Misfolding and Amyloid Aggregation of Apomyoglobin

Apomyoglobin is an excellent example of a monomeric all α-helical globular protein whose folding pathway has been extensively studied and well characterized. Structural perturbation induced by denaturants or high temperature as well as amino acid substitution have been described to induce misfolding and, in some cases, aggregation. In this article, we review the molecular mechanism of the aggregation process through which a misfolded form of a mutated apomyoglobin aggregates at physiological pH and room temperature forming an amyloid fibril. The results are compared with data showing that either amyloid or aggregate formation occurs under particular denaturing conditions or upon cleavage of the residues corresponding to the C-terminal helix of apomyoglobin. The results are discussed in terms of the sequence regions that are more important than others in determining the amyloid aggregation process.     

蛋白质折叠与淀粉样沉积的产生

蛋白质是生物体内一切功能的执行者,而蛋白质正确折叠则是发挥其生理功能的一个重要前提条件。近年来研究发现,蛋白质的错误折叠可以形成淀粉样沉积进而导致一些疾病的产生。探讨了蛋白质折叠和淀粉样纤维产生的机制。     

The Effect of Octapeptide Repeats on Prion Folding and Misfolding

Misfolding of prion protein (PrP) into amyloid aggregates is the central feature of prion diseases. PrP has an amyloidogenic C-terminal domain with three α-helices and a flexible tail in the N-terminal domain in which multiple octapeptide repeats are present in most mammals. The role of the octapeptides in prion diseases has previously been underestimated because the octapeptides are not located in the amyloidogenic domain. Correlation between the number of octapeptide repeats and age of onset suggests the critical role of octapeptide repeats in prion diseases. In this study, we have investigated four PrP variants without any octapeptides and with 1, 5 and 8 octapeptide repeats. From the comparison of the protein structure and the thermal stability of these proteins, as well as the characterization of amyloids converted from these PrP variants, we found that octapeptide repeats affect both folding and misfolding of PrP creating amyloid fibrils with distinct structures. Deletion of octapeptides forms fewer twisted fibrils and weakens the cytotoxicity. Insertion of octapeptides enhances the formation of typical silk-like fibrils but it does not increase the cytotoxicity. There might be some threshold effect and increasing the number of peptides beyond a certain limit has no further effect on the cell viability, though the reasons are unclear at this stage. Overall, the results of this study elucidate the molecular mechanism of octapeptides at the onset of prion diseases.     

Transthyretin Misfolding,A Fatal Structural Pathogenesis Mechanism

Transthyretin (TTR) is an essential transporter of a thyroid hormone and a holo-retinol binding protein, found abundantly in human plasma and cerebrospinal fluid. In addition, this protein is infamous for its amyloidogenic propensity, causing various amyloidoses in humans, such as senile systemic amyloidosis, familial amyloid polyneuropathy, and familial amyloid cardiomyopathy. It has been known for over two decades that decreased stability of the native tetrameric conformation of TTR is the main cause of these diseases. Yet, mechanistic details on the amyloidogenic transformation of TTR were not clear until recent multidisciplinary investigations on various structural states of TTR. In this review, we discuss recent advancements in the structural understanding of TTR misfolding and amyloidosis processes. Special emphasis has been laid on the observations of novel structural features in various amyloidogenic species of TTR. In addition, proteolysis-induced fragmentation of TTR, a recently proposed mechanism facilitating TTR amyloidosis, has been discussed in light of its structural consequences and relevance to acknowledge the amyloidogenicity of TTR.     

Protein Folding and Mechanisms of Proteostasis

Highly sophisticated mechanisms that modulate protein structure and function, which involve synthesis and degradation, have evolved to maintain cellular homeostasis. Perturbations in these mechanisms can lead to protein dysfunction as well as deleterious cell processes. Therefore in recent years the etiology of a great number of diseases has been attributed to failures in mechanisms that modulate protein structure. Interconnections among metabolic and cell signaling pathways are critical for homeostasis to converge on mechanisms associated with protein folding as well as for the preservation of the native structure of proteins. For instance, imbalances in secretory protein synthesis pathways lead to a condition known as endoplasmic reticulum (ER) stress which elicits the adaptive unfolded protein response (UPR). Therefore, taking this into consideration, a key part of this paper is developed around the protein folding phenomenon, and cellular mechanisms which support this pivotal condition. We provide an overview of chaperone protein function, UPR via, spatial compartmentalization of protein folding, proteasome role, autophagy, as well as the intertwining between these processes. Several diseases are known to have a molecular etiology in the malfunction of mechanisms responsible for protein folding and in the shielding of native structure, phenomena which ultimately lead to misfolded protein accumulation. This review centers on our current knowledge about pathways that modulate protein folding, and cell responses involved in protein homeostasis.     

Identification of Natural Products as Potential Pharmacological Chaperones for Protein Misfolding Diseases

Defective protein folding and accumulation of misfolded proteins is associated with neurodegenerative, cardiovascular, secretory, and metabolic disorders. Efforts are being made to identify small-molecule modulators or structural-correctors for conformationally destabilized proteins implicated in various protein aggregation diseases. Using a metastable-reporter-based primary screen, we evaluated pharmacological chaperone activity of a diverse class of natural products. We found that a flavonoid glycoside ( C-10 , chrysoeriol-7-O-β-D-glucopyranoside) stabilizes metastable proteins, prevents its aggregation, and remodels the oligomers into protease-sensitive species. Data was corroborated with additional secondary screen with disease-specific pathogenic protein. In vitro and cell-based experiments showed that C-10 inhibits α-synuclein aggregation which is implicated in synucleinopathies-related neurodegeneration. C-10 interferes in its structural transition into β-sheeted fibrils and mitigates α-synuclein aggregation-associated cytotoxic effects. Computational modeling suggests that C-10 binds to unique sites in α-synuclein which may interfere in its aggregation amplification. These findings open an avenue for comprehensive SAR development for flavonoid glycosides as pharmacological chaperones for metastable and aggregation-prone proteins implicated in protein conformational diseases.     

Lysozyme Amyloid Fibril Structural Variability Dependence on Initial Protein Folding State

Amyloid fibril formation is associated with several amyloidoses, including neurodegenerative Alzheimer’s or Parkinson’s diseases. The process of such fibrillar structure formation is still not fully understood, with new mechanistic insights appearing on a regular basis. This, in turn, has limited the development of potential anti-amyloid compounds, with only a handful of effective cures or treatment modalities available. One of the multiple amyloid aggregation factors that requires further examination is the ability of proteins to form multiple, structurally distinct aggregates, based on the environmental conditions. In this work, we examine how the initial folding state affects the fibrilization of lysozyme—an amyloidogenic protein, often used in protein aggregation studies. We show that there is a correlation between the initial state of the protein and the aggregate formation lag time, rate of elongation, resulting aggregate structural variability and dye-binding properties, as well as formation lag time and rate of elongation.     

Amyloid Aggregates of Smooth-Muscle Titin Impair Cell Adhesion

Various amyloid aggregates, in particular, aggregates of amyloid β-proteins, demonstrate in vitro and in vivo cytotoxic effects associated with impairment of cell adhesion. We investigated the effect of amyloid aggregates of smooth-muscle titin on smooth-muscle-cell cultures. The aggregates were shown to impair cell adhesion, which was accompanied by disorganization of the actin cytoskeleton, formation of filopodia, lamellipodia, and stress fibers. Cells died after a 72-h contact with the amyloid aggregates. To understand the causes of impairment, we studied the effect of the microtopology of a titin-amyloid-aggregate-coated surface on fibroblast adhesion by atomic force microscopy. The calculated surface roughness values varied from 2.7 to 4.9 nm, which can be a cause of highly antiadhesive properties of this surface. As all amyloids have the similar structure and properties, it is quite likely that the antiadhesive effect is also intrinsic to amyloid aggregates of other proteins. These results are important for understanding the mechanisms of the negative effect of amyloids on cell adhesion.     

Folding Mechanism and Aggregation Propensity of the KH0 Domain of FMRP and Its R138Q Pathological Variant

The K-homology (KH) domains are small, structurally conserved domains found in proteins of different origins characterized by a central conserved βααβ “core” and a GxxG motif in the loop between the two helices of the KH core. In the eukaryotic KHI type, additional αβ elements decorate the “core” at the C-terminus. Proteins containing KH domains perform different functions and several diseases have been associated with mutations in these domains, including those in the fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein crucial for the control of RNA metabolism whose lack or mutations lead to fragile X syndrome (FXS). Among missense mutations, the R138Q substitution is in the KH0 degenerated domain lacking the classical GxxG motif. By combining equilibrium and kinetic experiments, we present a characterization of the folding mechanism of the KH0 domain from the FMRP wild-type and of the R138Q variant showing that in both cases the folding mechanism implies the accumulation of an on-pathway transient intermediate. Moreover, by exploiting a battery of biophysical techniques, we show that the KH0 domain has the propensity to form amyloid-like aggregates in mild conditions in vitro and that the R138Q mutation leads to a general destabilization of the protein and to an increased fibrillogenesis propensity.     

Protein Folding and Aggregation into Amyloid: The Interference by Natural Phenolic Compounds

Amyloid aggregation is a hallmark of several degenerative diseases affecting the brain or peripheral tissues, whose intermediates (oligomers, protofibrils) and final mature fibrils display different toxicity. Consequently, compounds counteracting amyloid aggregation have been investigated for their ability (i) to stabilize toxic amyloid precursors; (ii) to prevent the growth of toxic oligomers or speed that of fibrils; (iii) to inhibit fibril growth and deposition; (iv) to disassemble preformed fibrils; and (v) to favor amyloid clearance. Natural phenols, a wide panel of plant molecules, are one of the most actively investigated categories of potential amyloid inhibitors. They are considered responsible for the beneficial effects of several traditional diets being present in green tea, extra virgin olive oil, red wine, spices, berries and aromatic herbs. Accordingly, it has been proposed that some natural phenols could be exploited to prevent and to treat amyloid diseases, and recent studies have provided significant information on their ability to inhibit peptide/protein aggregation in various ways and to stimulate cell defenses, leading to identify shared or specific mechanisms. In the first part of this review, we will overview the significance and mechanisms of amyloid aggregation and aggregate toxicity; then, we will summarize the recent achievements on protection against amyloid diseases by many natural phenols.     

Protein Misfolding and Aggregation in the Brain: Common Pathogenetic Pathways in Neurodegenerative and Mental Disorders

Mental disorders represent common brain diseases characterized by substantial impairments of social and cognitive functions. The neurobiological causes and mechanisms of psychopathologies still have not been definitively determined. Various forms of brain proteinopathies, which include a disruption of protein conformations and the formation of protein aggregates in brain tissues, may be a possible cause behind the development of psychiatric disorders. Proteinopathies are known to be the main cause of neurodegeneration, but much less attention is given to the role of protein impairments in psychiatric disorders’ pathogenesis, such as depression and schizophrenia. For this reason, the aim of this review was to discuss the potential contribution of protein illnesses in the development of psychopathologies. The first part of the review describes the possible mechanisms of disruption to protein folding and aggregation in the cell: endoplasmic reticulum stress, dysfunction of chaperone proteins, altered mitochondrial function, and impaired autophagy processes. The second part of the review addresses the known proteins whose aggregation in brain tissue has been observed in psychiatric disorders (amyloid, tau protein, α-synuclein, DISC-1, disbindin-1, CRMP1, SNAP25, TRIOBP, NPAS3, GluA1, FABP, and ankyrin-G).     

Protein GB1 Folding and Assembly from Structural Elements

Folding of the Protein G B1 domain (PGB1) shifts with increasing salt concentration from a cooperative assembly of inherently unstructured subdomains to an assembly of partly pre-folded structures. The salt-dependence of pre-folding contributes to the stability minimum observed at physiological salt conditions. Our conclusions are based on a study in which the reconstitution of PGB1 from two fragments was studied as a function of salt concentrations and temperature using circular dichroism spectroscopy. Salt was found to induce an increase in β-hairpin structure for the C-terminal fragment (residues 41 – 56), whereas no major salt effect on structure was observed for the isolated N-terminal fragment (residues 1 – 41). In line with the increasing evidence on the interrelation between fragment complementation and stability of the corresponding intact protein, we also find that salt effects on reconstitution can be predicted from salt dependence of the stability of the intact protein. Our data show that our variant (which has the mutations T2Q, N8D, N37D and reconstitutes in a manner similar to the wild type) displays the lowest equilibrium association constant around physiological salt concentration, with higher affinity observed both at lower and higher salt concentration. This corroborates the salt effects on the stability towards denaturation of the intact protein, for which the stability at physiological salt is lower compared to both lower and higher salt concentrations. Hence we conclude that reconstitution reports on molecular factors that govern the native states of proteins.     

蛋白质体外折叠技术研究现状与展望

蛋白质折叠技术是生物工程的新“单元操作”。本文阐述了蛋白质体外折叠研究的重要理论意义和应用价值，综述了目前蛋白质体外折叠技术研究进展，最后对蛋白质折叠技术的研究进行了展望。     

Macromolecule-Assisted de novo Protein Folding

In the processes of protein synthesis and folding, newly synthesized polypeptides are tightly connected to the macromolecules, such as ribosomes, lipid bilayers, or cotranslationally folded domains in multidomain proteins, representing a hallmark of de novo protein folding environments in vivo. Such linkage effects on the aggregation of endogenous polypeptides have been largely neglected, although all these macromolecules have been known to effectively and robustly solubilize their linked heterologous proteins in fusion or display technology. Thus, their roles in the aggregation of linked endogenous polypeptides need to be elucidated and incorporated into the mechanisms of de novo protein folding in vivo. In the classic hydrophobic interaction-based stabilizing mechanism underlying the molecular chaperone-assisted protein folding, it has been assumed that the macromolecules connected through a simple linkage without hydrophobic interactions and conformational changes would make no effect on the aggregation of their linked polypeptide chains. However, an increasing line of evidence indicates that the intrinsic properties of soluble macromolecules, especially their surface charges and excluded volume, could be important and universal factors for stabilizing their linked polypeptides against aggregation. Taken together, these macromolecules could act as folding helpers by keeping their linked nascent chains in a folding-competent state. The folding assistance provided by these macromolecules in the linkage context would give new insights into de novo protein folding inside the cell.     

Evaluation of nanoparticle tracking for characterization of fibrillar protein aggregates

Amyloidogenesis is the process of formation of protein aggregates with fibrillar morphology. Because amyloidogenesis is linked to neurodegenerative disease, there is interest in understanding the mechanism of fibril growth. Kinetic models of amyloidogenesis require data on the number concentration and size distribution of aggregates, but this information is difficult to obtain using conventional methods. Nanoparticle tracking analysis (NTA) is a relatively new technique that may be uniquely suited for obtaining these data. In NTA, the two‐dimensional (2‐D) trajectory of individual particles is tracked, from which the diffusion coefficient, and, hence, hydrodynamic radius is obtained. Here we examine the validity of NTA in tracking number concentration and size of DNA, as a model of a fibrillar macromolecule. We use NTA to examine three amyloidogenic materials: beta‐amyloid, transthyretin, and polyglutamine‐containing peptides. Our results are instructive in demonstrating the advantages and some limitations of single‐particle diffusion measurements for investigating aggregation in protein systems. © 2014 American Institute of Chemical Engineers AIChE J, 60: 1236–1244, 2014     

A Brief Journey through Protein Misfolding in Transthyretin Amyloidosis (ATTR Amyloidosis)

Transthyretin (TTR) amyloidogenesis involves the formation, aggregation, and deposition of amyloid fibrils from tetrameric TTR in different organs and tissues. While the result of amyloidoses is the accumulation of amyloid fibrils resulting in end-organ damage, the nature, and sequence of the molecular causes leading to amyloidosis may differ between the different variants. In addition, fibril accumulation and toxicity vary between different mutations. Structural changes in amyloidogenic TTR have been difficult to identify through X-ray crystallography; but nuclear magnetic resonance spectroscopy has revealed different chemical shifts in the backbone structure of mutated and wild-type TTR, resulting in diverse responses to the cellular conditions or proteolytic stress. Toxic mechanisms of TTR amyloidosis have different effects on different tissues. Therapeutic approaches have evolved from orthotopic liver transplants to novel disease-modifying therapies that stabilize TTR tetramers and gene-silencing agents like small interfering RNA and antisense oligonucleotide therapies. The underlying molecular mechanisms of the different TTR variants could be responsible for the tropisms to specific organs, the age at onset, treatment responses, or disparities in the prognosis.     

Molecular Mechanisms of Inhibition of Protein Amyloid Fibril Formation: Evidence and Perspectives Based on Kinetic Models

Inhibition of fibril formation is considered a possible treatment strategy for amyloid-related diseases. Understanding the molecular nature of inhibitor action is crucial for the design of drug candidates. In the present review, we describe the common kinetic models of fibril formation and classify known inhibitors by the mechanism of their interactions with the aggregating protein and its oligomers. This mechanism determines the step or steps of the aggregation process that become inhibited and the observed changes in kinetics and equilibrium of fibril formation. The results of numerous studies indicate that possible approaches to antiamyloid inhibitor discovery include the search for the strong binders of protein monomers, cappers blocking the ends of the growing fibril, or the species absorbing on the surface of oligomers preventing nucleation. Strongly binding inhibitors stabilizing the native state can be promising for the structured proteins while designing the drug candidates targeting disordered proteins is challenging.     

The Finite Size Effects and Two-State Paradigm of Protein Folding

The coil to globule transition of the polypeptide chain is the physical phenomenon behind the folding of globular proteins. Globular proteins with a single domain usually consist of about 30 to 100 amino acid residues, and this finite size extends the transition interval of the coil-globule phase transition. Based on the pedantic derivation of the two-state model, we introduce the number of amino acid residues of a polypeptide chain as a parameter in the expressions for two cooperativity measures and reveal their physical significance. We conclude that the k2 measure, defined as the ratio of van ’t Hoff and calorimetric enthalpy is related to the degeneracy of the denatured state and describes the number of cooperative units involved in the transition; additionally, it is found that the widely discussed k2=1 is just the necessary condition to classify the protein as the two-state folder. We also find that Ωc, a quantity not limited from above and growing with system size, is simply proportional to the square of the transition interval. This fact allows us to perform the classical size scaling analysis of the coil-globule phase transition. Moreover, these two measures are shown to describe different characteristics of protein folding.