular context remain poorly understood. In-cell NMR spectroscopy enables investigation of IDPs directly in the living eukaryotic cell enabling investigation of its intermolecular interactions and ensuing modifications at an unprecedented atomic resolution. In the following protocol, we describe how to prepare in-cell NMR samples of IDPs within eukaryotic cells and how to measure these in-cell NMR samples of an IDP in its natural environment, the living mammalian cell. Furthermore, we outline a procedure to assess the intracellular recombinant protein concentration of the studied IDP based on in-cell NMR methods. We use α-synuclein as a model protein, but the presented approach is highly modular and therefore should be easily adapted and altered to the desired needs for the studies of different IDPs.
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| 9. |
- Okeke, Damasus, 1985, et al.
(författare)
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Thumb-domain dynamics modulate the functional repertoire of DNA-Polymerase IV (DinB).
- 2023
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Ingår i: Nucleic acids research. - 1362-4962. ; 51:13, s. 7036-52
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Tidskriftsartikel (refereegranskat)abstract
- In order to cope with the risk of stress-induced mutagenesis, cells in all kingdoms of life employ Y-family DNA polymerases to resolve resulting DNA lesions and thus maintaining the integrity of the genome. In Escherichia coli,the DNA polymerase IV, or DinB, plays this crucial role in coping with these type of mutations via the so-called translesion DNA synthesis. Despite the availability of several high-resolution crystal structures, important aspects of the functional repertoire of DinB remain elusive. In this study, we use advanced solution NMR spectroscopy methods in combination with biophysical characterization to elucidate the crucial role of the Thumb domain within DinB's functional cycle. We find that the inherent dynamics of this domain guide the recognition of double-stranded (ds) DNA buried within the interior of the DinB domain arrangement and trigger allosteric signals through the DinB protein. Subsequently, we characterized the RNA polymerase interaction with DinB, revealing an extended outside surface of DinB and thus not mutually excluding the DNA interaction. Altogether the obtained results lead to a refined model of the functional repertoire of DinB within the translesion DNA synthesis pathway.
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| 10. |
- Quint, Wim Hendricus, et al.
(författare)
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Bispecific Tau Antibodies with Additional Binding to C1q or Alpha-Synuclein.
- 2021
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Ingår i: Journal of Alzheimer's disease : JAD. - 1875-8908. ; 80:2, s. 813-829
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer's disease (AD) and other tauopathies are neurodegenerative disorders characterized by cellular accumulation of aggregated tau protein. Tau pathology within these disorders is accompanied by chronic neuroinflammation, such as activation of the classical complement pathway by complement initiation factor C1q. Additionally, about half of the AD cases present with inclusions composed of aggregated alpha-synuclein called Lewy bodies. Lewy bodies in disorders such as Parkinson's disease and Lewy body dementia also frequently occur together with tau pathology.Immunotherapy is currently the most promising treatment strategy for tauopathies. However, the presence of multiple pathological processes within tauopathies makes it desirable to simultaneously target more than one disease pathway.Herein, we have developed three bispecific antibodies based on published antibody binding region sequences. One bispecific antibody binds to tau plus alpha-synuclein and two bispecific antibodies bind to tau plus C1q.Affinity of the bispecific antibodies to their targets compared to their monospecific counterparts ranged from nearly identical to one order of magnitude lower. All bispecific antibodies retained binding to aggregated protein in patient-derived brain sections. The bispecific antibodies also retained their ability to inhibit aggregation of recombinant tau, regardless of whether the tau binding sites were in IgG or scFv format. Mono- and bispecific antibodies inhibited cellular seeding induced by AD-derived pathological tau with similar efficacy. Finally, both Tau-C1q bispecific antibodies completely inhibited the classical complement pathway.Bispecific antibodies that bind to multiple pathological targets may therefore present a promising approach to treat tauopathies and other neurodegenerative disorders.
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| 11. |
- Cicognola, Claudia, et al.
(författare)
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Tauopathy-Associated Tau Fragment Ending at Amino Acid 224 Is Generated by Calpain-2 Cleavage.
- 2020
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Ingår i: Journal of Alzheimer's disease : JAD. - 1875-8908. ; 74:4, s. 1143-1156
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Tidskriftsartikel (refereegranskat)abstract
- Tau aggregation in neurons and glial cells characterizes tauopathies as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Tau proteolysis has been proposed as a trigger for tau aggregation and tau fragments have been observed in brain and cerebrospinal fluid (CSF). Our group identified a major tau cleavage at amino acid (aa) 224 in CSF; N-terminal tau fragments ending at aa 224 (N-224) were significantly increased in AD and lacked correlation to total tau (t-tau) and phosphorylated tau (p-tau) in PSP and CBD.Previous studies have shown cleavage from calpain proteases at sites adjacent to aa 224. Our aim was to investigate if calpain-1 or -2 could be responsible for cleavage at aa 224.Proteolytic activity of calpain-1, calpain-2, and brain protein extract was assessed on a custom tau peptide (aa 220-228), engineered with fluorescence resonance energy transfer (FRET) technology. Findings were confirmed with in-gel trypsination and mass spectrometry (MS) analysis of brain-derived bands with proteolytic activity on the FRET substrate. Finally, knock-down of the calpain-2 catalytic subunit gene (CAPN2) was performed in a neuroblastoma cell line (SH-SY5Y).Calpain-2 and brain protein extract, but not calpain-1, showed proteolytic activity on the FRET substrate. MS analysis of active gel bands revealed presence of calpain-2 subunits, but not calpain-1. Calpain-2 depletion and chemical inhibition suppressed proteolysis of the FRET substrate. CAPN2 knock-down caused a 76.4% reduction of N-224 tau in the cell-conditioned media.Further investigation of the calpain-2 pathway in the pathogenesis of tauopathies is encouraged.
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