Pathology of Protein Misfolding Diseases in Animals

Abstract

Protein misfolding diseases are the diseases, which cause transformation of proteins into beta-sheets, forming amyloid fibrils and resulting in aggregate formations and plaques. A wide horizon for occurrence of protein misfolding diseases, includes temperature, pH, surfactant, hydrophobic interaction etc. plays important role. Extensive studies on pathways for protein misfolding converge to mechanism of seed nucleation hypothesis for protein aggregation and misfolding within the cells. Correct folding of proteins is required for normal functioning of the cells and this is accomplished by presence of protein quality control (PQC) system, which make use of endoplasmic reticulum-associated degradation (ERAD), ubiquitin pathway, autophagy, and molecular chaperones. In addition, extrinsic and intrinsic alteration, however, causes misfolding of the protein. Pathological conditions, such as prion diseases, amyloidosis, lung diseases, cancer occurrences, Tay Sach’s disease, epidermolysis bullosa, and cataract, are repercussion of protein misfolding. Moreover, the diagnosis of protein aggregates and plaques at an initial stage is challenging. Diagnostic techniques Congo red assay, Thioflavin T binding assay, ANS fluorescence assay, antibody dot blot assay, magnetic resonance imaging, and positron emission tomography are applied but are not routinely used. Although newer techniques are being investigated, lack of suitable biomarkers limits the diagnosis for protein fibril deposition.

1. Introduction

Proteins are large biomolecules and macromolecules nexus, comprising long chains of different amino acids, thereby conferring proteins specific roles to perform forming essential component of working machinery. Proteins perform vital functions including, catalyzing metabolic reactions, DNA replication, normal structure of the cellular components, response to stimuli, catalyzes, enzymes, performing metabolic activities, and as transport molecules [1]. Although proteins are similar in function yet differ in configuration owing to particular encoding gene of nucleotide sequence and further ensembles protein folding and achieving its characteristic 3D structure or typical native conformation of protein. Protein primarily exist in four forms: primary, secondary, tertiary, and quaternary, whereas some researchers have reported the fifth form of protein structure called quinary structure [2]. The primary structure comprises of linear chain of amino acids. Secondary structure contains amino acid chains stabilized by hydrogen bonds, forming the polypeptide backbone creating alpha-helix and beta-pleated sheets of the secondary structure. Tertiary structure is determined by the interactions of side chains from the polypeptide backbone [3]. The quaternary structure is formed via side-chain interactions between two or more polypeptides [4]. Quinary structure has been identified and refers to the features of protein surfaces that are shaped by evolutionary adaptation [2].

Proteins are dynamic objects, and they arrange themselves in certain conformations to perform correct functions. With respect to structural rearrangements, the structures are referred to as conformation and transitions in between are called conformational changes [5]. Protein folding is ineluctable phenomenon to generate biologically active protein achieving its characteristic structure. Molecular interactions, such as hydrophobic effects, Vander Waals forces, H-bonds, and hydrostatic interactions, exist stably maintaining folded proteins in position and structure. These interactions may either be favorable or unfavorable [6]. The favorable interactions include primarily the enthalpy from Vander Waals packing interactions; secondly, hydrophobic effect or entropy; thirdly, gain of protein-protein Hydrogen bonds; and lastly, electrostatic effects. Unfavorable interaction series include protein conformational entropy and loss of protein-water H-bonding [4, 7]. The correct folding of protein is necessary to perform particular function. Ribosomes and endoplasmic reticulum plays role in synthesizing of proteins and ensures proper folding of the proteins and degrades unfolded protein by various mechanisms (Figure 1) [8, 9, 10].

Figure 1.

Depicting the normal binding of the protein when correctly folded and protein structure when misfolding of protein occurs.

2. Mechanism of protein folding and misfolding

Protein folding implies nucleation condensation mechanism, wherein interaction between the residues chiefly leads to the first stage of folding process forming various transition states subsequently resulting in folding of proteins leading to formation of a stable native state [3, 11, 12]. Minimum energy levels are effective for protein folding mechanism, where the lowest energy level helps protein to achieve their stable native state [13, 14]. Initiation of the nucleation process or first phase of the nucleation condensation mechanism prevails from optimum hydrophobic and polar interactions between the formed residues. These interaction results in formation of stable globular structure of protein and serves as quality control system avoiding protein misfolding [10, 15]. Moreover, depending upon the sizes of proteins slightly different mechanisms are adopted for smaller and larger proteins. Smaller proteins undergo two-state mechanisms unlikely to larger protein, where the procedure is more complex with formation of transition states or intermediates or oligomers between the unfolded and fully folded state [16].

Various locations utilized for protein folding, such as in the cells, ribosomes are responsible for protein synthesis in a mechanism called co-translational folding, some folds in cytoplasm after complete protein synthesis, and some folds in endoplasmic reticulum (ER) or mitochondria during translocation [10]. However, some of the polypeptide regions during folding, which are otherwise buried if exposed, lead to formation of unfavorable interactions with other molecules causing protein misfolding. This explains that in protein-folding pathways transient non-native states develop to hide the regions of protein chain, which can cause aggregates on interaction with other molecules commonly the hydrophobic patches [17].

Protein misfolding mechanism and aggregation follow similar trend of seeding nucleation model [3]. This involves two processes or phases, the former phase is called the lag phase, which is responsible for formation of oligomers causing misfolding to occur, and the later phase is called the elongation phase or exponential or polymerization phase [18]. Elaborated studies on the misfolding mechanism reveal that the initial steps are thermodynamically unfavorable and progress slowly until the minimum stable oligomeric unit is formed referred to as seeds [19]. After this step, exponential increase in the rate of formation is observed from oligomers to fibers [20]. The rate of formation can be altered by addition of preformed seeds, which minimizes the lag phase and enhances the polymerization phase. Oligomers are, thereby, considered as best seeds to propagate the misfolding process in an exponential manner (Figure 2) [22].

Figure 2.

The picture represents the process that occurs, leading to protein misfolding. Picture courtsey: [21].

3. Protein quality control system (PQC)

Protein quality control system ensures proper folding of proteins and degradation of the unfolded and partially folded proteins. Usually, the PQC system has the ability to eliminate defective ribosomal products, which have been synthesized as a result of errors in translation or post-translational processes or proteins. Protein quality control system comprises of endoplasmic reticulum-associated degradation, ubiquitin protease pathway, autophagy, and chaperones, which are activated countering misfolded protein and either slow their formation in order to either correct the misfolded protein and refold them or destruct the misfolded protein.

3.1 Endoplasmic reticulum (ER)

Endoplasmic reticulum (ER), besides being a major site for protein production, is one of the major cellular organelles involved in protein homeostasis and quality control. Cellular proteins utilize the ER to attain their folded and posttranslationally modified active state [23]. Although the ER is well furnished for synthesis and folding of significantly high amount of proteins, genetic or environmental alterations are known to stress out the ER promoting misfolding and accumulation of proteins. Unfolded protein response (UPR) mechanism utilized by ER to combat against protein misfolding. UPR is composed of three different transmembrane proteins, including ATF6 (activated transcription factor 6), PERK (double-stranded RNA activated protein kinase, such as ER kinase), and IRE1 (inositol-requiring transmembrane kinase and endonuclease). PERK blocks protein translation by phosphorylating eukaryotic translation initiation (eIF2), and ATF6 (p50ATF6) acts as transcription factor to induce expression of ER-resident chaperones, such as binding protein (BiP). IRE1 alternatively splices XBP1 mRNA. The activation of all three proximal sensors results in the attenuation of protein synthesis through eukaryotic initiation factor-2 (eIF2) kinase and increases protein-folding capacity of the ER. The spliced gene product induces transcription of different genes involved in the ER-associated degradation (ERAD) pathway [24].

The goals of the UPR involve shutting down further protein synthesis to reduce the overload of the ER, secondly, induce ER-resident chaperones to prevent misfolding, and lastly activate ER-associated degradation (ERAD) (IRE1pathway) system to shed off misfolded protein burden using the proteasome. While temporary stress is effectively handled by the UPR, chronic stress leads to continuous accumulation of misfolded protein beyond the capacity of the UPR regulation.

3.2 Ubiquitin (Ub): proteasome pathway (UPP)

Intracellular proteins are degraded by the ubiquitin (Ub)–proteasome pathway (UPP). The UPP consists of enzymes, which attach polypeptide cofactor, Ub onto proteins, and tags them for degradation. This tagging process enables their recognition by the 26S proteasome (large multicatalytic protease complex that degrades ubiquitinated proteins to small peptides). The UPP selectively eliminates misfolded and damaged proteins that arise by missense or nonsense mutations, biosynthetic errors, or damage by oxygen radicals or by denaturation [25]. Three enzymatic components E1, E2, and E3 are required to link chains of Ub onto proteins destined for degradation. E1 or Ub-activating enzyme and E2s or Ub-carrier or conjugating proteins prepare Ub for conjugation and E3 or Ub-protein ligase, recognize specific protein substrate, and catalyze the transfer of activated Ub to it. The initial step in conjugation is activation of Ub at its C-terminus by the enzyme E1. After activation, Ub bound to E1 through thioester linkage is transferred to a sulfhydryl group. The E2s generally are small proteins, containing the cysteine that forms a thioester linkage with the activated Ub. The large number of E2s helps to generate the specificity of the ubiquitination system because specific E2s function in the degradation of various types of substrates, and they can conjugate with various E3s (Figure 3) [24, 26, 27].

Figure 3.

Picture depicting the process of misfolding protein correction using the ubiquitin (Ub)–proteasome pathway (UPP).

3.3 Autophagy

Autophagy is a clearance mechanism that degrades damaged organelles and proteins. Normally, it is activated under stress conditions as a protective mechanism to ensure survival and cellular homeostasis by protein turnover. Autophagy is classified in three different categories, chaperone-mediated autophagy (CMA), microautophagy, and macroautophagy, depending on the mechanism used for the capture and degradation of substrates.

In CMA, proteins contain the pentapeptide KFERQ , which is recognized by the chaperone viz., heat shock protein 70 and transported to the lysosome for its hydrolysis. Lysosomes are sites for intracellular protein degradation, which involves uptake of secretory vesicles, portions of the cytoplasm, or whole organelles by lysosomes followed by enzymatic degradation. Microautophagy refers to a process in which some portions of the cytosol are trapped directly by the lysosome without the intervention of chaperones and macroautophagy involves sequestration of damaged organelles or large protein aggregates into cargo vesicles known as autophagosomes that transport the contents to the lysosome for its degradation (Figure 4) [28].

Figure 4.

The picture depicting the role of chaperones or heat shock protein in encountering the misfolding of protein and resolving it through different pathway.

3.4 Chaperones

In general, molecular chaperones are proteins that recognize and bind polypeptides to expose surfaces with specific physicochemical properties, thereby minimizing the potential for aggregation and protecting against attack by proteases [29]. Two different categories of chaperones are identified, folding helper, and holding-type chaperones. Folding helper chaperones comprises of ubiquitous Hsp70/Hsp40/GrpE chaperone system (eukaryotic homologs of the DnaK/DnaJ/GrpE system in Escherichia coli) and the large barrel chaperonin complex Hsp60/Hsp10 (eukaryotic homologs) [30, 31, 32, 33]. Lectin chaperones calreticulin and calnexin are family of folding helper chaperones without ATPase domain [34]. CLIPs (chaperones involved in protein synthesis) constitute a large family of proteins, and evidence suggests that various CLIPs are associated with different classes of proteins. CLIPs are physically linked to translation mechanisms to control the quality control of newly translated proteins [35, 36]. Chaperones utilize ATP binding and hydrolysis cycles to target nonnatural polypeptides for folding and unfolding. Several ATP-dependent chaperones, also called protein remodeling factors, mediate target degradation, unfolding, or reversal of aggregation [8, 21]. Chaperones target unfolded and partially folded proteins. In particular, it showed a separated hydrophobic region at the center of the folded protein, preventing aggregation by interacting with other molecules [30, 31].

Besides molecular chaperones, other types of folding catalysts that accelerate steps in the folding process, which can otherwise be very slow include protein disulfide isomerases. Protein disulfide isomerases enhance the rate of formation and reorganization of disulfide bonds within proteins and peptidylprolyl isomerases that increase the rate of cis/trans isomerization of peptide bonds involving proline residues [15]. Dysfunction of any of these pathways can, unsurprisingly, lead to protein misfolding diseases (Figure 5) [1, 34, 37].

Figure 5.

Summary of factors that are responsible for protein misfolding.

4. Factors affecting protein misfolding

Protein misfolding is a mutation in the gene in question, which results in the misfolding of an amino acid. These mutations in the genetic code are consequently directly related to abnormalities in protein folding, which are either a decrease (loss of function) in the presence of certain proteins that have never been folded into a functional state, or a misfolded protein inside or outside the cell. This situation can always be an igniting point for various diseases as these misfolded proteins are usually insoluble and tend to form aggregates (gains of function). The insoluble nature of protein aggregates results in recessive structures due to their high propensity for intermolecular hydrogen bonding. These are called amyloid fibrils, and as they accumulate, they form amyloid plaques [19, 38].

Incorrect protein folding can occur for a number of reasons, including internal and external factors. The internal factors broadly include, first, somatic mutations in the gene sequence resulting in transformation of proteins that cannot accommodate their native folding. Secondly, errors in the transcription or translation process result in modified proteins that cannot fold correctly. Third criteria include failure of folding and chaperones protective responses. Fourthly, posttranslational modifications of proteins and errors in protein delivery mechanism, and lastly, structural modifications due to environmental changes and induction of protein misfolding by seeding and cross-seeding mechanisms. The most common fate of misfolded proteins is self-aggregation due to exposure of fragments, which are otherwise hidden inside the protein and generate a high level of stickiness [39].

Protein folding is a fine-tuned process that is influenced by several external factors, including electric fields, magnetic fields, temperature, pH, chemicals, space constraints, and molecular density. These factors lead to improper folding of proteins, which leads to proteinopathy. Proteins become unstable at extreme temperatures and become denatured. Likewise, excessive alteration in pH, mechanical forces, and chemical denaturants denature proteins [4]. Denaturation leads to the loss of the tertiary structure of the protein and not the formation of aggregates, but mainly the beta layer of amyloid or amyloid fibrils, which subsequently forms amyloid plaques [7].

High temperature directly affects the conformation of proteins and causes irreversible sometimes reversible denaturation of proteins, which leads to aggregation. High temperature enhances oxidation and deamination reactions and also increases the frequency of hydrophobic interactions, which may lead to protein aggregation. Protein aggregation changes with the pH of the protein, resulting in partial unfolding of the protein and affecting the electrostatic interactions of protein. Aggregation occurs due to neutralization of charged molecules with enhancement of hydrophobic interactions. The presence of various surfactant molecules, for example, cationic (CTAB, CPC, DTAB), anionic (SDS, SLES, AOT), and nonionic causes protein aggregation. They have a strong effect on protein conformation as they destabilize the protein or stabilize it with subsequent aggregation. Aggregation of proteins occurs due to the interaction of surfactants with opposite charge centers of protein molecules and repulsion of water molecules by hydrophilic tails. Chemical modification is another technique that plays an important role in protein aggregation. Chemical reactions, such as hydrolysis, oxidation, isomerization, and deamidation, can destabilize protein structures and promote aggregation [29]. In addition, the induction of aggregation will be induced by photolytic degradation of proteins, including oxidation of aromatic residues, including histidine, cystine, and methionine.

Posttranslational modifications affect the structure and function of proteins, usually promoting proper folding or leading to improper folding and accumulation. Reducing sugars plays an important role in posttranslational protein modification, forming advanced glycosylation end products (AGEs) in a nonenzymatic process called glycosylation. Protein glycosylation depends on the influence of free amino groups on the polypeptide chain, sugar concentration, and oxidative conditions. It has been noted that amyloid deposits of β-amyloid, tau, prion, transthyretin, and β(2) microglobulin contain glycosylated proteins [40]. The mechanism behind aggregation-promoting glycosylation is that it stabilizes protein aggregates by promoting the formation of covalent cross-links that accumulate over a period of time and are not frequently removed. Proteins also undergo glycosylation at exposed lysine residues, which are also ubiquitination sites, which send proteins to the proteasome for degradation, resulting in clearance damage by the ubiquitin-proteasome system. Thus, the accumulation of proteins in the form of aggregates or in the form of deposits or inclusions in tissues may be beneficial after glycosylation. Various factors that cause protein folding include protein concentration, salinity, and ionic strength.

5. Diseases due to protein misfolding

Diseases, due to protein misfolding, can be broadly categorized into two categories basically the loss of function and the gain of function. Diseases arise because specific protein becomes unfunctional when adopting a misfolded state or undergoes severe impairment of protein trafficking [41]. This is observed in autosomal recessive disorders with loss of function pathology. This type of misfolding comprises of cystic fibrosis, Phenylketonuria, and short chain acyl Co A dehydrogenase. Second type of misfolding occurs by the gain of function, wherein the pathological state originates because of underlying aggregates or concomitant aggregation of proteins [7, 42]. This is further subcategorized into two subtypes. First, dominant inherited diseases [43]. Some researchers illustrate protein misfolding diseases under five different categories namely, improper degradation, mislocalization, dominant-negative mutations, structural alterations with novel toxic functions, and amyloid accumulation [14].

5.1 Prion disease and protein misfolding

Prions cause spongiform encephalopathy known as scrapie in sheep and bovine spongiform encephalopathy or mad cow disease in cattle. Transmissible spongiform encephalopathy is a protein-folding disease that causes fatal neurodegeneration characterized by vacuoles in the brain. The misfolded protein, named PrPSc, is derived from the endogenous cellular prion protein PrPC. PrPC is a glycoprotein of approximately 231 amino acid residues, which is fixed in plasma. Mature PrPC protein residue is 23–231 amino acid long composed of an independent and flexible N-terminal region (residues 23–120) and a C-terminal globular domain (residues 121–231), via the glycolipid anchor phosphatidylinositol that physically interact with each other. Globular domain contains two short leaf-forming antiparallel filaments (aa 128–130 and 160–162 aa for mouse PrPC) and three helices [44, 45]. The hypothesis is that amyloid-like fibrils are formed by two tightly staggered plates in the form of zippers, allowing nucleation to form fibrillar-forming aggregates [36, 46, 47].

Prime event within pathogenesis is the misfolding of the regular shape of the prion protein, PrPC, into the generally protease-resistant-sheet rich isoform, described because the scrapie prion protein (PrPSc), via way of means of a conformational rearrangement. The PrPSc constitutes the transmissible agent (“prion”), capable of recruit and convert natively folded PrPC into de novo PrPSc through an autocatalytic process. It is the PrP-TSE protein that can shape amyloid protein aggregates. The purpose for the two absolutely special configurations of the identical protein is not known; however, a vital commentary is if a small quantity of PrP-TSE is brought to a bigger quantity of PrP-C, the “healthy” protein is transformed to the TSE shape [48]. The two variations have wonderful traits glaring of their secondary and tertiary structures. The PrPC is constructed from 40% α-helical and 3% β-helical folds, while PrPSc is folded right into a parallel left-exceeded β-helical shape that has a 30% α-helical and 40% β-helical conformation [45]. There are at the least two proposed fashions for PrPSc autocatalytic propagation. The refolding version assumes that an electricity barrier precludes the preliminary conversion of PrPC to PrPS. The seeding version asserts that PrPC and PrPSc exist in thermodynamic equilibrium, and PrPSc starts to mixture while a particularly ordered monomeric PrPSc (the seed) stabilizes and recruits greater monomeric PrPSc to shape large aggregates [49, 50].

Elucidation of the high-decision shape of prions and aggregated prions has been hard due to problems related to the inherent chemical houses of the proteins. Two mechanisms, the cloud speculation and deformed-template speculation, were proposed for the genesis of prion lines related to classical scrapie. Although the two ideas are noticeably described, they may be now no longer collectively exclusive. The cloud speculation assumes that isolates are constructed from a heterogenous combination of PrPSc conformations (lines), and over time, a permissive conformer arises to turn out to be the fundamental variant [51]. The deformed-template speculation posits that, initially, there may be a fundamental conformer instead of a combination of PrPSc conformations, and adjustments within side the replication surroundings cause trial-and-mistakes seeding activities that generate a brand-new dominant conformer. Both hypotheses postulate the life of more than one conformer inside an isolate that makes a contribution to the discovered variations in sickness phenotype [52].

The key event in pathogenesis is the aberrant folding of the normal form of the prion protein, PrP C, into a commonly protease-resistant leaf-rich isoform, defined as prion protein scrapie (PrPSc) by structural rearrangement. PrPSc is an infectious agent (“prion”) that can naturally recruit folded PrPCs and convert them to novel PrPSc through an autocatalytic process. It is the PrP TSE protein that can form an aggregate of amyloid proteins. The reasons for the two completely different configurations of the same protein are unknown, but the normal prion protein PrP C undergoes a conformational change into a self-replicating, misfolded PrPSc conformer. On the other hand, in the genetic type of disease, a change in the PrPC form may occur due to a genetic mutation of the PRNP gene [44, 53]. The processes that promote development are not fully understood. Indeed, pathogenic mutations, such as G113V and A116V in the N-terminal domain, can induce prion pathogenesis by accelerating misfolding and aggregation and modifying the structure of the palindromic region, which appears to be the intermolecular binding site in oligomers [44].

The defining step in prion infection is the transformation of the PrPC form into the protease-resistant β-sheet. Prion disease is caused by the accumulation of a misfolded form of PrPC called PrPSc. At this time, PrPC expression is necessary and rate-limiting. Transformation of PrPC into the pathological conformer PrPSc is characterized by a significant increase in the secondary structure of the β-sheet [5, 48]. The conversion of PrPC to PrPSc is accompanied by significant structural and biophysical changes in the molecule. When improperly folded, PrPC, rich in α-helices, which normally attaches to cell membranes via glycosyl phosphatidylinositol (GPI) anchors, is converted into predominantly single β-sheets [5, 45]. These changes increase resistance to heat and degradation by proteases. Indeed, it has been suggested that prion proteins can experience an environment that can reconstitute disulfide bonds in the endosome, which has been shown to enhance the transition to a fibrillar state [5, 48]. In sheep, the highest concentrations of PRNP mRNA transcripts are found in the thalamus and brain, followed by the cerebellum, spinal cord, spleen, other lymphoid tissues, brainstem, gastrointestinal tract, and reproductive organs.

5.2 Amyloidosis and protein misfolding

Amyloidosis belongs to the group of protein-folding disorders. Various proteins that are soluble under physiological conditions can undergo conformational changes in their β-layer-rich structures, which can then self-assemble into highly insoluble amyloid fibrils. Proteins in a partially folded or misfolded state due to loss of function of the protein’s quality control system and various external factors contain open hydrophobic and unstructured regions that contribute to the formation of aggregates [36, 46, 49]. Amyloidosis can be divided into two main classes: localized or focal and systemic. In focal amyloidosis, amyloid fibrils deposit in organs, such as the brain and pancreas, where precursor proteins are synthesized [9, 54]. On the other hand, in systemic amyloidosis, serum precursor proteins, such as immunoglobulin light chain in amyloid amyloidosis (AL), transthyretin in familial amyloid polyneuropathy, and β2-microglobulin in dialysis amyloidosis circulate and polymerize to form amyloid fibrils then settles all over the tissue surface [40, 55]. In addition, the formation of stable aggregate structures can also occur due to hydrophobic decay due to the presence of extraneous debris in solution that changes conditions. On the other hand, electrostatic interaction with hydrophobic forces plays a crucial role in the formation of the complex amyloid fibrils [40].

5.3 Cancer occurence and protein misfolding

The maximum often altered gene in tumors is TP53 encoding the p53 protein. TP53 mutations are related to unfavorable diagnosis in lots of sporadic cancers. The initial stage of TP53 mutations is the loss of wild-kind p53 functions, which represents an essential gain for the duration of most cancers improvement through depriving cells of intrinsic tumor suppressive responses, along with senescence and apoptosis [56]. The tumor suppressor p53, a transcription element that regulates the cell cycle and apoptosis, is likewise amyloidogenic. In tumor models, each wildkind and mutant p53 protein displays aggregation kinetics and morphology just like the ones of classical amyloidogenic proteins, along with β-amyloid peptide and α- synuclein [14]. Wild type p53 loses its anticancer maneuver, while p53 mutants with enhanced amyloidogenicity show accelerated aggregation. The majority of TP53 mutations are missense, producing single residue substitutions within the protein’s DNA-binding domain when compared with most other tumor suppressor genes. However, p53 missense mutant proteins (mutp53) lose the ability to activate canonical p53 target genes, and some mutants exert trans-dominant repression over the wild-type counterpart. The cancer cells are supposed to gain selective advantages by retaining only the mutant form of the p53 protein. This can be explained by the ability of different p53 mutants to reshape the tumor cell’s transcriptome and proteome, by virtue of newly established interactions with transcription regulators, enzymes, and other cellular proteins.

Based on this, it has been reported that specific missense mutations in p53 disrupt important cellular pathways, promote proliferation and survival of cancer cells, and promote invasion, migration, metastasis, and chemical resistance. Some of the tumor suppressive activity of wild-type p53 is related to its ability to help cells adapt and survive in moderately stressful conditions, including oxidative and metabolic stress [38]. Mutant p53, similar to wild type, stabilizes and activates in response to tumor-associated stress conditions and may provide cancer cells with the ability to cope with difficult conditions encountered during tumor development, including DNA associated with hyperproliferation. Mutant p53 supports tumor progression by promoting an adaptive response to cancer-associated stress conditions. An oncogenic missense mutant form of p53 (mutp53) can recognize multiple stress effects and act as homeostatic factors triggering adaptive mechanisms. Mutant p53 has been shown to induce a survival response to oxidative stress, promoting protein folding and increasing proteasome activity. The mutant p53 protein is inherently unstable due to proteasome-mediated degradation induced by the E3 ubiquitin ligase MDM2 and CHIP. However, mutp53 protein accumulates in higher amounts in tumor tissues, and this stabilization is necessary to realize pleiotropic oncogenic activity [57].

5.4 Epidermolysis bullosa simplex and protein misfolding

EB simplex results from mutations affecting either keratin 14 (K14) or K5, the type I and type II intermediate filament (IF) proteins. Mutations in the gene encoding collagen, type XVII, alpha 1 (COL17A1), a hemidesmosomal plaque protein required for tight adherence of basal keratinocytes to the basal lamina, account for a special subset of patients with elements typical of both EB simplex and EB junctional dominantly disrupting keratin IF structure [58, 59]. Mutations in the K14 rod domain elicit the formation of aggregates of amorphous proteins in the cytoplasm. Such aggregates are diagnostic of the most severe form of EB simplex. In an experiment with mice, homozygous null for K14, K5, or plectin displayed the key features of EB simplex revealing that cell fragility is largely a loss of function phenotype, containing a K14 mutation causing simple EB compared to in vitro reconstituted filaments in wild-type K5 and K14 proteins contains an Arg125 → Cysor K5 mutation, 1649delG exhibiting significantly lower elasticity in low (linear) strain mode is easily broken. If the response of misfolded proteins cannot resolve these aggregates, the cell’s protein homeostasis machinery is overloaded. This induces cellular stress and can affect the phenotype of cells and tissues in vivo [60].

5.5 Lung diseases and protein misfolding

UPR activation is induced by several pathogens associated with respiratory diseases, including cystic fibrosis, asthma, and COPD. Toll-like receptor (TLR) activation and bacterial infection can trigger UPR. TLR2 and TLR4, specifically activate IRE1 to promote the release of inflammatory mediators. Respiratory pathogens can also interfere with UPR. Intracellular pathogens, such as bacteria, replicate in ER-associated compartments and selectively block IRE1 pathway activation [61]. Secreted bacterial toxins can modulate the UPR. For example, pyocyanin from Pseudomonas aeruginosa induces a similar response in the alveoli evidenced by XBP1 splicing and BiP induction. Aspergillus fumigatus is a fungal pathogen that interacts with airway protection in a variety of ways to cause broncho-pulmonary aspergillosis. We found that the expression of BiP was increased in lung tissue. Administration of A. fumigatus to rats induced pulmonary UPR and airway hyper-responsiveness. Although the mechanisms are not fully elucidated, they include the generation of mitochondrial reactive oxygen species (ROS) and impaired PDI function leading to ER stress. Many viruses cause ER stress, including RSV, influenza A virus (IAV), Coxsackie virus A16, SARSCoV1, and SARSCoV2 (COVID-19). The mechanism of virus-induced ER stress may involve abundant translation of viral proteins that inhibit their ability to fold. It has been reported that IAV induces inflammation and apoptosis in primary human bronchial epithelial cells by activating IRE1 with little or no activation of PERK or ATF6. Picornaviruses, such as rhinoviruses, can benefit from IRE1 activation because they can promote autophagy, and picornaviruses use autophagosomes as RNA replication sites. In contrast, RSV was reported to induce noncanonical UPR activation with IRE1 and ATF6 activation but not PERK, whereas IRE1 inhibits RSV replication, suggesting that inhibition of this UPR arm may be detrimental to RSV infection [62].

5.6 Taysach’s disease and protein misfolding

Lysosomal storage diseases (LSDs) are comprised magnificence group of rare diseases of numerous pathologies. Their distinctive characteristics include dysfunction of the endosome–lysosome system, which in lots of instances ends in the accumulation of toxic metabolites and death at molecular level. A subset of LSD includes GM2 gangliosidoses. GM2 gangliosidoses are a series of associated disorders resulting from insufficiency of active β-hexosaminidase A (HexA). HexA enzyme processes GM2 ganglioside to GM3 ganglioside in the lysosome. Inactivation or loss of HexA causes toxic metabolite buildup of GM2, leading to disease formation and cell death. Tay–Sachs disease (TSD) is clinically described with the aid of using mutations with HEXA gene. The HexA enzyme is made from the HEXA and HEXB genes, which encode α and β subunits, respectively with 60% similarity on the amino acid. They are synthesized on the endoplasmic reticulum (ER), wherein they are glycosylated and form intramolecular disulfide linkages and dimerize. The structural similarities among the chains form more than one isozyme through differential affiliation such as HexA (αβ), HexS (αα), and HexB (ββ). HexB is the most stable of the complexes and HexA is the only species capable of processing GM2 ganglioside. This led to hypothesis development stating low β production promotes heterodimerization over β homodimerization. In the Golgi apparatus, specific glycans are modified with mannose 6-phosphate (M6P), allowing for the trafficking of Hex enzymes to lysosomes. In the lysosome, presentation of the GM2 ganglioside substrate from the bilayer to the active site of HexA additionally requires the adaptor protein GM2-activator. Loss of function in either subunit of HexA or its adaptor protein can lead to GM2 gangliosidosis [63].

5.7 Miscellaneous conditions due to protein misfolding

5.7.1 Hypoxia

Hypoxia induces UPR at several targets, including airway epithelial cells and the mechanisms by which this occurs are not fully understood. Therefore, there are several ways, in which hypoxia can cause ER and UPR stress. First of all, reactive oxygen species formed under hypoxia can modulate UPR activation by directly or indirectly affecting BiP and interfering with the formation of disulfide bonds [64]. Redox-sensitive PDI chaperones are restored during protein folding. It is reduced by electron transfer to ER oxido-reduction (ERO1), which then requires molecular oxygen for reoxidation and restoration of function. In addition, since ERO1 is a target of hypoxia-inducing factor (HIF), hypoxia may modulate disulfide bond formation in several ways.

5.7.2 Idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a scarring disease histologically characterized by the presence of fibroblastic lesions. Despite antifibrotic therapy, the prognosis is grave. Fewer researchers have shown that ER stress plays an important role; however, the biological mechanisms that cause the condition are still unclear. Alveolar type II (AT2) cells secrete surfactant protein C (SFTPC) and mutations in surfactant protein C (SFTPC) are associated with familial IPF and profound UPR activation. Many of these mutations disrupt the BRICHOS SFTPC domain, which acts as a distinct chaperone to promote SFTPC folding. Infringement of SFTPC folding can lead to protein aggregation and activation of all UPR cancers. In a mouse model, these mutations interfere with lung morphogenesis, either directly leading to fibrosis or increasing the lung’s sensitivity to secondary infections that lead to pulmonary fibrosis [62].

5.7.3 Cataract

Cataract is defined as a clouding of the transparent lens inside the eye, reducing the amount of light and subsequently reducing vision. The natural lens is a crystalline substance and the precise structure of water and protein creates a clear pathway for light to pass through. Crystallin, the major protein of the mammalian eye lens, exists in the α-polydisperse B-crystallin form, and each with a molecular weight of approximately 20 kDa. Members of the alpha (α) and β γ crystallin family are the major soluble lens proteins. Α- crystalline is an ATP-independent chaperone that effectively binds to damaged or partially unfolded proteins and dissociates them to prevent large-scale protein aggregation. Α- crystalline comprises of αA and αB subunits and belongs to family of heat shock proteins [34, 65, 66]. Αlpha-crystallin depends on external conditions, such as pH and temperature, quaternary structure, ionic strength, and concentration [67]. In addition to the lens, it is widely found in many other tissues, including the brain, lung, spleen, heart, and skeletal muscle, where it acts as a chaperone and interacts with several partially folded target proteins. Alpha-crystallins prohibit the formation and precipitation of αB- and α-crystallin, as well as ordered protein aggregates (amyloid fibrils).

Proteomic analysis of lens proteins revealed deamidation, oxidation, glycosylation, and shortening as various factors associated with damage. Deamidation is one of the most common damages to crystallins, introducing a negative charge to proteins by converting residues of glutamine to glutamate. Asparagine is also susceptible to deamidation, and both residues are transformed into cataract aggregates. Several oxidation sites targeting tryptophan, cysteine, and methionine residues have been identified in crystallin. Deamination decreases the stability of βA3 and βB1 crystallins and increases the tendency to aggregation. Destabilization, due to lifetime accumulation of covalent modifications/alteration, can lead to partial unfolding of proteins, which can lead to the formation of intermediate conformations exposing previously hidden hydrophobic residues. Hence, proving that destabilization of the native state of the lens protein due to covalent bond damage leads to aggregation [66].

6. Diagnosis

Identification of protein aggregates can be divided into three classes: (i) visualization of protein aggregates in biopsies, (ii) monitoring of marker peptides in body fluids, and (iii) visualization of protein aggregates in vivo using imaging techniques [68].

6.1 Thioflavin T binding assay

The use of Thioflavin T (ThT) and its derivatives is perhaps the simplest and most widely used method for monitoring the aggregation of amyloidogenic proteins. ThT is a low molecular weight dye exhibiting fluorescence emission when fibrillar protein aggregates bind to the β-layer groove structure. Traditionally, ThT has been used to detect amyloid fibrils because of the characteristic sigmoidal increase in fluorescence that occurs between the monomeric state and the ends of the fibrils [69, 70] beside ThT also binds to fibrillar aggregates containing β-sheet groove binding sites (fibrillar oligomers and fibrils). Prefibrillar aggregates of β-sheet structure contain few binding sites than fibril thereby showing low frequency of fluorescence [71]. Thus, ThT could indicate the presence of toxic circular fibrin and fibrin oligomers, but not the presence of pre-fibrin oligomers that do not have a clear β-sheet structure [72].

6.2 Congo red binding assay

Congo red (CR) is a small molecule probe traditionally used to identify amyloid fibrils, especially in the form of brain tissue or in vitro deposits. CR binds to β-rich structures present in amyloid fibrils and exhibits a characteristic green birefringence with cross-polarization. Newer studies have revealed the use of CR to study fibrillar aggregates [5, 55, 71, 72].

6.3 ANS fluorescence analysis

1-anilinonaphthalene-8-sulfonate (ANS) is one of the most commonly used fluorescent probes for characterization. ANS provides an assessment of surface hydrophobicity, depicting increase in fluorescence intensity and a blue shift (decrease in wavelength) when exposed to hydrophobic regions of the protein surface [73]. The interaction and subsequent destruction of the bilayer of the hydrophobic lipid membrane is considered as one of the main mechanisms conferring toxicity to cells in diseases involving prefibrillar aggregation. Therefore, assessment of the surface hydrophobicity of protein aggregates could potentially be very useful for the study of fibrillar protein aggregates [74]. Indeed, a recent study of prefibrillar oligomers of Aβ42 peptide showed an increase in fluorescence and a change in blue color upon exposure to the ANS compared to fibrils and monomers, as well as a correlation between increased ANS fluorescence and toxicity. In certain areas of monitoring prefibrillar protein aggregates, ANS is used less frequently than CR and ThT [72, 75].

6.4 Antibody dot blot assay

Due to the difficulty to obtain high-resolution crystal structures of protein aggregates (especially fibrillar aggregates), structure-specific antibodies that help identify and control the state of amyloidogenic protein aggregates have been developed in the past decade. In a study by Glabe [76] have developed three conformation-specific antibodies important for the detection of physiologically relevant fibrillar aggregates: A11 (recognizing fibrillar oligomers but not fibrillar conformers) and OC (fibrillar oligomers, fibrillar conformers) [77]. These conformation-specific antibodies have the inhibitory ability of Aβ aggregation modulators, inhibition of toxic A11-reactive Aβ aggregation formation by diamond blue G (BBG), and low molecular weight inhibitor [78]. Although the application of fibrillar protein aggregates has provided an important understanding of the properties of fibrillar protein aggregates and the effectiveness of potential therapeutics, recent studies suggest caution should be exercised in the use and interpretation of results. First, due to the transient nature of the pre-fibrillar aggregates compared to the final-state conformers, it is very difficult to prepare homogeneous samples of pre-fibrillar aggregates that react exclusively with A11, OC, or αAPF (no cross-reactivity) in vitro [79]. It has proven difficult. Preparation of homogeneous prefibrillar aggregates. Second, when testing the inhibitory/modulatory activity of foreign compounds on protein aggregates in several study groups, false-positive antibody reactivity was observed in some cases. Because of these two factors, care must be taken when designing experiments and interpreting the results of these antibodies [72].

Direct observation of amyloid plaques in vivo is also used as a diagnostic tool for protein aggregation. Although this direct observation is attractive for clinical use, it is not routinely practiced. Technologies such as ELISA, magnetic resonance imaging (MRI), positron emission tomography (PET), and diffusion tensor imaging are being developed for the direct diagnosis of amyloid plaques based on visual inspection of enhanced images [80, 81]. However, all these methods are based solely on a qualitative approach and rely on the detection of visible changes in the central nervous system. Although some work has been done to quantify amyloid load in PET image analysis, the results are very limited. More recent advances in MRI are primarily based on the use of nanoparticles to localize plaques [82, 83]. For example, the use of magnetic nanoparticles bound to curcumin or hollow manganese oxide nanoparticles bound to specific antibodies. These two nanoparticle methods increase the specificity and sensitivity of the method to protein aggregates. However, these approaches are not routinely used and may not meet the need for diagnosis before irreversible tissue damage occurs [68].

7. Conclusion

Protein misfolding is the cause of many diseases, and new discoveries about protein misfolding have led to the study of another aspect of protein misfolding described as self-aggregation or self-activation. Various diagnostic approaches have been used to detect protein aggregates in vitro and in living cells. Traditional methods, dye-binding assays, TEM, CD, and FTIR analysis, have been widely used to monitor amyloid fibril formation in vitro. Recently, AFM and dot blot assays using conformation-specific antibodies have been used to characterize physiologically important pro fibrillar protein aggregates. A limitation of diagnostic techniques for dye-binding assays is the inadequate detection of prefibrous oligomers by current dyes. Lack of biomarkers and antibodies in protein aggregate detection prohibits prompt diagnosis of conditions relative to protein misfolding. Therefore, as discussed the main theme in therapeutic approach for misfolding of proteins relies on most effective procedure for early diagnosis and understanding the fundamental mechanisms of protein aggregation. Chaperone-related therapeutic measures are being explored for treatment of protein misfolding diseases. Also, the ubiquitin protease system and unfolded protein responses are being explored molecularly to achieve new insight into misfolding of proteins.

Authors’ contributions

Dr. Deepika Lather, Dr. Vikas Nehra has contributed toward the design, proper arrangement, and sectioning of the review topic. Dr. Babulal Jangir contributed in recent approaches and diagnostics in protein misfolding diseases. Diksha Kandpal has reviewed the articles and data available and written and compiled the manuscript.

Funding

No funding received.

Competing interests

Authors declare that there are no competing interests.

Ethical approval

No ethical approval needed as it is a review-based article.

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