Open Access

Mechanisms of protein aggregation and inhibition

Mohammad Khursheed Siddiqi1,Parvez Alam1,Sumit Kumar Chaturvedi1,Yasser E. Shahein2,Rizwan Hasan Khan1,*
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh - 200202, India
Molecular Biology Department, Genetic Engineering and Biotechnology Division, National Research Centre, Dokki, Cairo, Egypt
DOI: 10.2741/E781 Volume 9 Issue 1, pp.1-20
Published: 01 January 2017
(This article belongs to the Special Issue The wheezing illness in children)
*Corresponding Author(s):  
Rizwan Hasan Khan

Protein and peptide aggregation raises keen interest due to their involvement in number of pathological conditions ranging from neurodegenerative disorders to systemic amyloidosis. Here, we have reviewed recent advances in mechanisms of aggregation, emerging technologies towards exploration, characterization of aggregate structures, detection at molecular level and the strategies to combat the phenomenon of aggregation both in cellular and in vitro conditions. In consistence, we have illustrated almost all factors that influence the protein aggregation both in vitro and in vivo environments. In addition, we have discussed a detailed journey of protein aggregation phenomenon that starts with very first events of protein aggregation. We had also described advancement in current scenarios, present aspects of fibril association to several life threatening disorders and current experimental strategies that are employed to oppose or reverse the amyloid formation.

Key words

Protein Aggregation; Protein Misfolding, Amyloid, Review

2. Introduction

Protein misfolding and its aggregation are emerged as one of the most rousing new edge in molecular medicine as well as in protein chemistry. The contemporaneous curiosity in this topic arises from several considerations; it is believed that the physicochemical features of protein folding may be elucidated; it is also expected to shed light on various human related pathological conditions. Protein misfolding and aggregation usually associated with group of diseases, known as amyloidoses which mesmerize a great deal of recent attention. In amyloidoses, fibrillar aggregates deposit in tissues as intracellular inclusion or extracellular plaques (amyloid). Amyloidoses may be either systemic or localized. Systemic amyloidosis affects multiple organs whereas localized amyloidosis is limited to one organ or tissue type. When such proteinaceous deposit occurs in neuronal cell, results in its degeneration and manifest as neurodegenerative diseases. Neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Amyotrophic lateral Sclerosis, Huntington and other protein aggregate related diseases are categorized as localized amyloidosis (1, 2). Almost all recognized neurodegenerative diseases are associated with accumulation of specific insoluble protein aggregates that are lethal to the cell (3).

Neuron related diseases are ubiquitous, especially Alzheimer’s disease (AD) which emerged as the most common form of dementia and their frequency of occurrence somewhat lie at the beginning of exponential phase and is in continuous increasing mode. AD is recently recognized as ‘twenty-first century plague (4) and its incident rate is 1% at the age of 60 years and after each fifth years incidence rate gets double. Currently, 5.3. million people in US are affected with it and are expected to increase up to 13.5. million by 2050 (5). Parkinson’s disease (PD), a disorder of brain, is the second most common form of dementia characterized by difficulty in walking and movement. Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a neuromuscular disease, affects the nerve that controlling the voluntary muscles (6). Accumulation of insoluble protein aggregate in central nervous system is supposed to be common cause of all neurodegenerative diseases. Still, causative link and effects leading to protein aggregation are largely unknown but ultimately protein aggregates are diagnostically appeared to be involved in it.

How protein forms aggregate is still not fully deciphered and needs some better comprehensive insights. In this review, we have delineated almost all possible ways or mechanisms by which protein aggregation may occur and in addition, narrated factors and forces that may involve in either aggregate formation or inhibition, both in vivo and in vitro conditions. At last, a journey with full description of protein aggregation has been described in more detail.

3. Protein folding, misfolding and aggregation

Protein folding is a process in which a polypeptide must undergo folding process to obtain its functionallyactive three dimensional structure. Anfinsen experiment on ribonuclease A supports this notion which states that the information required to form folded conformation resides in its polypeptide amino acid sequence as the denatured enzyme refolds into native conformation without assistance of any other protein (7). Such studies showed that folding may be initiated by either hydrophobic collapse of non-polar chain of amino acid residues, or formation of secondary structures that provide base for subsequent folding, or formation of covalent bond like disulfide bond.

Before acquiring a biologically active conformation, proteins must undergo through several intermediate structures. Some of these intermediates are more susceptible to form misfolded conformation (Figure 1), a problem attributed to the highly crowded cellular environment (8). Under stress conditions, native proteins are also prone to form misfolded conformers because the energy difference barrier that separates the native and misfolded proteins are too small (9, 10). Remarkably, folding intermediates or misfolded conformers have surface exposed hydrophobic amino acid side chains rather than buried inside as in natively folded proteins (11). Such exposition of hydrophobic residues provides an opportunity for conformers to interact with other molecules especially via hydrophobic interactions, leading to the formation of protein aggregates. Protein aggregation is self-assembly process in which protein molecule with altered, misfolded or partially unfolded conformations associates in specific manner to form higher order structure with low solubility. These structures are often termed as amyloid fibrils. During aggregation, one species can influence the aggregation of another, and also interestingly, different protein species can also co-aggregate together as was for protein containing polyglutamine stretch (12).

Figure 1. Illustration of kinetic competition between aggregation and folding.

Amyloid fibrils are highly ordered structures with characteristic cross β-sheet structural motifs. In cross β-sheet structure, β-sheet structure runs perpendicular to the fibril axis. Amyloid fibrils are 7-12nm in diameter (13). The major β-sheet region of amyloid fibrils is formed by hydrophobic residues (14). Numerous studies have provided some clue that amyloid fibrillogenes is involves secondary structure transition from α-helix to β-sheet (15,16). Amyloid hypothesis states that amyloid fibrils or its formation acts as causative agent for disease progression or its onset and appears as a major inducement for characterization of amyloid fibrils in vitro (17). More recently, amyloid hypothesis stated that intermediate entities formed during amyloidogenesis are toxic to the neuronal cell (18). In vitro study of amyloids provides links to monitor associated disease progression but certainly also has other applications (19, 20).

4. Mechanisms of protein aggregation

Not only single but several pathways or mechanisms culminate to the protein aggregate formation (Figure 2). These mechanisms are not independent to each other but have several processes in common. In Table 1, different mechanisms utilized by protein to aggregate are summarized in chronological order and subsequent paragraphs deal with some of these mechanisms.

Table 1. Summary of proposed mechanisms and involved proteins in chronological order
S. No.Proposed mechanismsProteinsYear
1.End to end or Side by side addition of monomerAlbumins1953 (22)
2.Reversible growth mechanismGlutamate dehydrogenase1970 (111)
3.Monomer additionSickle cell hemoglobin1974 (112)
4.Nucleation mechanismActin1975 (113)
5.Homo- and Heterogeneous mechanismSickle cell hemoglobin1980 (114)
6.Irreversible growth mechanismLiving polymers1983 (115)
7.Prion aggregation mechanismPrion1991 (116)
8.Two steps model of nucleationAmyloid β1997 (117)
9.Association of conformationally altered monomerAmyloid β protein2004 (118)
10.Secondary nucleation mechanismAmyloid β2013 (27)

Figure 2. Schematic presentation showing possible different mechanisms of protein aggregation.

4.1. Reversible association of monomeric proteins

This mechanism states that native protein has an inherent capacity to self-associate in a reversible manner. Native monomer of therapeutic protein insulin molecules were shown to have self-complementary surfaces that force the protein to associate reversibly to form small and larger sized oligomers, and over time these reversible oligomers become irreversible aggregates (21). This mechanism to form aggregate was well reported since 1953, it was said that unfolding of protein is not a necessary condition to form fibrillar structure instead side by side or end to end union of protein molecules brought about aggregation of protein (22).

4.2. Aggregate formation by conformationally altered proteins

A native protein does not always have tendency to self-associate. Instead, protein with altered conformation or in partially unfolded state have strong propensity to form higher order oligomers. So the initial step in this mechanism is transition from native to non-native structure which distinguishes this mechanism from previous one. Such aggregation prone of protein non-native state may be achieved by stresses such as heat or shear. The condition or excipients that stabilize the native state of protein may be helpful in proposing anti-aggregating agent. Interferon-ϒ (23) and Granulocyte-colony stimulating factor (G-CSF) (24) have been reported to favor this mechanism.

4.3. Primary nucleation mechanism

However, native monomer alone is unable to initiate the process of fibril formation but aggregate of sufficient size favors the formation of larger size aggregate by the addition of monomer to it. This is so called as critical nucleus. This process of aggregation generally exhibits a lag phase during which no visible precipitate appears for a long period of time (perhaps a month) but after such critical period and suddenly, a much larger species appear and accumulate(25).Such process is termed as homogenous nucleation. Another variant of this mechanism is heterogeneous nucleation in which the impurity or contaminant acts as the nucleus. Some examples of impurities are silica particles shed by product vials(26), steel particles shed by a piston pump used for filling vials etc. Generally, amyloid formation occurs via nucleation-dependent oligomerization.This mechanism is widely used to illustrate the phenomenon of protein aggregation.

4.4. Secondary nucleation mechanism

Primary nucleation was supposed to be preceding step of secondary nucleation mechanism. In secondary nucleation mechanism, fibrils of smaller sizes (formed through primary nucleation of critical concentration) act as nucleus for further oligomerization with enhanced propensity. Morphological structure and accessible surface areas of amyloid fibrils affect the kinetics of oligomerization through secondary nucleation mechanism (27).

4.5. 3D domain swapping mechanism

Another striking mechanism for amyloid fibril formation is 3D domain swapping. In this mechanism, identical proteins replace their domain. The result is an intertwined dimer or higher order oligomer, with one domain of each subunit replaced by the identical domain of other subunit. The swapped domain may be an alpha helix or a β-sheet or an entire tertiary globular domain. Ribonuclease A has been shown to adopt this mechanism to form amyloid like fibrils (28).

5. Forces involved in folding and aggregation of proteins

We all are familiar with the process that how polypeptide attain a functionally active native protein conformation through protein folding process. But during folding, amino acid residues which are present far apart in polypeptide chain, come close together (29). Such close proximity of amino acid residues involves different intramolecular interactions that favor the folding of proteins. During folding, these interactions play their role in an efficiently regulated manner. Disturbance to any of these interactions leads the several non-desirable associations of amino acid residues and in turn responsible for the formation of various partially unfolded or misfolded proteins (30). Alteration to interactions results not only in development of partially unfolded or misfolded protein but also provides opportunity for conformationally altered conformer to interact with other protein molecules through intermolecular interactions. These opportunistic interactions lead to the formation of oligomers and continuing addition will result in higher order oligomeric aggregates. These interactions may include hydrogen bonding, hydrophobic interaction, vander Waals interaction, electrostatic interaction etc. (30).

Hydrogen bonding in α-helix chain is known to be formed between oxygen of carbonyl group(C=O) and hydrogen of N-H group which determines its cylindrical structure. In β-sheets, intermolecular hydrogen bonding occurs between carbonyl oxygen of one β-sheet to amino hydrogen of another β-sheet which determines it’s parallel and antiparallel orientation structures. It also occurs between α-helix and occurs when they are at its minimum critical distance (31). Non-polar side chain of amino acid residues interacts with other non-polar amino acid side chain through hydrophobic interaction. This is supposed to be major driving force for proteins to attain three dimensional conformations (32). In protein aggregation, these interactions are supposed to play major role and not surprisingly, a major portion of research work in field of protein aggregation is devoted towards inhibition of such interaction (33).

6. Factors responsible for protein aggregation

Protein aggregation can be induced by wide variety of factors both in vitro and in vivo environments. Physical parameters such as temperature (34), pH (35), co-solvent (36), metal ions (37), freezing and thawing (38), agitation stress (39), surfactants (40)etc, that have potential to partially unfold the protein or increase the propensity of folding intermediates, lead to protein aggregation (Figure 3).

Figure 3. Schematic illustration of amyloid formation by various factors in in vitro conditions.

As protein synthesis begins, number of chaperones work to fold the polypeptide properly to be functionally active. Since some chaperone functions are sequence specific, any change or mutation in the polypeptide chain may prevent its binding and abort its function. This inability of chaperone leads to partial folding/misfolding/improper folding of protein and consequently results in a conformation which is more prone to aggregation (41). Ability of protein biogenesis system to synthesize defective protein may also be considered as aggregate producing source. These defects may include improper translation, wrong incorporation of amino acid etc. (42).

During ageing, the cellular proteasome ability to eliminate impaired or misfolded proteins is reduced. Therefore, these proteins tend to produce aggregates resulting in fatal consequences. Mutant form(s) of human superoxide dismutase 1 has more exposed hydrophobic surfaces and hence more chaperones are bound, representing the situation where protein homeostasis is not maintained (43).

7. Analytical techniques: To study protein aggregation

Protein aggregates possess especially amyloid fibrils of highly ordered structures. These structures made unavoidable requirements of sensitive techniques which are not only merely detect the presence of aggregate fibrils but also to monitor the aggregation kinetics. Some of these techniques are described here and is summarized in Table 2.

Table 2. Techniques used for characterization of protein aggregates
S. No.CategoryTechniquesApplications
1.Separative methodsGel Electrophoresis, Size Exclusion Chromatography, UltracentrifugationAssembly size/size distribution
2.Spectroscopic techniquesCircular dichroism, UV-spectroscopy, Fluorescence spectroscopy, Fourier transform infrared Spectroscopy’ Mass spectrometry, Dynamic Light Scattering,Structural change to protein, turbidity, scattering, to identify co-existing protein conformers, structural detail, size, structure of aggregates
3.DyesCongo Red, 1-Anilinonaphthalene-8-Sulfonate (ANS), Bis ANS, Thioflavin T, Nile RedQualitative and quantitative estimation of amyloid formation and Assessment of surface hydrophobicity
4.NMR and X ray crystallographySolid and solution state NMR, X ray diffraction, X ray crystallographyTo get insight into the atomic structure of aggregates

Protein molecules, which are susceptible to form amyloid fibrils, undergo structural rearrangement in such a manner that brought about increment in β-sheet content as compared to native monomer. And such transition of protein secondary structures towards β-sheet enrichment are analyzed by monitoring the change in peak position and ellipticity of Circular Dichroism (CD) spectrum (44, 45). Fourier transform infrared (FTIR) spectroscopy is a measurement of wavelength and intensity of the absorption of infrared radiation (IR) by a sample. In FTIR of protein, strong absorption band appears at 1600-1700 cm-1. It occurs due to stretching vibrations of the C=O bond. Since, vibrators exhibit the phenomenon of transition dipole coupling; it should indicate the relationship between the peak position and type of secondary structure. By employing different empirical relationships, different secondary structures have been identified. Non-alpha-non-beta structure, both alpha helix and random coil; and beta sheet structure appear at >1660, 1660-1640, 1640-1620 cm-1 respectively (46). These frequencies are closely correlated with different secondary structures of proteins.

Gel electrophoresis is a frequently used protean technique for qualitative and quantitative analysis of proteins. The size of aggregates/proteins detected through gel electrophoresis ranges from 5-500 kDa as shown by separation of amyloid β(Aβ) monomers and oligomers (47). Native gel electrophoresis exploits entirely different principle than in SDS PAGE. Instead of denaturing or reducing the molecule, it separates the molecule as it is. Therefore, aggregates are apparently more sTable in non-denaturing PAGE than in denaturing PAGE as exemplified by Aβ, yeast prion aggregates(48) and oligomer of neuroserpin variants(49). SDS PAGE is quiet useful for studying highly complex protein aggregates. It is frequently used to characterize soluble Aβ aggregates and SDS resistant prion protein aggregates (50).

Thioflavin T, a fluorescent dye, which has been widely used to monitor the presence of amyloid fibrils. It displays a mark increase in fluorescence upon binding to amyloid fibrils with an excitation maximum at around 450nm and emission maximum at around 485nm. It specifically binds to the beta sheet groove structure present in amyloid protein aggregate (51).Another dye, congo red is also used to detect the presence of amyloid fibrils both in tissue sections and in vitro. It produces blue-green birefringence upon complexation with amyloid fibrils. It is also characterized by shift in wavelength of maximal absorption from 490 to 540 nm (52).

Direct visualization of three-dimensional structure of various fibrous systems of biological importance including collagen, keratin and slightly twisted paired helical filaments that act as nucleus for Alzheimer like diseases and morphology of amyloidogenic protein aggregate samples in ambient environment was achieved by Atomic Force Microscopy (AFM). AFM elucidated the basic structural features of protein aggregates at nano meter level like curvature, width, length both qualitatively and quantitatively(53).

X ray diffraction (XRD) is one of the most sensitive techniques for the detection of amyloid fibrils at high resolution. It resolves the cross β-sheet pattern of amyloid. It is easy, fast, non-destructive and used to find the structure of unknown crystals. It gives information about thickness, size, strain and atomic arrangement(54). In XRD, structure is obtained due the interference pattern of the X-rays scattered by the crystal. Diffracted x-ray is detected by the diffractometer (55).

The two dimensional visualization of the protein self-assembly into oligomers, protofibrils and mature fibrils have been facilitated by high-resolution microscopic techniques such as high resolution transmission electron microscopy (HRTEM). HRTEM provides images with high resolution of prefibrillar aggregates, circular species and mature fibrils. It is easy to perform and confirms the fibrillar morphology of amyloid(56). Likewise, field emission surface electron microscopy (FESEM) also used to visualize the three dimensional structure of amyloid fibrils. FESEM provides images with comparatively high resolution but it is not prevalent as other techniques(57, 58).

8. Functional Amyloids

A large number of proteins have been reported that are not associated with amyloidosis but form amyloid fibrils with characteristic morphology, structure and tinctorial properties. These findings support the idea that propensity to form amyloid is an inherent property of polypeptide chain but extent of amyloidogenesis depends on polypeptide sequence. This ability to form amyloid has been exploited by different living systems to perform their normal physiological cycle as some organisms have been shown to convert their endogenous proteins into amyloid fibrils that have beneficial functions rather than disease associated properties(59, 60). The associated roles of functional amyloids are listed in Table 3.

Table 3. Functional amyloid with specific functional roles
Escherichia coli (bacterium)CurliBiofilm formation and facilitate binding to host proteins(119)
Streptomyces coelicolor (bacterium)ChaplinDevelopment of aerial hyphae(120)
Saccharomyces cerevisiae (fungus)URE2pCatabolism of nitrogen(121)
Neurosporacrassa (fungus)HydrophobinDevelopment of aerial hyphae(122)
Podosporaanserina (fungus)HET-s (prion)Regulation of heterokaryon formation(123)
Nephilaedulis (spider)SpidroinFormation of silk fibers of the web(124)
Aplisiacalifornica (marine snail)Neuron-speci?c isoform of CPEB (prion)Maintenance of synaptic changes associated with memory storage(125)
Homo sapiensIntralumenal domain of Pmel17Forms fibrous striation upon which melanin granules forms(126)

One of the examples of functional amyloids in bacteria Escherichia coli is the curli proteins which are used to colonize inert surfaces and mediate binding to host proteins such as fibronectin, laminin, plasminogen, tissue plasminogen activator, and H-kininogen. In addition, the production of melanin in melanocytes is characterized by the presence of intralumenal fibrous striation upon which melanin is formed. This fibrous material is formed from the intralumenal domain of the membrane protein Pmel17, in mammalian system(61).

9. Strategies for aggregation inhibition

Still it is topic of debate and discussion that whether soluble monomers, or oligomers or larger aggregates are greatly associated with toxicity to the cell that results in various neuron related disorders. In higher eukaryotes, proteins have limited ability to refold into native forms, which increases the possibility of proteins to aggregate. Here, we have described the pathways that prevent the formation or accumulation of aggregates and Table 4 accounts for great details of these approaches.

Table 4. Anti-amyloidogenic agents and related proteins of study
CategoryAnti amyloidogenic agentsProteins
Huntingtin (127)
Transthyretin (128)
(−)-epigallocatechin 3-gallate
β-amyloid (129)
Islet amyloid peptides (130)
ChaperonesAlpha S β
Casein (91)
Casein (91)
β-amyloid (101)
β-amyloid (101)
Congo red
Casein (91)
β-amyloid (92)
SurfactantsCommercial (SDS)
Synthetic (Gemini)
Papain (131)
Serum albumin (132)
Beta amyloid 40 & 42 (95)
β-amyloid (133)
β-amyloid (99)
β-lactoglobulin (134)
OsmolytesTrimetylamine N-oxside
P39 cellular retinoic acid-binding protein (96)
β-amyloid (135)
Synthetic Drugs2-phenylbenzofuran derivatives
Isoliquiritigenin derivatives
β-amyloid (136)
β-amyloid (137)
Metal ionsCopper (II)β-amyloid (94)
Nucleic acidsSingle Stranded Syntheticβ-amyloid (138)

Inspite of intense research, the mechanism of inhibition is poorly understood but several formulations or drug molecules are proven to be effective against amyloid fibrillation process and have potential to either prevent the aggregate formation or to some extent reverse the process of protein aggregation (62-64). These molecules with the ability to impede the process of aggregation are designed on the basis of concepts that were utilized by the protein to form aggregates. Therefore, such molecules are more potent to hinder the self-assembly process of proteins. Apart from inhibition, molecules which stabilize the native state of proteins and minimize the possibility of protein to aggregate are also reported (Figure 4). This carries a great hope for development of anti-amyloid agents.

Figure 4. Schematic illustrations of pathway that inhibit amyloid fibril formation (A)-(E) represent the molecules used to either inhibition or stabilization of protein. (A) Native state stabilization (B) Refolding of polypeptide (C) Diversion from oligomerization pathway (D) Inhibition of fibril elongation by β-sheet breakers (E) Disaggregation of amyloid aggregate.

Proteasome is a barrel shaped multiprotein complex and believed to be central cellular machinery for degradation of various misfolded and aggregated proteins (Figure 5A)(65).Impairment in proteasome system may results in neuro-degeneration, indicating the crucial role of proteasome for clearance of aggregates(66, 67). The protein which is susceptible to degradation by proteasome machinery is marked by its association with ubiquitin. Another process, autophagy, emerges as major contributor for degradation of misfolded and aggregated proteins in the cytosol of mammalian cells(68). It uses specialized double membrane structure that engulfs the substrate to form autophagosomes (Figure 5B). Autophagosomes ultimately fuse with lysosomes that form autophagolysosomes to degrade their contents by acidic hydrolases present in lysosomes(69). Autophagy is regarded as a backup system to complement proteasomal degradation when it is exhausted or overwhelmed of dealing with aggregates. In agreement with this, it also has role in clearance of aggresomes(70, 71).

Figure 5. (5A) Schematic diagram showing key aspects of ubiquitin-proteasome mediated degradation and (5B) autophagy.

10. Ajourney from origin to current scenario of protein aggregation

Initially it was thought that protein molecules exist as single entity i.e.,they were unable to interact with each other. But later in 1948, a research group showed that protein molecules may associate as it was agreed by evaluating the molecular weight of egg albumin (72). In agreement with this, in 1952, a result was reported that a number of globular proteins like heamocyanin, horse hemoglobin, insulin, fibrinogen, ovalbumin possess the ability to interact with each other when provided with different conditions (73). These conditions may include pressure, salt concentration, heat treatment and were studied by techniques such as electron microscopy, viscosity, electrophoresis, ultracentrifugation, paper electrophoresis, fractional precipitation, streaming birefringence (22). Till now, the causative link for aggregation was unknown but a ray of hope was came when denaturation of proteins was blamed for their aggregation (74). In 1992, this process was further supported by the fact that partial unfolding of protein (e.g.transthyretin) was a necessary requirement for protein to aggregate (75).

Revolution to protein biophysics came into existence when protein aggregates were reported to be associated with diseases (76). This attracts the attention of core group of protein biophysicists to solve the puzzle of protein aggregation and its association with several diseases. Some of these include fibrinogen amyloidosis, lysozyme amyloidosis etc. (41). In addition to this, protein aggregates were also found to be deposited in neurons, leads to their degeneration, a prerequisite condition for dementia or mental retardation. Alzheimer, Parkinson, Huntingtin disease etc. were known to be the pathogenic consequences of neuron degeneration (41). Further research on aggregates leads categorization of aggregates into two either random type-amorphous or well-ordered amyloid structure (77).

Curiosity to know the organization of aggregate, structure, its physical appearance is made possible by utilization of several sensitive techniques to explore it at atomic detail both in vitro and in vivo. In 1997, with the aim to explore its structure, application of synchrotron x-ray diffraction method reveals the common core structure of amyloid aggregate which consist ofhelical array of β-sheets parallel to the fiber long axis, with the strands perpendicular to this axis (78). Apart from its characterization, detection of both amyloid as well as amorphous protein aggregate poses great challenge. In consistence, in vitro detection may include turbidity measurement (e.g.Catalase)(79), Light scattering(e.g.,IgG) (79), Thioflavin T assay(e.g.α-synuclein), and imaging techniques like AFM, Transmission electron microscopy (TEM), Scanning electron microscopy (SEM) etc. In vivo detection may include fluorescence correlation spectroscopy (e.g.amyloid β-peptide) (80), magnetic resonance imaging, positron emission tomography, single photon emission computed tomography, multiphoton microscopy etc. (81). But these techniques have some advantages and disadvantages also. Although ThioflavinT is widely used to diagnose the amyloid fibrils not amorphous aggregates, both in vitro and in vivo, but its sensitivity depends on pH. pH provides positive charge or prevents the formation of Thioflavin T micelle which causes several fold decrease in fluorescence (82).

As mentioned earlier, it was believed that aggregation is simple association of denatured or partially unfolded protein molecules. But intensive research on protein aggregates leads to the development of pathways which result in protein aggregates (Figure 6). A conformation which is different from native one i.e.,misfolded conformation is shown to associate more rapidly to form aggregates(83). During the process of aggregation, initially there were no appearance of visible aggregates but after some critical period, a large clump of proteins were appeared. This mystery was solved by a hypothesis stated that a small preformed aggregate of sufficient size with the ability to nucleate the process of aggregate formation is prerequisite (84). Next to it was the proposition of mechanism of fibril elongation (84). Thereafter, it was also reported that not only change in conformation but also chemical modification to the protein structure is associated with fibril formation (85).

Figure 6.Simplified view of protein aggregation phenomenon and its associated aspects.

Once the mechanism of formation was explored, then to determine the various factors/conditions (both in vitro and in vivo) which induce or inhibit the aggregation process was emerged as topic of keen interest. In vitro aggregate inducing factors may include temperature, pH, cosolvent, metal ions, surfactants etc. and in vivo may include mutation in polypeptide chain(86), translational error, aging, inability of cellular machinery to clear the aggregate,(87) etc.

Now days, majority of works in field of protein aggregation are carried out to develop or propose an effective formulation/inhibitor which is able to prevent the process of fibril formation. In this context, polyphenols were tested which possess potential to inhibit its formation (88). β-cyclodextrin (a cyclic glucopyranose) and its derivatives inhibits the formation of β amyloid peptide (89). Several other compounds with antioxidant activity were also capable of it (90). Not surprisingly, dyes like ANS (91), Congo red (92), methylene blue (93) etc. which are used to detect presence of protein aggregates, also shown to prevent aggregate formation. Metal ions like Cu (II) (94), sugars like trehalose (95), osmolytes like trimethylamine N-oxide(96, 97), and surfactants (79) also stand in front to abort aggregation process. Nanoparticle (such as copolymeric NiPAM:BAM)(98), synthesized chemicals and drugs also made contributory successful attempts towards inhibition of aggregation process. Plant derived products including Curcumin (99, 100) are claimed for curing protein aggregation. Interestingly, protein molecules itself inhibit the aggregation of other proteins. In this regard, chaperones were initially shown to possess such inhibitory function. Chaperones like Alpha S and β-casein inhibit the casein aggregation (91). But recently, non-chaperone proteins like catalase, pyruvate kinase, albumin, lysozyme, α-lactalbumin, and β-lactoglobulin were also reported to suppress the fibrillation of β-amyloid peptides (101). Intracellularly, cells have been provided with systems which deal with protein aggregates and have capacitiesto clear the aggregates. Autophagy (102) and proteasome (103) constitute two systems which are vigorously involved in clearance activity inside the cell.

A wide range of scientific disciplines working with protein aggregation and amyloids have been stimulated by their association with several debilitating medical disorders, from Alzheimer’s disease to type II diabetes, and many of which are responsible for major threats to human health and welfare in the modern world. Alzheimer’s disease is associated with deposition of β-amyloid and tau in neuronal cells extracellularly and intracellularly, respectively. β-amyloid peptides are generated from amyloid precursor protein by the action of secretases. Hyperphosphorylation of tau protein causes its dissociation from cytoskeletons, a known factor for its deposition. With the aim to screen for an effective compound, different approaches were employed. Some research works are directed towards inhibition of ϒ-secretase activity(104) and some towards how to inhibit the association of β-amyloid peptide(105) and tau (106).

Although the nature of aggregates is still unclear, but recently it has been shown that there is association of amyloid aggregates with cancer. p53 i.e.,tumor suppressor, is frequently mutated in nearly all types of cancers. Such mutations produce conformational alterations which, in several cases are accumulated as intracellular aggregates (107). Such results inferred that cancer could be considered as protein conformation-disease.

Awareness to the deleterious effects of amyloid aggregates is now well known. But beside this, protein oligomers and aggregates are reported to have some positive aspects also. Supramolecular insulin (insulin oligomer) assembly have been proven to be effective in treatment of type 1 diabetes mellitus. Authors have experimentally proved that oligomerization or aggregates of insulin causes sustained release of insulin monomer in blood on injection which has been retained for longer time and has excluded the condition of multiple insulin injections method to cure diabetes (108). Moreover, supramolecular polymeric structure of proteins is also used as drug delivery system. Ulyana Shimanovich et al. (109)have shown that nanofibrils can be used to form protein micro gels and demonstrated the controlled release of encapsulated drugs. Further these gels were found to be nontoxic to human cells and show relatively more efficacy as compared in homogenous solution (110).

11. Conclusion and future perspective

Now we are at the level from which we can strongly underscore the relevance that propensity to form fibril is a reflection of its polypeptide sequence, modulated by environmental conditions. Aggregates are formed by the interaction of partially unfolded intermediates containing significant amount of native like structures and rich in β-sheet secondary structures. Both amorphous as well as amyloids possess significant amount of β-sheet structures. These structures require techniques with high resolution and sensitivity to explore its pattern of association and mechanism. Apprehension of kinetics of amyloid formation and pathways may help in designing strategies and approaches that either lead to inhibition or reverse the process of aggregate formation.

It is now more evident that protein aggregates formed in cells are also due to malfunctioning of protein quality control systems like proteasome machinery and autophagy. Insight into the molecular aspects involved behind protein quality control systems may explain the facets of various diseases associated with protein aggregation and hence open way for therapeutic intervention. Further research will be needed to establish the biochemical strategies and genetic manipulation that explore the additional structural components, deposition sites, molecular architectures of protein aggregates. These studies will be a prerequisite for tackling other problems relating to regulation of this process.

Interestingly, beneficial facet of amyloid fibrils to form different nanostructure made them of great value. Nanoparticles or supramolecular polymeric structures formed by amyloid fibrils with remarkable sustained release of monomers may be exploited in near future to treat different human associated diseases. Similarly, protein mircogels as drug delivery systems carry great hope for delivery of therapeutic drugs (110).

There are still many outstanding and critical questions regarding protein aggregation. These include detailed mechanisms of aggregate formation, factors influencing the kinetics of aggregation, nature of molecular interaction and how aggregates are effectively and efficiently prevented, particularly in vivo. Moreover, revealing the pathway that leads to the protein misfolding which is thought as overture to the fibril formation will help in studying normal protein folding and the evolution of protein folding and aggregation. In parallel, co-aggregation of protein molecules raised excellent challenges and has not yet been fully understood.

12. Acknowledgements

Facilities provided by IBU, Aligarh Muslim University, Aligarh are gratefully acknowledged. M.K.S is highly thankful to Department of Biotechnology (DBT), New Delhi, for providing fellowship in the form of junior research fellowship. P.A and S.K.C are highly thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi, for financial assistance in the form of junior research fellowship (JRF) and senior research fellowship (SRF), respectively.


    1. R. H. Falk, R. L. Comenzo and M. Skinner: The systemic amyloidoses. New Eng J Med., 337(13), 898-909 (1997).

    2. M. L. Biewend, D. M. Menke and K. T. Calamia: The spectrum of localized amyloidosis: a case series of 20 patients and review of the literature. Amyloid, 13 (3), 135-142 (2006)

    3. M. Renner and R. Melki: Protein aggregation and prionopathies. Pathologie Biologie (2014)

    4. T. P. J. Knowles, M. Vendruscolo and C. M. Dobson: The amyloid state and its association with protein misfolding diseases. Nat Rev Mol Cell Biology, 15(6), 384-396 (2014)

    5. A. s. Association: 2010 Alzheimer’s disease facts and figures. Alz Dement, 6(2), 158-94 (2010) doi:S1552-5260(10)00014-2 (pii)

    6. D. Majoor-Krakauer, P. J. Willems and A. Hofman: Genetic epidemiology of amyotrophic lateral sclerosis. Clin Genet, 63(2), 83-101 (2003)

    7. E. M. Sivertsson and L. S. Itzhaki: Protein folding: When ribosomes pick the structure. Nat chem, 6(5), 378-379 (2014)

    8. R. J. Ellis and A. P. Minton: Protein aggregation in crowded environments. J Biolog Chem, 387(5), 485-97 (2006) doi:10.1.515/BC.2006.0.64

    9. C. M. Dobson: Protein folding and misfolding. Nature, 426 (6968), 884-890 (2003)

    10. T. R. Jahn and S. E. Radford: The Yin and Yang of protein folding. Febs J, 272(23), 5962-5970 (2005)

    11. J. M. Khan, A. Qadeer, E. Ahmad, R. Ashraf, B. Bhushan, S. K. Chaturvedi, G. Rabbani and R. H. Khan: Monomeric banana lectin at acidic pH overrules conformational stability of its native dimeric form. PloS one, 8(4), e62428 (2013)

    12. R. S. Rajan, M. E. Illing, N. F. Bence and R. R. Kopito: Specificity in intracellular protein aggregation and inclusion body formation. Pro Nat Acad Sci, 98(23), 13060-13065 (2001)

    13. M. Gopalswamy, A. Kumar, J. Adler, M. Baumann, M. Henze, S. T. Kumar, M. Fandrich, H. A. Scheidt, D. Huster and J. Balbach: Structural characterization of amyloid fibrils from the human parathyroid hormone. Biochim Biophy Acta (BBA)-Proteins and Proteomics, 1854(4), 249-257 (2015)

    14. H. Inouye, P. E. Fraser and D. A. Kirschner: Structure of beta-crystallite assemblies formed by Alzheimer beta-amyloid protein analogues: analysis by x-ray diffraction. Biophy J, 64(2), 502 (1993)

    15. J. M. Khan, A. Qadeer, S. K. Chaturvedi, E. Ahmad, S. A. Rehman, S. Gourinath and R. H. Khan: SDS can be utilized as an amyloid inducer: a case study on diverse proteins. PloS one, 7(1), e29694 (2012)

    16. S. K. Chaturvedi, P. Alam, J. M. Khan, M. K. Siddiqui, P. Kalaiarasan, N. Subbarao, Z. Ahmad and R. H. Khan: Biophysical insight into the anti-amyloidogenic behavior of taurine. Inter J bio macro, 80, 375-384 (2015)

    17. J. W. Kelly: Towards an understanding of amyloidogenesis. Nat Struct Biol, 9(5), 323-5 (2002)

    18. S. Barghorn, V. Nimmrich, A. Striebinger, C. Krantz, P. Keller, B. Janson, M. Bahr, M. Schmidt, R. S. Bitner and J. Harlan: Globular amyloid beta-peptide1-42 oligomer-a homogenous and stable neuropathological protein in Alzheimer’s disease. J of neurochem, 95(3), 834-847 (2005)

    19. C. M. Dobson: The structural basis of protein folding and its links with human disease. Philos Trans R Soc Lond B BiolSci, 356 (1406), 133-45 (2001)

    20. R. Wetzel: For protein misassembly, it’s the “I” decade. Cell, 86(5), 699-702 (1996)

    21. A. H. Pekar and B. H. Frank: Conformation of proinsulin. A comparison of insulin and proinsulin self-association at neutral pH.Biochemistry, 11(22), 4013-6 (1972)

    22. E. Barbu and M. Joly: The globular-fibrous protein transformation. Discuss. Faraday Soc., 13, 77-93 (1953)

    23. B. S. Kendrick, J. F. Carpenter, J. L. Cleland and T. W. Randolph: A transient expansion of the native state precedes aggregation of recombinant human interferon-gamma. Proc Nat Acad Sci U S A, 95(24), 14142-6 (1998)

    24. S. W. Raso, J. Abel, J. M. Barnes, K. M. Maloney, G. Pipes, M. J. Treuheit, J. King and D. N. Brems: Aggregation of granulocyte-colony stimulating factor in vitro involves a conformationally altered monomeric state. Protein Sci, 14(9), 2246-57 (2005)

    25. S. J. Shire, Z. Shahrokh and J. Liu: Challenges in the development of high protein concentration formulations. J Pharm Sci, 93(6), 1390-402 (2004)

    26. E. Y. Chi, J. Weickmann, J. F. Carpenter, M. C. Manning and T. W. Randolph: Heterogeneous nucleation-controlled particulate formation of recombinant human platelet-activating factor acetylhydrolase in pharmaceutical formulation. J of pharma sci, 94(2), 256-274 (2005)

    27. S. I. A. Cohen, S. Linse, L. M. Luheshi, E. Hellstrand, D. A. White, L. Rajah, D. E. Otzen, M. Vendruscolo, C. M. Dobson and T. P. J. Knowles: Proliferation of amyloid-beta 42 aggregates occurs through a secondary nucleation mechanism. Pro of Nat Acad Sci, 110(24), 9758-9763 (2013)

    28. S. Sambashivan, Y. Liu, M. R. Sawaya, M. Gingery and D. Eisenberg: Amyloid-like fibrils of ribonuclease A with three-dimensional domain-swapped and native-like structure. Nature, 437 (7056), 266-269 (2005)

    29. F. U. Hartl, A. Bracher and M. Hayer-Hartl: Molecular chaperones in protein folding and proteostasis. Nature, 475(7356), 324-332 (2011)

    30. Y. E. Kim, M. S. Hipp, A. Bracher, M. Hayer-Hartl and F. Ulrich Hartl: Molecular chaperone functions in protein folding and proteostasis. Ann rev of biochem, 82, 323-355 (2013)

    31. J. C. Faver, M. L. Benson, X. He, B. P. Roberts, B. Wang, M. S. Marshall, C. D. Sherrill and K. M. Merz Jr: The energy computation paradox and ab initio protein folding. PloS one, 6(4), e18868 (2011)

    32. C. N. Pace, H. Fu, K. L. Fryar, J. Landua, S. R. Trevino, B. A. Shirley, M. M. Hendricks, S. Iimura, K. Gajiwala and J. M. Scholtz: Contribution of hydrophobic interactions to protein stability. J mol bio, 408(3), 514-528 (2011)

    33. A. W. Fitzpatrick, T. P. J. Knowles, C. A. Waudby, M. Vendruscolo and C. M. Dobson: Inversion of the balance between hydrophobic and hydrogen bonding interactions in protein folding and aggregation. PLoS comput bio, 7(10), e1002169 (2011)

    34. W. Wang: Instability, stabilization, and formulation of liquid protein pharmaceuticals. Int J Pharm, 185(2), 129-88 (1999)

    35. R. C. Elgersma, L. M. Kroon-Batenburg, G. Posthuma, J. D. Meeldijk, D. T. Rijkers and R. M. Liskamp: pH-controlled aggregation polymorphism of amyloidogenic Abeta(16-22): Insights for obtaining peptide tapes and peptide nanotubes, as function of the N-terminal capping moiety. Eur J Med Chem (2014)

    36. W. Dzwolak, S. Grudzielanek, V. Smirnovas, R. Ravindra, C. Nicolini, R. Jansen, A. Loksztejn, S. Porowski and R. Winter: Ethanol-perturbed amyloidogenic self-assembly of insulin: looking for origins of amyloid strains. Biochemistry, 44(25), 8948-58 (2005)

    37. F. Hane, G. Tran, S. J. Attwood and Z. Leonenko: Cu(2+) affects amyloid-beta (1-42) aggregation by increasing peptide-peptide binding forces. PLoS One, 8(3), e59005 (2013)

    38. E. Cao, Y. Chen, Z. Cui and P. R. Foster: Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions. Biotechnol Bioeng, 82(6), 684-90 (2003)

    39. H. C. Mahler, R. Muller, W. Friess, A. Delille and S. Matheus: Induction and analysis of aggregates in a liquid IgG1-antibody formulation. Eur J Pharm Biopharm, 59(3), 407-17 (2005)

    40. J. M. Khan, S. K. Chaturvedi, S. K. Rahman, M. Ishtikhar, A. Qadeer, E. Ahmad and R. H. Khan: Protonation favors aggregation of lysozyme with SDS. Soft matter, 10(15), 2591-2599 (2014)

    41. F. Chiti and C. M. Dobson: Protein misfolding, functional amyloid, and human disease. Ann Rev Biochem, 75, 333-366 (2006)

    42. D. A. Drummond and C. O. Wilke: Mistranslation-induced protein misfolding as a dominant constraint on coding-sequence evolution. Cell, 134(2), 341-52 (2008)

    43. C. Munch and A. Bertolotti: Exposure of hydrophobic surfaces initiates aggregation of diverse ALS-causing superoxide dismutase-1 mutants. J mol bio, 399(3), 512-525 (2010)

    44. L. Breydo, K. D. Reddy, A. Piai, I. C. Felli, R. Pierattelli and V. N. Uversky: The crowd you’re in with: effects of different types of crowding agents on protein aggregation. Biochim Biophy Acta (BBA)-Proteins and Proteomics, 1844(2), 346-357 (2014)

    45. S. K. Chaturvedi, E. Ahmad, J. M. Khan, P. Alam, M. Ishtikhar and R. H. Khan: Elucidating the interaction of limonene with bovine serum albumin: a multi-technique approach. Mol BioSystems, 11(1), 307-316 (2015)

    46. D. M. Byler and H. Susi: Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymers, 25(3), 469-487 (1986)

    47. H. LeVine, 3rd: Alzheimer’s beta-peptide oligomer formation at physiologic concentrations. Anal Biochem, 335(1), 81-90 (2004) doi:S0003-2697(04)00664-5 (pii)

    48. S. N. Bagriantsev, V. V. Kushnirov and S. W. Liebman: Analysis of amyloid aggregates using agarose gel electrophoresis. Methods Enzymol, 412, 33-48 (2006)

    49. E. Miranda, I. MacLeod, M. J. Davies, J. Perez, K. Romisch, D. C. Crowther and D. A. Lomas: The intracellular accumulation of polymeric neuroserpin explains the severity of the dementia FENIB. Hum Mol Genet, 17(11), 1527-39 (2008)

    50. K. A. Coalier, G. S. Paranjape, S. Karki and M. R. Nichols: Stability of early-stage amyloid-beta (1-42) aggregation species. Biochim Biophy Acta (BBA)-Proteins and Proteomics, 1834(1), 65-70 (2013)

    51. A. A. Reinke and J. E. Gestwicki: Structure-activity relationships of amyloid beta-aggregation inhibitors based on curcumin: influence of linker length and flexibility. Chem Biol Drug Des, 70(3), 206-15 (2007)

    52. M. R. Nilsson: Techniques to study amyloid fibril formation in vitro. Methods, 34(1), 151-160 (2004)

    53. A. K. Chamberlain, C. E. MacPhee, J. s. Zurdo, L. A. Morozova-Roche, H. A. O. Hill, C. M. Dobson and J. J. Davis: Ultrastructural Organization of Amyloid Fibrils byAtomic Force Microscopy. Biophy J, 79(6), 3282-3293 (2000)

    54. M. Biancalana and S. Koide: Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochim Biophy Acta (BBA)-Proteins and Proteomics, 1804(7), 1405-1412 (2010)

    55. I. W. Hamley, V. Castelletto, C. Moulton, D. Myatt, G. Siligardi, C. L. P. Oliveira, J. S. Pedersen, I. Abutbul and D. Danino: Self-Assembly of a Modified Amyloid Peptide Fragment: pH-Responsiveness and Nematic Phase Formation. Macromol biosci, 10(1), 40-48 (2010)

    56. S. L. Gras, L. J. Waddington and K. N. Goldie: Transmission electron microscopy of amyloid fibrils. In: Protein Folding, Misfolding, and Disease. Springer, (2011)

    57. H. Seiler: Secondary electron emission in the scanning electron microscope. J App Phy, 54(11), R1-R18 (1983)

    58. L. Hughes, C. Hawes, S. Monteith and S. Vaughan: Serial block face scanning electron microscopy-the future of cell ultrastructure imaging. Protoplasma, 251(2), 395-401 (2014)

    59. V. N. Uversky and A. L. Fink: Conformational constraints for amyloid fibrillation: the importance of being unfolded. Biochim Biophy Acta (BBA)-Proteins and Proteomics, 1698(2), 131-153 (2004)

    60. M. Stefani and C. M. Dobson: Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J mol med, 81(11), 678-699 (2003)

    61. D. M. Fowler, A. V. Koulov, W. E. Balch and J. W. Kelly: Functional amyloid-from bacteria to humans. Trends in biocheml sci, 32(5), 217-224 (2007)

    62. M. Masuda, N. Suzuki, S. Taniguchi, T. Oikawa, T. Nonaka, T. Iwatsubo, S. Hisanaga, M. Goedert and M. Hasegawa: Small molecule inhibitors of alpha-synuclein filament assembly. Biochemistry, 45(19), 6085-94 (2006)

    63. D. E. Ehrnhoefer, J. Bieschke, A. Boeddrich, M. Herbst, L. Masino, R. Lurz, S. Engemann, A. Pastore and E. E. Wanker: EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nat Struct Mol Biol, 15(6), 558-66 (2008)

    64. P. Frid, S. V. Anisimov and N. Popovic: Congo red and protein aggregation in neurodegenerative diseases. Brain Res Rev, 53(1), 135-60 (2007)

    65. A. Ciechanover: The ubiquitin proteolytic system: from a vague idea, through basic mechanisms, and onto human diseases and drug targeting. Neurology, 66(2 Suppl 1), S7-19 (2006)

    66. U. B. Pandey, Z. Nie, Y. Batlevi, B. A. McCray, G. P. Ritson, N. B. Nedelsky, S. L. Schwartz, N. A. DiProspero, M. A. Knight, O. Schuldiner, R. Padmanabhan, M. Hild, D. L. Berry, D. Garza, C. C. Hubbert, T. P. Yao, E. H. Baehrecke and J. P. Taylor: HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS. Nature, 447(7146), 859-63 (2007)

    67. L. Bedford, D. Hay, A. Devoy, S. Paine, D. G. Powe, R. Seth, T. Gray, I. Topham, K. Fone, N. Rezvani, M. Mee, T. Soane, R. Layfield, P. W. Sheppard, T. Ebendal, D. Usoskin, J. Lowe and R. J. Mayer: Depletion of 26S proteasomes in mouse brain neurons causes neurodegeneration and Lewy-like inclusions resembling human pale bodies. J Neurosci, 28(33), 8189-98 (2008)

    68. H. Nakatogawa, K. Suzuki, Y. Kamada and Y. Ohsumi: Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat Rev Mol Cell Bio, 10(7), 458-467 (2009)

    69. C. He and D. J. Klionsky: Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet, 43, 67-93 (2009) doi:10.1.146/annurev-genet-102808-114910

    70. J. Fortun, W. A. Dunn, S. Joy, J. Li and L. Notterpek: Emerging role for autophagy in the removal of aggresomes in Schwann cells. The J neurosci, 23(33), 10672-10680 (2003)

    71. J. A. Olzmann and L.-S. Chin: Parkin-mediated K63-linked polyubiquitination: a signal for targeting misfolded proteins to the aggresome-autophagy pathway. Autophagy, 4(1), 85 (2008)

    72. M. Bier and F. F. Nord: Aggregation phenomena in egg albumin solutions as determined by light scattering measurements. Proc Nat Aca Sci, 35(1), 17 (1949)

    73. L. Pauling: Protein interactions. Aggregation of globular proteins. Discuss. Faraday Soc., 13, 170-176 (1952)

    74. E. V. Jensen: Sulfhydryl-Disulfide Interchange This biological chain reaction explains aspects of protein denaturation, blood clotting, and mitosis. Sci, 130(3385), 1319-1323 (1959)

    75. W. Colon and J. W. Kelly: Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro. Biochemistry, 31(36), 8654-8660 (1992)

    76. K. Halverson, P. E. Fraser, D. A. Kirschner and P. T. Lansbury Jr: Molecular determinants of amyloid deposition in Alzheimer’s disease: conformational studies of synthetic. beta.-protein fragments. Biochemistry, 29(11), 2639-2644 (1990)

    77. A. L. Fink: Protein aggregation: folding aggregates, inclusion bodies and amyloid. Folding and design, 3(1), R9-R23 (1998)

    78. M. Sunde, L. C. Serpell, M. Bartlam, P. E. Fraser, M. B. Pepys and C. C. F. Blake: Common core structure of amyloid fibrils by synchrotron X-ray diffraction. J mol bio, 273(3), 729-739 (1997)

    79. J. M. Khan, A. Qadeer, S. K. Chaturvedi, E. Ahmad, S. A. A. Rehman, S. Gourinath and R. H. Khan: SDS can be utilized as an amyloid inducer: a case study on diverse proteins. PloS one, 7(1), e29694 (2012)

    80. M. Pitschke, R. Prior, M. Haupt and D. Riesner: Detection of single amyloid beta-protein aggregates in the cerebrospinal fluid of Alzheimer’s patients by fluorescence correlation spectroscopy. Nat med, 4(7), 832-834 (1998)

    81. B. J. Bacskai, W. E. Klunk, C. A. Mathis and B. T. Hyman: Imaging Amyloid-&bgr; Deposits In vivo. J Cereb Blood Flow & Metab, 22(9), 1035-1041 (2002)

    82. R. Khurana, C. Coleman, C. Ionescu-Zanetti, S. A. Carter, V. Krishna, R. K. Grover, R. Roy and S. Singh: Mechanism of thioflavin T binding to amyloid fibrils. J str bio, 151(3), 229-238 (2005)

    83. I. V. Baskakov, G. Legname, M. A. Baldwin, S. B. Prusiner and F. E. Cohen: Pathway complexity of prion protein assembly into amyloid. J Biolog Chem, 277(24), 21140-21148 (2002)

    84. C.-C. Lee, A. Nayak, A. Sethuraman, G. Belfort and G. J. McRae: A three-stage kinetic model of amyloid fibrillation. Biophy j, 92(10), 3448-3458 (2007)

    85. P. H. Axelsen, H. Komatsu and I. V. J. Murray: Oxidative stress and cell membranes in the pathogenesis of Alzheimer’s disease. Physiology, 26(1), 54-69 (2011)

    86. P. Cao, L.-H. Tu, A. Abedini, O. Levsh, R. Akter, V. Patsalo, A. M. Schmidt and D. P. Raleigh: Sensitivity of amyloid formation by human islet amyloid polypeptide to mutations at residue 20. J mol bio, 421(2), 282-295 (2012)

    87. J. Tyedmers, A. Mogk and B. Bukau: Cellular strategies for controlling protein aggregation. Nat rev Mol cell biology, 11(11), 777-788 (2010)

    88. Q. I. Churches, J. Caine, K. Cavanagh, V. C. Epa, L. Waddington, C. E. Tranberg, A. G. Meyer, J. N. Varghese, V. Streltsov and P. J. Duggan: Naturally occurring polyphenolic inhibitors of amyloid beta aggregation. Bioorganic & med chem lett (2104)

    89. Z. Wang, L. Chang, W. L. Klein, G. R. J. Thatcher and D. L. Venton: Per-6-substituted-per-6-deoxy beta-cyclodextrins inhibit the formation of beta-amyloid peptide derived soluble oligomers. J med chem, 47(13), 3329-3333 (2004)

    90. J. Jayamani and G. Shanmugam: Gallic acid, one of the components in many plant tissues, is a potential inhibitor for insulin amyloid fibril formation. Europ j med chem (2014)

    91. D. C. Thorn, S. Meehan, M. Sunde, A. Rekas, S. L. Gras, C. E. MacPhee, C. M. Dobson, M. R. Wilson and J. A. Carver: Amyloid fibril formation by bovine milk kappa-casein and its inhibition by the molecular chaperones alphaS-and beta-casein. Biochemistry, 44(51), 17027-36 (2005)

    92. A. Lorenzo and B. A. Yankner: Beta-amyloid neurotoxicity requires fibril formation and is inhibited by congo red. Pro Nat Aca Sci, 91(25), 12243-12247 (1994)

    93. C. M. Wischik, P. C. Edwards, R. Y. Lai, M. Roth and C. R. Harrington: Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines. Pro Nat Aca Sci, 93(20), 11213-11218 (1996)

    94. K. Suzuki, T. Miura and H. Takeuchi: Inhibitory effect of copper (II) on zinc (II)-induced aggregation of amyloid beta-peptide. Biochem biophys res comm, 285(4), 991-996 (2001)

    95. R. Liu, H. Barkhordarian, S. Emadi, C. B. Park and M. R. Sierks: Trehalose differentially inhibits aggregation and neurotoxicity of beta-amyloid 40 and 42. Neurobio of disease, 20(1), 74-81 (2005)

    96. Z. Ignatova and L. M. Gierasch: Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant. Pro Nat Aca Sci, 103(36), 13357-13361 (2006)

    97. J. Wawer, J. Krakowiak, M. Szocinski, Z. Lustig, M. Olszewski and K. Szostak: Inhibition of amyloid fibril formation of hen egg white lysozyme by trimethylamine N-oxide at low pH. Inter j bio mac, 70, 214-221 (2014)

    98. C. Cabaleiro-Lago, F. Quinlan-Pluck, I. Lynch, S. Lindman, A. M. Minogue, E. Thulin, D. M. Walsh, K. A. Dawson and S. Linse: Inhibition of amyloid beta protein fibrillation by polymeric nanoparticles. J American Chem Soc, 130(46), 15437-15443 (2008)

    99. S. Palmal, A. R. Maity, B. K. Singh, S. Basu, N. R. Jana and N. R. Jana: Inhibition of Amyloid Fibril Growth and Dissolution of Amyloid Fibrils by Curcumin-Gold Nanoparticles. Chem-A Europ J, 20(20), 6184-6191 (2014)

    100. F. Yang, G. P. Lim, A. N. Begum, O. J. Ubeda, M. R. Simmons, S. S. Ambegaokar, P. P. Chen, R. Kayed, C. G. Glabe and S. A. Frautschy: Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo. J Biolog Chem, 280(7), 5892-5901 (2005)

    101. J. Luo, S. K. T. S. Warmlander, A. Graslund and J. P. Abrahams: Non-chaperone Proteins Can Inhibit Aggregation and Cytotoxicity of Alzheimer Amyloid beta Peptide. J Biolog Chem, 289(40), 27766-27775 (2014)

    102. T. Jiang, J. T. Yu, X. C. Zhu, M.-S. Tan, H. F. Wang, L. Cao, Q. Q. Zhang, J. Q. Shi, L. Gao and H. Qin: Temsirolimus promotes autophagic clearance of amyloid-beta and provides protective effects in cellular and animal models of Alzheimer’s disease. Pharmacol Res, 81, 54-63 (2014)

    103. L. Hong, H.-C. Huang and Z.-F. Jiang: Relationship between amyloid-beta and the ubiquitin-proteasome system in Alzheimer’s disease. Neurolog res, 36(3), 276-282 (2014)

    104. H. F. Dovey, V. John, J. P. Anderson, L. Z. Chen, P. de Saint Andrieu, L. Y. Fang, S. B. Freedman, B. Folmer, E. Goldbach and E. J. Holsztynska: Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain. J neurochem, 76(1), 173-181 (2001)

    105. K. Rezai-Zadeh, D. Shytle, N. Sun, T. Mori, H. Hou, D. Jeanniton, J. Ehrhart, K. Townsend, J. Zeng and D. Morgan: Green tea epigallocatechin-3-gallate (EGCG) modulates amyloid precursor protein cleavage and reduces cerebral amyloidosis in Alzheimer transgenic mice. J neurosci, 25(38), 8807-8814 (2005)

    106. G. T. Bramblett, M. Goedert, R. Jakes, S. E. Merrick, J. Q. Trojanowski and V. M. Y. Lee: Abnormal tau phosphorylation at Ser 396 in Alzheimer’s disease recapitulates development and contributes to reduced microtubule binding. Neuron, 10(6), 1089-1099 (1993)

    107. C. A. Lasagna-Reeves, A. L. Clos, D. Castillo-Carranza, U. Sengupta, M. Guerrero-Munoz, B. Kelly, R. Wagner and R. Kayed: Dual role of p53 amyloid formation in cancer; loss of function and gain of toxicity. Biochem biophys res comm, 430(3), 963-968 (2013)

    108. S. Gupta, T. Chattopadhyay, M. P. Singh and A. Surolia: Supramolecular insulin assembly II for a sustained treatment of type1 diabetes mellitus. Pro Nat Aca Sci, 107(30), 13246-13251 (2010)

    109. U. Shimanovich, I. Efimov, T. O. Mason, P. Flagmeier, A. K. Buell, A. Gedanken, S. Linse, K. S. A kerfeldt, C. M. Dobson and D. A. Weitz: Protein Microgels from Amyloid Fibril Networks. ACS nano, 9(1), 43-51 (2015)

    110. U. Shimanovich, I. Efimov, T. O. Mason, P. Flagmeier, A. K. Buell, A. Gedanken, S. A. Linse, Karin S., C. M. Dobson and D. A. Weitz: Protein Microgels from Amyloid Fibril Networks. ACS nano, 9(1), 43-51 (2015)

    111. E. Reisler, J. Pouyet and H. Eisenberg: Molecular weights, association, and frictional resistance of bovine liver glutamate dehydrogenase at low concentrations. Equilibrium and velocity sedimentation, light-scattering studies, and settling experiments with macroscopic models of the enzyme oligomer. Biochemistry, 9(15), 3095-102 (1970)

    112. J. Hofrichter, P. D. Ross and W. A. Eaton: Kinetics and mechanism of deoxyhemoglobin S gelation: a new approach to understanding sickle cell disease. Pro Nat Aca Sci, 71(12), 4864-4868 (1974)

    113. A. Wegner and J. Engel: Kinetics of the cooperative association of actin to actin filament. Biophy chem, 3(3), 215-225 (1975)

    114. F. A. Ferrone, J. Hofrichter, H. R. Sunshine and W. A. Eaton: Kinetic studies on photolysis-induced gelation of sickle cell hemoglobin suggest a new mechanism. Biophy j, 32(1), 361-380 (1980)

    115. M. P. Firestone, R. De Levie and S. K. Rangarajan: On one-dimensional nucleation and growth of “living” polymers I. Homogeneous nucleation. J theoret bio, 104(4), 535-552 (1983)

    116. S. B. Prusiner: Molecular biology of prion diseases. Science, 252 (5012), 1515-1522 (1991)

    117. M. A. Watzky and R. G. Finke: Transition metal nanocluster formation kinetic and mechanistic studies. A new mechanism when hydrogen is the reductant: slow, continuous nucleation and fast autocatalytic surface growth. J American Chem Soc, 119(43), 10382-10400 (1997)

    118. K. F. DuBay, A. P. Pawar, F. Chiti, J. s. Zurdo, C. M. Dobson and M. Vendruscolo: Prediction of the absolute aggregation rates of amyloidogenic polypeptide chains. J mol bio, 341(5), 1317-1326 (2004)

    119. M. M. Barnhart and M. R. Chapman: Curli biogenesis and function. Ann rev microbio, 60, 131 (2006)

    120. M. F. B. G. Gebbink, D. Claessen, B. Bouma, L. Dijkhuizen and H. A. B. Wosten: Amyloids-a functional coat for microorganisms. Nat Rev Microbio, 3(4), 333-341 (2005)

    121. P. Chien, J. S. Weissman and A. H. DePace: Emerging principles of conformation-based prion inheritance. Ann rev biochem, 73(1), 617-656 (2004)

    122. A. H. Y. Kwan, R. D. Winefield, M. Sunde, J. M. Matthews, R. G. Haverkamp, M. D. Templeton and J. P. Mackay: Structural basis for rodlet assembly in fungal hydrophobins. Pro Nat Aca Sci, 103(10), 3621-3626 (2006)

    123. S. J. Saupe: Molecular genetics of heterokaryon incompatibility in filamentous ascomycetes. Microbio and mol bio rev, 64(3), 489-502 (2000)

    124. J. M. Kenney, D. Knight, M. J. Wise and F. Vollrath: Amyloidogenic nature of spider silk. Europ J Biochem, 269(16), 4159-4163 (2002)

    125. K. Si, S. Lindquist and E. R. Kandel: A neuronal isoform of the aplysia CPEB has prion-like properties. Cell, 115(7), 879-891 (2003)

    126. J. F. Berson, A. C. Theos, D. C. Harper, D. Tenza, G. Raposo and M. S. Marks: Proproteinconvertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis. J cell bio, 161(3), 521-533 (2003)

    127. S. A. Hudson, H. Ecroyd, F. C. Dehle, I. F. Musgrave and J. A. Carver: (-)-epigallocatechin-3-gallate (EGCG) maintains kappa-casein in its pre-fibrillar state without redirecting its aggregation pathway. J Mol Bio, 392(3), 689-700 (2009)

    128. N. Ferreira, M. J. o. Saraiva and M. R. r. Almeida: Natural polyphenols inhibit different steps of the process of transthyretin (TTR) amyloid fibril formation. FEBS letters, 585(15), 2424-2430 (2011)

    129. M. Hirohata, K. Hasegawa, S. Tsutsumi-Yasuhara, Y. Ohhashi, T. Ookoshi, K. Ono, M. Yamada and H. Naiki: The anti-amyloidogenic effect is exerted against Alzheimer’s beta-amyloid fibrils in vitro by preferential and reversible binding of flavonoids to the amyloid fibril structure. Biochemistry, 46(7), 1888-99 (2007)

    130. F. Meng, A. Abedini, A. Plesner, C. B. Verchere and D. P. Raleigh: The flavanol (-)-epigallocatechin 3-gallate inhibits amyloid formation by islet amyloid polypeptide, disaggregates amyloid fibrils, and protects cultured cells against IAPP-induced toxicity. Biochemistry, 49(37), 8127-8133 (2010)

    131. A. Qadeer, M. Zaman and R. H. Khan: Inhibitory effect of post-micellar SDS concentration on thermal aggregation and activity of papain. Biochemistry (Moscow), 79(8), 785-796 (2014)

    132. N. Gull, M. A. Mir, J. M. Khan, R. H. Khan, G. M. Rather and A. A. Dar: Refolding of bovine serum albumin via artificial chaperone protocol using gemini surfactants. J coll interf sci, 364(1), 157-162 (2011)

    133. Y. Miura, K. Yasuda, K. Yamamoto, M. Koike, Y. Nishida and K. Kobayashi: Inhibition of Alzheimer amyloid aggregation with sulfated glycopolymers. Biomac, 8(7), 2129-2134 (2007)

    134. S. Sardar, S. Pal, S. Maity, J. Chakraborty and U. C. Halder: Amyloid fibril formation by beta-lactoglobulin is inhibited by gold nanoparticles. Inter j bio macromol (2014)

    135. M. Kanapathipillai, G. Lentzen, M. Sierks and C. B. Park: Ectoine and hydroxyectoine inhibit aggregation and neurotoxicity of Alzheimer’s beta-amyloid. FEBS letters, 579(21), 4775-4780 (2005)

    136. Y. S. Lee, H. Y. Kim, H. M. Youn, J. H. Seo, Y. Kim and K. J. Shin: 2-Phenylbenzofuran derivatives alleviate mitochondrial damage via the inhibition of beta-amyloid aggregation. Bioorganic & med chem lett, 23(21), 5882-5886 (2013)

    137. Y.-P. Chen, Z.-Y.Zhang, Y.-P. Li, D. Li, S.-L. Huang, L.-Q.Gu, J. Xu and Z.-S. Huang: Syntheses and evaluation of novel isoliquiritigenin derivatives as potential dual inhibitors for amyloid-beta aggregation and 5-lipoxygenase. Europ j med chem, 66, 22-31 (2013)

    138. J. N. Abraham, D. Kedracki, E. Prado, C. Gourmel, P. Maroni and C. Nardin: Effect of the Interaction of the Amyloid beta (1-42) Peptide with Short Single Stranded Synthetic Nucleotide Sequences: Morphological Characterization of the Inhibition of Fibrils Formation and Fibrils Disassembly. Biomac (2014)

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Mohammad Khursheed Siddiqi, Parvez Alam, Sumit Kumar Chaturvedi, Yasser E. Shahein, Rizwan Hasan Khan. Mechanisms of protein aggregation and inhibition. Frontiers in Bioscience-Elite. 2017. 9(1); 1-20.