Copper-induced structural conversion templates prion protein oligomerization and neurotoxicity

Prion protein (PrP) misfolding and oligomerization are key pathogenic events in prion disease. Copper exposure has been linked to prion pathogenesis; however, its mechanistic basis is unknown. We resolve, with single-molecule precision, the molecular mechanism of Cu2+-induced misfolding of PrP under physiological conditions. We also demonstrate that misfolded PrPs serve as seeds for templated formation of aggregates, which mediate inflammation and degeneration of neuronal tissue. Using a single-molecule fluorescence assay, we demonstrate that Cu2+ induces PrP monomers to misfold before oligomer assembly; the disordered amino-terminal region mediates this structural change. Single-molecule force spectroscopy measurements show that the misfolded monomers have a 900-fold higher binding affinity compared to the native isoform, which promotes their oligomerization. Real-time quaking-induced conversion demonstrates that misfolded PrPs serve as seeds that template amyloid formation. Finally, organotypic slice cultures show that misfolded PrPs mediate inflammation and degeneration of neuronal tissue. Our study establishes a direct link, at the molecular level, between copper exposure and PrP neurotoxicity.

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Diversity in prion protein oligomerization pathways results from domain expansion as revealed by hydrogen/deuterium exchange and disulfide linkage

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0607745104 ◽

2007 ◽

Vol 104 (18) ◽

pp. 7414-7419 ◽

Cited By ~ 108

Author(s):

Frederic Eghiaian ◽

Thorsten Daubenfeld ◽

Yann Quenet ◽

Marieke van Audenhaege ◽

Anne-Pascale Bouin ◽

...

Keyword(s):

Prion Protein ◽

Conformational Changes ◽

Present Report ◽

Deuterium Exchange ◽

Amyloid Formation ◽

Protein Oligomerization ◽

Hydrogen Deuterium Exchange ◽

High Concentration ◽

Disulfide Linkage ◽

Hydrogen Deuterium

The prion protein (PrP) propensity to adopt different structures is a clue to its biological role. PrP oligomers have been previously reported to bear prion infectivity or toxicity and were also found along the pathway of in vitro amyloid formation. In the present report, kinetic and structural analysis of ovine PrP (OvPrP) oligomerization showed that three distinct oligomeric species were formed in parallel, independent kinetic pathways. Only the largest oligomer gave rise to fibrillar structures at high concentration. The refolding of OvPrP into these different oligomers was investigated by analysis of hydrogen/deuterium exchange and introduction of disulfide bonds. These experiments revealed that, before oligomerization, separation of contacts in the globular part (residues 127–234) occurred between the S1–H1–S2 domain (residues 132–167) and the H2–H3 bundle (residues 174–230), implying a conformational change of the S2–H2 loop (residues 168–173). The type of oligomer to be formed depended on the site where the expansion of the OvPrP monomer was initiated. Our data bring a detailed insight into the earlier conformational changes during PrP oligomerization and account for the diversity of oligomeric entities. The kinetic and structural mechanisms proposed here might constitute a physicochemical basis of prion strain genesis.

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3D domain swapping, protein oligomerization, and amyloid formation.

Acta Biochimica Polonica ◽

10.18388/abp.2001_3849 ◽

2001 ◽

Vol 48 (4) ◽

pp. 807-827 ◽

Cited By ~ 43

Author(s):

M Jaskólski

Keyword(s):

Cystatin C ◽

Single Molecule ◽

Amyloid Fibrils ◽

Domain Swapping ◽

Amyloid Formation ◽

Protein Oligomerization ◽

Molecular Architecture ◽

Structural Element ◽

Human Cystatin C ◽

Human Cystatin

In 3D domain swapping, first described by Eisenberg, a structural element of a monomeric protein is replaced by the same element from another subunit. This process requires partial unfolding of the closed monomers that is then followed by adhesion and reconstruction of the original fold but from elements contributed by different subunits. If the interactions are reciprocal, a closed-ended dimer will be formed, but the same phenomenon has been suggested as a mechanism for the formation of open-ended polymers as well, such as those believed to exist in amyloid fibrils. There has been a rapid progress in the study of 3D domain swapping. Oligomers higher than dimers have been found, the monomer-dimer equilibrium could be controlled by mutations in the hinge element of the chain, a single protein has been shown to form more than one domain-swapped structure, and recently, the possibility of simultaneous exchange of two structural domains by a single molecule has been demonstrated. This last discovery has an important bearing on the possibility that 3D domain swapping might be indeed an amyloidogenic mechanism. Along the same lines is the discovery that a protein of proven amyloidogenic properties, human cystatin C, is capable of 3D domain swapping that leads to oligomerization. The structure of domain-swapped human cystatin C dimers explains why a naturally occurring mutant of this protein has a much higher propensity for aggregation, and also suggests how this same mechanism of 3D domain swapping could lead to an open-ended polymer that would be consistent with the cross-beta structure, which is believed to be at the heart of the molecular architecture of amyloid fibrils.

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Faculty Opinions recommendation of Cytotoxicity of prion protein-derived cell-penetrating peptides is modulated by pH but independent of amyloid formation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726937665.793537425 ◽

2017 ◽

Author(s):

Chandra Verma ◽

Suryani Lukman

Keyword(s):

Prion Protein ◽

Cell Penetrating Peptides ◽

Amyloid Formation ◽

Cell Penetrating

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Analyses of p73 Protein Oligomerization and p73–MDM2 Interaction in Single Living Cells Using In Situ Single Molecule Spectroscopy

Analytical Chemistry ◽

10.1021/acs.analchem.0c03521 ◽

2021 ◽

Author(s):

Fucai Li ◽

Zhixue Du ◽

Xiangyi Huang ◽

Chaoqing Dong ◽

Jicun Ren

Keyword(s):

Single Molecule ◽

Living Cells ◽

Protein Oligomerization ◽

Single Molecule Spectroscopy ◽

Single Living Cells ◽

Single Living

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The Prion Protein Octarepeat Domain Forms Transient β-sheet Structures Upon Residue-Specific Cu(II) and Zn(II) Binding

10.1101/2021.12.12.472308 ◽

2021 ◽

Author(s):

Maciej Gielnik ◽

Aneta Szymanska ◽

Xiaolin Dong ◽

Jyri Jarvet ◽

Zeljko M. Svedruzic ◽

...

Keyword(s):

Metal Ions ◽

Prion Protein ◽

Metal Binding ◽

Cd Spectroscopy ◽

Cellular Prion Protein ◽

Transmissible Spongiform Encephalopathies ◽

Amyloid Formation ◽

Spectroscopy Data ◽

Spongiform Encephalopathies ◽

Β Sheet

Misfolding of the cellular prion protein (PrPC) is associated with the development of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). Metal ions appear to play a crucial role in the protein misfolding, and metal imbalance may be part of TSE pathologies. PrPC is a combined Cu(II) and Zn(II) metal binding protein, where the main metal binding site is located in the octarepeat (OR) region. Here, we used biophysical methods to characterize Cu(II) and Zn(II) binding to the isolated OR region. Circular dichroism (CD) spectroscopy data suggest that the OR domain binds up to four Cu(II) ions or two Zn(II) ions. Upon metal binding, the OR region seems to adopt a transient antiparallel β-sheet hairpin structure. Fluorescence spectroscopy data indicates that under neutral conditions, the OR region can bind both Cu(II) and Zn(II) ions, whereas under acidic conditions it binds only Cu(II) ions. Molecular dynamics simulations suggest that binding of both metal ions to the OR region results in formation of β-hairpin structures. As formation of β-sheet structures is a first step towards amyloid formation, we propose that high concentrations of either Cu(II) or Zn(II) ions may have a pro-amyloid effect in TSEs.

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Biochemistry: one molecule at a time

Essays in Biochemistry ◽

10.1042/ebc20210015 ◽

2021 ◽

Vol 65 (1) ◽

pp. 1-3

Author(s):

Dominika T. Gruszka

Keyword(s):

Complex Networks ◽

Single Molecule ◽

Population Level ◽

Early Career ◽

Molecular Heterogeneity ◽

Biological Processes ◽

Biological Macromolecule ◽

Exciting Field ◽

Early Career Researchers ◽

Mechanistic Basis

Abstract Biological processes are orchestrated by complex networks of molecules. Conventional approaches for studying the action of biomolecules operate on a population level, averaging out any inhomogeneities within the ensemble. Investigating one biological macromolecule at a time allows researchers to directly probe individual behaviours, and thus characterise the intrinsic molecular heterogeneity of the system. Single-molecule methods have unravelled unexpected modes of action for many seemingly well-characterised biomolecules and often proved instrumental in understanding the intricate mechanistic basis of biological processes. This collection of reviews aims to showcase how single-molecule techniques can be used to address important biological questions and to inspire biochemists to ‘zoom in’ to the population and probe individual molecular behaviours, beyond the ensemble average. Furthermore, this issue of Essays in Biochemistry is the very first written and edited entirely by early career researchers, and so it also highlights the strength, diversity and excellence of the younger generation single-molecule scientists who drive this exciting field of research forward.

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On demand MyD88 oligomerization is controlled by IRAK4 during Myddosome signaling

10.1101/2020.09.03.280917 ◽

2020 ◽

Author(s):

Rafael Deliz-Aguirre ◽

Fakun Cao ◽

Fenja H. U. Gerpott ◽

Nichanok Auevechanichkul ◽

Mariam Chupanova ◽

...

Keyword(s):

Self Assembly ◽

Intracellular Signaling ◽

Innate Immune ◽

Live Cells ◽

Signaling Proteins ◽

Protein Oligomerization ◽

Downstream Signaling ◽

Oligomeric Complex ◽

Mechanistic Basis ◽

Cell Signaling Pathways

AbstractA recurring feature of innate immune receptor signaling is the self-assembly of signaling proteins into oligomeric complexes. The Myddosome is an oligomeric complex that is required to transmit inflammatory signals from TLR/IL1Rs and consists of MyD88 and IRAK family kinases. However, the molecular basis for how Myddosome proteins self-assemble and regulate intracellular signaling remains poorly understood. Here, we developed a novel assay to analyze the spatiotemporal dynamics of IL1R and Myddosome signaling in live cells. We found that MyD88 oligomerization is inducible and initially reversible. Moreover, the formation of larger, stable oligomers consisting of more than 4 MyD88s triggers the sequential recruitment of IRAK4 and IRAK1. Notably, genetic knockout of IRAK4 enhanced MyD88 oligomerization, indicating that IRAK4 controls MyD88 oligomer size and growth. MyD88 oligomer size thus functions as a physical threshold to trigger downstream signaling. These results provide a mechanistic basis for how protein oligomerization might function in cell signaling pathways.

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Zn(II) binding causes interdomain changes in the structure and flexibility of the human prion protein

Scientific Reports ◽

10.1038/s41598-021-00495-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Maciej Gielnik ◽

Michał Taube ◽

Lilia Zhukova ◽

Igor Zhukov ◽

Sebastian K. T. S. Wärmländer ◽

...

Keyword(s):

Prion Protein ◽

Metal Ion ◽

Structural Transition ◽

Cellular Prion Protein ◽

Zinc Homeostasis ◽

Transmissible Spongiform Encephalopathies ◽

Spongiform Encephalopathies ◽

Structural Conversion ◽

Protein Size ◽

Dynamics Simulations

AbstractThe cellular prion protein (PrPC) is a mainly α-helical 208-residue protein located in the pre- and postsynaptic membranes. For unknown reasons, PrPC can undergo a structural transition into a toxic, β-sheet rich scrapie isoform (PrPSc) that is responsible for transmissible spongiform encephalopathies (TSEs). Metal ions seem to play an important role in the structural conversion. PrPC binds Zn(II) ions and may be involved in metal ion transport and zinc homeostasis. Here, we use multiple biophysical techniques including optical and NMR spectroscopy, molecular dynamics simulations, and small angle X-ray scattering to characterize interactions between human PrPC and Zn(II) ions. Binding of a single Zn(II) ion to the PrPC N-terminal domain via four His residues from the octarepeat region induces a structural transition in the C-terminal α-helices 2 and 3, promotes interaction between the N-terminal and C-terminal domains, reduces the folded protein size, and modifies the internal structural dynamics. As our results suggest that PrPC can bind Zn(II) under physiological conditions, these effects could be important for the physiological function of PrPC.

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Abrogation of prenucleation, transient oligomerization of the Huntingtin exon 1 protein by human profilin I

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922264117 ◽

2020 ◽

Vol 117 (11) ◽

pp. 5844-5852 ◽

Cited By ~ 1

Author(s):

Alberto Ceccon ◽

Vitali Tugarinov ◽

Rodolfo Ghirlando ◽

G. Marius Clore

Keyword(s):

Single Molecule ◽

Chemical Exchange ◽

Coiled Coil ◽

Relaxation Dispersion ◽

Line Broadening ◽

Exon 1 ◽

Nmr Techniques ◽

Mechanistic Basis ◽

Kinetics Of ◽

Micromolar Range

Human profilin I reduces aggregation and concomitant toxicity of the polyglutamine-containing N-terminal region of the huntingtin protein encoded by exon 1 (httex1) and responsible for Huntington’s disease. Here, we investigate the interaction of profilin with httex1using NMR techniques designed to quantitatively analyze the kinetics and equilibria of chemical exchange at atomic resolution, including relaxation dispersion, exchange-induced shifts, and lifetime line broadening. We first show that the presence of two polyproline tracts in httex1, absent from a shorter huntingtin variant studied previously, modulates the kinetics of the transient branched oligomerization pathway that precedes nucleation, resulting in an increase in the populations of the on-pathway helical coiled-coil dimeric and tetrameric species (τex≤ 50 to 70 μs), while leaving the population of the off-pathway (nonproductive) dimeric species largely unaffected (τex∼750 μs). Next, we show that the affinity of a single molecule of profilin to the polyproline tracts is in the micromolar range (Kdiss∼ 17 and ∼ 31 μM), but binding of a second molecule of profilin is negatively cooperative, with the affinity reduced ∼11-fold. The lifetime of a 1:1 complex of httex1with profilin, determined using a shorter huntingtin variant containing only a single polyproline tract, is shown to be on the submillisecond timescale (τex∼ 600 μs andKdiss∼ 50 μM). Finally, we demonstrate that, in stable profilin–httex1complexes, the productive oligomerization pathway, leading to the formation of helical coiled-coil httex1tetramers, is completely abolished, and only the pathway resulting in “nonproductive” dimers remains active, thereby providing a mechanistic basis for how profilin reduces aggregation and toxicity of httex1.

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