Modulating the aggregation of human prion protein PrP106-126 by an indole-based cyclometallated palladium complex.

The spontaneous aggregation of infectious or misfolded forms of prion protein is known to be responsible for neurotoxicity in brain cells, which ultimately leads to the progression of prion disorders. Bovine spongiform encephalopathy (BSE) in animals and Creutzfeldt-Jakob disease (CJD) in humans are glaring examples in this regard. Square-planar complexes with labile ligands and indole-based compounds are found to be efficiently inhibitory against protein aggregation. Herein, we report the synthesis of an indole-based cyclometallated palladium complex. The ligand and complex were characterized by various spectroscopic techniques such as UV-visible, NMR, IR, and HRMS. The molecular structure of the complex was confirmed by single-crystal X-ray crystallography. The interaction of the complex with PrP106-126 was studied using UV-visible spectroscopy, CD spectroscopy, MALDI-TOF MS, and molecular docking. The inhibition effects of the complex on the PrP106-126 aggregation, fibrillization and amyloid formation phenomena were analysed through the ThT assay, CD, TEM and AFM. The effect of the complex on the aggregation process of PrP106-126 was determined kinetically through the ThT assay. The complex presented high binding affinity with the peptide and influenced the peptide's conformation and aggregation in different modes of binding. Furthermore, the MTT assay on neuronal HT-22 cells showed considerable protective properties of the complex against PrP106-126-mediated cytotoxicity. These findings suggest that the compound influences peptide aggregation in different ways, and the anti-aggregation action is primarily associated with the metal's physicochemical properties and the reactivity rather than the ligand. As a result, we propose that this compound be investigated as a potential therapeutic molecule in metallopharmaceutical research to treat prion disease (PD).

S100A9 inhibits and redirects prion protein 89-230 fragment amyloid aggregation.

Protein aggregation in the form of amyloid fibrils has long been associated with the onset and development of various amyloidoses, including Alzheimer's, Parkinson's or prion diseases. Recent studies of their fibril formation process have revealed that amyloidogenic protein cross-interactions may impact aggregation pathways and kinetic parameters, as well as the structure of the resulting aggregates. Despite a growing number of reports exploring this type of interaction, they only cover just a small number of possible amyloidogenic protein pairings. One such pair is between two neurodegeneration-associated proteins: the pro-inflammatory S100A9 and prion protein, which are known to co-localize in vivo. In this study, we examined their cross-interaction in vitro and discovered that the fibrillar form of S100A9 modulated the aggregation pathway of mouse prion protein 89-230 fragment, while non-aggregated S100A9 also significantly inhibited its primary nucleation process. These results complement previous observations of the pro-inflammatory protein's role in amyloid aggregation and highlight its potential role against neurodegenerative disorders.

In silico analysis of fungal prion-like proteins for elucidating their role in plant-fungi interactions.

Prion-like proteins (PrLPs) have emerged as beneficial molecules with implications in adaptive responses. These proteins possess a conserved prion-like domain (PrLD) which is an intrinsically disordered region capable of adopting different conformations upon perceiving external stimuli. Owing to changes in protein conformation, functional characteristics of proteins harboring PrLDs get altered thereby, providing a unique mode of protein-based regulation. Since PrLPs are ubiquitous in nature and involved in diverse functions, through this study, we aim to explore the role of such domains in yet another important physiological process viz. plant-microbe interactions to get insights into the mechanisms dictating cross-kingdom interactions. We have evaluated the presence and functions of PrLPs in 18 different plant-associated fungi of agricultural importance to unravel their role in plant-microbe interactions. Of the 241,997 proteins scanned, 3,820 (~ 1.6%) were identified as putative PrLPs with pathogenic fungi showing significantly higher PrLP density than their beneficial counterparts. Further, through GO enrichment analysis, we could predict several PrLPs from pathogenic fungi to be involved in virulence and formation of stress granules. Notably, PrLPs involved in (retro)transposition were observed exclusively in pathogenic fungi. We even analyzed publicly available data for the expression alterations of fungal PrLPs upon their interaction with their respective hosts which revealed perturbation in the levels of some PrLP-encoding genes during interactions with plants. Overall, our work sheds light into the probable role of prion-like candidates in plant-fungi interaction, particularly in context of pathogenesis, paving way for more focused studies for validating their role.

Liquid-liquid phase separation of the prion protein is regulated by the octarepeat domain independently of histidines and copper.

Liquid-liquid phase separation (LLPS) of the mammalian prion protein is mainly driven by its intrinsically disordered N-terminal domain (N-PrP). However, the specific intermolecular interactions that promote LLPS remain largely unknown. Here, we used extensive mutagenesis and comparative analyses of evolutionarily distant PrP species to gain insight into the relationship between protein sequence and phase behavior. LLPS of mouse PrP is dependent on two polybasic motifs in N-PrP that are conserved in all tetrapods. A unique feature of mammalian N-PrP is the octarepeat domain with four histidines that mediate binding to copper ions. We now show that the octarepeat is critical for promoting LLPS and preventing the formation of PrP aggregates. Amphibian N-PrP, which contains the polybasic motifs but lacks a repeat domain and histidines, does not undergo LLPS and forms nondynamic protein assemblies indicative of aggregates. Insertion of the mouse octarepeat domain restored LLPS of amphibian N-PrP, supporting its essential role in regulating the phase transition of PrP. This activity of the octarepeat domain was neither dependent on the four highly conserved histidines nor on copper binding. Instead, the regularly spaced tryptophan residues were critical for regulating LLPS, presumably via cation-π interactions with the polybasic motifs. Our study reveals a novel role for the tryptophan residues in the octarepeat in controlling phase transition of PrP and indicates that the ability of mammalian PrP to undergo LLPS has evolved with the octarepeat in the intrinsically disordered domain but independently of the histidines.

The Positively Charged Cluster in the N-terminal Disordered Region may Affect Prion Protein Misfolding: Cryo-EM Structure of Hamster PrP(23-144) Fibrils.

Prions, the misfolding form of prion proteins, are contagious proteinaceous macromolecules. Recent studies have shown that infectious prion fibrils formed in the brain and non-infectious fibrils formed from recombinant prion protein in a partially denaturing condition have distinct structures. The amyloid core of the in vitro-prepared non-infectious fibrils starts at about residue 160, while that of infectious prion fibrils formed in the brain involves a longer sequence (residues ∼90-230) of structural conversion. The C-terminal truncated prion protein PrP(23-144) can form infectious fibrils under certain conditions and cause disease in animals. In this study, we used cryogenic electron microscopy (cryo-EM) to resolve the structure of hamster sHaPrP(23-144) fibrils prepared at pH 3.7. This 2.88 Å cryo-EM structure has an amyloid core covering residues 94-144. It comprises two protofilaments, each containing five ß-strands arranged as a long hairpin plus an N-terminal ß-strand. This N-terminal ß-strand resides in a positively charged cluster region (named PCC2; sequence 96-111), which interacts with the turn region of the opposite protofilaments' hairpin to stabilize the fibril structure. Interestingly, this sHaPrP(23-144) fibril structure differs from a recently reported structure formed by the human or mouse counterpart at pH 6.5. Moreover, sHaPrP(23-144) fibrils have many structural features in common with infectious prions. Whether this structure is infectious remains to be determined. More importantly, the sHaPrP(23-144) structure is different from the sHaPrP(108-144) fibrils prepared in the same fibrillization buffer, indicating that the N-terminal disordered region, possibly the positively charged cluster, influences the misfolding pathway of the prion protein.

The structural line between prion and "prion-like": Insights from prion protein and tau.

The concept of 'prion-like' behavior has emerged in the study of diseases involving protein misfolding where fibrillar structures, called amyloids, self-propagate and induce disease in a fashion similar to prions. From a biological standpoint, in order to be considered 'prion-like,' a protein must traverse cells and tissues and further propagate via a templated conformational change. Since 2017, cryo-electron microscopy structures from patient-derived 'prion-like' amyloids, in particular tau, have been presented and revealed structural similarities shared across amyloids. Since 2021, cryo-EM structures from prions of known infectivity have been added to the ex vivo amyloid structure family. In this review, we discuss current proposals for the 'prion-like' mechanisms of spread for tau and prion protein as well as discuss different influencers on structures of aggregates from tauopathies and prion diseases. Lastly, we discuss some of the current hypotheses for what may distinguish structures that are 'prion-like' from transmissible prion structures.

Ultrasensitive Detection of Lipid-Induced Misfolding of the Prion Protein at the Aqueous-Liquid Crystal Interface.

The misfolding of the α-helical cellular prion protein into a self-propagating ß-rich aggregated form is a key pathogenic event in fatal and transmissible neurodegenerative diseases collectively known as prion diseases. Herein, we utilize the interfacial properties of liquid crystals (LCs) to monitor the lipid-membrane-induced conformational switching of prion protein (PrP) into ß-rich amyloid fibrils. The lipid-induced conformational switching resulting in aggregation occurs at the nanomolar protein concentration and is primarily mediated by electrostatic interactions between PrP and lipid headgroups. Our LC-based methodology offers a potent and sensitive tool to detect and delineate molecular mechanisms of PrP misfolding mediated by lipid-protein interactions at the aqueous interface under physiological conditions.

Experiment and molecular dynamics simulations reveal proanthocyanidin B2 and B3 can inhibit prion aggregation by different mechanisms.

Prion diseases are a group of fatal neurodegenerative diseases caused by the misfolding and aggregation of prion protein (PrP), and the inhibition of PrP aggregation is one of the most effective therapeutic strategies. Proanthocyanidin B2 (PB2) and B3 (PB3), the effective natural antioxidants have been evaluated for the inhibition of amyloid-related protein aggregation. Since PrP has similar aggregation mechanism with other amyloid-related proteins, will PB2 and PB3 affect the aggregation of PrP? In this paper, experimental and molecular dynamics (MD) simulation methods were combined to investigate the influence of PB2 and PB3 on PrP aggregation. Thioflavin T assays showed PB2 and PB3 could inhibit PrP aggregation in a concentrate-dependent manner in vitro. To understand the underlying mechanism, we performed 400 ns all-atom MD simulations. The results suggested PB2 could stabilize the α2 C-terminus and the hydrophobic core of protein by stabilizing two important salt bridges R156-E196 and R156-D202, and consequently made global structure of protein more stable. Surprisingly, PB3 could not stabilize PrP, which may inhibit PrP aggregation through a different mechanism. Since dimerization is the first step of aggregation, will PB3 inhibit PrP aggregation by inhibiting the dimerization? To verify our assumption, we then explored the effect of PB3 on protein dimerization by performing 800 ns MD simulations. The results suggested PB3 could reduce the residue contacts and hydrogen bonds between two monomers, preventing dimerization process of PrP. The possible inhibition mechanism of PB2 and PB3 on PrP aggregation could provide useful information for drug development against prion diseases.Communicated by Ramaswamy H. Sarma.

Bacteriophage-encoded chaperonins stimulate prion protein fibrillation in an ATP-dependent manner.

The pathogenesis of the various prion diseases is based on the conformational conversion of the prion protein from its physiological cellular form to the insoluble scrapie isoform. Several chaperones, including the Hsp60 family of group I chaperonins, are known to contribute to this transformation, but data on their effects are scarce and conflicting. In this work, two GroEL-like phage chaperonins, the single-ring OBP and the double-ring EL, were found to stimulate monomeric prion protein fibrillation in an ATP-dependent manner. The resulting fibrils were characterised by thioflavin T fluorescence, electron microscopy, proteinase K digestion assay and other methods. In the presence of ATP, chaperonins were found to promote the conversion of prion protein monomers into short amyloid fibrils with their further aggregation into less toxic large clusters. Fibrils generated with the assistance of phage chaperonins differ in morphology and properties from those formed spontaneously from monomeric prion in the presence of denaturants at acidic pH.

The role of cellular prion protein in immune system.

Numerous studies have investigated the cellular prion protein (PrPC) since its discovery. These investigations have explained that its structure is predominantly composed of alpha helices and short beta sheet segments, and when its abnormal scrapie isoform (PrPSc) is infected, PrPSc transforms the PrPC, leading to prion diseases, including Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. Given its ubiquitous distribution across a variety of cellular types, the PrPC manifests a diverse range of biological functions, including cell-cell adhesion, neuroprotection, signalings, and oxidative stress response. PrPC is also expressed in immune tissues, and its functions in these tissues include the activation of immune cells and the formation of secondary lymphoid tissues, such as the spleen and lymph nodes. Moreover, high expression of PrPC in immune cells plays a crucial role in the pathogenesis of prion diseases. In addition, it affects inflammation and the development and progression of cancer via various mechanisms. In this review, we discuss the studies on the role of PrPC from various immunological perspectives. [BMB Reports 2023; 56(12): 645-650].

The Smallest Infectious Substructure Encoding the Prion Strain Structural Determinant Revealed by Spontaneous Dissociation of Misfolded Prion Protein Assemblies.

It is commonly accepted that the prion replicative propensity and strain structural determinant (SSD) are encoded in the fold of PrPSc amyloid fibril assemblies. By exploring the quaternary structure dynamicity of several prion strains, we revealed that all mammalian prion assemblies exhibit the generic property of spontaneously generating two sets of discreet infectious tetrameric and dimeric species differing significantly by their specific infectivity. By using perturbation approaches such as dilution and ionic strength variation, we demonstrated that these two oligomeric species were highly dynamic and evolved differently in the presence of chaotropic agents. In general, our observations of seven different prion strains from three distinct species highlight the high dynamicity of PrPSc assemblies as a common and intrinsic property of mammalian prions. The existence of such small infectious PrPSc species harboring the SSD indicates that the prion infectivity and the SSD are not restricted only to the amyloid fold but can also be encoded in other alternative quaternary structures. Such diversity in the quaternary structure of prion assemblies tends to indicate that the structure of PrPSc can be divided into two independent folding domains: a domain encoding the strain structural determinant and a second domain whose fold determines the type of quaternary structure that could adopt PrPSc assemblies.

Y225A induces long-range conformational changes in human prion protein that are protective in Drosophila.

Prion protein (PrP) misfolding is the key trigger in the devastating prion diseases. Yet the sequence and structural determinants of PrP conformation and toxicity are not known in detail. Here, we describe the impact of replacing Y225 in human PrP with A225 from rabbit PrP, an animal highly resistant to prion diseases. We first examined human PrP-Y225A by molecular dynamics simulations. We next introduced human PrP in Drosophila and compared the toxicity of human PrP-WT and Y225A in the eye and in brain neurons. Y225A stabilizes the ß2-α2 loop into a 310-helix from six different conformations identified in WT and lowers hydrophobic exposure. Transgenic flies expressing PrP-Y225A exhibit less toxicity in the eye and in brain neurons and less accumulation of insoluble PrP. Overall, we determined that Y225A lowers toxicity in Drosophila assays by promoting a structured loop conformation that increases the stability of the globular domain. These findings are significant because they shed light on the key role of distal α-helix 3 on the dynamics of the loop and the entire globular domain.

Loss of Residues 119-136, Including the First ß-strand of Human Prion Protein, Generates an Aggregation-competent Partially "Open" Form.

In prion replication, the cellular form of prion protein (PrPC) must undergo a full conformational transition to its disease-associated fibrillar form. Transmembrane forms of PrP have been implicated in this structural conversion. The cooperative unfolding of a structural core in PrPC presents a substantial energy barrier to prion formation, with membrane insertion and detachment of parts of PrP presenting a plausible route to its reduction. Here, we examined the removal of residues 119-136 of PrP, a region which includes the first ß-strand and a substantial portion of the conserved hydrophobic region of PrP, a region which associates with the ER membrane, on the structure, stability and self-association of the folded domain of PrPC. We see an "open" native-like conformer with increased solvent exposure which fibrilises more readily than the native state. These data suggest a stepwise folding transition, which is initiated by the conformational switch to this "open" form of PrPC.

Insight into the conserved structural dynamics of the C-terminus of mammal PrPC identifies structural core and possible structural role of pharmacological chaperones.

Misfolding of the prion protein is central to prion disease aetiology. Although understanding the dynamics of the native fold helps to decipher the conformational conversion mechanism, a complete depiction of distal but coupled prion protein sites common across species is lacking. To fill this gap, we used normal mode analysis and network analysis to examine a collection of prion protein structures deposited on the protein data bank. Our study identified a core of conserved residues that sustains the connectivity across the C-terminus of the prion protein. We propose how a well-characterized pharmacological chaperone may stabilize the fold. Also, we provide insight into the effect on the native fold of initial misfolding pathways identified by others using kinetics studies.

The seeding barrier between human and Syrian hamster prion protein amyloid fibrils is determined by ß2-α2 loop sequence elements.

Transmissive spongiform encephalopathies (TSE) are a group of neurodegenerative diseases caused by infectious protein particles, known as prions. Prions are formed from cellular prion proteins (PrP) and can be transmitted between different mammalian species. Subsequently, the host's PrPs are then converted to prions, followed by the onset of TSE. Interspecies prion infectivity is governed by the amino acid sequence differences of PrPs and prions' inability to replicate in a host is termed a species barrier. Here, we investigated the amino acid sequence determinants of species barrier between recombinant human (rHuPrP) and hamster (rShaPrP) prion protein amyloid fibrils. We discovered that a unidirectional species barrier between rShaPrP and rHuPrP amyloid fibrils exists. This barrier stems from the difference of amino acid sequences in the conserved ß2-α2 loop region. Our results revealed that individual amino acids in the ß2-α2 loop region are critical for overcoming the barrier between human and hamster prion protein amyloid fibrils in vitro. Furthermore, the barrier was only possible to observe through aggregation kinetics, as the secondary structure rHuPrP fibrils was not affected by the cross-seeding. Overall, we demonstrated the mechanistic pathway behind this interspecies barrier phenomenon, which increases our understanding of prion-related disease development.

Simulation analysis of selective alanine mutation effect on stability of human prion protein.

Prion diseases are neurodegenerative disorders caused by spongiform degeneration of the brain. Understanding the fundamental mechanism of prion protein aggregation caused by mutations is very crucial to resolve the pathology of prion diseases. To help understand the roles of individual residues on the stability of the human prion protein, the computational method of free energy simulations based on atomistic molecular dynamics trajectories is applied to Phe175 → Ala, Val180 → Ala, and Val209 → Ala mutations of the human prion protein. The simulations show that all three alanine mutations destabilize the human prion protein. The calculated free energy change differences, ΔΔG, for the Phe175 → Ala, Val180 → Ala, and Val209 → Ala mutations are in good agreement with the experimental values. The significant destabilizing effects on the mutants relative to the wild-type protein arise from van der Waals terms. Furthermore, our free energy decomposition analysis shows that the major contribution to destabilizing the V180A and V209A mutants relative to the wild-type protein is originated from van der Waals interactions from residues near the mutation sites. In contrast, the contribution to destabilizing the F175A mutant is mainly caused by van der Waals interactions from residues near and far away from the mutation site. Our results show that the free energy simulation with a thermodynamic integration approach for selected alanine scanning mutations is beneficial for understanding the detailed mechanism of human prion protein destabilization, specific residues' role, and the hydrophobic effect on protein stability.Communicated by Ramaswamy H. Sarma.

Investigation of the conformation of human prion protein in ethanol solution using molecular dynamics simulations.

When the conformation of protein is changed from its natural state to a misfolded state, some diseases will happen like prion disease. Prion diseases are a set of deadly neurodegenerative diseases caused by prion protein misfolding and aggregation. Monohydric alcohols have a strong influence on the structure of protein. However, whether monohydric alcohols inhibit amyloid fibrosis remains uncertain. Here, to elucidate the effect of ethanol on the structural stability of human prion protein, molecular dynamics simulations were employed to analyze the conformational changes and dynamics characteristics of human prion proteins at different temperatures. The results show that the extension of ß-sheet occurs more easily and the α-helix is more easily disrupted at high temperatures. We found that ethanol can destroy the hydrophobic interactions and make the hydrogen bonds stable, which protects the secondary structure of the protein, especially at 500 K.Communicated by Ramaswamy H. Sarma.

Prion Propagation is Dependent on Key Amino Acids in Charge Cluster 2 within the Prion Protein.

To dissect the N-terminal residues within the cellular prion protein (PrPC) that are critical for efficient prion propagation, we generated a library of point, double, or triple alanine replacements within residues 23-111 of PrP, stably expressed them in cells silenced for endogenous mouse PrPC and challenged the reconstituted cells with four common but biologically diverse mouse prion strains. Amino acids (aa) 105-111 of Charge Cluster 2 (CC2), which is disordered in PrPC, were found to be required for propagation of all four prion strains; other residues had no effect or exhibited strain-specific effects. Replacements in CC2, including aa105-111, dominantly inhibited prion propagation in the presence of endogenous wild type PrPC whilst other changes were not inhibitory. Single alanine replacements within aa105-111 identified leucine 108 and valine 111 or the cluster of lysine 105, threonine 106 and asparagine 107 as critical for prion propagation. These residues mediate specific ordering of unstructured CC2 into ß-sheets in the infectious prion fibrils from Rocky Mountain Laboratory (RML) and ME7 mouse prion strains.

Development of Alkaline Phosphatase-Fused Mouse Prion Protein and Its Application in Toxic Aß Oligomer Detection.

Amyloid ß (Aß) oligomers play a key role in the progression of Alzheimer's disease (AD). Multiple forms of Aß assemblies have been identified by in vitro and in vivo analyses; however, it is uncertain which oligomer is highly neurotoxic. Thus, understanding the pathogenesis of AD by detecting toxic Aß oligomers is crucial. In this study, we report a fusion protein of cellular prion protein (PrPc) and alkaline phosphatase (ALP) from Escherichia coli as a sensing element for toxic Aß oligomers. Since the N-terminus domain of PrPc (residue 23-111) derived from mice is known to bind to toxic Aß oligomers in vitro, we genetically fused PrPc23-111 to ALP. The developed fusion protein, PrP-ALP, retained both the binding ability of PrPc and enzymatic activity of ALP. We showed that PrP-ALP strongly bound to high molecular weight (HMW) oligomers but showed little or no affinity toward monomers. The observation that PrP-ALP neutralized the toxic effect of Aß oligomers indicated an interaction between PrP-ALP and toxic HMW oligomers. Based on ALP activity, we succeeded in detecting Aß oligomers. PrP-ALP may serve as a powerful tool for detecting toxic Aß oligomers that may be related to AD progression.

Molecular insights into the critical role of gallate moiety of green tea catechins in modulating prion fibrillation, cellular internalization, and neuronal toxicity.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative diseases with no approved therapeutics. TSE pathology is characterized by abnormal accumulation of amyloidogenic and infectious prion protein conformers (PrPSc) in the central nervous system. Herein, we examined the role of gallate group in green tea catechins in modulating the aggregation of human prion protein (HuPrP) using two green tea constituents i.e., epicatechin 3-gallate (EC3G; with intact gallate ring) and epigallocatechin (EGC; without gallate ring). Molecular docking indicated distinct differences in hydrogen bonding and hydrophobic interactions of EC3G and EGC at the ß2-α2 loop of HuPrP. These differences were substantiated by 44-fold higher KD for EC3G as compared to EGC with the former significantly reducing Thioflavin T (ThT) binding aggregates of HuPrP. Conformational alterations in HuPrP aggregates were validated by particle sizing, AFM analysis and A11 and OC conformational antibodies. As compared to EGC, EC3G showed relatively higher reduction in toxicity and cellular internalization of HuPrP oligomers in Neuro-2a cells. Additionally, EC3G also displayed higher fibril disaggregating properties as observed by ThT kinetics and electron microscopy. Our observations were supported by molecular dynamics (MD) simulations that showed markedly reduced α2-α3 and ß2-α2 loop mobilities in presence of EC3G that may lead to constriction of HuPrP conformational space with lowered ß-sheet conversion. In totality, gallate moiety of catechins play key role in modulating HuPrP aggregation, and toxicity and could be a new structural motif for designing therapeutics against prion diseases and other neurodegenerative disorders.