In many amyloid diseases, toxic functions have been attributed to protein aggregation in the extracellular space ^[1][48]. Precise coordination of the process of protein biogenesis, traffic, and homeostasis spatially in the early secretory compartment is very important for the maintenance of normal protein function. Disturbances in these processes may result in diseases ^[2][3][44,49]. Therapeutic approaches to this protein folding problem include molecules targeting different aspects that favor protein misfolding and aggregation ^[4][5][36,50]. Therapeutic molecules can be used to enhance the stability of the native conformation of proteins widening the thermodynamic barrier to protein misfolding and miss-assembly ^[6][7][8][9][51,52,53,54]. Another approach can focus on the clearance of preformed misassembled and misfolded aggregates of the proteins. Under certain conditions such as denaturation conditions, it becomes thermodynamically favorable for a protein to move into an aggregated state more stable than the native state under prevailing conditions.

Factors affecting protein folding include errors in post-translational modifications, protein environment alterations ^[10][56], increased degradation and accumulation of degradation products, and oxidative stress. Aggregation-prone proteins show increased aggregation at high protein concentrations ^[11][57]. Partially folded or misfolded intermediates have patches of surface hydrophobicity which make assembly a more easy and energetically feasible process. This assembly grows to oligomers, protofibrils, and fibrils of aggregated protein ^[12][58]. The amyloid fibrils are essentially an unbranched bundle of 2–6 filaments twisted together. These fibrils can interfere with biological functions and have been implicated in many neurodegenerative and cognition diseases ^[13][59]. Recently aggregated oligomers have also been linked to the pathogenicity of protein aggregation. Protein misfolding occurs when molecules are trapped in local energy minima with non-native architecture and properties ^[14][60]. The native structure is the most stable folded form for a protein under normal conditions. The folding pathway can be represented as an energy funnel with minimum energy and almost fixed conformation compared to other states. The native state of protein lies at the bottom of the funnel ^[15][16][17][61,62,63]. The energy of the intermediates decrease as they gain more ordered structure and approach the native state. The native structure generally has a packed structure with secondary elements, and tertiary structure. The achievement of native structure type depends on sequence and length of polypeptide. As the folding proceeds the number of molecules in different structural states decreases as most of them attain a native-like state, and finally, they all conclude in the native state. The native state is the most stable minimum energy state under a set of conditions. Small proteins fold by nucleation condensation mechanisms where small regions fold first, and then it guides the folding of the rest of the structure. In such cases, the nucleus of condensation or the region of the protein that guides the rest of the polypeptide to fold is important. If by any means this nucleation is affected, it can result in misfolding and aggregation of protein ^[18][19][64,65]. Protein misfolding and aggregation are linked to numerous human diseases and the aggregation of therapeutic proteins produced by recombinant techniques. Bioanalytical techniques based on optical readout of aggregation process and fibril formation have been developed. Recent times have witnessed tremendous growth in analytical method development for studying equilibrium between soluble and insoluble protein and aggregates within living systems. The field of protein aggregation and inclusion body formation has witnessed numerous conceptual turns. Natural and functional amyloid has emerged as separate domains of the protein aggregation phenomenon. There is immense possibility of exploiting amyloids for beneficial purposes such as natural biomaterials generation materials for various possible applications in industry and medicine. Some ambitious and highly desired recent research shows possible application of amyloids in water filtration technology ^[20][21][22][66,67,68]. Amyloids showed promising application possibilities in water purification. Including the removal of micro pollutants from water resources to produce potable water to quench the thirst of millions. To fully exploit aggregation, it is equally important to understand the process of aggregation itself. The creation of deviant interactions is caused by misfolding and mis-binding and starts the process of protein aggregation. These events expose beforehand hidden regions in polypeptides establishing unwanted contacts. These interactions result in self-assembly and the formation of large and insoluble aggregates.

Morphologically, amyloids exist as amorphous aggregates and fibrils ^[23][24][25][69,70,71]. Amorphous aggregates have a granular appearance in electron microscopy and are formed mainly by disordered polypeptide chains ^[26][27][72,73].

Amyloid fibrils are ordered and have repetitive structure elements. Such peculiar morphology is important in the context of the pathogenic and functional roles of amyloids. An important protein in the context of human diseases is the prion protein, and there is a strong link between misfolding and aggregation of this human protein in pathogenesis. The prion is an anchored helical protein in the cell membrane. The functions of prion protein include modulation of signal pathways, defense against oxidative stress, embryonic cell adhesion, copper binding, and maintenance of peripheral myelin.

Functional protein aggregates and functional amyloids have been discovered in various organisms. Prokaryotes, such as bacteria, and modern animals, including humans, have functional amyloids. During the evolutionary course, functional amyloids are optimized to self-assemble. They are conformationally more stable and resistant to damage than pathological amyloids. In addition, their assembly is faster. Functional amyloids are kinetically and thermodynamically favored to form ordered aggregated structures, impeding the buildup of highly harmful small metastable aggregates before reaching the final aggregated state ^[28][74].

Neurodegenerative conditions currently lack effective pharmaceutical treatment. Identifying and applying compounds that can target the multistep protein aggregation process is complicated and difficult. Therapeutics capable of perturbing the protein aggregation process are sought-after drugs ^[29][75]. Pathological protein aggregation of peptides and proteins can cause molecular and cellular pathogenicity and hence need attention by researchers to generate possible treatment plans for curing and treating these conditions.

There are reports of the aggregation of numerous peptides and proteins involved in important diseases. This problem is aggravated when it comes to aggregation-prone proteins. Such aggregation-prone proteins have been linked to the onset of many neurodegenerative diseases causing increased burden to caregivers and decreased quality of life for patients. Alzheimer’s disease, Parkinson’s disease, and Type 2 diabetes are some widely discussed and known names in this category ^[30][31][32][76,77,78]. Various amyloidogenic proteins can be trimmed to as few as five residue self-recognition elements. These self-recognition element retains the ability to aggregate and can serve as a starting point for aggregation. These self-recognition elements are important targets for aggregation inhibitors, as recent studies point to their significance in amyloid generation ^[33][34][15,79].

Amyloid formation is a peculiar feature noticed in all amyloid diseases. Over 30 diseases have been reported to be caused by the deposition of insoluble aggregates. Analysis shows amyloid deposits bind congo red and have cross-β X-ray diffraction patterns ^[35][80]. Electron microscopy shows the morphology of amyloids as straight, unbranched fibrillar structures. There are various methods and techniques developed to study amyloid structure. Under suitable inducing conditions, any protein can form amyloid. It has been suggested that aggregation is a primordial event, a highly stable polypeptide form that is intentionally and resourcefully overcome to create globular functional proteins. It is very interesting to know that non-disease fibrils formed by proteins also do not show cellular toxicity ^[36][37][38][81,82,83]. Thus it is imperative to study the structures and assembly of amyloids. Mechanisms of globular protein conversion to amyloid precursors and amyloid fibril structures need to be elucidated to understand the molecular events resulting in the pathogenicity of amyloids.

The cellular protein quality check system continuously works, although its efficiency depends on many factors. Cellular, bodily, and environmental factors can affect the protein degradation system ^[39][85]. Protein homeostasis and proteostasis are two important interlinked phenomena in the protein quality control of cells ^[40][86]. The cellular environment is complex and crowded. The cellular mechanism to facilitate correct folding and removal of misfolded proteins is important ^[41][42][87,88]. Cellular protein quality control mechanisms aim to reinforce proper protein folding, prevent folding peptides trapped in misfolded structures, and remove and clean up already misfolded peptides from the system. Protein quality control sorts out proteins that have an increased possibility of aggregation and include mostly misfolded proteins. Cells have to spend energy for this process. Most of the time protein quality systems are ATP consuming processes for the cell. In eukaryotic cell there is compartmentalisation and protein folding is organelle specific. Different micro environments in different cell compartments result in a variety of challenges to protein folding, and thus different issues related to folding in organelle-specific chemical milieu exist ^[43][89]. Protein quality control in mitochondria is complicated as about 1500 proteins are in the mitochondria, most of which are imported from the cytosol. Only a few, less than 20, are encoded by the mitochondrial genome ^[44][45][90,91]. Mitochondrial proteins imported from cytosol are mostly post-translationally modified and move into mitochondria via the membrane pores in the inner and outer mitochondrial membrane ^[46][92]. Mostly proteins are imported in an unfolded state. This protein transport generally occurs at adhesion sites of the outer and inner mitochondrial membranes, and the transport process is governed by chemical gradient or ATP hydrolysis ^[47][93]. Similar mechanisms operate in the transport of nucleus-derived proteins to chloroplasts in plants. This process is generally ATP or GTP driven. N-terminal signal sequences in such peptides help them to be translocated to specific organelles. In some proteins, there also exists an internal signal sequence which is revealed when the terminal sequence is removed ^[48][94]. This internal signal sequence also helps in translocation. Mitochondrial chaperone HSP 70 binds incoming peptides and helps them pull into the organelle. Mitochondrial stress can develop because of accumulated and misfolded proteins, and damaged translocated proteins. Dysregulation in the translocation of nuclear-encoded mitochondrial proteins, disturbances in gene regulation of mitochondrial expressed proteins, and damage to mitochondrial DNA or its localized proteins can serve as a reason for mitochondrial stress. The endoplasmic reticulum is also an important site in cells where proteins are synthesized, and they attain specific functions after post-translational modification. The ER also plays an important role in protein sorting in the cell ^[49][95]. The ER lumen is a hub of molecular machines. Most importantly, various chaperones help the protein to gain its biologically active form while the protein gains post-translation modifications to prepare to perform some of the critical functions in the cell. In the ER, proteins are folded, co- and post-transnationally modified, and sorted for delivery into different cell compartments and cell locations. The ER has a specialized chaperone system assisting with the processing and post-translational protein achievement into the active state ^[50][96]. Intra luminal ER calcium reserves are important for proper ER functions. Perturbations in ER homeostasis impact protein misfolding and accumulation in the ER. Several ER chaperones need optimum calcium levels to retain their protein folding activity. The ER is a protein-folding factory for secretory pathways ^[51][97]. The ER hosts protein quality control mechanisms that are unique to it, important protein modifications such as glycosylation, disulphide formation, and other specialised modifications occur here. Quality control, in the ER protects cells from the accumulation of aberrant proteins and a specialised type of endoplasmic reticulum fusion system operates with lysosome for lysosomal destruction of the part of ER containing aggregated proteins ^[52][98]. Additional mechanism operates to check on the fidelity of post translational processes and survey proteins at early stages of synthesis and modification. Generally, the physical state of proteins such as hydrophobic patch exposure, glycan immature trimming, content of unpaired cysteine, and disulphide bond formation affects the tendency of proteins toward aggregation and are important recognition elements in ER protein quality control. Proteins interact with ER quality control components and are targeted for elimination or expulsion to cytosol where they are degraded by cytosolic proteasome machinery. Cell viability depends on proteome integrity. Changes in cells during ageing, cancer, stress conditions, unique metabolic changes, mutations and other stochastic events can affect the ER protein quality system. ^[53][99]. Protein misfolding and aggregation has emerged as a major mechanism for human diseases. The protein conformational disease list is growing day by day. With aging and other factors, cell’s ability to deal with the proteome decreases and is a major cause of late-onset diseases. Cytosolic protein quality components regularly search for possible substrates by binding to them in equilibrium of assembly and disassembly to prevent nascent proteins from misfolding and aggregation. Both nascent and pre-existing proteins are exposed to the aggregation process in the cytosol ^[54][100]. These events of protein aggregation can perturb cellular functioning and can enhance aging. Irreparable proteins are identified and subjected to elimination by quality control mechanisms. Mutations and adverse physiological conditions can lead to protein disorders due to protein aggregation. The difficulty for a protein to attain its native state can cause misfolding. Small molecules binding specifically to the folded state of a protein and stabilizing the structure are important candidates for the development of therapeutics. As protein aggregation prevents protein molecules from folding properly, these small molecules bind specifically to proteins or their intermediate folding states. They help stabilize the structure and are a good hope for the treatment of spatially misfolding-prone proteins related to a variety of human maladies.

In eukaryotes, the ubiquitin-proteasome system and macroautophagy are important protein cleanup mechanisms. Proteins destined for destruction are targeted with small proteins called ubiquitin ^[55][102]. Energy consuming degradative process then starts the cascade of events that transfer ubiquitinated substrate to the 26 S proteasome system, and then degradation proceeds. In eukaryotes, compartmentalization makes some organelles more prone to the aggregation of proteins. Proteins in the ER are more prone to errors of glycosylation and disulphide bond formation, so ER and cytosolic quality control for protein differ in dealing with protein damage. Malfunctioning protein quality control mechanisms allow proteins to aggregate and deposit in cells as soluble oligomers and monomers. Later these oligomers can form amyloid fibrils ^[56][103]. The transport system in the cell transports aggregates in the cell through microtubular tracks. Amyloid structures are tubular structures generated by globular beta sheet-rich oligomer structures. The inherited pathogenicity in oligomers has been reported widely. However, large aggregates can be eliminated by the autophagy pathway and are cleared from the cell. Polypeptides with exposed hydrophobic stretches are prone to aggregation and play an important role in aggregation assembly ^[57][104].

3. Approaches to Targeted Protein Aggregation

The strategies are concentrated mainly on the following three points.

• preventing aggregation by stabilizing the native state
• refolding of misfolded proteins
• reinforcing or modifying proteins preventing aggregation, such as post-translational modifications