Protein misfolding and aggregation: Mechanism

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Accepted Manuscript Title: Protein misfolding and aggregation: Mechanism, factors and detection Author: Sumit Kumar Chaturvedi Mohammad Khursheed Siddiqi Parvez Alam Rizwan Hasan Khan PII: DOI: Reference:

S1359-5113(16)30138-6 http://dx.doi.org/doi:10.1016/j.procbio.2016.05.015 PRBI 10689

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Process Biochemistry

Received date: Revised date: Accepted date:

30-7-2015 5-5-2016 16-5-2016

Please cite this article as: Chaturvedi Sumit Kumar, Siddiqi Mohammad Khursheed, Alam Parvez, Khan Rizwan Hasan.Protein misfolding and aggregation: Mechanism, factors and detection.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.05.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Protein misfolding and aggregation: Mechanism, factors and detection Sumit

Kumar

Chaturvedi,

Mohammad

Khursheed

Siddiqi,

Parvez

Alam,

Khan*[email protected] [email protected] Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India *Corresponding author at: Interdisciplinary Biotechnology Unit, Aligarh Muslim University Aligarh 202002 India. Tel.: +91 571 2720388; fax: +91 571 2721776.

Rizwan

Hasan

Highlights   

Mechanisms involved in protein aggregation have been delineated. Factors for inducing the formation of aggregate have been described. Evolving techniques towards the study of protein aggregates have been explained

Abstract Amyloidogenic diseases are characterised by the formation of amyloid aggregates inside or outside the cell. Amyloid-associated human diseases include Alzheimer’s disease, Parkinson’s disease, prion diseases and type II diabetes. Currently, these diseases are incurable; thus, detailed insight into the mechanism of amyloid formation, deposition and inhibition is required to develop treatment strategies. Herein, we have described the mechanism of amyloidogenesis in detail highlighting the major events including the association of native monomers into higher-ordered fibrillar structures. A review of the modern technologies that aid characterisation of amyloid aggregates is also discussed. Further, we have described the factors influencing the microenvironment of protein, which in turn promotes amyloidosis. Keywords: protein misfolding; protein aggregation; amyloid fibril; diseases.

1. Introduction Proteins perform several different biological functions with high fidelity ascribed to their specific threedimensional (3D) structures. Generally, misfolded or partially folded proteins are degraded by cellular quality control systems such as proteasome and autophagy, but failure of this system or overloading results in protein aggregation, in turn leading to protein-misfolding disorders.1 Amyloidogenesis is a process wherein peptide or protein molecules self-associate to form dimers and oligomers, subsequently transforming into mature fibrillar amyloid aggregates. In contrast to amorphous protein aggregates, amyloids have a quasicrystalline structure and possess characteristic properties.2 During amyloidogenesis, misfolded monomers give rise to toxic protein fibrils whose deposition in tissue or cells leads to various serious pathological consequences. The term ‘amyloidosis’ is widely used to denote the lethal consequences of amyloid deposition.3 Particularly, its deposition in neuronal cells leads to neuron degeneration (neurodegenerative diseases), which manifests symptoms such as memory loss and dementia. Some of the prevalent neurodegenerative diseases include Alzheimer’s disease and Parkinson’s disease, which remain incurable. The lack of detailed elucidation of self-assembly mechanisms is one of the possible reasons for the failure of the therapies. 1.1 Protein misfolding and aggregation Living systems are composed of four key macromolecules: proteins, carbohydrates, nucleic acids and lipids. Proteins perform a wide array of functions within living organisms. The life of every living organism depends on chemical reactions controlled by about 100,000 types of protein. Each protein can be distinguished from the other based on its constituent polymeric sequence of amino acids.5 Proteins are synthesised on ribosomes within the cell.6 In some cases, protein folding starts immediately, while the nascent polypeptide chain is still attached to the ribosomes in a co-translational manner. Other proteins follow a folding pathway in the endoplasmic reticulum after the translation process. Organisms have evolved various methods of controlling the folding process of proteins such as the use of folding catalysts and chaperones.7In adverse environments such as stress condition, mutation and ageing, proteins may lose their ability to fold properly and start to misfold (Fig.1). Generally, misfolded proteins trigger a complicated biological response such as unfolding of protein and heat shock, which help in protein folding and protein degradation.8 Incorrect folding of proteins may lead to their degradation by the proteasome machinery of the

cell or aggregation, thus leading to various pathogenic conditions. Figure 1 shows a schematic diagram of misfolding and the aggregation process. The various intermediate steps involved in the aggregation process are shown in Fig. 2AII. Failure of any of these processes may lead to the loss of particular protein function, overabundance or aggregation of misfolded proteins, subsequently leading to pathogenic conditions.4 The factors underlying protein misfolding include loss of cellular protein quality control system, inability of the ubiquitin– proteasome complex to degrade and eliminate misfolded aggregation-prone molecules, inefficient functioning of the molecular chaperone machinery, obstruction of normal cellular transport of protein, production of amyloidogenic fragments of protein due to inappropriate protease activity, destabilising mutations, etc.9-18 The protein aggregates may be deposited both intracellularly and extracellularly. Deposition of ordered structures also known as ‘amyloids’ results in >20 diseases in humans such as Alzheimer’s disease, Parkinson’s disease, type II diabetes and systemic amyloidosis (Table 1). Proteins that are not associated with any pathological conditions may also form amyloids under in vitro conditions. This indicates that amyloid formation is not restricted to any specific protein, but the propensity to form amyloids is modulated by the amino acid sequence of polypeptides.19 The fibrils are usually toxic to cells possibly giving rise to some of the most debilitating pathological conditions.20 The amorphous aggregates and the related cytotoxicity are discussed in supplementary section S1. 1.2 Classification of protein aggregates Various groups have classified protein aggregates in different ways, as no precise definition exists. Two broad categories of protein aggregates include in vivo versus in vitro and fibrillar versus amorphous. For example, amyloid fibrils are fibrillar or ordered aggregates that are observed both in vivo and in vitro, whereas inclusion bodies are amorphous or disordered aggregates that are formed in vivo.21 Similarly, in vitro aggregates formed during refolding at high protein concentration are disordered aggregates. Other classifications have also been proposed such as physical (or non-covalent) versus chemical (or covalent) aggregates, reversible versus irreversible aggregates and soluble oligomers (dimer to decamer) versus insoluble particles. 1.3 Structure and morphology

The morphology of protein aggregates is independent of the protein sequence; it is determined primarily by the solution conditions. This is because the same protein can form both fibrillar and amorphous types of aggregates based on the environmental conditions. pH has been found to play a key role in determining the aggregate morphology as it affects the charge distribution and the degree of structural perturbation to the protein as well. Krebs et al.,22 in a study on seven vastly different proteins, revealed that a pH that provides a high net charge to the protein favours the formation of fibrillar aggregates, whereas a low net charge on protein is likely to yield aggregates with amorphous morphology.23 Although it is difficult to study the pathogenic amyloid fibrils because of their large size, poor solubility and non-crystalline structure, the structure of aggregates has been extensively studied at the molecular level. Much of the advancement in the knowledge of fibril structures is facilitated by solid-state nuclear magnetic resonance (NMR), electron microscopy (EM) and X-ray diffraction.24 The amyloid fibrils formed from different unrelated peptides and proteins are characterised as having wellordered, elongated and relatively straight fibrillar structures. EM and atomic force microscopy (AFM) revealed that fibrils are composed of substructures called ‘protofilaments’. A single protofilament is either straight or curved with a diameter of 2–5 nm. About two to six of such protofilaments join by either twisting together like a rope or arranging themselves laterally in the form of a ribbon to construct a fibril with a diameter of 7–15 nm. X-ray diffraction studies reveal the presence of a common ‘cross-β-sheet’ structure in which β-sheets run parallel to the axis of the fibril, while β-strands in an individual sheet are arranged perpendicular. The distance between the β-strands is 4.8 Å, whereas the separation between β-sheets is ~10 Å. It should be noted that the morphology of the fibrils generated in vitro also varies with the solution conditions including protein concentration, pH, temperature, composition of buffer and the presence of additives. 2. Mechanism of protein aggregation The formation of protein aggregates begins from partially unfolded conformation of protein. The partially unfolded protein structures facilitate specific intermolecular interactions that are prerequisites for protein aggregation.25,26 Direct structural information on monomeric partially unfolded conformation competent for aggregations is rarely available because it is difficult to trap partially unfolded intermediates. Amyloid formation by tetrameric transthyretin starts only after its dissociation into monomers.27 Factors that stabilise

the native state retard the process of protein aggregation, and those that destabilise the native state promote protein aggregation. In the case of beta lactoglobulin, aggregation propensity was found to be maximum at urea concentration corresponding to the mid-point of unfolding transition. Partial unfolding in protein can be induced by several factors such as pH, temperature, modification, mutation and oxidative stress. It should be noted that in the case of globular proteins aggregation may be induced in the native state without attaining the precursor state.28 In the case of globular proteins, partially unfolded segments of proteins may help in the formation of aggregates. In the case of native unfolded proteins such as alpha synuclein and amyloid beta (Aβ), aggregation is promoted by factors such as low pH and high temperature.29 The prevalence and continuing irremediability of amyloid-associated disorders necessitate the understanding of protein self-assembly mechanisms and amyloidogenesis, which involve more than a single pathway to come into existence (Table 2).30–35 The mechanisms involved in amyloid formation are not unique but have several steps in common. The following section discusses some of these mechanisms (Fig.2). 2.1 Self-assembly of monomeric protein Native monomers may have an inherent propensity to self-associate in a reversible manner. The surfaces of native monomers are self-complementary in nature and favour the intermolecular interactions to form small reversible oligomers. Progressively larger oligomers are formed with the increase in protein concentration, but over time, these reversible oligomers are transformed into irreversible aggregates, which are attributed to the formation of covalent bonds such as disulphide bonds (Fig.2AI). Therapeutic proteins such as insulin have been shown to exhibit reversible oligomer formation.36,37 In 1952, it was proposed that unfolding of proteins is not a prerequisite condition to form amyloid aggregates; instead, side-by-side or end-to-end union of protein molecules results in the aggregation of globular proteins.30 2.2 Aggregation of conformationally altered monomeric protein The native monomer does not always lose the tendency to associate reversibly; instead, a conformationally altered monomer or the partially unfolded state has a strong tendency to form protein aggregates (Fig. 2AIII). Therefore, the first key step in this mechanism is a conformational change to the non-native state, which distinguishes it from the previous mechanisms. Stress such as heat or shear forces the native monomer to adopt a conformation that is more prone to aggregation. The condition or excipients that stabilise the native-

state conformation may be helpful in suggesting the appropriate inhibitory agents. Interferon-γ and granulocyte colony-stimulating factor (G-CSF) undergo amyloidosis through this mechanism.38-40 2.3 Nucleation and seeding mechanism This mechanism is widely used in the formation of visible aggregates or precipitates.41 In this mechanism, the native monomer alone is unable to seed the phenomenon of fibrillation, but aggregates of critical size promote the formation of aggregates of progressively larger size by adding monomers (Fig. 2AIII). Therefore, this is called a ‘critical nucleus’. The nucleation mechanism usually exhibits a lag phase without any visible precipitate for a long period of time (perhaps a month), but after this critical period a much larger species appears instantly. This process of aggregation is termed as ‘homogeneous nucleus’ wherein the critical nucleus is itself the product aggregate. However, in the case of ‘heterogeneous nucleation’, a nucleus is formed from moieties other than protein aggregates. Some examples of impurities are silica particles shed by product vials, steel particle shed by a piston pump used for filling vials, etc.42 Generally, amyloid formation occurs via nucleation-dependent oligomerisation. In order to start the process, an ordered nucleus formed in the supersaturated protein solution exceeds the critical concentration of the amyloidogenic protein. After the establishment of the nucleus, the fibril growth occurs very rapidly. The time gap between the formation of monomer and nucleus is known as the lag phase. Seeding or supply of nucleus to the supersaturated protein solution enhances the fibrillation process by eliminating the lag phase. Figure 2C shows the typical growth curve obtained during seeding of the protein solution. Reports that advocate the seeded fibrillation of Aβ, α synuclein, islet amyloid polypeptide precursor (IAPP) and prion proteins in vitro are available.43 2.4 Surface-induced aggregation Surface-induced aggregation is initiated with the binding of the native monomer to the surface of the container or flask. Binding would probably be driven by hydrophobic interaction in an air–liquid interface, but electrostatic interaction may also be involved in the container or flask. The interaction of the monomer with the surface brings about a conformational change that increases the propensity to aggregate either at the surface or when the altered monomer is released back into the solution (Fig. 2D). Apart from the four mechanisms discussed earlier, globular, thermophilic and prion proteins may follow a different mode of aggregation, which is discussed in detail in supplementary section S2.

3. Factors affecting protein aggregation Why proteins aggregate instead of reaching their minimum energy landscape is a critical question in protein chemistry. Under suitable conditions, proteins with an intrinsic capacity to fold into their well-stable native state can form amyloids both in vitro and in vivo due to their marginal stability and dynamic nature. The major reason underlying aggregation is protein unfolding or misfolding.44 Loss in any of the quality control systems of protein or external factors leads to the generation of either misfolded or partially folded states, which have exposed hydrophobic patches and unstructured regions that are normally buried inside in the native state of protein.45 In order to protect themselves from the solvent, the partially folded structures undergo hydrophobic collapse leading to the formation of a stable aggregated structure.46 Thus, we can say that hydrophobic forces drive the aggregation process. For example, non-covalent interactions such as electrostatic and van der Waals interaction drive the aggregation process in Aβ protein and monoclonal antibodies.47-49 The various factors responsible for aggregation are discussed in detail in supplementary section S3 and presented schematically in Fig.3. 4. Analytical methods to study protein aggregation The characterisation of protein aggregates using a combination of analytical methods is essential for understanding the mechanism of aggregation and designing potential drugs to combat amyloid-associated pathogenesis. It also helps in evaluating the protein structure, biological integrity and product-related impurities during the manufacturing and storage of biopharmaceuticals. The salient features of some of the methods commonly used to characterise protein aggregates including amyloid fibrils, the underlying principles and detailed methodology are discussed in the following sections.50 4.1 Turbidity measurements Turbidity is cloudiness or haziness of fluids caused by individual particles of various sizes. A number of particles scattering or absorbing light causes the optical property of a solution. Protein aggregation is marked by an increase in turbidity with time. The turbidity can be suitably examined spectrophotometrically as the recorded optical density at λmax, which refers to the wavelength at which maximum absorption occurs, corresponding to the particle size in the solution. The time-dependent change in turbidity has also been taken into account for studying the formation or inhibition of aggregates under various conditions.

4.2 Rayleigh scattering measurements The optical properties of a solution are a result of the ability of molecules or particles present therein to absorb and scatter light. Rayleigh’s theory of light scattering states that particles with diameter lesser than the wavelength of incident light are able to scatter light. The upper limit of the size of particles is about onetenth of the wavelength, which implies that scattering does not take the shape of the particle into account. Light scattering is essentially used in the investigation of protein aggregation, and it can be monitored using a fluorescence spectrophotometer by setting identical excitation and emission wavelengths, usually at 350 nm. This method has been considerably employed to detect the formation of protein aggregates.51, 52 4.3 Dye-binding assays Various dye-binding assays are performed to characterise the aggregates, be they amorphous or amyloid. The details of the dye-binding assays are discussed in supplementary section S4 (Table 3). 4.4 Circular dichroism spectroscopy Circular dichroism (CD) spectroscopy is a very important tool in structural biology and protein chemistry.53 For determination of the secondary and tertiary structure of proteins and peptides, far-ultraviolet (UV) CD (190–250 nm) and near-UV CD (250–320 nm) are used, respectively. Aggregation of amyloid proteins into protofibrils and fibrils is accompanied by the formation of β-sheet conformation, which gives the characteristic minima at 215–218 nm in the far-UV CD region. Qualitative information on amyloid assemblies and kinetics of conformational transition associated with aggregation can be obtained by CD.54, 55 4.5 Intrinsic fluorescence measurements Intrinsic fluorescence parameters such as fluorescence intensity (FI) and emission maxima (λmax) are very sensitive to the changes in the structural dynamics and polarity of protein chromophores such as Trp and Tyr; therefore, they can be used to study protein folding and assembly.56, 57 The intrinsic fluorescence of aromatic residues has been used to probe conformational dynamics and the self-assembly of several amyloidogenic proteins such as Aβ, IAPP, prion, α-synuclein and immunoglobulin G (IgG) light chains.58, 59 4.6 Transmission electron microscopy The direct visualisation of the protein self-assembly into oligomers, protofibrils and mature fibrils has been facilitated by high-resolution microscopic techniques such as transmission electron microscopy (TEM). It

provides only qualitative information about the aggregates. Protein aggregates such as Aβ, insulin, lysozyme and α-synuclein have been characterised by TEM.60, 61 4.7Atomic Force Microscopy Biological sciences have exploited the ability of AFM to give 3D structural details of numerous proteinbased samples or fibrils with very high resolution. AFM does not require special sample preparation that would otherwise damage the sample. Its principle involves scanning of the tip over the samples, and the laser beam detects the deflection. It can be used in the examination of supramolecular structures of living cells and the growth of amyloid fibrils. AFM provides both qualitative and quantitative information at the nanometer level including length, width and surface characteristic of protein aggregates.62,63 It can also be used to monitor hydrated or dehydrated samples under atmospheric conditions. 4.8 High-Resolution Transmission Electron Microscopy High-resolution transmission electron microscopy (HRTEM) is a useful technique for monitoring the formation of amyloid fibrils in vitro and providing images of prefibrillar aggregates, circular species and mature fibrils at high resolution. Moreover, it also provides information on protein aggregates formed by other misfolding pathways such as amorphous aggregate formation. HRTEM has the advantage of quick performance, which allows researchers to assess the fibril formation rapidly. HRTEM confirms the fibrillar morphology of protein aggregates, but it does not confirm the presence of the characteristic cross-β-sheet structure. Thus, ancillary techniques are required in addition to HRTEM for proving the fibrillar morphology or any other configuration of amyloid. It provides greater resolution than other techniques such as confocal and scanning electron microscopy (SEM).64 4.9 Field-Emission Scanning Electron Microscopy Amyloid fibrils have a characteristic cross-β-sheet structure that may be formed in both in vitro and in vivo environments. An SEM is used to obtain the 3D image of an amyloid, but it is not as prevalent as other techniques although it possesses relatively higher resolution up to the nanometer level. In SEM, secondary electrons emitted from the specimen are used to construct the final image. In field-emission scanning electron microscopy (FESEM), a field-emission cathode provides narrower probing beams with high as well as low electron energy, which is responsible for the enhanced resolution and minimised sample damage of images.65, 66

4.10 Fluorescence Microscopy Fluorescence microscopy is an invaluable tool for detection and characterisation of protein aggregates. 67 Dyes such as thioflavin T, Congo red and Nile Red are used as extrinsic fluorophores to monitor the protein aggregates; Congo red shows an apple green birefringence, and fluorescence of the thioflavin T increases in the presence of amyloid deposits.68 Fluorescence microscopy has been efficiently utilised even at high protein concentrations, minimising changes in the protein local environment for the detection and characterisation of aggregates. Nile Red is a low molecular weight phenoxazone dye, binds to hydrophobic surfaces of proteins and exhibits a strong fluorescence.67 Recent approaches include confocal and two- or multi-photon fluorescence microscopy and total internal reflection. Confocal fluorescence microscopy is well suited for 3D resolution investigation. Two- or multi-photon fluorescence microscopy has been used for the analysis of UV-excitable fluorescence, which is not possible with confocal fluorescence microscopy. Total internal reflection fluorescence microscopy (TIRFM) has been used to study the plasma membrane and single-molecule methods such as fibril progress.69 4.11 Bioinformatics as a tool to study protein aggregation Any protein can aggregate under suitable conditions, but the propensity is modulated by the sequence of amino acids. Certain regions in the protein known as ‘hotspots’ are more prone to form aggregates. Hotspots are rich in hydrophobic and aliphatic amino acid residues. Generally, surrounded by amino acid residues that enhance the folding capacity of polypeptides, and amino acids present in these regions are polar in nature. Information on the structure of amyloids and factors that retard/induce the formation of aggregates has led to the development of several algorithms that help us identify amyloid-prone regions in the polypeptides. Amyloid prediction software is divided into two categories. The first category is empirical, based on the theoretical and experimental analysis of the contribution of individual amino acids to the amyloid formation. The second category is structural, based on the well-defined 3D structure of the fibrillar conformations of polypeptides. A list of software and their main application is given in Table 4.70–78 4.11 Immunohistochemistry Immunohistochemistry (IHC) involves the study of immunological, anatomical and biochemical aspects to detect the desired protein in cells of a tissue section using a labelled antibody–antigen complex.79 Prior to IHC, only histological staining was used to locate and visualise the aggregates, which includes eosin,

haematoxylin and silver staining. Amyloids are classified based on formalin-fixed and paraffin-embedded tissue sections in the IHC using monoclonal and polyclonal antibodies.80 The demerits of IHC include the difficulty of application in minor biopsy samples that contain a low amount of amyloids as well as a very advanced set-up that includes laser micro-dissection, immuno electron microscopy and proteomics (mass spectroscopy). Schonland et al. conducted a systemic examination of 117 patients and classified the systemic forms of amyloid by IHC.81 4.12 Other techniques to study protein aggregation In general, other techniques used to analyse protein aggregates include SDS-polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography, Fourier transform infrared (FTIR) spectroscopy, differential light scattering spectroscopy, analytical ultracentrifugation, NMR spectroscopy, X-ray crystallography, electron spin resonance and mass spectrometry. These techniques are used to determine the shape, size and atomic structure of amyloids. 5. Therapeutic strategies for aggregation inhibition Living systems have adopted various strategies to avoid unwanted protein aggregation and amyloid formation at high internal protein concentrations. In a study on the role of protein sequence in aggregation, Dobson et al. demonstrated that peptides/proteins reduce or arrest the process at different stages of the amyloid formation pathway such as the native state, partially unfolded state, oligomers, amyloid seeds and fibrils. The inhibition of protein self-assembly is important for two reasons: developing strategies to combat protein aggregation in general (situation encountered during protein expression, refolding, manufacturing and processing of biopharmaceuticals), and identifying or designing inhibitors that specifically target amyloid aggregates (associated with several pathogenic conditions). To develop therapies against protein aggregation is very challenging in the modern world. A range of molecules has been tested both in vitro and in vivo to evaluate their anti-amyloidogenic effects. In this context, natural polyphenols, flavonoids, chaperones, nanoparticles, surfactants, vitamins, small organic molecules, antibiotics, drugs, etc.3, 54, 82-88 have been extensively shown to disrupt the amyloid fibrillation of proteins. In particular, synthetic single-stranded nucleic acids have been reported to possess the capability of inhibiting β-amyloid aggregation.89 Dyes such as Congo red, which is widely used to characterise protein molecules, also inhibit the process of protein self-assembly.90 Amphipathic molecules such as surfactants are

prevalent not only for their cleaning property but also for their dual role as inducer and inhibitor of amyloid formation. While revolutionising nearly all science-related fields, the impact of nanoparticles is also clearly visible on amyloid aggregates, for instance, curcumin–gold nanoparticles having potentially higher antiamyloidogenic property.54 Recently, researchers have reported that even native proteins act as chaperones and inhibit protein aggregation, in addition to assisting in protein folding. Figure 4 shows the design of antiamyloidogenic compounds that target the process of aggregation in the following stages: 1. Inhibiting the expression of amyloidogenic proteins. 2. Stabilising native proteins. 3. Inhibiting protein aggregation either directly using small molecules or indirectly by vaccination. 4. Alleviating the effect of post-pathological conditions. Recently, ionic liquids such as ethyl ammonium nitrate (EAN) and non-detergent surfactants such as sulfobetaines (NDSBs) have been shown to effectively inhibit heat- and chemical-induced aggregation. Some amino acids, metal ions, drugs and imidazole derivatives are reported to exhibit potent inhibitory action against the aggregation of several proteins.91 Besides, polyamines such as putrescines, spermines and spermidines; amphiphilic polymers such as polyethylene glycol (PEGs) and polyvinylpyrrolidone (PVP); and osmolytes have been proven to efficiently suppress aggregation in several cases.92 Artificial chaperones have also been used to increase the refolding efficiency of several proteins and to inhibit aggregation.93, 94

Conclusion Conformational alterations of protein molecules are considered the foremost causative link leading to aggregation. These alterations to conformation result in exposition of potent amyloidogenic amino acid residues to the surrounding environment, which are otherwise buried inside the core of the protein molecule. In summary, the information reviewed in this article signifies that a specific conformational structure is necessary to form entities rich in cross-β-sheets, and that inhibition of amyloid aggregation generally involved disruption of hydrophobic interactions that act as a predominant stabilising force for amyloids. This conceptual information provides us with a practical conclusion that strategies or inhibitors directed at inhibiting the protein self-assembly process should be designed to prevent the hydrophobic interaction of aromatic or hydrophobic amino acid residues.

Acknowledgements SKC and PA are grateful to the Council of Scientific and Industrial Research, New Delhi, for providing fellowship in the form of SRF and JRF, respectively. MKS is grateful to the Department of Biotechnology, New Delhi, for providing fellowship in the form of JRF. The authors would like to thank anonymous reviewers for critical revision and valuable suggestions.

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Figure Captions

Figure 1. Schematic representation of protein misfolding and aggregation.

Figure 2. A (I) Mechanism showing oligomerisation of monomeric protein into mature amyloid fibril. (II) Schematic representation showing intermediate formation during protein aggregation process. (III) Mechanism showing self-assembly of conformationally altered monomer protein into mature amyloid fibril.

Figure 2. (B) Mechanism showing nucleation-dependent fibril formation.

Figure 2. (C) Graph showing fibril formation via nucleation-dependent manner or seeding.

Figure 2. (D) Mechanism showing surface-induced polymerisation leading to aggregation.

Figure 3. Factors affecting the protein aggregation.

Figure 4. Alternative therapeutic strategies to block amyloidosis (source: Current Opinion in Structural Biology 2003, 13:1-7).

Tables Table 1. Proteins involved in human diseases caused by amyloid formation. Disease conditions Protein involved

Disease conditions

Alzheimer's disease Beta amyloid

Finnish amyloidosis

Proteins involved Gelsolin

Systemic amyloidosi s

Immunoglobulin light chain AL

Medullary carcinoma of the thyroid

Calcitonin

Prolactinomas

Prolactin

Hereditary nonneuropathic systemic amyloidosis

Lysozym e

Rheumatoid arthritis

Serum amyloid A

Dialysis-related amyloidosis β2 microglobulin

Parkinson's disease Alpha-synuclein

Fatal familial Insomnia

Huntington's Disease

Huntingtin

Familial amyloid polyneuropathy

Diabetes mellitus type 2 (Amylin)

IAPP

PrPSc Transthyretin

Table 2. List include proteins and their amyloid formation process

S. No. Mechanisms End-to-end or Side1. by-side addition of monomer Reversible 2. growth mechanism 3. 4.

5. 6.

Nucleation mechanism Irreversible growth mechanism Prion aggregation mechanism Secondary nucleation mechanism

Proteins of study Albumins

Year 1953 [23]

Glutamate dehydrogenase 1970 [24]

Actin, lysozyme

1975 [25,26]

Living polymers

1983

[27]

Prion

1991 2013 [29]

[28]

Amyloid

β

Table 3. Summary of dyes used for the detection of protein aggregation Dyes Solvents Experimental Extinction Concentration Coefficients (μM) (M-1cm-1) ThT Aqueous 5-40 36,000 Congo Red

Aqueous

Applications

Amyloid detection

10-300

45,000

Amyloid detection Surface hydrophobicity, unfolding/folding intermediates and aggregates detection Surface hydrophobicity, unfolding/folding intermediates and aggregates detection Detection of Viscosity of protein solvent

ANS and Bis-ANS

Aqueous and alcohol

1-30

5000 (ANS) 16,790 (Bis-ANS)

Nile Red

Di-methylsulfoxide (DMSO) and alcohol

0.5-20

19,600

DCVJ and CCVJ

Di-methylsulfoxide (DMSO) and alcohol

5

659,000

Table 4. Servers/Software for studying protein aggregation phenomenon. AGGRESCAN Predict aggregation hotspots in the polypeptides

TANGO

Prediction of aggregation regions in unfolded proteins

[65]

PASTA

Prediction of protein aggregation

[66]

AMYPRED

Prediction of aggregation-prone regions in proteins

[67]

RAFIG

Stable β-strand

WALTZ

Distinguish true amyloids from amorphous aggregates

FOLDAMYLOID

Prediction of amyloid-forming regions from the protein sequence Prediction of aggregation propensities [69] Prediction of amyloid-forming regions in [70] prion sequences

ZYGGREGATOR SECSTR

NetCSSP

Betascan

Predict the changes in native and nonnative state secondary-structure propensities due to mutation Prediction of amyloidogenic regions in the polypeptides

[68]

[71] [14]

[64]

ZipperDB

Predict amyloid-forming regions in proteins [70]

31