| 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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| 11. |
- Blockhuys, Stephanie, 1983, et al.
(författare)
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Second harmonic generation for collagen I characterization in rectal cancer patients with and without preoperative radiotherapy
- 2017
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Ingår i: Journal of Biomedical Optics. - : SPIE-Intl Soc Optical Eng. - 1560-2281 .- 1083-3668. ; 22:10
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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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| 12. |
- Blockhuys, Stephanie, 1983, et al.
(författare)
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Single-cell tracking demonstrates copper chaperone Atox1 to be required for breast cancer cell migration
- 2020
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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
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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.
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| 13. |
- Blomberg, Jeanette, et al.
(författare)
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Interactions between DNA, transcriptional regulator Dreb2a and the Med25 mediator subunit from Arabidopsis thaliana involve conformational changes
- 2012
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Ingår i: Nucleic Acids Research. - Oxford : Oxford University Press. - 0305-1048 .- 1362-4962. ; 40:13, s. 5938-5950
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Tidskriftsartikel (refereegranskat)abstract
- Mediator is a multiprotein coregulatory complex that conveys signals from DNA-bound transcriptional regulators to the RNA polymerase II transcription machinery in eukaryotes. The molecular mechanisms for how these signals are transmitted are still elusive. By using purified transcription factor Dreb2a, mediator subunit Med25 from Arabidopsis thaliana, and a combination of biochemical and biophysical methods, we show that binding of Dreb2a to its canonical DNA sequence leads to an increase in secondary structure of the transcription factor. Similarly, interaction between the Dreb2a and Med25 in the absence of DNA results in conformational changes. However, the presence of the canonical Dreb2a DNA-binding site reduces the affinity between Dreb2a and Med25. We conclude that transcription regulation is facilitated by small but distinct changes in energetic and structural parameters of the involved proteins.
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| 14. |
- Bosaeus, Niklas, 1982, et al.
(författare)
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A stretched conformation of DNA with a biological role?
- 2017
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Ingår i: Quarterly Reviews of Biophysics. - 1469-8994 .- 0033-5835. ; 50, s. UNSP e11-e11
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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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| 15. |
- Celauro, Emanuele, 1980, et al.
(författare)
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Copper chaperone ATOX1 regulates pluripotency factor OCT4 in preimplantation mouse embryos
- 2017
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 491:1, s. 147-153
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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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| 16. |
- Chen, Eefei, et al.
(författare)
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Effects of macromolecular crowding on burst phase kinetics of cytochrome c folding
- 2012
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:49, s. 9836-9845
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Tidskriftsartikel (refereegranskat)abstract
- Excluded volume and viscosity effects of crowding agents that mimic crowded conditions in vivo on "classical" burst phase folding kinetics of cytochrome c are assessed in vitro. Upon electron transfer-triggered folding of reduced cytochrome c, far-UV time-resolved circular dichroism (TRCD) is used to monitor folding under different conditions. Earlier work has shown that folding of reduced cytochrome c from the guanidinium hydrochloride-induced unfolded ensemble in dilute phosphate buffer involves kinetic partitioning: one fraction of molecules folds rapidly, on a time scale identical to that of reduction, while the remaining population folds more slowly. In the presence of 220 mg/mL dextran 70, a synthetic macromolecular crowding agent that occupies space but does not interact with proteins, the population of the fast folding step for cytochrome c is greatly reduced. Increasing the viscosity with sucrose to the same microviscosity exhibited by the dextran solution showed no significant decrease in the amplitude of the fast-folding phase of cytochrome c. Experiments show that the unfolded-state heme ligation remains bis-His in the presence of dextran 70, but coarse-grained simulations suggest that the unfolded-state ensemble becomes more compact in the presence of crowders. We conclude that excluded volume effects alter unfolded cytochrome c such that access to fast-folding conformations is reduced.
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| 17. |
- Chen, Mingzhi, et al.
(författare)
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Predicting protein folding cores by empirical potential functions
- 2009
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Ingår i: Archives of Biochemistry and Biophysics. - New York : Elsevier. - 0003-9861 .- 1096-0384. ; 483:1, s. 16-22
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Tidskriftsartikel (refereegranskat)abstract
- Theoretical and in vitro experiments suggest that protein-folding cores form early in the process of folding, and that proteins may have evolved to optimize both folding speed and native-state stability. In our previous work (Chen et al., Structure, 14, 1401 (2006)), we developed a set of empirical potential functions and used them to analyze interaction energies among secondary-structure elements in two β-sandwich proteins. Our work on this group of proteins demonstrated that the predicted folding core also harbors residues that form native-like interactions early in the folding reaction. In the current work, we have tested our empirical potential functions on structurally-different proteins for which the folding cores have been revealed by protein hydrogen-deuterium exchange experiments. Using a set of 29 unrelated proteins, which have been extensively studied in the literature, we demonstrate that the average prediction result from our method is significantly better than predictions based on other computational methods. Our study is an important step towards the ultimate goal of understanding the correlation between folding cores and native structures.
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| 18. |
- Chen, Xun, et al.
(författare)
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Frustration Dynamics and Electron-Transfer Reorganization Energies in Wild-Type and Mutant Azurins
- 2022
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 144:9, s. 4178-4185
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Tidskriftsartikel (refereegranskat)abstract
- Long-range electron tunneling through metalloproteins is facilitated by evolutionary tuning of donor-acceptor electronic couplings, formal electrochemical potentials, and active-site reorganization energies. Although the minimal frustration of the folding landscape enables this tuning, residual frustration in the vicinity of the metallocofactor can allow conformational fluctuations required for protein function. We show here that the constrained copper site in wild-type azurin is governed by an intricate pattern of minimally frustrated local and distant interactions that together enable rapid electron flow to and from the protein. In contrast, sluggish electron transfer reactions (unfavorable reorganization energies) of active-site azurin variants are attributable to increased frustration near to as well as distant from the copper site, along with an exaggerated oxidation-state dependence of both minimally and highly frustrated interaction patterns.
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| 19. |
- Chorell, Erik, et al.
(författare)
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Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts.
- 2015
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 10:10, s. e0140194-
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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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| 20. |
- Christiansen, Alexander, 1982-, et al.
(författare)
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Effects of macromolecular crowding agents on protein folding in vitro and in silico
- 2013
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Ingår i: Biophysical Reviews. - : Springer. - 1867-2450 .- 1867-2469. ; 5:2, s. 137-145
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Forskningsöversikt (refereegranskat)abstract
- Proteins fold and function inside cells which are environments very different from that of dilute buffer solutions most often used in traditional experiments. The crowded milieu results in excluded-volume effects, increased bulk viscosity and amplified chances for inter-molecular interactions. These environmental factors have not been accounted for in most mechanistic studies of protein folding executed during the last decades. The question thus arises as to how these effects-present when polypeptides normally fold in vivo-modulate protein biophysics. To address excluded volume effects, we use synthetic macromolecular crowding agents, which take up significant volume but do not interact with proteins, in combination with strategically selected proteins and a range of equilibrium and time-resolved biophysical (spectroscopic and computational) methods. In this review, we describe key observations on macromolecular crowding effects on protein stability, folding and structure drawn from combined in vitro and in silico studies. As expected based on Minton's early predictions, many proteins (apoflavodoxin, VlsE, cytochrome c, and S16) became more thermodynamically stable (magnitude depends inversely on protein stability in buffer) and, unexpectedly, for apoflavodoxin and VlsE, the folded states changed both secondary structure content and, for VlsE, overall shape in the presence of macromolecular crowding. For apoflavodoxin and cytochrome c, which have complex kinetic folding mechanisms, excluded volume effects made the folding energy landscapes smoother (i. e., less misfolding and/or kinetic heterogeneity) than in buffer.
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| 21. |
- Christiansen, Alexander, 1982-, et al.
(författare)
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Factors defining effects of Macromolecular crowding on protein stability : an in vitro/in silico case study using cytochrome c
- 2010
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 49:31, s. 6519-6530
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Tidskriftsartikel (refereegranskat)abstract
- Previous experiments with two single-domain proteins showed that macromolecular crowding can stabilize dramatically toward heat perturbation and modulate native-state structure and shape. To assess the generality of this, we here tested the effects of the synthetic crowding agents on cytochrome c, a small single-domain protein. Using far-UV circular dichroism (CD), we discovered that there is no effect on cytochrome c's secondary structure upon addition of Ficoll or dextran (0-400 mg/mL, pH 7). Thermal experiments revealed stabilizing effects (5-10 degrees C) of Ficoll 70 and dextran 70; this effect was enhanced by the presence of low levels of guanidine hydrochloride (GuHCl) that destabilize the protein. When using a smaller dextran, dextran 40, the thermal effects were larger (10-20 degrees C). In silico analysis, using structure-based (Go-like) interactions for cytochrome c, is in excellent agreement with the in vitro thermodynamic data and also agrees with scaled particle theory. Simulations of a range of crowder size and shape demonstrated that the smaller the crowder the larger the favorable effect on cytochrome c's folded-state stability. Together with previous data, we conclude that protein size, stability, conformational malleability, and folding routes, as well as crowder size and shape, are key factors that modulate the net effect of macromolecular crowding on proteins.
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| 22. |
- Christiansen, Alexander, 1982-, et al.
(författare)
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Quantification of excluded volume effects on the folding landscape of Pseudomonas aeruginosa Apoazurin In Vitro
- 2013
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Ingår i: Biophysical Journal. - : Elsevier. - 0006-3495 .- 1542-0086. ; 105:7, s. 1689-1699
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Tidskriftsartikel (refereegranskat)abstract
- Proteins fold and function inside cells that are crowded with macromolecules. Here, we address the role of the resulting excluded volume effects by in vitro spectroscopic studies of Pseudomonas aeruginosa apoazurin stability (thermal and chemical perturbations) and folding kinetics (chemical perturbation) as a function of increasing levels of crowding agents dextran (sizes 20, 40, and 70 kDa) and Ficoll 70. We find that excluded volume theory derived by Minton quantitatively captures the experimental effects when crowding agents are modeled as arrays of rods. This finding demonstrates that synthetic crowding agents are useful for studies of excluded volume effects. Moreover, thermal and chemical perturbations result in free energy effects by the presence of crowding agents that are identical, which shows that the unfolded state is energetically the same regardless of method of unfolding. This also underscores the two-state approximation for apoazurin’s unfolding reaction and suggests that thermal and chemical unfolding experiments can be used in an interchangeable way. Finally, we observe increased folding speed and invariant unfolding speed for apoazurin in the presence of macromolecular crowding agents, a result that points to unfolded-state perturbations. Although the absolute magnitude of excluded volume effects on apoazurin is only on the order of 1–3 kJ/mol, differences of this scale may be biologically significant.
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| 23. |
- Christiansen, Alexander, 1982-, et al.
(författare)
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Synthetic crowding agent dextran causes excluded volume interactions exclusively to tracer protein apoazurin
- 2014
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Ingår i: FEBS Letters. - : Elsevier. - 0014-5793 .- 1873-3468. ; 588:5, s. 811-814
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Tidskriftsartikel (refereegranskat)abstract
- To understand protein biophysics in crowded cellular environments, researchers often use synthetic polymers as 'crowding agents' in vitro. The idea is that these agents will occupy space and reproduce the in vivo scenario in terms of excluded volume. However, recent work has challenged this concept and pointed out that attractive interactions between protein and crowding agent will provide an enthalpic contribution to the overall effect on protein thermodynamics. Here we use a typical synthetic crowding agent and a well-studied model protein to demonstrate in a window of 50 K that the presence of dextran 20 affects apoazurin by steric repulsion. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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| 24. |
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| 25. |
- 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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| 26. |
- 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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| 27. |
- 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-
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 28. |
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| 29. |
- 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.
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| 30. |
- Haaima, G., et al.
(författare)
-
Peptide nucleic acids (PNA) derived from N-(N-methylaminoethyl)glycine. Synthesis, hybridization and structural properties
- 1999
-
Ingår i: New Journal of Chemistry. - 1369-9261 .- 1144-0546. ; 23:8, s. 833-840
-
Tidskriftsartikel (refereegranskat)abstract
- Backbone N-methylated peptide nucleic acids (PNAs) containing the four nucleobases adenine, cytosine, guanine and thymine were synthesized via solid phase peptide oligomerization. The oligomers bind to their complementary target with a thermal stability that is 1.5-4.5 degrees C lower per "N-methyl nucleobase unit" [dependent on the number and position(s) of the N-methyl] than that of unmodified PNA. However, even fully N-methyl modified PNAs bind as efficiently to DNA or RNA targets as DNA itself. Furthermore, the hybridization efficiency per N-methyl unit in a PNA decreased with increasing N-methyl content, and the effect was more pronounced when the N-methyl backbone units are present in the Hoogsteen versus the Watson-Crick strand in (PNA)(2)-DNA triplexes. Interestingly, CD spectral analyses indicate that 30% (3 out of ten) substitution with N-methyl nucleobases did not alter the structure of PNA-DNA (or RNA) duplexes or (PNA)(2)-DNA triplexes, and likewise CD spectroscopy and X-ray crystallography showed no major structural differences between N-methylated (30%) and unmodified PNA-PNA duplexes. However, PNA-DNA duplexes as well as triplexes adopted a different conformation when formed with all-Ai-methyl PNAs.
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| 31. |
- Hannestad, Jonas, 1981, et al.
(författare)
-
Single-vesicle imaging reveals lipid-selective and stepwise membrane disruption by monomeric α-synuclein
- 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. ; 117:25, s. 14178-14186
-
Tidskriftsartikel (refereegranskat)abstract
- The interaction of the neuronal protein α-synuclein with lipid membranes appears crucial in the context of Parkinson's disease, but the underlying mechanistic details, including the roles of different lipids in pathogenic protein aggregation and membrane disruption, remain elusive. Here, we used single-vesicle resolution fluorescence and label-free scattering microscopy to investigate the interaction kinetics of monomeric α-synuclein with surface-tethered vesicles composed of different negatively charged lipids. Supported by a theoretical model to account for structural changes in scattering properties of surface-tethered lipid vesicles, the data demonstrate stepwise vesicle disruption and asymmetric membrane deformation upon α-synuclein binding to phosphatidylglycerol vesicles at protein concentrations down to 10 nM (∼100 proteins per vesicle). In contrast, phosphatidylserine vesicles were only marginally affected. These insights into structural consequences of α-synuclein interaction with lipid vesicles highlight the contrasting roles of different anionic lipids, which may be of mechanistic relevance for both normal protein function (e.g., synaptic vesicle binding) and dysfunction (e.g., mitochondrial membrane interaction).
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| 32. |
- Homouz, Dirar M., et al.
(författare)
-
Role of Zero-Order Loop in Protein Unfolding Case Study with Apoazurin
- 2020
-
Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 118:3, Supplement 1, s. 197A-197A
-
Konferensbidrag (refereegranskat)abstract
- Pseudomonas aeruginosa apoazurin (apo, without the copper cofactor) is a two-state folder that has a single disulfide bond between residues 3 and 26. This bond covalently connects the N-termini of beta-strands 1 and 3; thereby it creates a zero-order loop. The loop restricts the conformational space for the apoazurin polypeptide. In order to understand the role played by the zero-order loop, we used molecular dynamics (MD) simulations to compare two variants of apoazurin; one variant called “loop” which contained the disulfide and another called “open” in which the disulfide bond between residues 3 and 26 was removed. MD simulations were performed to probe the unfolding pathway and stability of the two apoazurin variants at different urea concentrations and temperatures. Our results show that the folded structure apoazurin is somewhat more stable due to the presence of the disulfide bond. However, the disulfide bond plays a prominent role in the apoazurin unfolding mechanism: we find that it changes both the folding-transition state and the unfolded-state ensemble of conformations.
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| 33. |
- Homouz, Dirar, et al.
(författare)
-
Macromolecular Crowding Modulates Folding Mechanism of α/β Protein Apoflavodoxin
- 2009
-
Ingår i: Biophysical Journal. - : Elsevier Inc. - 0006-3495. ; 96:2, s. 671-80
-
Tidskriftsartikel (refereegranskat)abstract
- Protein dynamics in cells may be different from those in dilute solutions in vitro, because the environment in cells is highly concentrated with other macromolecules. This volume exclusion because of macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, we investigated the folding energy landscape of an α/β protein, apoflavodoxin, in the presence of inert macromolecular crowding agents, using in silico and in vitro approaches. By means of coarse-grained molecular simulations and topology-based potential interactions, we probed the effects of increased volume fractions of crowding agents (c) as well as of crowding agent geometry (sphere or spherocylinder) at high c. Parallel kinetic folding experiments with purified Desulfovibro desulfuricans apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and Dextran (spherocylinder) synthetic crowding agents. In conclusion, we identified the in silico crowding conditions that best enhance protein stability, and discovered that upon manipulation of the crowding conditions, folding routes experiencing topological frustrations can be either enhanced or relieved. Our test-tube experiments confirmed that apoflavodoxin's time-resolved folding path is modulated by crowding agent geometry. Macromolecular crowding effects may be a tool for the manipulation of protein-folding and function in living cells.
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| 34. |
- 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.
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| 35. |
- Horvath, Istvan, 1979, et al.
(författare)
-
Amyloid Fibers of α-Synuclein Catalyze Chemical Reactions
- 2023
-
Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 14:4, s. 603-608
-
Tidskriftsartikel (refereegranskat)abstract
- Amyloid fibers of the protein α-synuclein, found in Lewy body deposits, are hallmarks of Parkinson’s disease. We here show that α-synuclein amyloids catalyze biologically relevant chemical reactions in vitro. Amyloid fibers, but not monomers, of α-synuclein catalyzed hydrolysis of the model ester para-nitrophenyl acetate and dephosphorylation of the model phosphoester para-nitrophenyl-orthophosphate. When His50 was replaced with Ala in α-synuclein, dephosphorylation but not esterase activity of amyloids was diminished. Truncation of the protein’s C-terminus had no effect on fiber catalytic efficiency. Catalytic activity of α-synuclein fibers may be a new gain-of-function that plays a role in Parkinson’s disease.
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| 36. |
- Horvath, Istvan, 1979, et al.
(författare)
-
Amyloids of α-Synuclein Promote Chemical Transformations of Neuronal Cell Metabolites
- 2023
-
Ingår i: International Journal of Molecular Sciences. - 1661-6596 .- 1422-0067. ; 24:16
-
Tidskriftsartikel (refereegranskat)abstract
- The assembly of α-synuclein into cross-β structured amyloid fibers results in Lewy body deposits and neuronal degeneration in Parkinson’s disease patients. As the cell environment is highly crowded, interactions between the formed amyloid fibers and a range of biomolecules can occur in cells. Although amyloid fibers are considered chemically inert species, recent in vitro work using model substrates has shown α-synuclein amyloids, but not monomers, to catalyze the hydrolysis of ester and phosphoester bonds. To search for putative catalytic activity of α-synuclein amyloids on biologically relevant metabolites, we here incubated α-synuclein amyloids with neuronal SH-SY5Y cell lysates devoid of proteins. LC-MS-based metabolomic (principal component and univariate) analysis unraveled distinct changes in several metabolite levels upon amyloid (but not monomer) incubation. Of 63 metabolites identified, the amounts of four increased (3-hydroxycapric acid, 2-pyrocatechuic acid, adenosine, and NAD), and the amounts of seventeen decreased (including aromatic and apolar amino acids, metabolites in the TCA cycle, keto acids) in the presence of α-synuclein amyloids. Many of these metabolite changes match what has been reported previously in Parkinson’s disease patients and animal–model metabolomics studies. Chemical reactivity of α-synuclein amyloids may be a new gain-of-function that alters the metabolite composition in cells and, thereby, modulates disease progression.
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| 37. |
- 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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| 38. |
- 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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| 39. |
- Horvath, Istvan, 1979, et al.
(författare)
-
Distinct growth regimes of α-synuclein amyloid elongation
- 2023
-
Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 122:12, s. 2556-2563
-
Tidskriftsartikel (refereegranskat)abstract
- Addition of amyloid seeds to aggregation-prone monomers allows for amyloid fiber growth (elongation) omitting slow nucleation. We here combine Thioflavin T fluorescence (probing formation of amyloids) and solution-state NMR spectroscopy (probing disappearance of monomers) to assess elongation kinetics of the amyloidogenic protein, α-synuclein, for which aggregation is linked to Parkinson's disease. We found that both spectroscopic detection methods give similar kinetic results, which can be fitted by applying double exponential decay functions. When the origin of the two-phase behavior was analyzed by mathematical modeling, parallel paths as well as stop-and-go behavior were excluded as possible explanations. Instead, supported by previous theory, the experimental elongation data reveal distinct kinetic regimes that depend on instantaneous monomer concentration. At low monomer concentrations (toward end of experiments), amyloid growth is limited by conformational changes resulting in β-strand alignments. At the higher monomer concentrations (initial time points of experiments), growth occurs rapidly by incorporating monomers that have not successfully completed the conformational search. The presence of a fast disordered elongation regime at high monomer concentrations agrees with coarse-grained simulations and theory but has not been detected experimentally before. Our results may be related to the wide range of amyloid folds observed.
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| 40. |
- 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. ; , 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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| 41. |
- 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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| 42. |
- Horvath, Istvan, 1979, et al.
(författare)
-
Macromolecular crowding modulates α-synuclein amyloid fiber growth
- 2021
-
Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 120:16, s. 3374-3381
-
Tidskriftsartikel (refereegranskat)abstract
- The crowdedness of living cells, hundreds of milligrams per milliliter of macromolecules, may affect protein folding, function, and misfolding. Still, such processes are most often studied in dilute solutions in vitro. To assess consequences of the in vivo milieu, we here investigated the effects of macromolecular crowding on the amyloid fiber formation reaction of α-synuclein, the amyloidogenic protein in Parkinson's disease. For this, we performed spectroscopic experiments probing individual steps of the reaction as a function of the macromolecular crowding agent Ficoll70, which is an inert sucrose-based polymer that provides excluded-volume effects. The experiments were performed at neutral pH at quiescent conditions to avoid artifacts due to shaking and glass beads (typical conditions for α-synuclein), using amyloid fiber seeds to initiate reactions. We find that both primary nucleation and fiber elongation steps during α-synuclein amyloid formation are accelerated by the presence of 140 and 280 mg/mL Ficoll70. Moreover, in the presence of Ficoll70 at neutral pH, secondary nucleation appears favored, resulting in faster overall α-synuclein amyloid formation. In contrast, sucrose, a small-molecule osmolyte and building block of Ficoll70, slowed down α-synuclein amyloid formation. The ability of cell environments to modulate reaction kinetics to a large extent, such as severalfold faster individual steps in α-synuclein amyloid formation, is an important consideration for biochemical reactions in living systems.
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| 43. |
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| 44. |
- 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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| 45. |
- 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.
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| 46. |
- 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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| 47. |
- 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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| 48. |
- Kamerlin, Shina Caroline Lynn, 1981-, et al.
(författare)
-
Female Faculty: Why So Few and Why Care?
- 2020
-
Ingår i: Chemistry - A European Journal. - : Wiley. - 1521-3765 .- 0947-6539. ; 26:38, s. 8319-8323
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Despite slow ongoing progress in increasing the representation of women in academia, women remain significantly under-represented at senior levels, in particular in the natural sciences and engineering. Not infrequently, this is downplayed by bringing forth arguments such as inherent biological differences between genders, that current policies are adequate to address the issue, or by deflecting this as being “not my problem” among other examples. In this piece we present scientific evidence that counters these claims, as well as a best-practice example, Genie, from Chalmers University of Technology, where one of the authors is currently employed. We also highlight particular challenges caused by the current COVID-19 pandemic. Finally, we conclude by proposing some possible solutions to the situation and emphasize that we need to all do our part, to ensure that the next generation of academics experience a more diverse, inclusive, and equitable working environment.
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| 49. |
- 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
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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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- Kiskis, Juris, 1987, et al.
(författare)
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Unraveling amyloid formation paths of Parkinson's disease protein alpha-synuclein triggered by anionic vesicles
- 2017
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Ingår i: Quarterly Reviews of Biophysics. - : Cambridge University Press (CUP). - 1469-8994 .- 0033-5835. ; 50, s. 1-9
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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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