| 1. |
- Ghaeidamini, Marziyeh, 1989, et al.
(author)
-
Graphene oxide sheets and quantum dots inhibit alpha-synuclein amyloid formation by different mechanisms
- 2020
-
In: Nanoscale. - : Royal Society of Chemistry (RSC). - 2040-3372 .- 2040-3364. ; 12:37, s. 19450-19460
-
Journal article (peer-reviewed)abstract
- Aggregation and amyloid formation of the 140-residue presynaptic and intrinsically disordered protein alpha-synuclein (alpha-syn) is a pathological hallmark of Parkinson's disease (PD). Understanding how alpha-syn forms amyloid fibrils, and investigations of agents that can prevent their formation is therefore important. We demonstrate herein that two types of graphene oxide nanoparticles (sheets and quantum dots) inhibit alpha-syn amyloid formation by different mechanisms mediatedviadifferential interactions with both monomers and fibrils. We have used thioflavin-T fluorescence assays and kinetic analysis, circular dichroism, dynamic light scattering, fluorescence spectroscopy and atomic force microscopy to asses the kinetic nature and efficiency of this inhibitory effect. We show that the two types of graphene oxide nanoparticles alter the morphology of alpha-syn fibrils, disrupting their interfilament assembly and the resulting aggregates therefore consist of single protofilaments. Our results further show that graphene oxide sheets reduce the aggregation rate of alpha-syn primarily by sequestering of monomers, thereby preventing primary nucleation and elongation. Graphene quantum dots, on the other hand, interact less avidly with both monomers and fibrils. Their aggregation inhibitory effect is primarily related to adsorption of aggregated species and reduction of secondary processes, and they can thus not fully prevent aggregation. This fine-tuned and differential effect of graphene nanoparticles on amyloid formation shows that rational design of these nanomaterials has great potential in engineering materials that interact with specific molecular events in the amyloid fibril formation process. The findings also provide new insight into the molecular interplay between amyloidogenic proteins and graphene-based nanomaterials in general, and opens up their potential use as agents to manipulate fibril formation.
|
|
| 2. |
- Horvath, Istvan, 1979, et al.
(author)
-
Amyloids of α-Synuclein Promote Chemical Transformations of Neuronal Cell Metabolites
- 2023
-
In: International Journal of Molecular Sciences. - 1661-6596 .- 1422-0067. ; 24:16
-
Journal article (peer-reviewed)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.
|
|
| 3. |
- Horvath, Istvan, 1979, et al.
(author)
-
Copper chaperone blocks amyloid formation via ternary complex
- 2018
-
In: Quarterly Reviews of Biophysics. - 1469-8994 .- 0033-5835. ; 51, s. e6-e6
-
Journal article (peer-reviewed)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.
|
|
| 4. |
- Horvath, Istvan, 1979, et al.
(author)
-
Interaction between Copper Chaperone Atox1 and Parkinson's Disease Protein α-Synuclein Includes Metal-Binding Sites and Occurs in Living Cells
- 2019
-
In: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 10:11, s. 4659-4668
-
Journal article (peer-reviewed)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.
|
|
| 5. |
- Horvath, Istvan, 1979, et al.
(author)
-
Macromolecular crowding modulates α-synuclein amyloid fiber growth
- 2021
-
In: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 120:16, s. 3374-3381
-
Journal article (peer-reviewed)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.
|
|
| 6. |
- Jiang, Kai, 1988, et al.
(author)
-
C-terminal truncation of α-synuclein alters DNA structure from extension to compaction
- 2021
-
In: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 568, s. 43-47
-
Journal article (peer-reviewed)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.
|
|
| 7. |
- Kanagarajan, Selvaraju, et al.
(author)
-
Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers
- 2021
-
In: International Journal of Biological Macromolecules. - : Elsevier BV. - 0141-8130 .- 1879-0003. ; 184, s. 955-966
-
Journal article (peer-reviewed)abstract
- Hemoglobin-based oxygen carriers have long been pursued to meet clinical needs by using native hemoglobin (Hb) from human or animal blood, or recombinantly produced Hb, but the development has been impeded by safety and toxicity issues. Herewith we report the successful production of human fetal hemoglobin (HbF) in Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transient expression. HbF is a heterotetrameric protein composed of two identical α- and two identical γ-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the α- and γ-subunits of HbF were fused in order to stabilize the α-subunits and facilitate balanced expression of α- and γ-subunits in N. benthamiana. Efficient extraction and purification methods enabled production of the recombinantly fused endotoxin-free HbF (rfHbF) in high quantity and quality. The transiently expressed rfHbF protein was identified by SDS-PAGE, Western blot and liquid chromatography-tandem mass spectrometry analyses. The purified rfHbF possessed structural and functional properties similar to native HbF, which were confirmed by biophysical, biochemical, and in vivo animal studies. The results demonstrate a high potential of plant expression systems in producing Hb products for use as blood substitutes.
|
|
| 8. |
- Kumar, Ranjeet, 1980-, et al.
(author)
-
Comparative analysis of stress induced gene expression in caenorhabditis elegans following exposure to environmental and lab reconstituted complex metal mixture
- 2015
-
In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:7
-
Journal article (peer-reviewed)abstract
- Metals are essential for many physiological processes and are ubiquitously present in the environment. However, high metal concentrations can be harmful to organisms and lead to physiological stress and diseases. The accumulation of transition metals in the environment due to either natural processes or anthropogenic activities such as mining results in the contamination of water and soil environments. The present study used Caenorhabditis elegans to evaluate gene expression as an indicator of physiological response, following exposure to water collected from three different locations downstream of a Swedish mining site and a lab reconstituted metal mixture. Our results indicated that the reconstituted metal mixture exerted a direct stress response in C. elegans whereas the environmental waters elicited either a diminished or abrogated response. This suggests that it is not sufficient to use the biological effects observed from laboratory mixtures to extrapolate the effects observed in complex aquatic environments and apply this to risk assessment and intervention.
|
|
| 9. |
- Kumar, Ranjeet, 1980, et al.
(author)
-
Disease-causing point-mutations in metal-binding domains of Wilson disease protein decrease stability and increase structural dynamics
- 2017
-
In: Biometals. - : Springer Science and Business Media LLC. - 0966-0844 .- 1572-8773. ; 30:1, s. 27-35
-
Journal article (peer-reviewed)abstract
- After cellular uptake, Copper (Cu) ions are transferred from the chaperone Atox1 to the Wilson disease protein (ATP7B) for incorporation into Cu-dependent enzymes in the secretory pathway. Human ATP7B is a large multi-domain membrane-spanning protein which, in contrast to homologues in other organisms, has six similar cytoplasmic metal-binding domains (MBDs). The reason for multiple MBDs is proposed to be indirect modulation of enzymatic activity and it is thus intriguing that point mutations in MBDs can promote Wilson disease. We here investigated, in vitro and in silico, the biophysical consequences of clinically-observed Wilson disease mutations, G85V in MBD1 and G591D in MBD6, incorporated in domain 4. Because G85 and G591 correspond to a conserved Gly found in all MBDs, we introduced the mutations in the well-characterized MBD4. We found the mutations to dramatically reduce the MBD4 thermal stability, shifting the midpoint temperature of unfolding by more than 20 °C. In contrast to wild type MBD4 and MBD4D, MBD4V adopted a misfolded structure with a large β-sheet content at high temperatures. Molecular dynamic simulations demonstrated that the mutations increased backbone fluctuations that extended throughout the domain. Our findings imply that reduced stability and enhanced dynamics of MBD1 or MBD6 is the origin of ATP7B dysfunction in Wilson disease patients with the G85V or G591D mutation.
|
|
| 10. |
- Lorentzon, Emma, 1995, et al.
(author)
-
Differential effects of Cu2+ and Fe3+ ions on in vitro amyloid formation of biologically-relevant α-synuclein variants
- 2020
-
In: Biometals. - : Springer Science and Business Media LLC. - 0966-0844 .- 1572-8773. ; 33:2-3, s. 97-106
-
Journal article (peer-reviewed)abstract
- Alterations in metal ion homeostasis appear coupled to neurodegenerative disorders but mechanisms are unknown. Amyloid formation of the protein α-synuclein in brain cells is a hallmark of Parkinson’s disease. α-Synuclein can bind several metal ions in vitro and such interactions may affect the assembly process. Here we used biophysical methods to study the effects of micromolar concentrations of Cu2+ and Fe3+ ions on amyloid formation of selected α-synuclein variants (wild-type and A53T α-synuclein, in normal and N-terminally acetylated forms). As shown previously, Cu2+ speeds up aggregation of normal wild-type α-synuclein, but not the acetylated form. However, Cu2+ has a minimal effect on (the faster) aggregation of normal A53T α-synuclein, despite that Cu2+ binds to this variant. Like Cu2+, Fe3+ speeds up aggregation of non-acetylated wild-type α-synuclein, but with acetylation, Fe3+ instead slows down aggregation. In contrast, for A53T α-synuclein, regardless of acetylation, Fe3+ slows down aggregation with the effect being most dramatic for acetylated A53T α-synuclein. The results presented here suggest a correlation between metal-ion modulation effect and intrinsic aggregation speed of the various α-synuclein variants.
|
|
