Aggregation Condition–Structure Relationship of Mouse Prion Protein Fibrils

Prion diseases are associated with conformational conversion of cellular prion protein into a misfolded pathogenic form, which resembles many properties of amyloid fibrils. The same prion protein sequence can misfold into different conformations, which are responsible for variations in prion disease phenotypes (prion strains). In this work, we use atomic force microscopy, FTIR spectroscopy and magic-angle spinning NMR to devise structural models of mouse prion protein fibrils prepared in three different denaturing conditions. We find that the fibril core region as well as the structure of its N- and C-terminal parts is almost identical between the three fibrils. In contrast, the central part differs in length of β-strands and the arrangement of charged residues. We propose that the denaturant ionic strength plays a major role in determining the structure of fibrils obtained in a particular condition by stabilizing fibril core interior-facing glutamic acid residues.     

Temperature-Dependent Structural Variability of Prion Protein Amyloid Fibrils

Prion protein aggregation into amyloid fibrils is associated with the onset and progression of prion diseases—a group of neurodegenerative amyloidoses. The process of such aggregate formation is still not fully understood, especially regarding their polymorphism, an event where the same type of protein forms multiple, conformationally and morphologically distinct structures. Considering that such structural variations can greatly complicate the search for potential antiamyloid compounds, either by having specific propagation properties or stability, it is important to better understand this aggregation event. We have recently reported the ability of prion protein fibrils to obtain at least two distinct conformations under identical conditions, which raised the question if this occurrence is tied to only certain environmental conditions. In this work, we examined a large sample size of prion protein aggregation reactions under a range of temperatures and analyzed the resulting fibril dye-binding, secondary structure and morphological properties. We show that all temperature conditions lead to the formation of more than one fibril type and that this variability may depend on the state of the initial prion protein molecules.     

Molecular Investigations of Protein Aggregation in the Pathogenesis of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disorder characterized by selective loss of lower and upper motor neurons (MNs) in the brain and spinal cord, resulting in paralysis and eventually death due to respiratory insufficiency. Although the fundamental physiological mechanisms underlying ALS are not completely understood, the key neuropathological hallmarks of ALS pathology are the aggregation and accumulation of ubiquitinated protein inclusions within the cytoplasm of degenerating MNs. Herein, we discuss recent insights into the molecular mechanisms that lead to the accumulation of protein aggregates in ALS. This will contribute to a better understanding of the pathophysiology of the disease and may open novel avenues for the development of therapeutic strategies.     

Protein Aggregation Landscape in Neurodegenerative Diseases: Clinical Relevance and Future Applications

Intrinsic disorder is a natural feature of polypeptide chains, resulting in the lack of a defined three-dimensional structure. Conformational changes in intrinsically disordered regions of a protein lead to unstable β-sheet enriched intermediates, which are stabilized by intermolecular interactions with other β-sheet enriched molecules, producing stable proteinaceous aggregates. Upon misfolding, several pathways may be undertaken depending on the composition of the amino acidic string and the surrounding environment, leading to different structures. Accumulating evidence is suggesting that the conformational state of a protein may initiate signalling pathways involved both in pathology and physiology. In this review, we will summarize the heterogeneity of structures that are produced from intrinsically disordered protein domains and highlight the routes that lead to the formation of physiological liquid droplets as well as pathogenic aggregates. The most common proteins found in aggregates in neurodegenerative diseases and their structural variability will be addressed. We will further evaluate the clinical relevance and future applications of the study of the structural heterogeneity of protein aggregates, which may aid the understanding of the phenotypic diversity observed in neurodegenerative disorders.     

Different Roles of p62 (SQSTM1) Isoforms in Keratin-Related Protein Aggregation

p62/Sequestosome-1 (p62) is a multifunctional adaptor protein and is also a constant component of disease-associated protein aggregates, including Mallory–Denk bodies (MDBs), in steatohepatitis and hepatocellular carcinoma. We investigated the interaction of the two human p62 isoforms, p62-H1 (full-length isoform) and p62-H2 (partly devoid of PB1 domain), with keratins 8 and 18, the major components of MDBs. In human liver, p62-H2 is expressed two-fold higher compared to p62-H1 at the mRNA level and is present in slightly but not significantly higher concentrations at the protein level. Co-transfection studies in CHO-K1 cells, PLC/PRF/5 cells as well as p62⁻ total-knockout and wild-type mouse fibroblasts revealed marked differences in the cytoplasmic distribution and aggregation behavior of the two p62 isoforms. Transfection-induced overexpression of p62-H2 generated large cytoplasmic aggregates in PLC/PRF/5 and CHO-K1 cells that mostly co-localized with transfected keratins resembling MDBs or (transfection without keratins) intracytoplasmic hyaline bodies. In fibroblasts, however, transfected p62-H2 was predominantly diffusely distributed in the cytoplasm. Aggregation of p62-H2 and p62ΔSH2 as well as the interaction with K8 (but not with K18) involves acquisition of cross-β-sheet conformation as revealed by staining with luminescent conjugated oligothiophenes. These results indicate the importance of considering p62 isoforms in protein aggregation disease.     

Cervid Prion Protein Polymorphisms: Role in Chronic Wasting Disease Pathogenesis

Chronic wasting disease (CWD) is a prion disease found in both free-ranging and farmed cervids. Susceptibility of these animals to CWD is governed by various exogenous and endogenous factors. Past studies have demonstrated that polymorphisms within the prion protein (PrP) sequence itself affect an animal’s susceptibility to CWD. PrP polymorphisms can modulate CWD pathogenesis in two ways: the ability of the endogenous prion protein (PrP^C) to convert into infectious prions (PrP^Sc) or it can give rise to novel prion strains. In vivo studies in susceptible cervids, complemented by studies in transgenic mice expressing the corresponding cervid PrP sequence, show that each polymorphism has distinct effects on both PrP^C and PrP^Sc. It is not entirely clear how these polymorphisms are responsible for these effects, but in vitro studies suggest they play a role in modifying PrP epitopes crucial for PrP^C to PrP^Sc conversion and determining PrP^C stability. PrP polymorphisms are unique to one or two cervid species and most confer a certain degree of reduced susceptibility to CWD. However, to date, there are no reports of polymorphic cervid PrP alleles providing absolute resistance to CWD. Studies on polymorphisms have focused on those found in CWD-endemic areas, with the hope that understanding the role of an animal’s genetics in CWD can help to predict, contain, or prevent transmission of CWD.     

Conformational Analysis of Misfolded Protein Aggregation by FRET and Live-Cell Imaging Techniques

Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). A hallmark of neurodegenerative disorders is formation of ubiquitin-positive inclusion bodies in neurons, suggesting that the aggregation process of misfolded proteins changes during disease progression. Hence, high-throughput determination of soluble oligomers during the aggregation process, as well as the conformation of sequestered proteins in inclusion bodies, is essential for elucidation of physiological regulation mechanism and drug discovery in this field. To elucidate the interaction, accumulation, and conformation of aggregation-prone proteins, in situ spectroscopic imaging techniques, such as Förster/fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and bimolecular fluorescence complementation (BiFC) have been employed. Here, we summarize recent reports in which these techniques were applied to the analysis of aggregation-prone proteins (in particular their dimerization, interactions, and conformational changes), and describe several fluorescent indicators used for real-time observation of physiological states related to proteostasis.     

Strategies for the Inhibition of Protein Aggregation in Human Diseases

Protein misfolding and aggregation has been related to several human disorders, generally termed protein aggregation diseases. These diseases include neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases and peripheral disorders such as systemic amyloidosis and type 2 diabetes. The complexity of the aggregation processes and the intertwined events account for the fact that no effective disease‐modifying treatments for these disorders are currently available. Nevertheless, in‐depth research into the aggregation processes has recently yielded major insights into some key mechanisms of aggregation‐mediated cell toxicity, offering new targets for drug development. In addition, recent findings in the field have identified similar features, revealing the possibility of shared mechanisms and hence potential common approaches for intervention. This review aims to give an overview of potential strategies for tackling protein aggregation and its associated toxicity, focusing on protein aggregation in human disease.     

What Macromolecular Crowding Can Do to a Protein

The intracellular environment represents an extremely crowded milieu, with a limited amount of free water and an almost complete lack of unoccupied space. Obviously, slightly salted aqueous solutions containing low concentrations of a biomolecule of interest are too simplistic to mimic the “real life” situation, where the biomolecule of interest scrambles and wades through the tightly packed crowd. In laboratory practice, such macromolecular crowding is typically mimicked by concentrated solutions of various polymers that serve as model “crowding agents”. Studies under these conditions revealed that macromolecular crowding might affect protein structure, folding, shape, conformational stability, binding of small molecules, enzymatic activity, protein-protein interactions, protein-nucleic acid interactions, and pathological aggregation. The goal of this review is to systematically analyze currently available experimental data on the variety of effects of macromolecular crowding on a protein molecule. The review covers more than 320 papers and therefore represents one of the most comprehensive compendia of the current knowledge in this exciting area.     

Combined Effects of Confinement and Macromolecular Crowding on Protein Stability

Confinement and crowding have been shown to affect protein fates, including folding, functional stability, and their interactions with self and other proteins. Using both theoretical and experimental studies, researchers have established the independent effects of confinement or crowding, but only a few studies have explored their effects in combination; therefore, their combined impact on protein fates is still relatively unknown. Here, we investigated the combined effects of confinement and crowding on protein stability using the pores of agarose hydrogels as a confining agent and the biopolymer, dextran, as a crowding agent. The addition of dextran further stabilized the enzymes encapsulated in agarose; moreover, the observed increases in enhancements (due to the addition of dextran) exceeded the sum of the individual enhancements due to confinement and crowding. These results suggest that even though confinement and crowding may behave differently in how they influence protein fates, these conditions may be combined to provide synergistic benefits for protein stabilization. In summary, our study demonstrated the successful use of polymer-based platforms to advance our understanding of how in vivo like environments impact protein function and structure.     

Extracellular Vesicles Physiological Role and the Particular Case of Disease-Spreading Mechanisms in Polyglutamine Diseases

Recent research demonstrated pathological spreading of the disease-causing proteins from one focal point across other brain regions for some neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease. Spreading mediated by extracellular vesicles is one of the proposed disease-spreading mechanisms. Extracellular vesicles are cell membrane-derived vesicles, used by cells for cell-to-cell communication and excretion of toxic components. Importantly, extracellular vesicles carrying pathological molecules, when internalized by “healthy” cells, may trigger pathological pathways and, consequently, promote disease spreading to neighboring cells. Polyglutamine diseases are a group of genetic neurodegenerative disorders characterized by the accumulation of mutant misfolded proteins carrying an expanded tract of glutamines, including Huntington’s and Machado–Joseph disease. The pathological spread of the misfolded proteins or the corresponding mutant mRNA has been explored. The understanding of the disease-spreading mechanism that plays a key role in the pathology progression of these diseases can result in the development of effective therapeutic approaches to stop disease progression, arresting the spread of the toxic components and disease aggravation. Therefore, the present review’s main focus is the disease-spreading mechanisms with emphasis on polyglutamine diseases and the putative role played by extracellular vesicles in this process.     

Effects of Molecular Crowding and Betaine on HSPB5 Interactions,with Target Proteins Differing in the Quaternary Structure and Aggregation Mechanism

The aggregation of intracellular proteins may be enhanced under stress. The expression of heat-shock proteins (HSPs) and the accumulation of osmolytes are among the cellular protective mechanisms in these conditions. In addition, one should remember that the cell environment is highly crowded. The antiaggregation activity of HSPB5 and the effect on it of either a crowding agent (polyethylene glycol (PEG)) or an osmolyte (betaine), or their mixture, were tested on the aggregation of two target proteins that differ in the order of aggregation with respect to the protein: thermal aggregation of glutamate dehydrogenase and DTT-induced aggregation of lysozyme. The kinetic analysis of the dynamic light-scattering data indicates that crowding can decrease the chaperone-like activity of HSPB5. Nonetheless, the analytical ultracentrifugation shows the protective effect of HSPB5, which retains protein aggregates in a soluble state. Overall, various additives may either improve or impair the antiaggregation activity of HSPB5 against different protein targets. The mixed crowding arising from the presence of PEG and 1 M betaine demonstrates an extraordinary effect on the oligomeric state of protein aggregates. The shift in the equilibrium of HSPB5 dynamic ensembles allows for the regulation of its antiaggregation activity. Crowding can modulate HSPB5 activity by affecting protein–protein interactions.     

Unveiling the Metal-Dependent Aggregation Properties of the C-terminal Region of Amyloidogenic Intrinsically Disordered Protein Isoforms DPF3b and DPF3a

Double-PHD fingers 3 (DPF3) is a BAF-associated human epigenetic regulator, which is increasingly recognised as a major contributor to various pathological contexts, such as cardiac defects, cancer, and neurodegenerative diseases. Recently, we unveiled that its two isoforms (DPF3b and DPF3a) are amyloidogenic intrinsically disordered proteins. DPF3 isoforms differ from their C-terminal region (C-TERb and C-TERa), containing zinc fingers and disordered domains. Herein, we investigated the disorder aggregation properties of C-TER isoforms. In agreement with the predictions, spectroscopy highlighted a lack of a highly ordered structure, especially for C-TERa. Over a few days, both C-TERs were shown to spontaneously assemble into similar antiparallel and parallel β-sheet-rich fibrils. Altered metal homeostasis being a neurodegeneration hallmark, we also assessed the influence of divalent metal cations, namely Cu²⁺, Mg²⁺, Ni²⁺, and Zn²⁺, on the C-TER aggregation pathway. Circular dichroism revealed that metal binding does not impair the formation of β-sheets, though metal-specific tertiary structure modifications were observed. Through intrinsic and extrinsic fluorescence, we found that metal cations differently affect C-TERb and C-TERa. Cu²⁺ and Ni²⁺ have a strong inhibitory effect on the aggregation of both isoforms, whereas Mg²⁺ impedes C-TERb fibrillation and, on the contrary, enhances that of C-TERa. Upon Zn²⁺ binding, C-TERb aggregation is also hindered, and the amyloid autofluorescence of C-TERa is remarkably red-shifted. Using electron microscopy, we confirmed that the metal-induced spectral changes are related to the morphological diversity of the aggregates. While metal-treated C-TERb formed breakable and fragmented filaments, C-TERa fibrils retained their flexibility and packing properties in the presence of Mg²⁺ and Zn²⁺ cations.     

The Cellular Prion Protein: A Promising Therapeutic Target for Cancer

Studies on the cellular prion protein (PrP^C) have been actively conducted because misfolded PrP^C is known to cause transmissible spongiform encephalopathies or prion disease. PrP^C is a glycophosphatidylinositol-anchored cell surface glycoprotein that has been reported to affect several cellular functions such as stress protection, cellular differentiation, mitochondrial homeostasis, circadian rhythm, myelin homeostasis, and immune modulation. Recently, it has also been reported that PrP^C mediates tumor progression by enhancing the proliferation, metastasis, and drug resistance of cancer cells. In addition, PrP^C regulates cancer stem cell properties by interacting with cancer stem cell marker proteins. In this review, we summarize how PrP^C promotes tumor progression in terms of proliferation, metastasis, drug resistance, and cancer stem cell properties. In addition, we discuss strategies to treat tumors by modulating the function and expression of PrP^C via the regulation of HSPA1L/HIF-1α expression and using an anti-prion antibody.     

Chemical Biology Toolbox to Visualize Protein Aggregation in Live Cells

Protein misfolding and aggregation is a complex biochemical process and has been associated with numerous human degenerative diseases. Developing novel chemical and biological tools and approaches to visualize aggregated proteins in live cells is in high demand for mechanistic studies, diagnostics, and therapeutics. In this review, we summarize the recent developments in the chemical biology toolbox applied to protein aggregation studies in live cells. These methods exploited fluorescent protein tags, fluorescent chemical tags, and small-molecule probes to visualize the protein-aggregation process, detect proteome stresses, and quantify the protein homeostasis network capacity. Inspired by these seminal works, we have generalized design principles for the development of new detection methods and probes in the future that will illuminate this important biological process.     

Expression of Huntingtin and TDP-43 Derivatives in Fission Yeast Can Cause Both Beneficial and Toxic Effects

Many neurodegenerative disorders display protein aggregation as a hallmark, Huntingtin and TDP-43 aggregates being characteristic of Huntington disease and amyotrophic lateral sclerosis, respectively. However, whether these aggregates cause the diseases, are secondary by-products, or even have protective effects, is a matter of debate. Mutations in both human proteins can modulate the structure, number and type of aggregates, as well as their toxicity. To study the role of protein aggregates in cellular fitness, we have expressed in a highly tractable unicellular model different variants of Huntingtin and TDP-43. They each display specific patterns of aggregation and toxicity, even though in both cases proteins have to be very highly expressed to affect cell fitness. The aggregation properties of Huntingtin, but not of TDP-43, are affected by chaperones such as Hsp104 and the Hsp40 couple Mas5, suggesting that the TDP-43, but not Huntingtin, derivatives have intrinsic aggregation propensity. Importantly, expression of the aggregating form of Huntingtin causes a significant extension of fission yeast lifespan, probably as a consequence of kidnapping chaperones required for maintaining stress responses off. Our study demonstrates that in general these prion-like proteins do not cause toxicity under normal conditions, and in fact they can protect cells through indirect mechanisms which up-regulate cellular defense pathways.     

Inhibition of Amyloid Protein Aggregation Using Selected Peptidomimetics

Aberrant protein aggregation leads to the formation of amyloid fibrils. This phenomenon is linked to the development of more than 40 irremediable diseases such as Alzheimer's disease, Parkinson's disease, type 2 diabetes, and cancer. Plenty of research efforts have been given to understanding the underlying mechanism of protein aggregation, associated toxicity, and the development of amyloid inhibitors. Recently, the peptidomimetic approach has emerged as a potential tool to modulate several protein-protein interactions (PPIs). In this review, we discussed selected peptidomimetic-based approaches for the modulation of important amyloid proteins (Islet Amyloid Polypeptide, Amyloid Beta, α-synuclein, mutant p53, and insulin) aggregation. This approach holds a powerful platform for creating an essential stepping stone for the vital development of anti-amyloid therapeutic agents.     

Mechanism of Suppression of Protein Aggregation by ��-Crystallin

This review summarizes experimental data illuminating the mechanism of suppression of heat-induced protein aggregation by α-crystallin, one of the small heat shock proteins. The dynamic light scattering data show that the initial stage of thermal aggregation of proteins is the formation of the initial aggregates involving hundreds of molecules of the denatured protein. Further sticking of the starting aggregates proceeds in a regime of diffusion-limited cluster-cluster aggregation. The protective effect of α-crystallin is due to transition of the aggregation process to the regime of reaction-limited cluster-cluster aggregation, wherein the sticking probability for the colliding particles becomes lower than unity.