| 1. |
- Grasso, Michael, et al.
(författare)
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The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation
- 2021
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 297:6
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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.
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| 2. |
- Matson Dzebo, Maria, 1985
(författare)
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Cellular Dyes and Bioactive Peptides - Cell Membrane Interactions and Cellular Uptake
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Knowledge about mechanisms behind interactions of molecules with biomembranes and cellular uptake is very important for understanding biological processes and for drug design. The work described in this thesis has focused on interactions of cellular dyes and bioactive peptides with cells and the cell membrane. Cellular dyes can be used to investigate processes occurring in the cell since they enhance the contrast of specific intracellular areas or molecules. Here, two types of dyes have been investigated; ruthenium dipyridophenazine complexes and voltage-sensitive dyes. Further, bioactive peptides have gained an increased attention for promising therapeutic applications. In this thesis, the cellular uptake of cell-penetrating peptides, interesting as drug delivery systems, and of the antisecretory peptide, AF-16, is examined. For the development of compounds of both categories, a large challenge is to overcome the poor cellular uptake, which is restricted by the cell membrane, and to understand the mechanisms of interactions with the cell membrane. The results show that ruthenium dipyridophenazine complexes, interesting as cellular dyes because of their low background emission, have tunable affinity for biomembranes and nucleic acids upon slight changes of their lipophilicity and stereochemistry. These complexes enter cells in two different ways, by endocytosis and by a photoactivated uptake mechanism. The voltage-sensitive dyes, which are used for visualization of the membrane potential variation by microscopy, seem to interact with lipid membranes in a dimeric form. For the studied series of arginine- and tryptophan-rich cell-penetrating peptides, the cellular uptake efficiency was found to be sequence specific, both regarding the number and the position of the tryptophan residues. Concerning the therapeutic peptide AF-16, its cellular uptake is mediated by endocytosis, which is enhanced by the presence of cell-surface proteoglycans. The results in this thesis give insights in membrane binding properties and cellular uptake of dyes and bioactive peptides as well as factors influencing these interactions, important knowledge for inspiring in future development of diagnostic and therapeutic molecules.
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| 3. |
- Matson Dzebo, Maria, 1985, et al.
(författare)
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Copper Chaperone Atox1 Interacts with Cell Cycle Proteins
- 2018
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Ingår i: Computational and Structural Biotechnology Journal. - : Elsevier BV. - 2001-0370. ; 16, s. 443-449
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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.
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| 4. |
- Matson Dzebo, Maria, 1985, et al.
(författare)
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Enhanced Cellular Uptake of Antisecretory Peptide AF-16 through Proteoglycan Binding
- 2014
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 53:41, s. 6566-6573
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Tidskriftsartikel (refereegranskat)abstract
- Peptide AF-16, which includes the active site of Antisecretory Factor protein, has antisecretory and antiinflammatory properties, making it a potent drug candidate for treatment of secretory and inflammatory diseases such as diarrhea, inflammatory bowel diseases, and intracranial hypertension. Despite remarkable physiological effects and great pharmaceutical need for drug discovery, very little is yet understood about AF-16 mechanism of action. In order to address interaction mechanisms, we investigated the binding of AF-16 to sulfated glycosaminoglycan, heparin, with focus on the effect of pH and ionic strength, and studied the influence of cell-surface proteoglycans on cellular uptake efficiency. Confocal laser scanning microscopy and flow cytometry experiments on wild type and proteoglycan-deficient Chinese hamster ovary cells reveal an endocytotic nature of AF-16 cellular uptake that is, however, less efficient for the cells lacking cell-surface proteoglycans. Isothermal titration calorimetry provides quantitative thermodynamic data and evidence for that the peptide affinity to heparinincreases at lower pH and ionic strength. Experimental data, supported by theoretical modeling, of peptide−glycosaminoglycan interaction indicate that it has a large electrostatic contribution, which will be enhanced in diseases accompanied by decreased pH and ionic strength. These observations show that cell-surface proteoglycans are of general and crucial importance for the antisecretory and anti-inflammatory activities of AF-16.
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| 5. |
- Matson Dzebo, Maria, 1985, et al.
(författare)
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Extended functional repertoire for human copper chaperones
- 2016
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Ingår i: Biomolecular Concepts. - : Walter de Gruyter GmbH. - 1868-5021 .- 1868-503X. ; 7:1, s. 29-39
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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.
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| 6. |
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| 7. |
- Vilhelmsson Wesén, Emelie, 1989, et al.
(författare)
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Endocytic uptake of monomeric amyloid-β peptides is clathrin- and dynamin-independent and results in selective accumulation of Aβ(1-42) compared to Aβ(1-40)
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Intraneuronal accumulation of amyloid-? (A?) peptides represent an early pathological feature in Alzheimer's disease. We have therefore utilized flow cytometry and confocal microscopy in combination with endocytosis inhibition to explore the internalisation efficiency and uptake mechanisms of A?(1-40) and A?(1-42) monomers in cultured SH-SY5Y cells. We find that both variants are constitutively internalised via endocytosis and that their uptake is proportional to cellular endocytic rate. Moreover, SH-SY5Y cells internalise consistently twice the amount of A?(1-42) compared to A?(1-40); an imaging-based quantification showed that cells treated with 1 ?M peptide for 8 h contained 800,000 peptides of A?(1-42) and 400,000 of A?(1-40). Both variants co-localised to >90% with lysosomes or other acidic compartments. Dynasore and chlorpromazine endocytosis inhibitors were both found to reduce uptake, particularly of A?(1-42). Overexpression of the C-terminal of the clathrin-binding domain of AP180, dynamin2 K44A, or Arf6 Q67L did however not reduce uptake of the A? variants. By contrast, perturbation of actin polymerisation and inhibition of macropinocytosis reduced A?(1-40) and A?(1-42) uptake considerably. This study clarifies mechanisms of A?(1-40) and A?(1-42) uptake, pinpoints differences between the two variants and highlights a common and putative role of macropinocytosis in the early accumulation of intraneuronal A? in AD. © 2017 The Author(s).
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| 8. |
- Wands, Amberlyn M., et al.
(författare)
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Fucosylated Molecules Competitively Interfere with Cholera Toxin Binding to Host Cells
- 2018
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Ingår i: ACS Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 4:5, s. 758-770
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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.
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