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1.
  • Ariöz, Candan, 1983, et al. (författare)
  • Folding of copper proteins: Role of the metal?
  • 2018
  • Ingår i: Quarterly Reviews of Biophysics. - 1469-8994 .- 0033-5835. ; 51
  • Forskningsöversikt (refereegranskat)abstract
    • Copper is a redox-active transition metal ion required for the function of many essential human proteins. For biosynthesis of proteins coordinating copper, the metal may bind before, during or after folding of the polypeptide. If the metal binds to unfolded or partially folded structures of the protein, such coordination may modulate the folding reaction. The molecular understanding of how copper is incorporated into proteins requires descriptions of chemical, thermodynamic, kinetic and structural parameters involved in the formation of protein- metal complexes. Because free copper ions are toxic, living systems have elaborate copper-transport systems that include particular proteins that facilitate efficient and specific delivery of copper ions to target proteins. Therefore, these pathways become an integral part of copper protein folding in vivo. This review summarizes biophysical-molecular in vitro work assessing the role of copper in folding and stability of copper-binding proteins as well as protein-protein copper exchange reactions between human copper transport proteins. We also describe some recent findings about the participation of copper ions and copper proteins in protein misfolding and aggregation reactions in vitro.
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2.
  • Ariöz, Candan, 1983, et al. (författare)
  • The six metal binding domains in human copper transporter, ATP7B: molecular biophysics and disease-causing mutations
  • 2017
  • Ingår i: Biometals. - : Springer Science and Business Media LLC. - 0966-0844 .- 1572-8773. ; 30:6, s. 823-840
  • Tidskriftsartikel (refereegranskat)abstract
    • Wilson Disease (WD) is a hereditary genetic disorder, which coincides with a dysfunctional copper (Cu) metabolism caused by mutations in ATP7B, a membrane-bound P-1B-type ATPase responsible for Cu export from hepatic cells. The N-terminal part (similar to 600 residues) of the multi-domain 1400-residue ATP7B constitutes six metal binding domains (MBDs), each of which can bind a copper ion, interact with other ATP7B domains as well as with different proteins. Although the ATP7B's MBDs have been investigated in vitro and in vivo intensively, it remains unclear how these domains modulate overall structure, dynamics, stability and function of ATP7B. The presence of six MBDs is unique to mammalian ATP7B homologs, and many WD causing missense mutations are found in these domains. Here, we have summarized previously reported in vitro biophysical data on the MBDs of ATP7B and WD point mutations located in these domains. Besides the demonstration of where the research field stands today, this review showcasts the need for further biophysical investigation about the roles of MBDs in ATP7B function. Molecular mechanisms of ATP7B are important not only in the development of new WD treatment but also for other aspects of human physiology where Cu transport plays a role. RAHAMS JP, 1994, NATURE, V370, P621
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3.
  • Blockhuys, Stephanie, 1983, et al. (författare)
  • Copper chaperone Atox1 plays role in breast cancer cell migration
  • 2017
  • Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 483:1, s. 301-304
  • Tidskriftsartikel (refereegranskat)abstract
    • Copper (Cu) is an essential transition metal ion required as cofactor in many key enzymes. After cell uptake of Cu, the metal is transported by the cytoplasmic Cu chaperone Atox1 to P1B-type ATPases in the Golgi network for incorporation into Cu-dependent enzymes in the secretory path. Cu is vital for many steps of cancer progression and Atox1 was recently suggested to have additional functionality as a nuclear transcription factor. We here investigated the expression level, cellular localization and role in cell migration of Atox1 in an aggressive breast cancer cell line upon combining immunostaining, microscopy and a wound healing assay. We made the unexpected discovery that Atox1 accumulates at lamellipodia borders of migrating cancer cells and Atox1 silencing resulted in migration defects as evidenced from reduced wound closure. Therefore, we have discovered an unknown role of the Cu chaperone Atox1 in breast cancer cell migration.
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4.
  • Blockhuys, Stephanie, 1983, et al. (författare)
  • Copper distribution in breast cancer cells detected by time-of-flight secondary ion mass spectrometry with delayed extraction methodology
  • 2018
  • Ingår i: Biointerphases. - : American Vacuum Society. - 1559-4106 .- 1934-8630. ; 13:6
  • Tidskriftsartikel (refereegranskat)abstract
    • Copper (Cu) is an essential transition metal ion that acts as a cofactor in many key enzymes. Cu is also needed for several hallmarks of cancer, and many copper-binding proteins are upregulated in various cancers. However, Cu-dependent cellular mechanisms and molecular pathways involved in cancer progression are not known. Fundamental to a better understanding of such phenomena is the investigation of the Cu subcellular distribution in cancer cells. The authors here show that Time-of-Flight Secondary Ion Mass Spectrometry combined with delayed extraction can be successfully applied to probe Cu localization in fixed MDA-MB-231 breast cancer cells providing subcellular resolution. Interestingly, the authors find Cu to be accumulated at nuclear regions of the cancer cells. Published by the AVS.
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5.
  • Blockhuys, Stephanie, 1983, et al. (författare)
  • Defining the human copper proteome and analysis of its expression variation in cancers.
  • 2017
  • Ingår i: Metallomics. - : Oxford University Press (OUP). - 1756-5901 .- 1756-591X. ; 9:2, s. 112-123
  • Tidskriftsartikel (refereegranskat)abstract
    • Copper (Cu) is essential for living organisms, and acts as a cofactor in many metabolic enzymes. To avoid the toxicity of free Cu, organisms have specific transport systems that 'chaperone' the metal to targets. Cancer progression is associated with increased cellular Cu concentrations, whereby proliferative immortality, angiogenesis and metastasis are cancer hallmarks with defined requirements for Cu. The aim of this study is to gather all known Cu-binding proteins and reveal their putative involvement in cancers using the available database resources of RNA transcript levels. Using the database along with manual curation, we identified a total of 54 Cu-binding proteins (named the human Cu proteome). Next, we retrieved RNA expression levels in cancer versus normal tissues from the TCGA database for the human Cu proteome in 18 cancer types, and noted an intricate pattern of up- and downregulation of the genes in different cancers. Hierarchical clustering in combination with bioinformatics and functional genomics analyses allowed for the prediction of cancer-related Cu-binding proteins; these were specifically inspected for the breast cancer data. Finally, for the Cu chaperone ATOX1, which is the only Cu-binding protein proposed to have transcription factor activities, we validated its predicted over-expression in patient breast cancer tissue at the protein level. This collection of Cu-binding proteins, with RNA expression patterns in different cancers, will serve as an excellent resource for mechanistic-molecular studies of Cu-dependent processes in cancer.
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6.
  • Blockhuys, Stephanie, 1983, et al. (författare)
  • Roles of copper-binding proteins in breast cancer
  • 2017
  • Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 18:4
  • Forskningsöversikt (refereegranskat)abstract
    • Copper ions are needed in several steps of cancer progression. However, the underlying mechanisms, and involved copper-binding proteins, are mainly elusive. Since most copper ions in the body (in and outside cells) are protein-bound, it is important to investigate what copper-binding proteins participate and, for these, how they are loaded with copper by copper transport proteins. Mechanistic information for how some copper-binding proteins, such as extracellular lysyl oxidase (LOX), play roles in cancer have been elucidated but there is still much to learn from a biophysical molecular viewpoint. Here we provide a summary of copper-binding proteins and discuss ones reported to have roles in cancer. We specifically focus on how copper-binding proteins such as mediator of cell motility 1 (MEMO1), LOX, LOX-like proteins, and secreted protein acidic and rich in cysteine (SPARC) modulate breast cancer from molecular and clinical aspects. Because of the importance of copper for invasion/migration processes, which are key components of cancer metastasis, further insights into the actions of copper-binding proteins may provide new targets to combat cancer.
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7.
  • Blockhuys, Stephanie, 1983, et al. (författare)
  • Second harmonic generation for collagen I characterization in rectal cancer patients with and without preoperative radiotherapy
  • 2017
  • Ingår i: Journal of Biomedical Optics. - : SPIE-Intl Soc Optical Eng. - 1560-2281 .- 1083-3668. ; 22:10
  • Tidskriftsartikel (refereegranskat)abstract
    • Rectal cancer is treated with preoperative radiotherapy (RT) to downstage the tumor, reduce local recurrence, and improve patient survival. Still, the treatment outcome varies significantly and new biomarkers are desired. Collagen I (Col-I) is a potential biomarker, which can be visualized label-free by second harmonic generation (SHG). Here, we used SHG to identify Col-I changes induced by RT in surgical tissue, with the aim to evaluate the clinical significance of RT-induced Col-I changes. First, we established a procedure for quantitative evaluation of Col-I by SHG in CDX2-stained tissue sections. Next, we evaluated Col-I properties in material from 31 non-RT and 29 RT rectal cancer patients. We discovered that the Col-I intensity and anisotropy were higher in the tumor invasive margin than in the inner tumor and normal mucosa, and RT increased and decreased the intensity in inner tumor and normal mucosa, respectively. Furthermore, higher Col-I intensity in the inner tumor was related to increased distant recurrence in the non-RT group but to longer survival in the RT group. In conclusion, we present a new application of SHG for quantitative analysis of Col-I in surgical material, and the first data suggest Col-I intensity as a putative prognostic biomarker in rectal cancer.
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8.
  • Bosaeus, Niklas, 1982, et al. (författare)
  • A stretched conformation of DNA with a biological role?
  • 2017
  • Ingår i: Quarterly Reviews of Biophysics. - 1469-8994 .- 0033-5835. ; 50, s. UNSP e11-e11
  • Tidskriftsartikel (refereegranskat)abstract
    • We have discovered a well-defined extended conformation of double-stranded DNA, which we call Sigma-DNA, using laser-tweezers force-spectroscopy experiments. At a transition force corresponding to free energy change Delta G = 1.57 +/- 0.12 kcal (mol base pair)(-1) 60 or 122 base-pair long synthetic GC-rich sequences, when pulled by the 3'-3' strands, undergo a sharp transition to the 1.52 +/- 0.04 times longer Sigma-DNA. Intriguingly, the same degree of extension is also found in DNA complexes with recombinase proteins, such as bacterial RecA and eukaryotic Rad51. Despite vital importance to all biological organisms for survival, genome maintenance and evolution, the recombination reaction is not yet understood at atomic level. We here propose that the structural distortion represented by Sigma-DNA, which is thus physically inherent to the nucleic acid, is related to how recombination proteins mediate recognition of sequence homology and execute strand exchange. Our hypothesis is that a homogeneously stretched DNA undergoes a 'disproportionation' into an inhomogeneous Sigma-form consisting of triplets of locally B-like perpendicularly stacked bases. This structure may ensure improved fidelity of base-pair recognition and promote rejection in case of mismatch during homologous recombination reaction. Because a triplet is the length of a gene codon, we speculate that the structural physics of nucleic acids may have biased the evolution of recombinase proteins to exploit triplet base stacks and also the genetic code.
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9.
  • Celauro, Emanuele, 1980, et al. (författare)
  • Copper chaperone ATOX1 regulates pluripotency factor OCT4 in preimplantation mouse embryos
  • 2017
  • Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 491:1, s. 147-153
  • Tidskriftsartikel (refereegranskat)abstract
    • Despite of the importance of copper (Cu) during pregnancy, the roles of Cu-binding proteins during early embryonic development are unknown. The Cu chaperone ATOX1 was recently suggested to have additional functions related to transcription and cancer. When we analyzed single-cell RNA transcript data from early mouse embryos, Atox1 transcript levels increased dramatically at the 8-cell stage and, at 16 and 32-cell embryo stages, matched those of Oct4 which expresses a transcription factor essential for pluripotency in the inner cell mass. To explore this, we probed Atox1 expression during the first week of development of mouse embryos. ATOX1 appeared ubiquitously expressed throughout the cells until compaction; in subsequent embryo stages, ATOX1 relocalized to cytoplasmic perinuclear domains in the inner cell mass. Silencing of Oct4 did not affect Atoxl expression, but silencing of Atoxl at the 2-cell stage strongly diminished Oct4 expression in 16-cell embryos.
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10.
  • Chorell, Erik, et al. (författare)
  • Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts.
  • 2015
  • Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 10:10, s. e0140194-
  • Tidskriftsartikel (refereegranskat)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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11.
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12.
  • Dedic, Jan, et al. (författare)
  • Membrane-Protein-Hydration Interaction of α-Synuclein with Anionic Vesicles Probed via Angle-Resolved Second-Harmonic Scattering
  • 2019
  • Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 123:5, s. 1044-1049
  • Tidskriftsartikel (refereegranskat)abstract
    • Amyloid formation of the protein α-synuclein promotes neurodegeneration in Parkinson's disease. The normal function of α-synuclein includes synaptic vesicle transport and fusion, and the protein binds strongly to negatively charged vesicles in vitro. Here, we demonstrate that nonresonant angle-resolved second-harmonic scattering detects α-synuclein binding to liposomes through changes in water orientational correlations and can thus be used as a high-accuracy and high-throughput label-free probe of protein-liposome interactions. The obtained results support a binding model in which the N-terminus of α-synuclein adopts an α-helical conformation that lies flat on the vesicle surface while the negatively charged C-terminus remains in solution.
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13.
  • Evans, Margery L, et al. (författare)
  • The bacterial curli system possesses a potent and selective inhibitor of amyloid formation
  • 2015
  • Ingår i: Molecular Cell. - : Elsevier BV. - 1097-4164 .- 1097-2765. ; 57:3, s. 445-455
  • Tidskriftsartikel (refereegranskat)abstract
    • Curli are extracellular functional amyloids that are assembled by enteric bacteria during biofilm formation and host colonization. An efficient secretion system and chaperone network ensures that the major curli fiber subunit, CsgA, does not form intracellular amyloid aggregates. We discovered that the periplasmic protein CsgC was a highly effective inhibitor of CsgA amyloid formation. In the absence of CsgC, CsgA formed toxic intracellular aggregates. In vitro, CsgC inhibited CsgA amyloid formation at substoichiometric concentrations and maintained CsgA in a non-β-sheet-rich conformation. Interestingly, CsgC inhibited amyloid assembly of human α-synuclein, but not Aβ42, in vitro. We identified a common D-Q-Φ-X0,1-G-K-N-ζ-E motif in CsgC client proteins that is not found in Aβ42. CsgC is therefore both an efficient and selective amyloid inhibitor. Dedicated functional amyloid inhibitors may be a key feature that distinguishes functional amyloids from disease-associated amyloids.
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14.
  • Gomes, Cláudio M., et al. (författare)
  • About the editors
  • 2016
  • Ingår i: Protein folding and metal ions. - : CRC Press. - 9781439809655 - 9781439809648 - 9780367655884 - 9780429148187 ; , s. xi-
  • Bokkapitel (övrigt vetenskapligt/konstnärligt)
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15.
  • Horvath, Istvan, 1979, et al. (författare)
  • Copper chaperone blocks amyloid formation via ternary complex
  • 2018
  • Ingår i: Quarterly Reviews of Biophysics. - 1469-8994 .- 0033-5835. ; 51, s. e6-e6
  • Tidskriftsartikel (refereegranskat)abstract
    • Protein misfolding in cells is avoided by a network of protein chaperones that detect misfolded or partially folded species. When proteins escape these control systems, misfolding may result in protein aggregation and amyloid formation. We here show that aggregation of the amyloidogenic protein alpha-synuclein (alpha S), the key player in Parkinson's disease, is controlled by the copper transport protein Atox1 in vitro. Copper ions are not freely available in the cellular environment, but when provided by Atox1, the resulting copper-dependent ternary complex blocks aS aggregation. Because the same inhibition was found for a truncated version of alpha S, lacking the C-terminal part, it appears that Atox1 interacts with the N-terminal copper site in alpha S. Metal-dependent chaperoning may be yet another manner in which cells control its proteome.
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16.
  • Horvath, Istvan, 1979, et al. (författare)
  • Cross-talk between amyloidogenic proteins in type-2 diabetes and Parkinson's disease
  • 2016
  • Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 113:44, s. 12473-12477
  • Tidskriftsartikel (refereegranskat)abstract
    • In type-2 diabetes (T2D) and Parkinson's disease (PD), polypeptide assembly into amyloid fibers plays central roles: in PD, alpha-synuclein (aS) forms amyloids and in T2D, amylin [islet amyloid polypeptide (IAPP)] forms amyloids. Using a combination of biophysical methods in vitro we have investigated whether aS, IAPP, and unprocessed IAPP, pro-IAPP, polypeptides can cross-react. Whereas IAPP forms amyloids within minutes, aS takes many hours to assemble into amyloids and pro-IAPP aggregates even slower under the same conditions. We discovered that preformed amyloids of proIAPP inhibit, whereas IAPP amyloids promote, aS amyloid formation. Amyloids of aS promote pro-IAPP amyloid formation, whereas they inhibit IAPP amyloid formation. In contrast, mixing of IAPP and aS monomers results in coaggregation that is faster than either protein alone; moreover, pro-IAPP can incorporate aS monomers into its amyloid fibers. From this intricate network of cross-reactivity, it is clear that the presence of IAPP can accelerate aS amyloid formation. This observation may explain why T2D patients are susceptible to developing PD.
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17.
  • Horvath, Istvan, 1979, et al. (författare)
  • In vitro analysis of α-synuclein amyloid formation and cross-reactivity
  • 2018
  • Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1940-6029 .- 1064-3745. ; 1779, s. 73-83
  • Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
    • In vitro time-resolved characterization of protein aggregation into amyloid fibers and the effects of other proteins on the aggregation process are fundamentally important measurements to obtain a better understanding of the mechanisms contributing to neurodegeneration, as well as other diseases involving amyloid formation. Here, we describe how to perform in vitro aggregation experiments with α-synuclein, the amyloidogenic protein involved in Parkinson’s disease, including how to assess the starting material, useful experimental/instrumental conditions, as well as how to set up cross-seeding and co-aggregation experiments. The high variability of data reported for in vitro α-synuclein amyloid formation may in part be explained by experimental differences.
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18.
  • Horvath, Istvan, 1979, et al. (författare)
  • Interaction between Copper Chaperone Atox1 and Parkinson's Disease Protein α-Synuclein Includes Metal-Binding Sites and Occurs in Living Cells
  • 2019
  • Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 10:11, s. 4659-4668
  • Tidskriftsartikel (refereegranskat)abstract
    • Alterations in copper ion homeostasis appear coupled to neurodegenerative disorders, but mechanisms are unknown. The cytoplasmic copper chaperone Atox1 was recently found to inhibit amyloid formation in vitro of α-synuclein, the amyloidogenic protein in Parkinson's disease. As α-synuclein may have copper-dependent functions, and free copper ions promote α-synuclein amyloid formation, it is important to characterize the Atox1 interaction with α-synuclein on a molecular level. Here we applied solution-state nuclear magnetic resonance spectroscopy, with isotopically labeled α-synuclein and Atox1, to define interaction regions in both proteins. The α-synuclein interaction interface includes the whole N-terminal part up to Gln24; in Atox1, residues around the copper-binding cysteines (positions 11-16) are mostly perturbed, but additional effects are also found for residues elsewhere in both proteins. Because α-synuclein is N-terminally acetylated in vivo, we established that Atox1 also inhibits amyloid formation of this variant in vitro, and proximity ligation in human cell lines demonstrated α-synuclein-Atox1 interactions in situ. Thus, this interaction may provide the direct link between copper homeostasis and amyloid formation in vivo.
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19.
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20.
  • Jiang, Kai, 1988, et al. (författare)
  • Alpha-Synuclein Modulates the Physical Properties of DNA
  • 2018
  • Ingår i: Chemistry - A European Journal. - : Wiley. - 1521-3765 .- 0947-6539. ; 24:58, s. 15685-15690
  • Tidskriftsartikel (refereegranskat)abstract
    • Published by Wiley-VCH Verlag GmbH & Co. KGaA. Fundamental research on Parkinson's disease (PD) most often focuses on the ability of α-synuclein (aS) to form oligomers and amyloids, and how such species promote brain cell death. However, there are indications that aS also plays a gene-regulatory role in the cell nucleus. Here, the interaction between monomeric aS and DNA in vitro has been investigated with single-molecule techniques. Using a nanofluidic channel system, it was discovered that aS binds to DNA and by studying the DNA–protein complexes at different confinements we determined that aS binding increases the persistence length of DNA from 70 to 90 nm at high coverage. By atomic force microscopy it was revealed that at low protein-to-DNA ratio, the aS binding occurs as small protein clusters scattered along the DNA; at high protein-to-DNA ratio, the DNA is fully covered by protein. As DNA-aS interactions may play roles in PD, it is of importance to characterize biophysical properties of such complexes in detail.
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21.
  • Kahra, Dana, et al. (författare)
  • Human Copper Chaperone Atox1 Translocates to the Nucleus but does not Bind DNA In Vitro.
  • 2015
  • Ingår i: Protein and peptide letters. - : Bentham Science. - 1875-5305 .- 0929-8665. ; 22:6, s. 532-8
  • Tidskriftsartikel (refereegranskat)abstract
    • After Ctr1-mediated cell uptake, copper (Cu) is transported by the cytoplasmic Cu chaperone Atox1 to P1B type ATPases ATP7A and ATP7B in the Golgi network, for incorporation into Cudependent enzymes. Atox1 is a small 68-residue protein that binds Cu in a conserved CXXC motif; it delivers Cu to target domains in ATP7A/B via direct protein-protein interactions. Specific transcription factors regulating expression of the human Cu transport proteins have not been reported although Atox1 was recently suggested to have dual functionality such that it, in addition to its cytoplasmic chaperone function, acts as a transcription factor in the nucleus. To examine this hypothesis, here we investigated the localization of Atox1 in HeLa cells using fluorescence imaging in combination with in vitro binding experiments to fluorescently labeled DNA duplexes harboring the proposed promotor sequence. We found that whereas Atox1 is present in the nucleus in HeLa cells, it does not bind to DNA in vitro. It appears that Atox1 mediates transcriptional regulation via additional (unknown) proteins.
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22.
  • Kahra, Dana, et al. (författare)
  • The C-Terminus of Human Copper Importer Ctr1 Acts as a Binding Site and Transfers Copper to Atox1
  • 2016
  • Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 110:1, s. 95-102
  • Tidskriftsartikel (refereegranskat)abstract
    • Uptake of copper (Cu) ions into human cells is mediated by the plasma membrane protein Ctr1 and is followed by Cu transfer to cytoplasmic Cu chaperones for delivery to Cu-dependent enzymes. The C-terminal cytoplasmic tail of Ctr1 is a 13-residue peptide harboring an HCH motif that is thought to interact with Cu. We here employ biophysical experiments under anaerobic conditions in peptide models of the Ctr1 C-terminus to deduce Cu-binding residues, Cu affinity, and the ability to release Cu to the cytoplasmic Cu chaperone Atox1. Based on NMR assignments and bicinchoninic acid competition experiments, we demonstrate that Cu interacts in a 1:1 stoichiometry with the HCH motif with an affinity, KD, of ∼10-14 M. Removing either the Cys residue or the two His residues lowers the Cu-peptide affinity, but site specificity is retained. The C-terminal peptide and Atox1 do not interact in solution in the absence of Cu. However, as directly demonstrated at the residue level via NMR spectroscopy, Atox1 readily acquires Cu from the Cu-loaded peptide. We propose that Cu binding to the Ctr1 C-terminal tail regulates Cu transport into the cytoplasm such that the metal ion is only released to high-affinity Cu chaperones.
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23.
  • Kiskis, Juris, 1987, et al. (författare)
  • The processes of α-synuclein amyloid protein complexes involved in the pathogenesis of Parkinson’s disease
  • 2018
  • Ingår i: Zhurnal Nevrologii i Psikhiatrii imeni S.S. Korsakova. - : Media Sphere Publishing Group. - 1997-7298. ; 118:8, s. 75-81
  • Tidskriftsartikel (refereegranskat)abstract
    • Objective. To analyze interactions between α-synuclein (αS) protein and lipids using biophysical methods. Material and methods. Recombinant α-synuclein synthesized in prokaryotic cells was used. To characterize the interaction of αS with negatively charged vesicles of DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine, sodium salt) and DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol), sodium salt) and the consequences of such interactions on αS amyloid formation, combined circular dichroism, fluorescence and imaging methods in vitro were applied. Results and conclusion. Lipid head-group chemistry modulates αS interactions and also affects amyloid fiber formation. Pre-formed αS oligomers, typically present in a small amount in the αS starting material, acted as templates for linear growth of anomalous amyloid fibers in the presence of vesicles. At the same time, the remaining αS monomers were restricted from vesicle-mediated nucleation of amyloid fibers. Although not a dominant process in bulk experiments, this hidden αS aggregation pathway may be of importance in vivo.
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24.
  • Kiskis, Juris, 1987, et al. (författare)
  • Unraveling amyloid formation paths of Parkinson's disease protein alpha-synuclein triggered by anionic vesicles
  • 2017
  • Ingår i: Quarterly Reviews of Biophysics. - : Cambridge University Press (CUP). - 1469-8994 .- 0033-5835. ; 50, s. 1-9
  • Tidskriftsartikel (refereegranskat)abstract
    • Amyloid formation of the synaptic brain protein alpha-synuclein (alpha S) is related to degeneration of dopaminergic neurons in Parkinson's disease patients. aS is thought to function in vesicle transport and fusion and it binds strongly to negatively charged vesicles in vitro. Here we combined circular dichroism, fluorescence and imaging methods in vitro to characterize the interaction of alpha S with negatively charged vesicles of DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine, sodium salt) and DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), sodium salt) and the consequences of such interactions on alpha S amyloid formation. We found that lipid head-group chemistry modulates alpha S interactions and also affects amyloid fiber formation. During the course of the experiments, we made the unexpected discovery that pre-formed alpha S oligomers, typically present in a small amount in the alpha S starting material, acted as templates for linear growth of anomalous amyloid fibers in the presence of vesicles. At the same time, the remaining alpha S monomers were restricted from vesicle-mediated nucleation of amyloid fibers. Although not a dominant process in bulk experiments, this hidden alpha S aggregation pathway may be of importance in vivo.
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25.
  • Kozak, John J., et al. (författare)
  • Geometrical Description of Protein Structural Motifs
  • 2018
  • Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 122:49, s. 11289-11294
  • Tidskriftsartikel (refereegranskat)abstract
    • We present a geometrical method that can identify secondary structural motifs in proteins via angular correlations. The method uses crystal structure coordinates to calculate angular and radial signatures of each residue relative to an external reference point as the number of nearest-neighbor residues increases. We apply our approach to the blue copper protein amicyanin using the copper cofactor as the external reference point. We define a signature termed Δβ which describes the change in angular correlation as the span of nearest neighbor residues increases. We find that three turn regions of amicyanin harbor residues with Δβ near zero, while residues in other secondary structures have Δβ greater than zero: for β-strands, Δβ changes gradually residue by residue along the strand. Extension of our analysis to other blue copper proteins demonstrated that the noted structural trends are general. Importantly, our geometrical description of the folded protein accounts for all forces holding the structure together. Through this analysis, we identified some of the turns in amicyanin as symmetrical anchor points.
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26.
  • Kumar, Ranjeet, 1980, et al. (författare)
  • Disease-causing point-mutations in metal-binding domains of Wilson disease protein decrease stability and increase structural dynamics
  • 2017
  • Ingår i: Biometals. - : Springer Science and Business Media LLC. - 0966-0844 .- 1572-8773. ; 30:1, s. 27-35
  • Tidskriftsartikel (refereegranskat)abstract
    • After cellular uptake, Copper (Cu) ions are transferred from the chaperone Atox1 to the Wilson disease protein (ATP7B) for incorporation into Cu-dependent enzymes in the secretory pathway. Human ATP7B is a large multi-domain membrane-spanning protein which, in contrast to homologues in other organisms, has six similar cytoplasmic metal-binding domains (MBDs). The reason for multiple MBDs is proposed to be indirect modulation of enzymatic activity and it is thus intriguing that point mutations in MBDs can promote Wilson disease. We here investigated, in vitro and in silico, the biophysical consequences of clinically-observed Wilson disease mutations, G85V in MBD1 and G591D in MBD6, incorporated in domain 4. Because G85 and G591 correspond to a conserved Gly found in all MBDs, we introduced the mutations in the well-characterized MBD4. We found the mutations to dramatically reduce the MBD4 thermal stability, shifting the midpoint temperature of unfolding by more than 20 °C. In contrast to wild type MBD4 and MBD4D, MBD4V adopted a misfolded structure with a large β-sheet content at high temperatures. Molecular dynamic simulations demonstrated that the mutations increased backbone fluctuations that extended throughout the domain. Our findings imply that reduced stability and enhanced dynamics of MBD1 or MBD6 is the origin of ATP7B dysfunction in Wilson disease patients with the G85V or G591D mutation.
  •  
27.
  • Köhn, Birgit, et al. (författare)
  • A Luminal Loop of Wilson Disease Protein Binds Copper and Is Required for Protein Activity
  • 2018
  • Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 115:6, s. 1007-1018
  • Tidskriftsartikel (refereegranskat)abstract
    • The copper-transporting ATPase ATP7B is essential for loading of copper ions to copper-dependent enzymes in the secretory pathway; its inactivation results in Wilson disease. In contrast to copper-ion uptake by the cytoplasmic domains, ATP7B-mediated copper-ion release in the Golgi has not been explored yet. We demonstrate here that a luminal loop in ATP7B, rich in histidine/methionine residues, binds reduced copper (Cu(I)) ions, and identified copper-binding residues play an essential role in ATP7B-mediated metal ion release. NMR experiments on short-peptide models demonstrate that three methionine and two histidine residues are specifically involved in Cu(I) ion binding; with these residues replaced by alanines, no Cu(I) ion interaction is detected. Although more than one Cu(I) ion can interact with the wild-type peptide, removing either all histidine or all methionine residues reduces the stoichiometry to one Cu(I) ion binding per peptide. Using a yeast complementation assay, we show that for efficient copper transport by full-length ATP7B, the complete set of histidine and methionine residues in the lumen loop are required. The replacement of histidine/methionine residues by alanines does not perturb overall ATP7B structure, as the localization of ATP7B variants in yeast cells matches that of the wild-type protein. Thus, in similarity to ATP7A, ATP7B also appears to have a luminal “exit” copper ion site.
  •  
28.
  • Matson Dzebo, Maria, 1985, et al. (författare)
  • Copper Chaperone Atox1 Interacts with Cell Cycle Proteins
  • 2018
  • Ingår i: Computational and Structural Biotechnology Journal. - : Elsevier BV. - 2001-0370. ; 16, s. 443-449
  • Tidskriftsartikel (refereegranskat)abstract
    • The anaphase-promoting complex (APC) is involved in several processes in the cell cycle, most prominently it facilitates the separation of the sister chromatids during mitosis, before cell division. Because of the key role in the cell cycle, APC is suggested as a putative target for anticancer agents. We here show that the copper chaperone Atox1, known for shuttling copper in the cytoplasm from Ctr1 to ATP7A/B in the secretory pathway, interacts with several APC subunits. Atox1 interactions with APC subunits were discovered by mass spectrometry of co-immunoprecipitated samples and further confirmed using proximity ligation assays in HEK293T cells. Upon comparing wild-type cells with those in which the Atox1 gene had been knocked out, we found that in the absence of Atox1 protein, cells have prolonged G2/M phases and a slower proliferation rate. Thus, in addition to copper transport for loading of copper-dependent enzymes, Atox1 may modulate the cell cycle by interacting with APC subunits.
  •  
29.
  • Matson Dzebo, Maria, 1985, et al. (författare)
  • Extended functional repertoire for human copper chaperones
  • 2016
  • Ingår i: Biomolecular Concepts. - : Walter de Gruyter GmbH. - 1868-5021 .- 1868-503X. ; 7:1, s. 29-39
  • Tidskriftsartikel (refereegranskat)abstract
    • Copper (Cu) ions are cofactors in many essential enzymes. As free Cu ions are toxic, most organisms have highly specialized Cu transport systems involving dedicated proteins. The human cytoplasmic Cu chaperone Atox1 delivers Cu to P1B-type ATPases in the Golgi network, for incorporation into Cu-dependent enzymes following the secretory path. Atox1 homologs are found in most organisms; it is a 68-residue ferredoxin-fold protein that binds Cu in a conserved surface-exposed CXXC motif. In addition to Atox1, the human cytoplasm also contains Cu chaperones for loading of superoxide dismutase 1 (i.e. CCS) and cytochrome c oxidase in mitochondria (i.e. Cox17). Many mechanistic aspects have been resolved with respect to how Cu ions are moved between these proteins. In addition to the primary cytoplasmic Cu chaperone function, all three cytoplasmic chaperones have been reported to have other interaction partners that are involved in signaling pathways that modulate cell growth and development. These new discoveries imply that humans have evolved a highly sophisticated network of control mechanisms that connect Cu transport with cell regulatory processes. This knowledge may eventually be exploited for future drug developments towards diseases such as cancer and neurodegenerative disorders.
  •  
30.
  • Mondol, Tanumoy, et al. (författare)
  • Copper binding triggers compaction in N-terminal tail of human copper pump ATP7B
  • 2016
  • Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 470:3, s. 663-669
  • Tidskriftsartikel (refereegranskat)abstract
    • Protein conformational changes are fundamental to biological reactions. For copper ion transport, the multi-domain protein ATP7B in the Golgi network receives copper from the cytoplasmic copper chaperone Atox1 and, with energy from ATP hydrolysis, moves the metal to the lumen for loading of copper dependent enzymes. Although anticipated, conformational changes involved in ATP7B's functional cycle remain elusive. Using spectroscopic methods we here demonstrate that the four most N-terminal metal binding domains in ATP7B, upon stoichiometric copper addition, adopt a more compact arrangement which has a higher thermal stability than in the absence of copper. In contrast to previous reports, no stable complex was found in solution between the metal-binding domains and the nucleotide-binding domain of ATP7B. Metal-dependent movement of the first four metal-binding domains in ATP7B may be a trigger that initiates the overall catalytic cycle.
  •  
31.
  • Niemiec, Moritz S., et al. (författare)
  • Enthalpy-entropy compensation at play in human copper ion transfer.
  • 2015
  • Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 5, s. 10518-
  • Tidskriftsartikel (refereegranskat)abstract
    • Copper (Cu) is an essential trace element but toxic in free form. After cell uptake, Cu is transferred, via direct protein-protein interactions, from the chaperone Atox1 to the Wilson disease protein (WD) for incorporation into Cu-dependent enzymes. Cu binds to a conserved C(1)XXC(2) motif in the chaperone as well as in each of the cytoplasmic metal-binding domains of WD. Here, we dissect mechanism and thermodynamics of Cu transfer from Atox1 to the fourth metal binding domain of WD. Using chromatography and calorimetry together with single Cys-to-Ala variants, we demonstrate that Cu-dependent protein heterocomplexes require the presence of C(1) but not C(2). Comparison of thermodynamic parameters for mutant versus wild type reactions reveals that the wild type reaction involves strong entropy-enthalpy compensation. This property is explained by a dynamic inter-conversion of Cu-Cys coordinations in the wild type ensemble and may provide functional advantage by protecting against Cu mis-ligation and bypassing enthalpic traps.
  •  
32.
  • Nors Perdersen, Martin, et al. (författare)
  • Direct Correlation Between Ligand-Induced α-Synuclein Oligomers and Amyloid-like Fibril Growth.
  • 2015
  • Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 5, s. 10422-
  • Tidskriftsartikel (refereegranskat)abstract
    • Aggregation of proteins into amyloid deposits is the hallmark of several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. The suggestion that intermediate oligomeric species may be cytotoxic has led to intensified investigations of pre-fibrillar oligomers, which are complicated by their transient nature and low population. Here we investigate alpha-synuclein oligomers, enriched by a 2-pyridone molecule (FN075), and the conversion of oligomers into fibrils. As probed by leakage assays, the FN075 induced oligomers potently disrupt vesicles in vitro, suggesting a potential link to disease related degenerative activity. Fibrils formed in the presence and absence of FN075 are indistinguishable on microscopic and macroscopic levels. Using small angle X-ray scattering, we reveal that FN075 induced oligomers are similar, but not identical, to oligomers previously observed during alpha-synuclein fibrillation. Since the levels of FN075 induced oligomers correlate with the amounts of fibrils among different FN075:protein ratios, the oligomers appear to be on-pathway and modeling supports an 'oligomer stacking model' for alpha-synuclein fibril elongation.
  •  
33.
  • Ohrvik, Helena, et al. (författare)
  • Identification of New Potential Interaction Partners for Human Cytoplasmic Copper Chaperone Atox1: Roles in Gene Regulation?
  • 2015
  • Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 16:8, s. 16728-39
  • Tidskriftsartikel (refereegranskat)abstract
    • The human copper (Cu) chaperone Atox1 delivers Cu to P1B type ATPases in the Golgi network, for incorporation into essential Cu-dependent enzymes. Atox1 homologs are found in most organisms; it is a 68-residue ferredoxin-fold protein that binds Cu in a conserved surface-exposed Cys-X-X-Cys (CXXC) motif. In addition to its well-documented cytoplasmic chaperone function, in 2008 Atox1 was suggested to have functionality in the nucleus. To identify new interactions partners of Atox1, we performed a yeast two-hybrid screen with a large human placenta library of cDNA fragments using Atox1 as bait. Among 98 million fragments investigated, 25 proteins were found to be confident interaction partners. Nine of these were uncharacterized proteins, and the remaining 16 proteins were analyzed by bioinformatics with respect to cell localization, tissue distribution, function, sequence motifs, three-dimensional structures and interaction networks. Several of the hits were eukaryotic-specific proteins interacting with DNA or RNA implying that Atox1 may act as a modulator of gene regulation. Notably, because many of the identified proteins contain CXXC motifs, similarly to the Cu transport reactions, interactions between these and Atox1 may be mediated by Cu.
  •  
34.
  • Petzoldt, Svenja, et al. (författare)
  • Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro.
  • 2015
  • Ingår i: Biometals. - : Springer Science and Business Media LLC. - 0966-0844 .- 1572-8773. ; 28:3, s. 577-85
  • Tidskriftsartikel (refereegranskat)abstract
    • After Ctr1-mediated copper ion (Cu) entry into the human cytoplasm, chaperones Atox1 and CCS deliver Cu to P1B-type ATPases and to superoxide dismutase, respectively, via direct protein-protein interactions. Although the two Cu chaperones are presumed to work along independent pathways, we here assessed cross-reactivity between Atox1 and the first domain of CCS (CCS1) using biochemical and biophysical methods in vitro. By NMR we show that CCS1 is monomeric although it elutes differently from Atox1 in size exclusion chromatography (SEC). This property allows separation of Atox1 and CCS1 by SEC and, combined with the 254/280 nm ratio as an indicator of Cu loading, we demonstrate that Cu can be transferred from one protein to the other. Cu exchange also occurs with full-length CCS and, as expected, the interaction involves the metal binding sites since mutation of Cu-binding cysteine in Atox1 eliminates Cu transfer from CCS1. Cross-reactivity between CCS and Atox1 may aid in regulation of Cu distribution in the cytoplasm.
  •  
35.
  • Pokrzywa, M., et al. (författare)
  • Effects of small-molecule amyloid modulators on a Drosophila model of Parkinson's disease
  • 2017
  • Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 12:9, s. Aet no e0184117-
  • Tidskriftsartikel (refereegranskat)abstract
    • Alpha-synuclein (aS) amyloid formation is involved in Parkinson's disease (PD); therefore, small molecules that target aS and affect its aggregation are of interest as future drug candidates. We recently reported modified ring-fused 2-pyridones that modulate aS amyloid formation in vitro. Here, we describe the effects of such molecules on behavioral parameters of a Drosophila model of PD (i.e., flies expressing human aS), using a new approach (implemented in a commercially available FlyTracker system) to quantify fly mobility. FlyTracker allows for automated analysis of walking and climbing locomotor behavior, as it collects large sequences of data over time in an unbiased manner. We found that the molecules per se have no toxic or kinetic effects on normal flies. Feeding aS-expressing flies with the amyloid-promoting molecule FN075, remarkably, resulted in increased fly mobility at early time points; however, this effect switched to reduced mobility at later time points, and flies had shorter life spans than controls. In contrast, an amyloid inhibitor increased both fly kinetics and life span. In agreement with increased aS amyloid formation, the FN075-fed flies had less soluble aS, and in vitro aS-FN075 interactions stimulated aS amyloid formation. In addition to a new quantitative approach to probe mobility (available in FlyTracker), our results imply that aS regulates brain activity such that initial removal (here, by FN075-triggered assembly of aS) allows for increased fly mobility.
  •  
36.
  • Ponnandai Schanmugavel, Kumaravel, 1991, et al. (författare)
  • Copper relay path through the N-terminus of Wilson disease protein, ATP7B
  • 2019
  • Ingår i: Metallomics. - : Oxford University Press (OUP). - 1756-5901 .- 1756-591X. ; 11:9, s. 1472-1480
  • Tidskriftsartikel (refereegranskat)abstract
    • In human cells, copper (Cu) ions are transported by the cytoplasmic Cu chaperone Atox1 to the Wilson disease protein (ATP7B) in the Golgi for loading of Cu-dependent enzymes. ATP7B is a membrane-spanning protein which, in contrast to non-mammalian homologs, has six cytoplasmic metal-binding domains (MBDs). To address the reason for multiple MBDs, we introduced strategic mutations in which one, two or three MBDs had been blocked for Cu binding via cysteine-to-serine mutations (but all six MBDs are present in all) in a yeast system that probes Cu flow through Atox1 and ATP7B. The results, combined with earlier work, support a mechanistic model in which MBD1-3 forms a regulatory unit of ATP7B Cu transport. Cu delivery via Atox1 to this unit, followed by loading of Cu in MBD3, promotes release of inhibitory interactions. Whereas the Cu site in MBD4 can be mutated without a large effect, an intact Cu site in either MBD5 or MBD6 is required for Cu transport. All MBDs, expressed as single-domain proteins, can replace Atox1 and deliver Cu to full-length ATP7B. However, only MBD6 can deliver Cu to truncated ATP7B where all six MBDs are removed, suggesting a docking role for this structural unit
  •  
37.
  • Ponnandai Schanmugavel, Kumaravel, 1991, et al. (författare)
  • Probing functional roles of Wilson disease protein (ATP7B) copper-binding domains in yeast
  • 2017
  • Ingår i: Metallomics. - : Oxford University Press (OUP). - 1756-5901 .- 1756-591X. ; 9:7, s. 981-988
  • Tidskriftsartikel (refereegranskat)abstract
    • After Ctr1-mediated uptake into human cells, copper (Cu) ions are transported by the cytoplasmic Cu chaperone Atox1 to the Wilson disease protein (ATP7B) in the Golgi network. Cu transfer occurs via direct protein-protein interactions and leads to incorporation of Cu into Cu-dependent enzymes. ATP7B is a large multi-domain membrane-spanning protein which, in contrast to homologs, has six cytoplasmic metal-binding domains (MBDs). The reason for multiple MBDs is proposed to be indirect modulation of activity but mechanistic studies of full-length ATP7B are limited. We here developed a system that probes Cu flow through human Atox1 and ATP7B proteins when expressed in yeast. Using this assay, we assessed the roles of the different MBDs in ATP7B and found that the presence of the most N-terminal MBD increased, whereas the third MBD decreased, overall ATP7B-mediated Cu transport activity. Upon removal of all MBDs in ATP7B, the ability to transport Cu disappeared. The designed system can be expanded to include other yeast viability parameters and will be a useful tool for further mechanistic insights on human Cu transport as well as diseases involving Cu imbalance.
  •  
38.
  • Ponnandai Schanmugavel, Kumaravel, 1991, et al. (författare)
  • Wilson disease missense mutations in ATP7B affect metal-binding domain structural dynamics
  • 2019
  • Ingår i: Biometals. - : Springer Science and Business Media LLC. - 0966-0844 .- 1572-8773. ; 32:6, s. 875-885
  • Tidskriftsartikel (refereegranskat)abstract
    • Wilson disease (WD) is caused by mutations in the gene for ATP7B, a copper transport protein that regulates copper levels in cells. A large number of missense mutations have been reported to cause WD but genotype–phenotype correlations are not yet established. Since genetic screening for WD may become reality in the future, it is important to know how individual mutations affect ATP7B function, with the ultimate goal to predict pathophysiology of the disease. To begin to assess mechanisms of dysfunction, we investigated four proposed WD-causing missense mutations in metal-binding domains 5 and 6 of ATP7B. Three of the four variants showed reduced ATP7B copper transport ability in a traditional yeast assay. To probe mutation-induced structural dynamic effects at the atomic level, molecular dynamics simulations (1.5 μs simulation time for each variant) were employed. Upon comparing individual metal-binding domains with and without mutations, we identified distinct differences in structural dynamics via root-mean square fluctuation and secondary structure content analyses. Most mutations introduced distant effects resulting in increased dynamics in the copper-binding loop. Taken together, mutation-induced long-range alterations in structural dynamics provide a rationale for reduced copper transport ability.
  •  
39.
  • Ribeiro, Diana, 1987, et al. (författare)
  • 3D-Models of Insulin-Producing β-Cells: from Primary Islet Cells to Stem Cell-Derived Islets
  • 2018
  • Ingår i: Stem Cell Reviews and Reports. - : Springer Science and Business Media LLC. - 1558-6804 .- 1550-8943. ; 14:2, s. 177-188
  • Forskningsöversikt (refereegranskat)abstract
    • There is a need for physiologically relevant assay platforms to provide functionally relevant models of diabetes, to accelerate the discovery of new treatment options and boost developments in drug discovery. In this review, we compare several 3D-strategies that have been used to increase the functional relevance of ex vivo human primary pancreatic islets and developments into the generation of stem cell derived pancreatic beta-cells (β-cells). Special attention will be given to recent approaches combining the use of extracellular matrix (ECM) scaffolds with pancreatic molecular memory, which can be used to improve yield and functionality of in vitro stem cell-derived pancreatic models. The ultimate goal is to develop scalable cell-based platforms for diabetes research and drug screening. This article will critically assess key aspects related to in vitro pancreatic 3D-ECM models and highlight the most promising approaches for future research.
  •  
40.
  • Ribeiro, Diana, 1987, et al. (författare)
  • Extracellular vesicles from human pancreatic islets suppress human islet amyloid polypeptide amyloid formation
  • 2017
  • Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:42, s. 11127-11132
  • Tidskriftsartikel (refereegranskat)abstract
    • Extracellular vesicles (EVs) are small vesicles released by cells to aid cell-cell communication and tissue homeostasis. Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in pancreatic islets of patients with type 2 diabetes (T2D). IAPP is secreted in conjunction with insulin from pancreatic beta cells to regulate glucose metabolism. Here, using a combination of analytical and biophysical methods in vitro, we tested whether EVs isolated from pancreatic islets of healthy patients and patients with T2D modulate IAPP amyloid formation. We discovered that pancreatic EVs from healthy patients reduce IAPP amyloid formation by peptide scavenging, but T2D pancreatic and human serum EVs have no effect. In accordance with these differential effects, the insulin: C-peptide ratio and lipid composition differ between EVs from healthy pancreas and EVs from T2D pancreas and serum. It appears that healthy pancreatic EVs limit IAPP amyloid formation via direct binding as a tissue-specific control mechanism.
  •  
41.
  • Rocha, Sandra, 1975, et al. (författare)
  • Synaptic vesicle mimics affect the aggregation of wild-type and A53T α-synuclein variants differently albeit similar membrane affinity
  • 2019
  • Ingår i: Protein engineering, design & selection : PEDS. - : Oxford University Press (OUP). - 1741-0134 .- 1741-0126. ; 32:2, s. 59-66
  • Tidskriftsartikel (refereegranskat)abstract
    • Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com. α-Synuclein misfolding results in the accumulation of amyloid fibrils in Parkinson's disease. Missense protein mutations (e.g. A53T) have been linked to early onset disease. Although α-synuclein interacts with synaptic vesicles in the brain, it is not clear what role they play in the protein aggregation process. Here, we compare the effect of small unilamellar vesicles (lipid composition similar to synaptic vesicles) on wild-type (WT) and A53T α-synuclein aggregation. Using biophysical techniques, we reveal that binding affinity to the vesicles is similar for the two proteins, and both interact with the helix long axis parallel to the membrane surface. Still, the vesicles affect the aggregation of the variants differently: effects on secondary processes such as fragmentation dominate for WT, whereas for A53T, fibril elongation is mostly affected. We speculate that vesicle interactions with aggregate intermediate species, in addition to monomer binding, vary between WT and A53T, resulting in different consequences for amyloid formation. © The Author(s) 2019.
  •  
42.
  • Sampson, Timothy R, et al. (författare)
  • Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson's Disease.
  • 2016
  • Ingår i: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 167:6, s. 1469-1480.e12
  • Tidskriftsartikel (refereegranskat)abstract
    • The intestinal microbiota influence neurodevelopment, modulate behavior, and contribute to neurological disorders. However, a functional link between gut bacteria and neurodegenerative diseases remains unexplored. Synucleinopathies are characterized by aggregation of the protein α-synuclein (αSyn), often resulting in motor dysfunction as exemplified by Parkinson's disease (PD). Using mice that overexpress αSyn, we report herein that gut microbiota are required for motor deficits, microglia activation, and αSyn pathology. Antibiotic treatment ameliorates, while microbial re-colonization promotes, pathophysiology in adult animals, suggesting that postnatal signaling between the gut and the brain modulates disease. Indeed, oral administration of specific microbial metabolites to germ-free mice promotes neuroinflammation and motor symptoms. Remarkably, colonization of αSyn-overexpressing mice with microbiota from PD-affected patients enhances physical impairments compared to microbiota transplants from healthy human donors. These findings reveal that gut bacteria regulate movement disorders in mice and suggest that alterations in the human microbiome represent a risk factor for PD.
  •  
43.
  • Sedlák, Erik, et al. (författare)
  • Synergistic effects of copper sites on apparent stability of multicopper oxidase, Fet3p
  • 2018
  • Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 19:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Saccharomyces cerevisiae Fet3p is a multicopper oxidase that contains three cupredoxin-like domains and four copper ions located in three distinct metal sites (T1 in domain 3; T2 and the binuclear T3 at the interface between domains 1 and 3). To probe the role of the copper sites in Fet3p thermodynamic stability, we performed urea-induced unfolding experiments with holo-, apoand three partially-metallated (T1, T2 and T1/T2 sites depleted of copper) forms of Fet3p. Using a combination of spectroscopic probes (circular dichroism, fluorescence intensity and maximum, 8-anilinonaphthalene-1-sulfonic acid (ANS) emission, oxidase activity and blue color), we reveal that all forms of Fet3p unfold in a four-state reaction with two partially-folded intermediates. Using phase diagrams, it emerged that Fet3p with all copper sites filled had a significantly higher stability as compared to the combined contributions of the individual copper sites. Hence, there is long-range inter-domain communication between distal copper sites that contribute to overall Fet3p stability.
  •  
44.
  • Sharma, Sandeep K, et al. (författare)
  • Insulin-degrading enzyme is activated by the C-terminus of α-synuclein.
  • 2015
  • Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 466:2, s. 192-5
  • Tidskriftsartikel (refereegranskat)abstract
    • The insulin-degrading enzyme (IDE) plays a key role in type-2 diabetes and typically degrades small peptides such as insulin, amyloid β and islet amyloid polypeptide. We recently reported a novel non-proteolytical interaction in vitro between IDE and the Parkinson's disease 140-residue protein α-synuclein that resulted in dual effects: arrested α-synuclein oligomers and, simultaneously, increased IDE proteolysis activity. Here we demonstrate that these outcomes arise due to IDE interactions with the C-terminus of α-synuclein. Whereas a peptide containing the first 97 residues of α-synuclein did not improve IDE activity and its aggregation was not blocked by IDE, a peptide with the C-terminal 44 residues of α-synuclein increased IDE proteolysis to the same degree as full-length α-synuclein. Because the α-synuclein C-terminus is acidic, the interaction appears to involve electrostatic attraction with IDE's basic exosite, known to be involved in activation.
  •  
45.
  • Sharma, Sandeep K, et al. (författare)
  • Insulin-degrading enzyme prevents α-synuclein fibril formation in a nonproteolytical manner.
  • 2015
  • Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 5, s. 12531-
  • Tidskriftsartikel (refereegranskat)abstract
    • The insulin-degrading enzyme (IDE) degrades amyloidogenic proteins such as Amyloid β (Αβ) and Islet Amyloid Polypeptide (IAPP), i.e. peptides associated with Alzheimer's disease and type 2 diabetes, respectively. In addition to the protease activity normally associated with IDE function an additional activity involving the formation of stable, irreversible complexes with both Αβ and α-synuclein, an amyloidogenic protein involved in Parkinson's disease, was recently proposed. Here, we have investigated the functional consequences of IDE-α-synuclein interactions in vitro. We demonstrate that IDE in a nonproteolytic manner and at sub-stoichiometric ratios efficiently inhibits α-synuclein fibril formation by binding to α-synuclein oligomers making them inert to amyloid formation. Moreover, we show that, within a defined range of α-synuclein concentrations, interaction with α-synuclein oligomers increases IDE's proteolytic activity on a fluorogenic substrate. We propose that the outcomes of IDE-α-synuclein interactions, i.e. protection against α-synuclein amyloid formation and stimulated IDE protease activity, may be protective in vivo.
  •  
46.
  • Singh, Pardeep, et al. (författare)
  • Synthesis of Multiring Fused 2-Pyridones via a Nitrene Insertion Reaction: Fluorescent Modulators of α-Synuclein Amyloid Formation
  • 2015
  • Ingår i: Organic Letters. - : American Chemical Society (ACS). - 1523-7052 .- 1523-7060. ; 17:24, s. 6194-6197
  • Tidskriftsartikel (refereegranskat)abstract
    • © 2015 American Chemical Society. An efficient, straightforward method for the synthesis of thiazolo-2-pyridone embedded peptidomimetic polyheterocycles via a catalyst-free, microwave-assisted, intramolecular C-H amination reaction is reported. All the synthesized polyheterocycles were evaluated for their fluorescent properties and effect on α-synuclein amyloid formation.
  •  
47.
  • Wands, Amberlyn M., et al. (författare)
  • Fucosylated Molecules Competitively Interfere with Cholera Toxin Binding to Host Cells
  • 2018
  • Ingår i: ACS Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 4:5, s. 758-770
  • Tidskriftsartikel (refereegranskat)abstract
    • Cholera toxin (CT) enters host intestinal epithelia cells, and its retrograde transport to the cytosol results in the massive loss of fluids and electrolytes associated with severe dehydration. To initiate this intoxication process, the B subunit of CT (CTB) first binds to a cell surface receptor displayed on the apical surface of the intestinal epithelia. While the monosialoganglioside GM1 is widely accepted to be the sole receptor for CT, intestinal epithelial cell lines also utilize fucosylated glycan epitopes on glycoproteins to facilitate cell surface binding and endocytic uptake of the toxin. Further, l-fucose can competively inhibit CTB binding to intestinal epithelia cells. Here, we use competition binding assays with l-fucose analogs to decipher the molecular determinants for l-fucose inhibition of cholera toxin subunit B (CTB) binding. Additionally, we find that mono- and difucosylated oligosaccharides are more potent inhibitors than l-fucose alone, with the LeY tetrasaccharide emerging as the most potent inhibitor of CTB binding to two colonic epithelial cell lines (T84 and Colo205). Finally, a non-natural fucose-containing polymer inhibits CTB binding two orders of magnitude more potently than the LeY glycan when tested against Colo205 cells. This same polymer also inhibits CTB binding to T84 cells and primary human jejunal epithelial cells in a dose-dependent manner. These findings suggest the possibility that polymeric display of fucose might be exploited as a prophylactic or therapeutic approach to block the action of CT toward the human intestinal epithelium.
  •  
48.
  • Werner, Tony, 1990, et al. (författare)
  • Abundant fish protein inhibits α-synuclein amyloid formation
  • 2018
  • Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 8:1
  • Tidskriftsartikel (refereegranskat)abstract
    • The most common allergen in fish, the highly-abundant protein β-parvalbumin, forms amyloid structures as a way to avoid gastrointestinal degradation and transit to the blood. In humans, the same amyloid structures are mostly associated with neurodegenerative disorders such as Alzheimer's and Parkinson's. We here assessed a putative connection between these amyloids using recombinant Atlantic cod β-parvalbumin and the key amyloidogenic protein in Parkinson's disease, α-synuclein. Using a set of in vitro biophysical methods, we discovered that β-parvalbumin readily inhibits amyloid formation of α-synuclein. The underlying mechanism was found to involve α-synuclein binding to the surface of β-parvalbumin amyloid fibers. In addition to being a new amyloid inhibition mechanism, the data suggest that health benefits of fish may be explained in part by cross-reaction of β-parvalbumin with human amyloidogenic proteins.
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49.
  • Wittung Stafshede, Pernilla, 1968 (författare)
  • A Copper Story: From Protein Folding and Metal Transport to Cancer
  • 2016
  • Ingår i: Israel Journal of Chemistry. - : Wiley. - 0021-2148. ; 56:9-10, s. 671-681
  • Forskningsöversikt (refereegranskat)abstract
    • Enzymes need to fold into unique three-dimensional structures in order to function. Copper ions are cofactors in many essential enzymes. Such enzymes need to couple polypeptide folding with metal incorporation, as the metal sites are often integrated within the folded structure. Since free copper ions are toxic, most organisms have highly specialized copper transport systems. The human cytoplasmic copper chaperone Atox1 delivers copper to P-1B-type ATPases in the Golgi, for incorporation into copper-dependent enzymes following the secretory path. Copper plays key roles in cancer development, as copper-dependent enzymes are needed for tumor growth and metastasis. In addition, platinum-based drugs are exported out of cells by the copper transport machinery. Recent findings also imply that some copper transport proteins regulate cell growth and development. In this brief journey of my later career, I will discuss the roles of copper in protein folding, mechanisms of copper ion transport, and cisplatin hitchhiking. The identification of new partners for Atox1 underscore the importance of further research in this area for combating cancer.
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50.
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