| 1. |
- 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.
|
|
| 2. |
- Blockhuys, Stephanie, 1983, et al.
(författare)
-
Single-cell tracking demonstrates copper chaperone Atox1 to be required for breast cancer cell migration
- 2020
-
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. ; 117:4, s. 2014-2019
-
Tidskriftsartikel (refereegranskat)abstract
- Copper ions are needed for several hallmarks of cancer. However, the involved pathways, mechanisms, and copper-binding proteins are mostly unknown. We recently found that cytoplasmic Antioxidant 1 copper chaperone (Atox1), which is up-regulated in breast cancer, is localized at the lamellipodia edges of aggressive breast cancer cells. To reveal molecular insights into a putative role in cell migration, we here investigated breast cancer cell (MDA-MB-231) migration by video microscopy as a function of Atox1. Tracking of hundreds of individual cells (per condition) over a 9-h time series revealed that cell migration velocity and directionality are significantly reduced upon Atox1 silencing in the cells. Because silencing of the copper transporter ATP7A also reduced cell migration, these proteins appear to be on the same pathway, suggesting that their well-known copper transport activity is involved. In-cell proximity ligation assays demonstrated that Atox1, ATP7A, and the proenzyme of lysyl oxidase (LOX; copper-loaded via ATP7A) are all in close proximity and that LOX activity is reduced upon Atox1 silencing in the cells. Since LOX is an established player in cancer cell migration, our results imply that Atox1 mediates breast cancer cell migration via coordinated copper transport in the ATP7A-LOX axis. Because individual cell migration is an early step in breast cancer metastasis, Atox1 levels in tumor cells may be a predictive measure of metastasis potential and serve as a biomarker for copper depletion therapy.
|
|
| 3. |
- Grasso, Michael, et al.
(författare)
-
The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation
- 2021
-
Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 297:6
-
Tidskriftsartikel (refereegranskat)abstract
- Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAFMEK- ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.
|
|
| 4. |
- Horn, Nina, et al.
(författare)
-
Atp7a-regulated enzyme metalation and trafficking in the menkes disease puzzle
- 2021
-
Ingår i: Biomedicines. - : MDPI AG. - 2227-9059. ; 9:4
-
Forskningsöversikt (refereegranskat)abstract
- Copper is vital for numerous cellular functions affecting all tissues and organ systems in the body. The copper pump, ATP7A is critical for whole-body, cellular, and subcellular copper homeostasis, and dysfunction due to genetic defects results in Menkes disease. ATP7A dysfunction leads to copper deficiency in nervous tissue, liver, and blood but accumulation in other tissues. Site-specific cellular deficiencies of copper lead to loss of function of copper-dependent enzymes in all tissues, and the range of Menkes disease pathologies observed can now be explained in full by lack of specific copper enzymes. New pathways involving copper activated lysosomal and steroid sulfatases link patient symptoms usually related to other inborn errors of metabolism to Menkes disease. Additionally, new roles for lysyl oxidase in activation of molecules necessary for the innate immune system, and novel adapter molecules that play roles in ERGIC trafficking of brain receptors and other proteins, are emerging. We here summarize the current knowledge of the roles of copper enzyme function in Menkes disease, with a focus on ATP7A-mediated enzyme metalation in the secretory pathway. By establishing mechanistic relationships between copper-dependent cellular processes and Menkes disease symptoms in patients will not only increase understanding of copper biology but will also allow for the identification of an expanding range of copper-dependent enzymes and pathways. This will raise awareness of rare patient symptoms, and thus aid in early diagnosis of Menkes disease patients.
|
|
| 5. |
- 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.
|
|
| 6. |
- 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.
|
|
| 7. |
- 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.
|
|
| 8. |
- Jiang, Kai, 1988, et al.
(författare)
-
C-terminal truncation of α-synuclein alters DNA structure from extension to compaction
- 2021
-
Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 568, s. 43-47
-
Tidskriftsartikel (refereegranskat)abstract
- Parkinson's disease (PD) is linked to aggregation of the protein α-synuclein (aS) into amyloid fibers. aS is proposed to regulate synaptic activity and may also play a role in gene regulation via interaction with DNA in the cell nucleus. Here, we address the role of the negatively-charged C-terminus in the interaction between aS and DNA using single-molecule techniques. Using nanofluidic channels, we demonstrate that truncation of the C-terminus of aS induces differential effects on DNA depending on the extent of the truncation. The DNA extension increases for full-length aS and the (1–119)aS variant, but decreases about 25% upon binding to the (1–97)aS variant. Atomic force microscopy imaging showed full protein coverage of the DNA at high aS concentration. The characterization of biophysical properties of DNA when in complex with aS variants may provide important insights into the role of such interactions in PD, especially since C-terminal aS truncations have been found in clinical samples from PD patients.
|
|
| 9. |
- 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.
|
|
| 10. |
- Lorentzon, Emma, 1995, et al.
(författare)
-
Differential effects of Cu2+ and Fe3+ ions on in vitro amyloid formation of biologically-relevant α-synuclein variants
- 2020
-
Ingår i: Biometals. - : Springer Science and Business Media LLC. - 0966-0844 .- 1572-8773. ; 33:2-3, s. 97-106
-
Tidskriftsartikel (refereegranskat)abstract
- Alterations in metal ion homeostasis appear coupled to neurodegenerative disorders but mechanisms are unknown. Amyloid formation of the protein α-synuclein in brain cells is a hallmark of Parkinson’s disease. α-Synuclein can bind several metal ions in vitro and such interactions may affect the assembly process. Here we used biophysical methods to study the effects of micromolar concentrations of Cu2+ and Fe3+ ions on amyloid formation of selected α-synuclein variants (wild-type and A53T α-synuclein, in normal and N-terminally acetylated forms). As shown previously, Cu2+ speeds up aggregation of normal wild-type α-synuclein, but not the acetylated form. However, Cu2+ has a minimal effect on (the faster) aggregation of normal A53T α-synuclein, despite that Cu2+ binds to this variant. Like Cu2+, Fe3+ speeds up aggregation of non-acetylated wild-type α-synuclein, but with acetylation, Fe3+ instead slows down aggregation. In contrast, for A53T α-synuclein, regardless of acetylation, Fe3+ slows down aggregation with the effect being most dramatic for acetylated A53T α-synuclein. The results presented here suggest a correlation between metal-ion modulation effect and intrinsic aggregation speed of the various α-synuclein variants.
|
|
