Misfolding Process Research Articles

Enhanced stabilisation and reduced fibril forming potential of an amyloidogenic light chain using a variable heavy domain to mimic the homodimer complex.

Light chain amyloidosis (AL), is classified as a plasma cell dyscrasia, whereby a mutant plasma cell multiplies uncontrollably and secretes enormous amounts of immunoglobulin-free light chain (FLC) fragments. These FLCs undergo a process of misfolding and aggregation into amyloid fibrils, that can cause irreversible system-wide damage. Current treatments that focus on depleting the underlying plasma cell clone are often poorly tolerated, particularly in patients with severe cardiac involvement, meaning patient prognosis is poor. An alternative treatment approach currently being explored is the inhibition of FLC aggregation by stabilisation of the native conformer. Here, we aimed to identify and characterise antibody fragments that target FLC domains and promote their stabilisation. Using phage-display screening methods, we identified a variable heavy (VH) domain, termed VH1, targeted towards the FLC. Using differential scanning fluorimetry and surface plasmon resonance, VH1 was characterised to bind and kinetically stabilise an amyloidogenic FLC, whereby a > 5.5 °C increase in thermal stability was noted. This improved stability corresponded to the inhibition of fibril formation, where 10 : 1 LC : VH1 concentration reduced aggregation to baseline levels. X-ray crystallographic structures of the LC : VH1 complex at atomic resolution revealed binding in a 1 : 1 ratio, mimicking the dimeric antigen binding sites of the native immunoglobulin molecule and the native LC homodimer.

Probing the Molecular Structure and Dynamics of Membrane-Bound Proteins during Misfolding Processes by Sum-Frequency Generation Vibrational Spectroscopy.

Protein misfolding and amyloid formation are implicated in the protein dysfunction, but the underlying mechanism remains to be clarified due to the lack of effective tools for detecting the transient intermediates. Sum frequency generation vibrational spectroscopy (SFG-VS) has emerged as a powerful tool for identifying the structure and dynamics of proteins at the interfaces. In this review, we summarize recent SFG-VS studies on the structure and dynamics of membrane-bound proteins during misfolding processes. This paper first introduces the methods for determining the secondary structure of interfacial proteins: combining chiral and achiral spectra of amide A and amide I bands and combining amide I, amide II, and amide III spectral features. To demonstrate the ability of SFG-VS in investigating the interfacial protein misfolding and amyloid formation, studies on the interactions between different peptides/proteins (islet amyloid polypeptide, amyloid β, prion protein, fused in sarcoma protein, hen egg-white lysozyme, fusing fusion peptide, class I hydrophobin SC3 and class II hydrophobin HFBI) and surfaces such as lipid membranes are discussed. These molecular-level studies revealed that SFG-VS can provide a unique understanding of the mechanism of interfacial protein misfolding and amyloid formation in real time, in situ and without any exogenous labeling.

Edge Substitution Effects of Histidine Tautomerization Behaviors on the Structural Properties and Aggregation Properties of Aβ(1-42) Mature Fibril.

Histidine behaviors play critical roles in folding and misfolding processes due to the changes in net charge and the various N/N-H orientations on imidazole rings. However, the effect of histidine tautomerization (HIE (Nε-H, ε) and HID (Nδ-H, δ) states) behaviors on the edge chain of Aβ mature fibrils remains inadequately understood, which is critical for finding a strategy to disturb fibril elongation and growth. In the current study, eight independent molecular dynamics simulations were conducted to investigate such impacts on the structural and aggregation properties. Our results from three different binding models revealed that the binding contributions of edge substitution effects are primarily located between chains 1 and 2. Histidine states significantly influence the secondary structure of each domain. Further analysis confirmed that the C1_H6//C1_E11 intrachain interaction is essential in maintaining the internal stability of chain 1, while the C1_H13//C2_H13 and C1_H14//C2_H13 interchain interactions are critical in maintaining the interchain stability of the fibril structure. Our subsequent analysis revealed that the current edge substitution leads to the loss of the C1_H13//C1_E11 intrachain and C1_H13//C2_H14 interchain interactions. The N-terminal regularity was significantly directly influenced by histidine states, particularly by the residue of C1_H13. Our study provides valuable insights into the effect of histidine behaviors on the edge chain of Aβ mature fibril, advancing our understanding of the histidine behavior hypothesis in misfolding diseases.

Screening of oxidative behavior in catalytic amyloid assemblies.

Once considered a thermodynamic minimum of the protein fold or as simply by-products of a misfolding process, amyloids are increasingly showing remarkable potential for promoting enzyme-like catalysis. Recent studies have demonstrated a diverse range of catalytic behaviors that amyloids can promote way beyond the hydrolytic behaviors originally reported. We and others have demonstrated the strong propensity of catalytic amyloids to facilitate redox reactions both in the presence and in the absence of metal cofactors. Here, we present a detailed protocol for measuring the oxidative ability of supramolecular peptide assemblies.

Detergent induced structural perturbations in peanut agglutinin: insights from spectroscopic and molecular dynamic simulation studies

The three dimensional structure of a protein is very important for its structure. Studies relating to protein structure have been numerous and the effect of denaturants on proteins can help understand the process of protein folding and misfolding. Detergents are important denaturants and play important roles in various fields. Here we explored the effect of sodium dodecyl sulphate (SDS) and cetyltrimethylammonium bromide (CTAB) on the structure of peanut agglutinin (PNA). The protein was purified from its natural source and impact of SDS and CTAB was studied by circular dichroism, intrinsic fluorescence, 8-anilino-1-napthalenesulfonic acid, molecular docking and molecular dynamics simulation. Pure peanut agglutinin showed a trough at 220 nm and positive ellipticity peak at 195 nm, specific for lectins. Results from the experimental and simulation studies suggest how oppositely charged detergents can interact differently and lead to varied structural perturbations in PNA. Both the surfactants induce all α protein-like circular dichroism in the protein, above its critical micelle concentrations, with significant change in accessible surface area that became more hydrophobic upon the treatment. Major interactions between the surfactants and protein, resulting in PNA conformational rearrangement, are electrostatic and van der Waals interactions. However, CTAB, a cationic surfactant, has similar effects as anionic surfactant (SDS) but at significantly very low concentration. Though the effects followed same pattern in both the surfactant treatment, i.e. above respective CMC, the surfactants were inducing all α protein-like conformation in PNA. Communicated by Ramaswamy H. Sarma

Advanced Techniques for Detecting Protein Misfolding and Aggregation in Cellular Environments.

Protein misfolding and aggregation, a key contributor to the progression of numerous neurodegenerative diseases, results in functional deficiencies and the creation of harmful intermediates. Detailed visualization of this misfolding process is of paramount importance for improving our understanding of disease mechanisms and for the development of potential therapeutic strategies. While in vitro studies using purified proteins have been instrumental in delivering significant insights into protein misfolding, the behavior of these proteins in the complex milieu of living cells often diverges significantly from such simplified environments. Biomedical imaging performed in cell provides cellular-level information with high physiological and pathological relevance, often surpassing the depth of information attainable through in vitro methods. This review highlights a variety of methodologies used to scrutinize protein misfolding within biological systems. This includes optical-based methods, strategies leaning on mass spectrometry, in-cell nuclear magnetic resonance, and cryo-electron microscopy. Recent advancements in these techniques have notably deepened our understanding of protein misfolding processes and the features of the resulting misfolded species within living cells. The progression in these fields promises to catalyze further breakthroughs in our comprehension of neurodegenerative disease mechanisms and potential therapeutic interventions.

Discontinuous Galerkin methods for Fisher–Kolmogorov equation with application to [formula omitted]-synuclein spreading in Parkinson’s disease

This spreading of prion proteins is at the basis of brain neurodegeneration. This paper deals with the numerical modelling of the misfolding process of α-synuclein in Parkinson’s disease. We introduce and analyse a discontinuous Galerkin method for the semi-discrete approximation of the Fisher–Kolmogorov (FK) equation that can be employed to model the process. We employ a discontinuous Galerkin method on polygonal and polyhedral grids (PolyDG) for space discretization, to accurately simulate the wavefronts typically observed in the prionic spreading and we prove stability and a priori error estimates. Next, we use a Crank–Nicolson scheme to advance in time. For the numerical verification of our numerical model, we first consider a manufactured solution, and then we consider a case with wavefront propagation in two-dimensional polygonal grids. Next, we carry out a simulation of α-synuclein spreading in a two-dimensional brain slice in the sagittal plane with a polygonal agglomerated grid that takes full advantage of the flexibility of PolyDG approximation. Finally, we present a simulation in a three-dimensional geometry reconstructed from magnetic resonance images of a patient’s brain.

Polarization-sensitive Two-photon Microscopy for a Label-free Amyloid Structural Characterization.

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue penetration, efficient operation in densely packed systems, and reduced angular photoselection of fluorophores. Thus, the introduction of polarization analysis in two-photon fluorescence microscopy (2PFM) provides a more precise determination of molecular organization in a sample compared to standard imaging methods based on linear optical processes. In this work, we focus on polarization-sensitive 2PFM (ps-2PFM) and its application in the determination of molecular ordering within complex bio-structures-amyloid spherulites. Neurodegenerative diseases such as Alzheimer's or Parkinson's are often diagnosed through the detection of amyloids-protein aggregates formed due to an impaired protein misfolding process. Exploring their structure leads to a better understanding of their creation pathway and consequently, to developing more sensitive diagnostic methods. This paper presents the ps-2PFM adapted for the determination of local fibril ordering inside the bovine insulin spherulites and spherical amyloidogenic protein aggregates. Moreover, we prove that the proposed technique can resolve the three-dimensional organization of fibrils inside the spherulite.

Mechanisms and pathology of protein misfolding and aggregation.

Despite advances in machine learning-based protein structure prediction, we are still far from fully understanding how proteins fold into their native conformation. The conventional notion that polypeptides fold spontaneously to their biologically active states has gradually been replaced by our understanding that cellular protein folding often requires context-dependent guidance from molecular chaperones in order to avoid misfolding. Misfolded proteins can aggregate into larger structures, such as amyloid fibrils, which perpetuate the misfolding process, creating a self-reinforcing cascade. A surge in amyloid fibril structures has deepened our comprehension of how a single polypeptide sequence can exhibit multiple amyloid conformations, known as polymorphism. The assembly of these polymorphs is not a random process but is influenced by the specific conditions and tissues in which they originate. This observation suggests that, similar to the folding of native proteins, the kinetics of pathological amyloid assembly are modulated by interactions specific to cells and tissues. Here, we review the current understanding of how intrinsic protein conformational propensities are modulated by physiological and pathological interactions in the cell to shape protein misfolding and aggregation pathology.

Extracellular protein homeostasis: The dawning of a new age for human disease therapies?

Extracellular protein homeostasis: The dawning of a new age for human disease therapies?

A comprehensive signature based on endoplasmic reticulum stress-related genes in predicting prognosis and immunotherapy response in melanoma

Melanoma is considered as one of the most invasion types of skin cancer with high mortality rates. Although combination of immune checkpoint therapy with local surgical excision provide a novel promising therapeutic strategies, the overall prognosis of melanoma patients remains unsatisfactory. Endoplasmic reticulum (ER) stress, a process of protein misfolding and undue accumulation, has been proven to play an indispensable regulatory role in tumor progression and tumor immunity. However, whether the signature based ER genes has predictive value for the prognosis and immunotherapy of melanoma has not been systematically manifested. In this study, the LASSO regression and multivariate Cox regression were applied to construct a novel signature for predicting melanoma prognosis both in the training and testing set. Intriguingly, we found that patients endowed with high- and low-risk scores displayed differences in clinicopathologic classification, immune cell infiltration level, tumor microenvironment, and immune checkpoint treatment response. Subsequently, based on molecular biology experiments, we validated that silencing the expression of RAC1, an ERG composed of the risk signature, could restrain the proliferation and migration, promote apoptosis, as well as increase the expression of PD-1/PD-L1 and CTLA4 in melanoma cells. Taken together, the risk signature was regarded as promising predictors for melanoma prognosis and might provide prospective strategies to ameliorate patients’ response to immunotherapy.

Histidine tautomerism-mediated transthyretin amyloidogenesis: A molecular insight

Characterization of the conformational alterations involved in monomer misfolding is essential for elucidating the molecular basis of the initial stage of protein accumulation. Here, we report the first structural analyses of transthyretin (TTR) (26−57) fragments with two histidine tautomeric states (δ; Nδ1H and ε; Nε2H) using replica-exchange molecular dynamics (REMD) simulations. Explaining the organizational properties and misfolding procedure is challenging because the δ and ε configurations can occur in the free neutral state. REMD revealed that β-sheet generation is favored for the δδ (16.8%) and εδ (6.7%) tautomeric isomers, showing frequent main-chain contacts between the stable regions near the head (N-terminus) and central (middle) part compared to the εε (4.8%) and δε (2.8%) isomers. The presence of smaller and wider local energy minima may be related to the structural stability and toxicity of δδ/εδ and εε/δε. Histidines31 and 56 were the parts of regular (such as β-strand) and nonregular (such as coil) secondary structures within the highly toxic TTR isomer. For TTR amyloidosis, focusing on hazardous isomeric forms with high sheet contents may be a potent treatment strategy. Overall, our findings support the tautomerism concept and aid in our comprehension of the basic tautomeric actions of neutral histidine throughout the misfolding process.

The α-Synuclein Monomer May Have Different Misfolding Mechanisms in the Induction of α-Synuclein Fibrils with Different Polymorphs

The aggregation of alpha-synuclein (α-Syn) is closely related to the occurrence of some neurodegenerative diseases such as Parkinson's disease. The misfolding of α-Syn monomer plays a key role in the formation of aggregates and extension of fibril. However, the misfolding mechanism of α-Syn remains elusive. Here, three different α-Syn fibrils (isolated from a diseased human brain, generated by in vitro cofactor-tau induction, and obtained by in vitro cofactor-free induction) were selected for the study. The misfolding mechanisms of α-Syn were uncovered by studying the dissociation of the boundary chains based on the conventional molecular dynamics (MD) and Steered MD simulations. The results showed that the dissociation paths of the boundary chains in the three systems were different. According to the reverse process of dissociation, we concluded that in the human brain system, the binding of the monomer and template starts from the C-terminal and gradually misfolds toward the N-terminal. In the cofactor-tau system, the monomer binding starts from residues 58-66 (contain β3), followed by the C-terminal coil (residues 67-79). Then, the N-terminal coil (residues 36-41) and residues 50-57 (contain β2) bind to the template, followed by residues 42-49 (contain β1). In the cofactor-free system, two misfolding paths were found. One is that the monomer binds to the N/C-terminal (β1/β6) and then binds to the remaining residues. The other one is that the monomer binds sequentially from the C- to N-terminal, similar to the human brain system. Furthermore, in the human brain and cofactor-tau systems, electrostatic interactions (especially from residues 58-66) are the main driving force during the misfolding process, whereas in the cofactor-free system, the contributions of electrostatic and van der Waals interactions are comparable. These results may provide a deeper understanding for the misfolding and aggregation mechanism of α-Syn.

AGEs RAGE Pathways: Alzheimer's Disease.

Neurofibrillary tangles and plaques containing tau serve as the biological markers for Alzheimer disease (AD) and pathogenesis is widely believed to be driven by the production and deposition of the β-amyloid peptide (Aβ). The β-amyloid peptide (Aβ) that results from the modification of the amyloid precursor protein (APP) by builds up as amyloid deposits in neuronal cells. Thus, a protein misfolding process is involved in the production of amyloid. In a native, aqueous buffer, amyloid fibrils are usually exceedingly stable and nearly insoluble. Although amyloid is essentially a foreign substance made of self-proteins, the immune system has difficulty identifying and eliminating it as such for unknown reasons. While the amyloidal deposit may have a direct role in the disease mechanism in some disease states involving amyloidal deposition, this is not always the case. Current research has shown that PS1 (presenilin 1) and BACE (beta-site APP-cleaving enzyme) have - and -secretase activity that increases β-amyloid peptide (Aβ). Wealth of data has shown that oxidative stress and AD are closely connected that causes the death of neuronal cells by producing reactive oxygen species (ROS). Additionally, it has been demonstrated that advanced glycation end products (AGEs) and β-amyloidal peptide (Aβ) together increase neurotoxicity. The objective of this review is to compile the most recent and intriguing data of AGEs and receptor for advanced glycation end products (RAGE) pathways which are responsible for AD.

Mapping the interactions between prion protein (PrPC) and prion protein fibrils (PrPSc).

Transmissible spongiform encephalopathies (TSEs), prion diseases, arise when normal prion protein (PrPC) misfolds and accumulates, forming prion fibrils (PrPSc) in the brain. Normal prion protein is GPI-anchored to the outer leaflet of the plasma membrane. Therefore, under pathological conditions, templated misfolding and aggregation occur on, or close to, the cell surface. However, this process of misfolding is distinct from neurotoxicity signaling pathways. Experimentally resolved three-dimensional structures of prion fibrils show at least two distinct populations: PIRIBS and 4-rungB. Although these structures provide valuable insight into fibril morphology, the mechanism responsible for templating prion protein misfolding largely remains unknown. In our project, we aim to characterize the modes of recognition between the prion proteins and fibrils on the surface of the cell membrane, using computational structural bioinformatics techniques. The results suggest that fibril-cell surface interactions are differentially driven by the electrostatics of the unique fibril morphologies. Our mapping of the binding interface of prion protein and fibril indicates a distribution of recognition sites, including the lateral surface and edge of the fibril. We will discuss our results considering the hypothesis that a distribution of protein-fibril recognition events leads to distinct outcomes, either scaffolding templated misfolding or triggering neurotoxic signaling. We will propose implications in terms of anti-prion druggable pathways as they relate to the potential mechanisms of neurotoxicity.

Prion receptors, prion internalization, intra- and inter-cellular transport.

Cellular prion protein (PrP C ) is a membrane bound protein that undergoes several post translational modifications. It can exert pleiotropic functions either in physiological and pathological conditions. The disease-causing isoform of the prion protein, called PrP Sc , acts as corruptive seed able to establish a process of misfolding and aggregation of further PrP Sc molecules. Therefore, PrP C acts as substrate for PrP Sc formation and as receptor to convey neurotoxic functions typical of other prion-like proteins involved in several neurodegenerative conditions. PrP C is transported from the outer leaflet of the cellular membrane to the intracellular compartments through several endocytic pathways, including clathrin-dependent and independent mechanisms and interactions with different adaptor proteins. During prion infection, the pathological counterpart PrP Sc , can be uptaken by the cells with mechanisms partially overlapping the ones of the physiological form, PrP C . The intercellular routing of both prion protein isoforms involves the release through vesicles and tunneling nanotubes. From the extracellular milieu, PrP C and PrP Sc can be uptaken by adjacent cells, thus exerting physiological and pathological functions.

Critically appraised topic on Rapid Eye Movement Behavior Disorder: From protein misfolding processes to clinical pathophysiology and conversion to neurodegenerative disorders

Critically appraised topic on Rapid Eye Movement Behavior Disorder: From protein misfolding processes to clinical pathophysiology and conversion to neurodegenerative disorders

Structural properties of Aβ (1–40) peptide in protonation stage of one, two, and three: New insights from the histidine protonation behaviors

Structural properties and aggregation tendency can be significantly influenced by histidine behaviors (histidine on Nδ-H state is defined as δ, likewise, Nε-H: ε and both Nδ-H and Nε-H: p). In current study, we investigated structural properties of Aβ(1–40) peptide during protonation evolution stage of one, two, and three by total 19 independent replica exchange molecular dynamics simulations using implicit solvent. Our results show that any kind of protonated state will promote β-sheet structure formation in comparison with deprotonated (εεε). With increase in number of protonation, the lowest β-sheet content increased. The highest averaged β-sheet structure content was detected in (δpδ) (46.0 %), (εpp) (36.8 %), and (ppp) (16.0 %) in each protonation stage. With three β-strand structures, (δpδ) shows more stable features and high hydrophobic properties. Further analysis confirmed that H13 and H14 are more important than H6. Specifically, H13 and H14 have a synergistic effect for structural formations by controlling H-bond networks in H13(p) with V39/V40 and H14(p/δ) with G37/G38. Finally, the Pearson correlation coefficient results confirmed that experimental result (ref. 44) is corresponding to our (εpp) system. Our current study will be conducive to understanding the effects of the histidine behaviors, it provides new insights for exploration protein folding and misfolding processes.

Evolutionarily Conserved Proline Residues Impede the Misfolding of the Mouse Prion Protein by Destabilizing an Aggregation-competent Partially Unfolded Form

The misfolding of the prion protein has been linked to several neurodegenerative diseases. Despite extensive studies, the mechanism of the misfolding process remains poorly understood. The present study structurally delineates the role of the conserved proline residues present in the structured C-terminal domain of the mouse prion protein (moPrP) in the misfolding process. It is shown that mutation of these Pro residues to Ala leads to destabilization of the native (N) state, and also to rapid misfolding. Using hydrogen–deuterium exchange (HDX) studies coupled with mass spectrometry (MS), it has been shown that the N state of moPrP is in rapid equilibrium with a partially unfolded form (PUF2*) at pH 4. It has been shown that the Pro to Ala mutations make PUF2* energetically more accessible from the N state by stabilizing it relative to the unfolded (U) state. The apparent rate constant of misfolding is found to be linearly proportional to the extent to which PUF2* is populated in equilibrium with the N state, strongly indicating that misfolding commences from PUF2*. It has also been shown that the Pro residues restrict the boundary of the structural core of the misfolded oligomers. Overall, this study highlights how the conserved proline residues control misfolding of the prion protein by modulating the stability of the partially unfolded form from which misfolding commences.

Applications of Single-Molecule Vibrational Spectroscopic Techniques for the Structural Investigation of Amyloid Oligomers.

Amyloid oligomeric species, formed during misfolding processes, are believed to play a major role in neurodegenerative and metabolic diseases. Deepening the knowledge about the structure of amyloid intermediates and their aggregation pathways is essential in understanding the underlying mechanisms of misfolding and cytotoxicity. However, structural investigations are challenging due to the low abundance and heterogeneity of those metastable intermediate species. Single-molecule techniques have the potential to overcome these difficulties. This review aims to report some of the recent advances and applications of vibrational spectroscopic techniques for the structural analysis of amyloid oligomers, with special focus on single-molecule studies.