| 1. |
- Aguilar, Ximena, 1978-, et al.
(författare)
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Macromolecular crowding extended to a heptameric system : the co-chaperonin protein 10
- 2011
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 50:14, s. 3034-3044
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Tidskriftsartikel (refereegranskat)abstract
- Experiments on monomeric proteins have shown that macromolecular crowding can stabilize toward heat perturbation and also modulate native-state structure. To assess the effects of macromolecular crowding on unfolding of an oligomeric protein, we here tested the effects of the synthetic crowding agent Ficoll 70 on human cpn10 (GroES in E. coli), a heptameric protein consisting of seven identical β-barrel subunits assembling into a ring. Using far-UV circular dichroism (CD), tyrosine fluorescence, nuclear magnetic resonance (NMR), and cross-linking experiments, we investigated thermal and chemical stability, as well as the heptamer-monomer dissociation constant, without and with crowding agent. We find that crowding shifts the heptamer-monomer equilibrium constant in the direction of the heptamer. The cpn10 heptamer is both thermally and thermodynamically stabilized in 300 mg/mL Ficoll 70 as compared to regular buffer conditions. Kinetic unfolding experiments show that the increased stability in crowded conditions, in part, is explained by slower unfolding rates. A thermodynamic cycle reveals that in presence of 300 mg/mL Ficoll the thermodynamic stability of each cpn10 monomer increases by over 30%, whereas the interfaces are stabilized by less than 10%. We also introduce a new approach to analyze the spectroscopic data that makes use of multiple wavelengths: this provides robust error estimates of thermodynamic parameters.
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| 2. |
- Ardhammar, Malin, 1970, et al.
(författare)
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In vitro membrane penetration of modified peptide nucleic acid (PNA)
- 1999
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Ingår i: Journal of Biomolecular Structure and Dynamics. - : Informa UK Limited. - 0739-1102 .- 1538-0254. ; 17:1, s. 33-40
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Tidskriftsartikel (refereegranskat)abstract
- Efficient cellular uptake is crucial for the success of any drug directed towards targets inside cells. Peptide nucleic acid (PNA), a DNA analog with a promising potential as a gene-directed drug, has been shown to display slow membrane penetration in cell cultures. We here used liposomes as an in vitro model of cell membranes to investigate the effect on penetration of a PNA molecule colvalently modified with a lipophilic group, an adamantyl moiety. The adamantyl attachment was found to increase the membrane-penetration rate of PNA three-fold, as compared to corresponding unmodified PNA. From the penetration behaviour of a number of small and large molecules we could conclude that passive diffusion is the mechanism for liposome-membrane passage. Flow linear dichroism (LD) of the modified PNA in presence of rod-shaped micelles, together with octanol-water distribution experiments. showed that the adamantyl-modified PNA is amphiphilic; the driving force behind the observed increased membrane-penetration rate appears to be an accumulation of the PNA in the lipid double layer.
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| 3. |
- Ariöz, Candan, 1983, et al.
(författare)
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Folding of copper proteins: Role of the metal?
- 2018
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Ingår i: Quarterly Reviews of Biophysics. - 1469-8994 .- 0033-5835. ; 51
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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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| 4. |
- Ariöz, Candan, 1983, et al.
(författare)
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The six metal binding domains in human copper transporter, ATP7B: molecular biophysics and disease-causing mutations
- 2017
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Ingår i: Biometals. - : Springer Science and Business Media LLC. - 0966-0844 .- 1572-8773. ; 30:6, s. 823-840
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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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| 5. |
- Blockhuys, Stephanie, 1983, et al.
(författare)
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Copper chaperone Atox1 plays role in breast cancer cell migration
- 2017
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 483:1, s. 301-304
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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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| 6. |
- Blockhuys, Stephanie, 1983, et al.
(författare)
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Copper distribution in breast cancer cells detected by time-of-flight secondary ion mass spectrometry with delayed extraction methodology
- 2018
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Ingår i: Biointerphases. - : American Vacuum Society. - 1559-4106 .- 1934-8630. ; 13:6
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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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| 7. |
- Blockhuys, Stephanie, 1983, et al.
(författare)
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Defining the human copper proteome and analysis of its expression variation in cancers.
- 2017
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Ingår i: Metallomics. - : Oxford University Press (OUP). - 1756-5901 .- 1756-591X. ; 9:2, s. 112-123
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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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| 8. |
- Blockhuys, Stephanie, 1983, et al.
(författare)
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Evaluation of ATOX1 as a potential predictive biomarker for tetrathiomolybdate treatment of breast cancer patients with high risk of recurrence
- 2021
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Ingår i: Biomedicines. - : MDPI AG. - 2227-9059. ; 9:12
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Tidskriftsartikel (refereegranskat)abstract
- Copper plays a key role in cancer metastasis, which is the most common cause of cancer death. Copper depletion treatment with tetrathiomolybdate (TM) improved disease-free survival in breast cancer patients with high risk of recurrence in a phase II clinical trial. Because the copper metallochaperone ATOX1 was recently reported to drive breast cancer cell migration and breast cancer migration is a critical factor in metastasis, we tested if ATOX1 expression levels in primary tumor tissue could predict the TM treatment outcome of breast cancer patients at high risk of recurrence. We performed ATOX1 immunohistochemical staining of breast tumor material (before TM treatment) of 47 patients enrolled in the phase II TM clinical trial and evaluated ATOX1 expression levels in relation with patient outcome after TM treatment. Our results show that higher ATOX1 levels in the tumor cell cytoplasm correlate with a trend towards better event-free survival after TM treatment for triple-negative breast cancer patients and patients at stage III of disease. In conclusion, ATOX1 may be a potential predictive biomarker for TM treatment of breast cancer patients at high risk of recurrence and should be tested in a larger cohort of patients.
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| 9. |
- Blockhuys, Stephanie, 1983, et al.
(författare)
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Evaluation of copper chaperone ATOX1 as prognostic biomarker in breast cancer
- 2020
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Ingår i: Breast Cancer. - : Springer Science and Business Media LLC. - 1880-4233 .- 1340-6868. ; 27:3, s. 505-509
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Tidskriftsartikel (refereegranskat)abstract
- Copper is involved in different hallmarks of cancer, including metastasis, but responsible copper-binding proteins and pathways are not clear. The copper chaperone ATOX1 was recently shown to play a role in breast cancer cell migration, which is a key step in metastasis. Since most cancer-related deaths are due to metastasis, we hypothesized that ATOX1 mRNA expression may be associated with breast cancer disease progression and thus, a prognostic biomarker in breast cancer. We therefore studied the association of ATOX1 expression levels with clinicopathological parameters and survival for 1904 breast cancer patients using the METABRIC data set. Our results indicate ATOX1 expression levels as a potential prognostic biomarker for ER-positive subtypes and early stages of breast cancer. Pre-clinical studies and clinical trials are desired to identify the molecular roles of ATOX1 in these conditions.
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| 10. |
- Blockhuys, Stephanie, 1983, et al.
(författare)
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Roles of copper-binding proteins in breast cancer
- 2017
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 18:4
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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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