**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) [30–33]. 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

#### **Figure 5.**

*Summary of factors that are responsible for protein misfolding.*

*Pathology of Protein Misfolding Diseases in Animals DOI: http://dx.doi.org/10.5772/intechopen.112405*

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].

### **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

#### *Pathology of Protein Misfolding Diseases in Animals DOI: http://dx.doi.org/10.5772/intechopen.112405*

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 trialand-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

#### *Pathology of Protein Misfolding Diseases in Animals DOI: http://dx.doi.org/10.5772/intechopen.112405*

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].
