Cellular and epigenetic perspective of protein stability and its implications in the biological system

Abstract

Protein stability is a fundamental prerequisite in both experimental and therapeutic applications. Current advancements in high throughput experimental techniques and functional ontology approaches have elucidated that impairment in the structure and stability of proteins is intricately associated with the cause and cure of several diseases. Therefore, it is paramount to deeply understand the physical and molecular confounding factors governing the stability of proteins. In this review article, we comprehensively investigated the evolution of protein stability, examining its emergence over time, its relationship with organizational aspects and the experimental methods used to understand it. Furthermore, we have also emphasized the role of Epigenetics and its interplay with post-translational modifications (PTMs) in regulating the stability of proteins.

Plain Language Summary

Proteins are essential for life and are used in many medical treatments. Understanding what makes proteins stable can help us use them more effectively. This review looks at how different things like temperature and pH affect protein stability. It also discusses how chemical changes in cells, called epigenetic modifications, can impact protein stability. Understanding these factors can help us develop better treatments and therapies.

Tweetable Abstract

Understanding protein stability is crucial in experiments and therapeutic implications. Our review explores how proteins evolve for stability, methods to study it and the influence of epigenetics and post-translational modifications.

Plain language summary

Article highlights.

The evolutionary basis of protein stability

• The composition of amino acids, their sequence, and their 3D folding pattern, played a key role in evolving the protein class in the cellular domain, conferring protein evolution.

• Intrinsically disordered proteins play a prominent role in regulating transcription, translation, cell cycle, and protein–protein interaction.

Confounding factors governing protein stability

• Covalent modifications of the side chains, disulphide bond formation and proteolytic cleavage are paramount for protein stability.

• Epigenetics play a crucial role in gene expression and thus regulates protein function.

• DNA methylation, noncoding RNA and histone modification (includes methylation, acetylation/deacetylation, ubiquitination and phosphorylation) play pivotal roles in regulating protein stability.

Epigenetic players play a key role in protein stability

• Lysine acetylation prevents the ubiquitylation of certain proteins by competing with the ubiquitin group at the same lysine site, enhancing the stability of target proteins.

• HDAC inhibitors like TSA or SAHA could potentially augment the stability of proteins in several diseases in clinical settings.

• Various PTMs on proteins induced by epigenetic players modulate the stability of proteins and determine their fate in different signaling conditions.

• N-terminal acetylation has been shown to play a pivotal role in controlling protein stability.

Fate of misfolded proteins: decision & process

• Misfolded proteins are tagged with ubiquitin groups by ubiquitin ligases (E1, E2 and E3) and are marked for proteasomal degradation.

• The autophagy pathway attempts to degrade the proteins that are not degraded by the conventional proteasomal degradation pathway.

Future perspective

• In addition to epigenetics, understanding gene mutation and designing novel proteins by site-directed or random mutagenesis is highly important for precision medicine.

• A practical computational approach and logarithm must be implemented to predict a booming immunotherapeutic target.

1. Introduction

Proteins are the most versatile and dynamic macromolecules that act as authentic executioners inside a cell and drive every possible cellular process [1]. Notably, the stability of proteins inside a cell is crucial to having a well-functional cell. Initializing the molecular avenue from replication of genetic material, transcription, and processing the generation of new proteins to degrading the superfluous proteins regulate their own pool in the platter of the cellular environment [2]. The interaction of a protein with other biomolecules is key to conducting the broad spectrum of biological functions [3]. Any dysregulation of the array of proteins in the cellular milieu can contribute to irregularities in the downstream pathways, leading to cell instability. This instability can be witnessed in a range of pathological conditions, primarily neurological disorders, cancer, and certain genetically inherited diseases like cystic fibrosis [4]. Previously, the finding has shown that the sequence and order of the protein play a crucial role in conferring its folding and thus determining its function. This scenario plays a significant role in drug discovery and translational research [5]. Biochemically, proteins are the polymer of basic monomeric units of amino acids exhibiting different orders of structural organization – primary, secondary, tertiary and some proteins have another highest level of organization, in other words, quaternary structure [6]. However, between secondary and tertiary levels, two different lower order structures, in other words, super secondary structure and domains, help define the protein's higher-order structure and function [7]. Any change in either order or sequence of protein structure leads to the formation of abnormal proteins with altered function, which can form abnormal intracellular and extracellular aggregates, initiating the progression of several diseases, for example, Alzheimer's, Parkinson's disease, etc. [8].

2. Protein stability: an evolutionary perspective

The essence of evolution lies in the acquisition of changes, adaptation, and selection of positive changes to improve cellular survival and function [9]. During the progression of evolution, many selections and mutations have acted upon the biological systems that have contributed to creating more adaptive versions of these systems [10]. In order to evolve, the class of proteins has been subjected to a number of selections. From a bird's eye view, a protein needs to fulfill specific parameters to get evolutionarily selected, which include the reasonable amount of time in its folding, the functioning ability of the folded structure, and the stability of the folded structure to achieve its function reliably without getting into aggregation or proteolysis [11]. Although the composition of amino acids, their sequence, and 3D folding pattern played a crucial role in the evolution of the protein, the stability of a protein is one of the major factors of its functionality and has a significant role in protein evolution [12,13]. It has served as the constant selection to shape the protein repertoire inside the cell the way it is today. However, does it confer a selective advantage or disadvantage; this question has been debated, as scientists have favored both statements. Traditionally, it is assumed that the proteins need to acquire only marginal stability to function well, and most globular proteins have been shown to have marginal stability only [14]. Since highly stable proteins tend to attain a rigid structure, while marginally stable proteins are pretty flexible in nature. This flexibility confers some advantages as well as disadvantages, thereby making stability a substantial factor in protein evolution.

The advantages of flexible proteins include enhanced catalysis in the case of enzymes and enhanced biological activity in general [15]. Moreover, the flexible proteins enable enhanced scope in structure-based drug discovery as flexibility allows enhanced affinity between the drug and its target [16]. The disadvantages of flexible proteins include their increased proneness to proteolysis due to low stability and denaturation, which ultimately leads to the non-functionality of the protein. Hence, this leads to degradation/aggregation of protein due to aberrant intermolecular linkage and polymer formation and paves its way to cellular dysfunction and the onset of numerous diseases, including Huntington's disease, Alzheimer's, and even dementia. A study by Lomas and Carrell (2002) showed how the aggregation of the serpin superfamily of serine protease inhibitors plays a significant role in dementia [17]. Also, when proteins functioning as enzymes become less stable; they undergo a detrimental conformational change. For instance, Guijarro et al. (1998) have shown that the PI3K protein gets denatured at low pH, and its SH3 domain forms a fibrils-like structure which is associated with many amyloid diseases. This fibril formation occurred due to faulty conformational change from soluble alpha-helical to aggregated beta-sheet conformation [18]. In addition, the less stable proteins require a good amount of strong interactions with their ligands due to less rigidness in the active site.

In contrast, stable proteins lose less entropy and do not require such energy levels to bind the ligand [19]. Apart from this, a previous study by Kamal et al. (2012) has established the role of rigidity of active sites in enzymatic activity and its function. They have shown that the rigidness in the enzyme's active site is positively correlated to the enzymatic activity. Proteins having less stability and more flexibility, exhibit a loss of active site integrity. They are believed to have less propensity to perform the designated catalytic reaction. At the same time, the more stable proteins have a more rigid active site that can bind to their ligand, specifically in a lock-and-key fashion. It also avoids the premature denaturation or aggregation of proteins and possesses extended biological activity [20]. Nevertheless, all these factors pose protein stability as a positive characteristic for the natural selection of proteins and indicate that proteins with higher stability are expected to be selected.

Furthermore, another study was put forward to explain the non-adaptive and neutral evolutionary basis of proteins to attain marginal stability, thereby questioning the general notion of positive selection of protein stability [11,21]. Simulation results from computational and analytical approaches have shown how nature decides a protein's functionality and stability ratio. As per their study, when a protein heads toward the goal of marginal stability, this process leads to a selection pressure that positively selects the functionality and marginal stability. As marginal stability is associated with increased plasticity of the protein structure, it aids the protein in gaining new functions. So, marginal stability can aptly be termed as a ‘spandrel’, a term perfectly defined and used by Gould and Lewontin (1979) to describe the characteristics that are generated due to non-adaptive and neutral reasons but used by the whole biological system for adaptive purposes. This idea helps us understand the biological system of quickly adapting to new roles and functions [11,22]. This theory, advocating the existence of a dominating, marginally stable, functional protein class, has compelled many protein scientists to explore the proteome for functional but improperly folded structures. These polypeptides were later termed intrinsically disordered proteins (IDPs) [23].

As the folding potential of a protein relies on its structural aspects, IDPs possess particular sequential preferences that are different from the customarily ordered proteins. On a majority scale, IDPs lack the hydrophobic core, which is the primary determinant of the proper 3D structure of a protein, leading to the decompaction of the protein. Also, lower proportions of the hydrophobic amino acid residues and higher proportions of charged and hydrophilic amino acid residues lead to a net high charge, thereby contributing to the electrostatic repulsion among the residues. These factors are responsible for the absence of a compact protein structure [24,25]. Figure 1 illustrates the 3D structure of common IDPs such as Alpha-synuclein, Microtubule-associated protein tau, and PUMA [26–29]. Despite being unstructured, this group of proteins plays a vital role in every essential process of the cell. IDPs have been observed to play a prominent role in regulating transcription, translation, cell cycle, and protein–protein interaction to carry out intricate cell signaling [25]. Recent studies have reported that the unfolded regions in the chaperones bind to other misfolded proteins and help unfold those intermediates [30]. Therefore, IDPs can also be considered in the context of marginal stability [31,32].

Figure 1.

Illustration of 3D structure of intrinsically disordered proteins. Protein structure of (A) alpha-synuclein (PDB ID: 1XQ8), (B) microtubule-associated protein tau (Alphafold), (C) PUMA (Alphafold).

3. Protein stability at different structural levels

Since the protein is organized into different structural orders, it becomes crucial to study each order in terms of stability. Each order confers a unique identity to the protein that helps us to characterize it. In biochemical terms, a protein is usually considered a polypeptide structure composed of one or more than one chain of amino acid residues [7,33]. Each protein has its unique sequence of amino acids [7]. A single alteration in the amino acid sequence can lead to various drastic consequences, including conformational changes, changes in functionality or protein destabilization due to protein stability change. Most of the time, these changes culminate in diseases [34]. Previous findings have shown that more than 50% of monogenic diseases arise due to single point mutations in the protein, and the leading reason and mechanism that makes these substituted amino acids cause human disease is protein stability change. Few studies have even given a quantitative estimation of the same. For instance, Yoe and Moult have widely studied the impact of amino acid substitution on protein stability and reported an estimation of about 25% non-synonymous single nucleotide polymorphism (nsSNPs) in the human population that has been proved deleterious to protein function [35]. Also, the study by Wang and Moult has revealed that about 83% of the known disease-causing missense mutations result in alterations in protein stability, thus shedding light on the importance of protein stability maintained at the primary structure of the protein [36]. Alpha-helix and beta-pleated sheets are the two majorly common folds found in the secondary structure of proteins [37]. Some selected amino acids correlate to appear in a specific type of fold that determines their propensity to be a part of that particular fold. For instance, alanine, glutamate and leucine appear more frequently in alpha-helices, while aspartic acid, glycine and proline are less frequent to be part of it [38]. The concept of amino acid propensities affecting protein stability at the secondary level has been well studied. Hermans et al. have reported that while glycine destabilizes the alpha helix due to enhanced conformational flexibility after folding [39], Proline has a destabilizing effect on the alpha helix of a protein due to the lack of H atom that prevents its participation in hydrogen bonding which is a prerequisite for stabilization. Besides, the bulky side chain of the proline creates a steric hindrance, resulting in a kink in the alpha-helix [40]. Hence, the secondary level stability depends on the amino acid composition in a specific fold.

Once the protein is stabilized at the secondary level, it acquires its final shape through interactions among the side chains of the amino acids. It is generally known as a tertiary structure of the protein [41]. In the cellular environment, where a protein can attain as many conformations, attaining the functionally active conformation for a protein encompasses a highly complex pathway. This task is successfully conducted by a particular class of proteins called molecular chaperones. These proteins are widely recognized as heat-shock proteins (HSPs) or stress proteins, as they get upregulated in stress conditions due to the accumulation of aggregate-prone folding intermediates [42]. This class of proteins is involved in maintaining the cell's proteome. These proteins help maintain other proteins throughout their life span, in other words, from synthesis to degradation. The process of protein folding is a multi-factorial process that includes conformational and compositional stability, primary and secondary structure, cellular microenvironment possessing temperature and pH, solvation, hydrophobic effect, solvation, Van der Wall forces, salt bridges, hydrogen bonding, chaperones, post-translational modifications (PTMs), ion binding and cofactor binding [43]. The stability of the 3D structure of a protein is primarily determined by a combination of intramolecular interactions comprising hydrogen bonds, Van der Waals interactions, and hydrophobic forces. Among these, hydrogen bonds contribute the most to maintaining stability, whereas hydrophobic forces contribute less [44]. The intra-molecular H bond plays a pivotal role in protein stability. These intramolecular H-bonds are formed when the unfolded nascent protein/peptide escapes from hydrogen bonding with the neighboring water molecules (polar) under solution conditions requiring entropy-enthalpy compensation. These interactions stabilize the folded conformation of the protein and bring down the folding enthalpy below zero upon the formation of stable and folded protein. Subsequently, a more ordered and folded faces the restriction of conformational freedom upon folding, leading to a decrease in the entropy to below zero [45].

Any physical factor that changes the overall fold of the protein or its conformation can disrupt its tertiary structure. These factors include the cellular environment and physical factors like pH and temperature. Any minute alteration in these factors yields changes in the hydrophobic interactions and hydrogen bonding within a protein that not only disturb its secondary structure and bring change in the binding of any ligand or cofactor and PTMs associated with it but also change the Gibbs free energy associated with it [43]. Once a protein has attained a completely folded tertiary structure, folded subunits reorganize themselves to form a quaternary structure, forming oligomeric proteins [46,47]. Furthermore, non-covalent protein-protein interaction can also create a spatial arrangement [46].

4. Factors affecting protein stability

Several factors affect the stability of proteins in the cellular milieu. In this review, these factors will be studied categorically based on their properties: biophysical factors such as pH, temperature, molecular crowding, PTMs, and epigenetic factors.

4.1. Biophysical factors

Each macromolecule or protein in our context has evolved to perform its function in a specific cellular environment. The biophysical properties of the environment affect the stability in addition to the activity of the protein.

4.1.1. pH

Each protein, be it in-vivo or in-vitro, has its profile of stability as well as activity at different pH. Every protein has an optimum pH to be stable enough to function well. Any fluctuation in pH that switches the cellular environment to any of the three states of an acidic, basic, or neutral medium changes the protein structure and stability and can lead to denaturation [48]. Several studies have been published concerning the effect of pH on a particular protein. Consider the classic example of Hexokinase A enzyme, where evidence from Kumar et al. shows the pH change of the medium plays a key role in affecting the protein's stability. When denatured in an acidic medium, a secondary structure with an inconspicuous amount of tertiary structure is predominant, while the alkali-denatured state is less structured than the acid-denatured state [49]. Proteins found in the highly acidic environment of lysosomes tend to have a median pI of about 6.5 due to a higher frequency of negatively charged, acidic amino acids, in other words, aspartate and glutamate. In contrast, the proteins found in the basic environment of mitochondria tend to have a median pI of about 8.0 due to a higher frequency of positively charged basic amino acids, in other words, lysine and arginine [50]. This pH-protein dynamics can be understood by the study of Russo et al. on NP4. This protein is responsible for nitric oxide (NO) release but in a pH-dependent manner. At an acidic pH of 5.5, NP4 is established in a closed conformation with NO tightly bound to it. Once this pH turns basic at about 7.5, aspartate at the 30th position becomes deprotonated, making the conformation change from the closed state to the open state; this aids the NO in being released. This example sheds a good amount of light on the role that pH can play in changing the stability as well as the activity of a protein [51].

4.1.2. Temperature

Cellular response to the temperature helps adapt the organism to the environment. The temperature has been shown to modulate the production and thermostability of the proteins. Upon sensing the temperature fluctuation, cells will start producing more proteins capable of thriving in that temperature. A class of proteins known as heat shock proteins will be expressed to act as a savior for cells after an increase in temperature, and the same goes for cold shock proteins in case of a decrease in temperature beyond a threshold [52]. At the molecular level, the thermal stability of a protein is studied by considering two factors: thermodynamic stability and thermal resistance, which is the melting temperature (T_m) [53]. Ideally, how fast the protein gets refolded upon unfolding or how slow the protein is getting unfolded due to temperature changes determines the stability of the protein thermodynamically. However, intramolecular aggregation or hydrolysis of peptide bonds, etc., and cooperativity of the shift between folded and unfolded states affect the thermodynamics of protein stability [54]. Besides, thermal resistance is the temperature upon which the concentration of the folded state of a protein is tantamount to its unfolded state [53]. This thermal resistance is directly correlated to the kinetic resistance to unfolding. As a result, hyperthermostable proteins sustain extreme temperatures due to slow unfolding rates compared with moderately stable proteins [55]. Having discussed the aspects of thermal stability and how its components respond to temperature, it is equally imperative to reflect upon the structural parameters of a protein that describes thermostability. Structurally thermostable proteins acquire increased ionic interactions, enhanced level of hydrophobic surface burial, and a large number of prolines while the decreased number of glutamines, enhanced core packing, and more rigidity with all these factors culminating in increased thermal resistance (TM). Along with this, shorter loops with an extended state of secondary structure and a higher state of oligomerization also contribute to the enhanced thermostability of a protein and help the protein bear the temperature changes [56].

Several biophysical techniques are employed and proven to be the gold standard in exploring protein stability (Figure 2). Table 1 enlists all the standard techniques and methods used to understand protein stability [57–65].

Figure 2.

Implication of different biophysical techniques to study protein structure and stability. Various biophysical techniques to study protein stability and structure based on protein secondary and 3D structure, protein folding/dynamics and thermal stability.

Table 1.

Study of protein stability: methods and techniques.

Method	Principle	Characteristics used for analysis	Parameters of protein analyzed	Ref.
Raman spectroscopy	Inelastic scattering of incident light after interacting with vibrating molecules	Amide I (80% C=O stretch, band near 1650 cm-1), Amide III (30% N-H bend and 40% C-N stretch, band near 1300 cm-1)	Analysis of protein secondary structure	[57]
FTIR	Measurement of wavelength and Intensity of the absorption of IR radiation by protein structural repeat units	Mainly Amide I (80% C=O Stretch, band near 1650 cm-1) Amide II (60% N-H bend and 40% C-N stretch, band near 1550 cm-1)	Estimation of protein secondary structure, Protein stability (folding and unfolding or altered 2° structure of protein in response to stress)	[58]
Circular dichroism (CD)	Measure the difference in absorbance of the L and R circularly polarized component of the plane polarized light by the folded and unfolded protein as a function of temperature	Peptide bond (absorption below 240 nm), Aromatic amino acid side chains (absorption from 260 nm to 320 nm) Disulfide bonds (absorption near 260 nm)	Secondary structure composition (% helix, sheet, turns, etc.) Tertiary structure characteristics, protein folding, conformational changes in proteins	[59]
NMR	Chemical shift is produced by the nuclei of atoms when a molecule is subjected to an external magnetic field	Chemical shift of 1Hα, 13C-, and 15N of the peptide backbone	Secondary structure composition, 3D structure of the protein Protein dynamics	[60]
X-ray crystallography	Diffraction of a beam of x-rays into many specific directions by a crystalline atom and the subsequent measurement of the intensities and angles of the diffracted beams that generate a 3D picture of the density of the electrons contained by the crystal	Electron density map of each amino acid	3D structure of the protein	[61]
smFRET	Involves the transfer of energy from a donor to an acceptor chromophore through dipole-dipole coupling in a non-radiative manner, which gives us the estimation of the intervening distance based on the ratio of acceptor to total emission intensity	Uses the distance between two sites of the protein that are conjugated with donor and acceptor fluorophores in a site-specific manner	Protein folding-unfolding, Protein conformation dynamics	[62]
Differential scanning calorimeter (DSC)	Upon temperature change, a sample may either radiate or absorb a particular heat quantity excessively. DSC measures this heat to give insights into changes in the physical properties of a sample along with temperature and time	Melting temperature or thermal transition temperature energy is needed to disrupt the stabilizing interactions of the tertiary structure of a protein	Thermal stability	[63]
Differential scanning fluorimetry (DSF)	An increase in energy or temperature leads to protein unfolding. This unfolding is measured by monitoring changes in fluorescence with respect to temperature	Measures fluorescence of either a hydrophobic fluorescent dye that binds the unfolded proteins or changes in intrinsic fluorescence of a protein upon unfolding	Thermal stability, protein folding state	[64]
Ion mobility mass spectrometry (MS)	Ions from the sample exhibit different size, shape and charge in gas phase and get separated accordingly	Mass to charge ratio of different ions that helps in characterization of the protein	Protein structure, protein folding-unfolding, protein conformation dynamics	[65]

4.1.3. Molecular crowding effect

Molecular crowding is a prevalent concept primarily considered while studying the various biochemical processes occurring in the cell. The cellular milieu comprises many macromolecules occupying around 40% of the cellular volume, limiting the accessible volume to carry out the functions. This concept is characterized as macromolecular crowding [66]. Besides macromolecules, a large amount of low molecular weight organic compounds called osmolytes also contribute to this ‘excluded volume effect’, most commonly known as molecular crowding in general terms [67,68]. This has been studied both in vivo as well as in vitro, taking proteins as the subject. For instance, ficoll70 has been reported to have increased protein compaction along with the enzymatic activity of PGK, while the addition of sucrose has led to significant thermodynamic stabilization [67]. Various other studies have been conducted experimentally to check the consequence of crowding agents on a structure of selected proteins, for example, on the oligomerization of GroEL subunits, folding of denatured lysozyme, on the self-assembly of FtsZ, etc [68]. During the folding process, these crowding agents are predicted to favor the dense folded state instead of the unfolded state in a correlated manner with the amount of the crowding agent [69]. One reason for this is that crowding increases the free energy of the decompacted unfolded state as compaction is quite unfavorable for unfolded protein; therefore, the relative stability of the folded protein increases [70]. Some studies have been conducted to assess the conformational change that crowding agents bring to a protein. For instance, at low pH and in the presence of crowding agents, unfolded Cytc acquires a molten globule state [68].

4.2. Post-translational modifications

PTMs are the covalent processing events that occur in a protein after its synthesis. PTM plays a key role in extending and diversifying the protein function beyond the role that gene transcripts dictate. They modify the structure and properties of the protein in either a reversible or irreversible manner through an array of biochemical reactions and help them to locate themselves in the correct cellular compartment. This regulates the cell's signaling and physiological state [71]. Generally, PTMs are categorized into three types: covalent modifications of the side chains of amino acids of a protein, marked by the addition of chemical moieties to them; disulfide bond formation, a covalent bond formed between two cysteine residues by removal of two hydrogen atoms, proteolytic cleavage, marked by breaking peptide bonds [72].

4.3. Covalent modifications of amino acids

Covalent modifications of amino acids account for a large portion of the PTMs that occur inside a cell. These biological mechanisms help the cell diversify its proteome and allow the cell to regulate its physiology dynamically. This involves the addition of small chemical moieties to the side chains of some selected amino acids, which can either be catalyzed by various enzymes or due to some non-enzymatic processes having reactive oxygen species as the mediators [73]. There are reportedly no known PTMs on isoleucine, leucine, valine, alanine, and phenylalanine [72]. Till now, more than 200 types of PTMs have been reported that have varying effects on cellular functions, but not all are very abundant [74].

4.3.1. Enzymatic

Most PTMs occurring inside the cells are catalyzed by specific enzymes at designated amino acid residues in the protein. One modification can occur at multiple amino acids, and multiple modifications can also modify one amino acid. For instance, two different PTMs, in other words, phosphorylation, and acetylation, can occur at Ser/Tyr/Thr [75,76]. These types of PTMs are generally reversible, in other words, another set of specific enzymes can remove these modifications. Phosphorylation, which involves the attachment of the phosphate group, is catalyzed by kinase enzymes, while phosphatases serve the opposite. Supplementary Table S1 enlists all the main enzymatic PTMs that affect protein stability and other cellular functions [75–83].

4.3.2. Non-enzymatic

Besides enzymatic PTMs, redox imbalances inside the cell also lead to reversible and non-reversible PTMs. These redox imbalances can occur due to environmental stress or as a part of natural cellular function and extracellular stimuli. PTMs on account of environmental stress are found to be irreversible, whereas PTMs on account of natural cellular function are generally reversible. Both of these comprise of oxidative stress induced by either reactive oxygen species (ROS) or reactive nitrogen species (RNS). These reactive species are responsible for modifications in amino acids and have a damaging impact on proteins. These PTMs lead to the generation of dysfunctional proteins and get translated into several neurodegenerative diseases [84]. Supplementary Table S2 enlists the major non-enzymatic PTMs induced due to these reactive species [85–92].

4.4. Disulfide bond

The amino acids within a protein interact via different interactions that help the protein fold and attain the required conformation. These interactions can either be the comparatively weaker non-covalent bonds, which include hydrogen and hydrophobic bonding, or the strong covalent bonds, which include disulfide bonds. These bonds are formed between the sulfur groups of two interacting cysteine residues [93]. These bonds make proteins less prone to denaturation and enhance their resistance to extreme physical factors like temperature and pH; all this is achieved by enhancing the thermodynamic stability of the proteins [94].

4.5. Proteolytic cleavage

It signifies the hydrolysis of peptide bonds that break the protein into peptides. This kind of post-translational processing of proteins converts the inactive form of proteins to their active form. It prevents the accumulation of non-functional, abnormal proteins inside the cell [95].

4.6. Epigenetics

Epigenetics involves the study of altered gene function/phenotype due to reversible DNA and histone protein modifications rather than DNA sequence alteration. The modification of DNA or DNA-associated chromatins results in gene silencing or activation. The epigenetic modifications are often conserved and can be transmitted from one generation to another. Therefore, epigenetic inheritance plays a vital role in controlling various genes apart from the conserved genetic information [96,97]. To date, DNA methylation (mainly cytosine methylation) and histone modification (includes methylation, acetylation/deacetylation, ubiquitination, and phosphorylation) are the two very important known epigenetic mechanisms responsible for altering gene-phenotype. However, some of the RNA-mediated processes (lncRNAs) also contribute to epigenetic-based gene expression [96–101]. Epigenetic modifications depend on various environmental factors such as drugs, exercise, stress, diet, environmental toxins, alcohol, weather, and pathogens [97,98,102–105]. Interestingly, the paternal and maternal factors also contribute to deciphering the phenotype of the various genes of the offspring apart from the plethora of environmental factors [103,104,106]. Epigenetic alterations are also key contributors to various human diseases such as diabetes, various cancers, numerous autoimmune diseases, and neurological disorders [107,108].

It has been well understood that epigenetics involves the modification of proteins such as histones and transcription factors such as p53. Histones are one of the important components of the chromatin. These histone proteins are highly basic and constitute globular domains. The DNA is wrapped around these globular domains, and some of the unstructured N-terminal domains project out from the nucleosome [109]. These N-terminal domains are the hotspots for major epigenetic PTMs such as phosphorylation, acetylation and methylation. In addition, sumoylation, ubiquitination, deamination and ADP ribosylation are a few other epigenetic modifications that are found mainly on H3 histones [110,111]. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) add and remove acetyl groups from lysines' ϵ amino group of histone tails, which results in transcription activation and repression, respectively [112,113]. Acetylation neutralizes the positive charge of lysine in histone tails, weakening DNA and histone tail interaction, which is essential for transcription. Like acetylation, histone phosphorylation/dephosphorylation is regulated by histone kinases/phosphatases at the serine, threonine and tyrosine hydroxyl groups present at the histone tails. This results in the alteration of the ionic states of these resides, ultimately leading to the alteration of histone–DNA interaction [114]. Histone acetylation is involved in regulating large arrays of cellular functions. On the other hand, histone phosphorylation is required for the regulation of transcription, DNA damage repair, chromatin compaction, and apoptosis [114].

On the other, the methylation by histone methyltransferase (HMT) at lysine (HKMT) and arginine (HRMT) results in gene silencing. Unlike the reversible nature of acetyl and phosphate histone modifications, histone methylation is usually found to be irreversible [114–116]. However, some histone demethylases are reported that can undo the gene silencing [112,117]. All these epigenetic modifications are required for the regulation of gene expression. It has been well established that dysregulation of these epigenetic modifications results in numerous diseases such as cancer, autoimmune disorders, etc [112,113,116–118]. Epigenetic changes regulate an organism's maintenance and development cascades throughout its lifetime. Epigenetics controls the function of a cell by modifying the DNA, DNA-related histone proteins, as well as transcription factors under various stimuli such as cellular stresses, drugs, etc. One of the most important transcription factors that undergoes frequent epigenetic alteration is p53, often called the guardian of the genome [119]. The p53 is a 393 amino acid protein, and 44 of these amino acids can be modified by post-translational modification (such as phosphorylation, acetylation, glycosylation, ADP-ribosylation, neddylation, ubiquitination, methylation, demethylation, poly-ubiquitination, and sumoylation) events under various stimuli. The PTMs in p53 play a deciding role in p53 performing a particular function, such as cell differentiation, cell death, cell growth, and senescence. Based on the PTMs and their localization, transcription activation, transcription suppression, DNA binding enhancement, promoter-specific DNA enhancement, p53 nuclear export, p53 degradation, p53 stabilization, and anti-repression pathways can take place [120–123]. It has been found that phosphorylation and acetylation are the major PTMS that stabilize the p53. A recent study showed the drug-mediated epigenetic modification restoration of p53 Y220C mutant in pancreatic adenocarcinoma. The curcumin treatment restores the distorted structure of p53 and stabilizes post-translational modifications such as P-Ser-15, P-Ser20, and Ac-Lys282 [124]. This restored mut-p53 has the capacity to transactivate the apoptotic genes just like the wild-type, therefore ultimately leading to apoptosis.

Moreover, another segment of epigenetic mechanism, in other words, noncoding RNAs, is being studied and established to regulate protein stability. Many lncRNAs have been reported to affect the stability of p53 through their epigenetic mechanism. A novel study by Deng et al. (2020) reported that lncRNA PiHL negatively regulates the stability of p53 by enhancing its ubiquitination through an axis of different other proteins GRWD1, RPL11, and MDMB2 in colorectal cancer [125]. Contrastingly, lncRNA PANDA has been observed to stabilize p53 through some undeciphered mechanism [126]. Also, the lncRNA DINO stabilizes p53 in DNA-damaged conditions by interacting with p53 itself [127]. lncRNA GUARDIN helps in maintaining genomic stability by stabilizing BRCA1 [128]. ANCR lncRNA facilitates the interaction of CDK1 and EZH2 thereby promoting the phosphorylation of EZH2 that leads to its ubiquitination and its degradation [129]. An overexpressed lncRNA-UICC in cervical cancer stabilizes p-STAT3 by interacting with it and preventing its proteasomal degradation [130]. Similarly, LSINCT5 stabilizes HMGA2 by inhibiting its proteasomal degradation and promoting malignancy in NSCLC (Non-small cell lung carcinoma) [131]. On similar lines, in hepatocellular carcinoma, LINC00473 stabilizes protein survivin by interacting with it and by recruiting a deubiquitinase USP9X [132]. Also, HULC stabilizes SIRT1 by reducing its ubiquitination levels, resulting in chemoresistance and autophagy in Hepatocellular carcinoma [133]. Correspondingly, many more lncRNAs have been established to regulate the stability of specific proteins and ultimately regulate the essential signaling mechanism of cells [134].

5. Interplay between epigenetic players & post-translational modifications play a crucial role in modulating the protein stability

Epigenetics has been shown to play a vital role in regulating the genome organization, host immune response, and protein expression [135–139. In addition to fine-tuning the transcriptional program, epigenetics has been shown to play an essential role in the stability of proteins. As discussed above DNA and histone methylation/demethylation and histone acetylation/deacetylation are the major types of chromatin modifications associated with the stability of proteins. The interplay of epigenetic players and PTMs provides insights into protein stability beyond their structural aspects.

Lysine acetylation prevents the ubiquitylation of specific proteins by competing with the ubiquitin group at the same lysine site and, thereby, enhances the stability of target proteins. p300 is a transcriptional co-activator and acts as a histone acetyltransferase (HAT). p300 has been shown to enhance the stability of various proteins by acetylating them, for example, HIF-1, Smad7, p53, WRN, etc. The finding shows that during hypoxia, p300 can acetylate the Lysine 709 of HIF1α, thus increasing its stability by decreasing the ubiquitination [140]. However, lysine deacetylases (class I-III HDACs), including HDAC-1, can interact with Lys-709 acetylation and decrease its activity [141]. It is imperative to note that HDAC inhibitors like TSA or SAHA could potentially augment the stability of proteins in several diseases in clinical settings [142]. Besides, it has been established to augment the stability of Smad7 by acetylating it at two lysine residues in its N-terminal and preventing its degradation from TGFß and Smurf ubiquitin ligase (Figure 3) [143]. This CBP/P300 acetylates p53 at various lysine sites in the C-terminal and prevents Mdm2 ubiquitin ligase from degrading p53 [144]. Another protein, WRN, is a target protein of CBP that acetylates WRN on 6 lysine sites and prevents its degradation by its E3 ubiquitin ligase MIB1 [145]. Apart from p300/CBP, PCAF has been reported to acetylate b-catenin and E2F1 and stabilize them by preventing their degradation by β-TrCP and SCF^skp2, respectively [146,147]. Above and beyond the lysine acetylation, N-terminal acetylation has been reported to control the stability of various proteins, including the intermediary proteins of the ubiquitination process, for example, E3 ubiquitin ligase, 26S proteasome, Hsp90 chaperone, etc. Met acetylation at the N terminal of the Ubc12 subunit of E2 ubiquitin ligase aids it in docking into the hydrophobic cleft of the Dcn1 subunit of Nedd8 E3 ligase and promotes neddylation to certain target culling proteins, leading to enhanced stability and activity of E3 ligases. N terminal-Acetylation even prevents Hsp90 and its clients from degradation. Loss of N terminal-acetylation of proteins causes the aggregation of various neurodegenerative proteins for example Huntington [148].

Figure 3.

Lysine acetylation mediated increase in protein stability. Lysine acetylation prevents the ubiquitination of protein by increasing protein stability. p300 has been shown to enhance the stability by acetylating HIF-1, Smad7, p53 and WRN. p300 can acetylate the Lysine 709 of HIF1α, thus increasing its stability by decreasing the ubiquitination. Acetylation can increase Smad7 protein stability and prevent its degradation from TGFß and Smurf ubiquitin ligase. CBP/P300 acetylates p53 at C-terminal and prevents Mdm2 ubiquitin ligase from degrading p53. WRN is a target protein of p300 and CBP complex, which acetylates WRN on 6 lysine sites and prevents its degradation by its E3 ubiquitin ligase MIB1. PCAF can acetylate β-catenin and E2F1 and prevent their degradation from β-TrCP and SCF^skp2.

Besides acetylation, methylation has also been a major regulator of protein stability. It mainly occurs on lysine and arginine residues by various methyl transferases. Methylation has been reported to regulate the protein stability in either manner, in other words, it may decrease or increase the stability of the target protein. Leng et al. reported that SET7/9 methylates DNMT1, E2F1 and R/K-S/T-K motif-containing proteins at lysine residue. A methyl-binding protein, L3MBTL3, recognizes this methylation mark at K142 in DNMT1. L3MBTL3 interacts with CRL4^DCAF E3 ubiquitin ligase that targets these proteins for ubiquitin-based proteolysis [149]. However, a methyl group is removed by a demethylase LSD1, whose primary function is to maintain the stability of DNMT1 and related proteins and subsequently help in sustaining the DNA methylation levels across the genome. Set7/9 has been shown to destabilize the p65 domain of NF-κB at lysines 314 and 315, leading to its ubiquitination and its degradation [150]. A similar trend is observed in the case of another DNA methyl transferase, UHRF1, which is methylated by SET8 protein methyl transferase at K385, leading to its ubiquitination while LSD1 stabilizes it by demethylating it [151]. On the contrary, Set7/9 was reported to stabilize a number of non-histone proteins, including p53 and estrogen receptor (ER). It methylates p53 at lysine 372 and ER at lysine 302 and stabilizes them (Figure 4) [150]. Aside from lysine methylation, arginine methylation modulates protein stability. Dong Hu et al. (2015) have provided evidence for the interplay between arginine methylation, its ubiquitylation and the stability of KLF4, an important player in DNA damage response and apoptosis of cells. PMRT5, an arginine methyl transferase, methylates KLF4 at arginine 374, 376 and 377. This methylation brings about a conformation change in the KLF4, thus preventing VHL, an E3 ubiquitin ligase, from binding and ubiquitinating it [152]. E2F1 is methylated by both PMRT1 and PMRT5 competitively. During DNA damage, PMRT1 stabilizes E2F1 and leads to apoptosis, while PMRT5 destabilizes it and leads to cell proliferation (Figure 6A) [153].

Figure 4.

Methylation-mediated regulation of protein stability. The methylation on lysine and arginine residues plays a crucial role in modulating protein stability. Set7/9, a methyltransferase, has been reported to regulate the stability of various non-histone proteins positively and negatively. Lysine methylation of proteins like DNMT1 and p65 domain of NF-κB leads to their proteasomal degradation, whereas in proteins like p53 and ER, lysine methylation leads to prevention of degradation and enhanced stability. Similarly, another methyltransferase SET8 mediated lysine methylation of UHRF1 leads to its proteasomal degradation.

Figure 6.

Interplay of polyubiquitination with other PTMs in regulating protein stability. (A) Role of polyubiquitination in inducing protein stability. PMRT5 causes KLF4 arginine methylation at 374th, 376th and 377th residues, thus preventing the degradation of KLF4 by the ubiquitin ligase VHL. PMRT1 arginine mediated methylation of E2F1 and its subsequent K-63 linked ubiquitination on lysine cluster 161/164 results in enhanced stability of E2F1 protein. Similarly, methylation of DUSP14 at 17th, 38th and 45th arginine residues by PMRT5 is recognized by an E3 ubiquitin ligase, TRAF2, which catalyzes the K63 linked ubiquitination of DUSP14 on its 103rd lysine residues and results in increased stability and activity of DUSP14. (B) Coordinated activity of different PTMs regulate protein stability. The coordinated activity of different PTMs can regulate the stability of proteins. Dynamics of increased stability of p53 involves recognition of methylation marks at 372nd lysine residue by Tip60, a chromodomain-containing histone acetyltransferase, which leads to acetylation of other lysine residues. Further acetylation of lysine residues can prevent ubiquitination and prevents its proteasomal degradation. PHD2-mediated hydroxylation of HIFα at 402 and 564 proline residues leads to its methylation at K391 by SET9 methyltransferase. This led to ARD1-induced acetylation of HIFα at K532. This signaling cascade can lead to VHL-induced degradation of HIFα.

PTM observed in proteins that regulate protein stability is phosphorylation, and it can modulate it in either way. Phosphorylation of IκB (inhibitor of NF-κB) by IκB Kinase complex (IKK) at two serine residues in N terminal leads to its ubiquitylation by E3RSIκB/β-TrCP, an SCF-type E3 ubiquitin ligase and subsequent degradation by 26S proteasome [154]. Similarly, in the absence of a WNT signal, phosphorylation of β-catenin by glycogen synthase kinase 3 (GSK3) and casein kinase I-alpha (CKIα) leads to its ubiquitylation by SKP1-cullin1-F-box (SCFβ-TrCP) E3 ligase complex and degradation by 26S proteasome [155]. CDK1-cyclinB phosphorylates UHRF1 at Serine 652, leading to its degradation (Figure 5A) [151]. The stability of the DUSP family is widely regulated by phosphorylation. On the one hand, phosphorylation of DUSP1 by ERK on serine 296 and 323 leads to its binding to ubiquitin E3 ligase CUL1/SKP2/CKS1 complex that marks DUSP1 for proteasomal degradation (Figure 5A) while on the other hand, phosphorylation of DUSP1 by ERK at Serine 359 and 364 enhance its stability. ERK-induced phosphorylation of DUSP4 at Serine 386 and 391 prevents it from proteasomal degradation. Phosphorylation of DUSP10 by mTORC2 at serine 224 and 230 leads to its stabilization. DUSP16 phosphorylation at Serine 446 by ERK leads to its stabilization by preventing ubiquitylation (Figure 5B) [156]. As reviewed by Chen et al. (2021), phosphorylation of an oncogene XBP1 at serine 212/217 leads to its interaction with FBW7, which acts as a substrate protein for cullin 1-RING E3 ligase (CRL1s) and leads to its degradation while the anti-apoptotic protein Bcl2 is phosphorylated by pyruvate kinase M2 isoform (PKM2) at Threonine 69 and stops cullin based E3 ubiquitin ligase from interacting with it and stabilizes it [157].

Figure 5.

Role of serine phosphorylation in regulating the protein stability. (A) Phosphorylation-mediated proteasomal degradation: phosphorylation of IκB (inhibitor of NF-κB) by IκB Kinase complex (IKK) at two serine residues in N terminal leads to its ubiquitination by E3RSIκB/β-TrCP, an SCF-type E3 ubiquitin ligase and subsequent degradation by 26S proteasome. Absence of WNT signal, phosphorylation of β-catenin by glycogen synthase kinase 3 (GSK3) and casein kinase I-alpha (CKIα) leads to its ubiquitinylation by SKP1-cullin1-F-box (SCFβ-TrCP) E3 ligase complex and degradation by 26S proteasome. CDK1-cyclinB phosphorylates UHRF1 at Serine 652 and leads to its degradation. Phosphorylation of DUSP1 by ERK on serine 296 and 323 leads to its binding to ubiquitin E3 ligase CUL1/SKP2/CKS1 complex, which marks DUSP1 for proteasomal degradation. Phosphorylation of XBP1 oncogene at serine 212/217 marks it as a substrate for CRL1s and leads to its degradation. (B) Phosphorylation-mediated protein stability: phosphorylation of DUSP10 by mTORC2 at serine 224 and 230 leads to its stabilization. Phosphorylation of DUSP1 by ERK at Serine 359 and 364 enhances its stability. DUSP16 phosphorylation at Serine 446 by ERK leads to its stabilization by preventing ubiquitination. ERK-induced phosphorylation of DUSP4 at Serine 386 and 391 prevents it from proteasomal degradation. Bcl2 is phosphorylated by Pyruvate kinase M2 isoform (PKM2) at Threonine 69 and prevents cullin E3 ubiquitin ligase mediated protein degradation.

Proteins ubiquitinated by E3 ligases are mostly rendered to degradation by proteasome assembly. However, polyubiquitination of a target protein may have other consequences, such as enhanced activity. For instance, as discussed above, E2F1 is stabilized by PMRT1 methylation. This is because of the cellular inhibitor of apoptosis 1 (cIAP1), an E3 ubiquitin ligase that ubiquitylates E2F1 through K63 ubiquitylation through lysine cluster 161/164 that results in its enhanced activity and stabilization [158]. PMRT5 causes KLF4 arginine methylation at 374, 376 and 377 residues, leading to its stability. VHL, a ubiquitin E3 ligase, causes ubiquitylation followed by degradation of KLF4. Similarly, Dual-specificity phosphatase (DUSP14) is methylated by PMRT5 at Arginine 17, 38 and 45. Methylated DUSP14 interacts with TRAF2, an E3 ubiquitin ligase, which binds to it and mediates K-63-linked ubiquitination on its 103rd lysine residue. This methylation and subsequent ubiquitylation lead to enhanced stability and phosphatase activity of DUSP14 in TCR signaling (Figure 6A) [156].

We have reviewed the role of individual PTM and their response with respect to ubiquitylation. However, the interplay of two or more PTMs of proteins has also been prominent in modulating the stability of proteins. As evidence, methylation of p53 by Set7/9 at K372 rendered its stabilization. This stability of methylated p53 is due to Tip60, a chromodomain-containing histone acetyltransferase that happens to recognize the methylation of lysine at 372 of p53 and leads to acetylation of other lysine residues that have propensities to be ubiquitinated thus preventing ubiquitylation [150]. In another classic case of HIFα, the crosstalk of hydroxylation, methylation, acetylation and ubiquitylation is explored by Lee et al. (2017). PHD2-mediated hydroxylation of HIFα at P402 and P564 leads to its methylation at K391 by SET9 methyltransferase. Hydroxylation of Proline in the ODDD region of HIFα is supposed to bring about a conformational change of the protein that enables SET9 to recognize the K391 methylation. Both these PTMs lead to ARD1-induced acetylation of HIFα at K532. This cascade of PTMs results in von Hippel-Lindau tumor suppressor (VHL) ubiquitin ligase-induced degradation of HIFα. All these PTMs are reversed by LSD1, which protects the HIFα from degradation (Figure 6B) [159].

6. Fate of misfolded proteins: decision & process

Protein repertoire is maintained with a balance of the rate of synthesis of new proteins and the rate of degradation of the existing ones that are no longer required by the cell. Degradation of proteins ensures the existence of only required proteins and serves as a quality control mechanism by eliminating damaged or misfolded proteins [160]. Any structural alteration that changes the conformation of a protein will subject the protein to rapid degradation [161]. Intracellular misfolded protein degradation is mainly carried out by the ubiquitin-proteasome system (UPS), which is responsible for carrying out 80% of total protein degradation, while the rest of it is taken care of by autophagy [162]. In the UPS mechanism, protein degradation is executed by the 26S proteasome. This proteasome utilizes ATP hydrolysis as an energy source to disrupt the protein structure and translocate the unfolded entity into a degradation chamber for proteolytic cleavage [163]. The proteins destined to be degraded by UPS are firstly modified by the covalent attachment of multiple, small 76 residues long ubiquitin protein molecules to the side chain of a lysine residue in the substrate protein [164]. A cascade of 3 enzymes catalyzes this modification. At first, this ubiquitin molecule is activated by the ubiquitin-activating enzyme E1. This enzyme converts the C-terminal glycine to a thioester intermediate. Once activated, this intermediate is carried about by another set of enzymes called E2 ubiquitin-conjugating enzymes to the E3 ubiquitin ligase enzyme. The target protein is already explicitly bound to the E3 enzyme. Last, the E3 ligase enzyme covalently attaches the ubiquitin molecule to the target protein. In this manner, a polyubiquitin chain is formed that serves as a marker for the proteasome assembly, thus degrading the protein [165].

When the amount of misfolded proteins goes beyond the capacity of the cell's degradation machinery, the residual misfolded proteins start making aggregates and accumulate inside the cell [161]. If misfolded proteins, tagged for degradation, anyhow become resistant to proteasome degradation due to their aggregation-forming nature or manage to escape the UPS degradation mechanism, autophagy will degrade them. It is another degradation system that relies on forming an autophagosome that ultimately fuses with a proteolytic enzyme carrying lysozyme [162]. This process of degradation of misfolded proteins can be triggered in two steps. These ubiquitin-tagged proteins are initially collected by autophagic adaptor proteins, namely NBR1 or p62. These adaptor proteins possess Ubiquitin binding domains (UBDs) that help them to bind the ubiquitinated substrate protein.

Similarly, these adaptors contain LC3 interacting region (LIR), which helps these proteins bind LC3 on autophagic vacuoles. Once these adaptors bind both substrate protein and LC3, the substrate proteins are sequestered into autophagosomes, and the lysosomal hydrolases degrade them [166]. However, if these aggregated misfolded proteins cannot be degraded immediately, then these proteins are stored in aggresomes by HDAC6. HDAC6 binds to the ubiquitin molecules chain using its UBDs. These stored aggregates are then hydrolyzed by lysosomal hydrolases [167]. A schematic diagram depicting the overall process of protein folding to unfolding to its degradation is shown in Figure 7.

Figure 7.

Fate of misfolded proteins. A schematic depiction of protein folding to its misfolding leading to proteasomal degradation and autophagy.

7. Protein stability: as a cause & cure of diseases

More than 1000 human genes have been identified where even single and more sequence modifications are directly responsible for disease. These mutations may impact protein function via numerous mechanisms, such as alterations in transcription, processing of RNA, folding and stability of the polypeptide chain, protein expression, post-translational modification, their interactions with other binding partners, and alterations to catalysis [168]. An exploration of the Human Gene Mutation Database (HGMD) shows that missense mutation is the major cause affecting more than 60% of monogenic disease mutations, affecting protein structure and leading to the impairment of protein stability. These mutations lead to the destabilization of correct fold and enhanced stabilization of misfolded proteins. Cells possess a protein quality control system (QCS) to maintain ‘proteostasis’. In other words, striking a balance between protein synthesis, folding, trafficking to its destined location, and degradation. To execute this QCS, cells deploy the chaperone molecules, the ubiquitin proteasomal degradation pathway, and autophagy [168]. So, if proteins are misfolded, they are subjected to the various chaperones inside the cell that assist in folding and aid misfolded proteins to re-attain their accurate conformation. But if misfolded proteins cannot be refolded, then the cell's degradation machinery, in other words, proteasome and autophagy, gets activated. However, when the homeostasis mechanisms of the cell cannot keep up with either of the two defense mechanisms, this leads to various severe diseases [169].

Loss of function due to misfolding proteins can lead to several lethal diseases, including cystic fibrosis, ß-Thalassemia, etc., and can make some crucial proteins, including p53, non-functional. Studies have shown that about 70% of patients suffering from cystic fibrosis have a mutation of a phenylalanine residue at the 508th CFTR protein position, leading to its premature proteolysis [168]. People suffering from ß-Thalassemia have a reduced rate of production of the beta-chain of hemoglobin. This results in premature destruction of RBC precursors and shortened life span of mature RBCs. This lethal phenomenon is attributed to the point mutations in the whole gene sequence of the beta chain, including the promoter, introns, and exons. Till now, 200 such mutations have been identified in the functional region of the beta chain [170]. In more than 50% of human cancers, the tumor suppressor protein p53, also known as the guardian of the genome, is mutated, which leads to the loss of function of p53. Hence, p53 is unable to restrict the uncontrollable growth of cancer cells [171]. Moreover, certain mutations causing proteins to misfold can lead to improper subcellular localization. A classic example of this phenomenon is α1-antitrypsin, a secreted protease inhibitor. Once mutated, the protein fails to fold correctly, and the misfolded protein thus formed is retained within the ER, which was supposed to be secreted otherwise. This misfolded protein is not degraded within the ER and starts accumulating in hepatocytes ER, leading to liver damage. Due to changes in the subcellular localization, it cannot perform its destined function: neutralize proteases for example, Neutrophil elastase, in the lung. Eventually, it damages the lungs along with the liver [169]. Furthermore, deficiency in the proteasome cellular machinery contributes to various amyloid diseases due to aggregation of misfolded proteins, the most prominent examples being neurodegenerative diseases enlisting Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and cystic fibrosis.

Hence, treatment of such diseases can be done either by rescuing the folded and stable conformation of these aggregated proteins, by enhanced degradation of such misfolded proteins so that their aggregation can be prevented or by using gene therapies to cure the mutations leading to misfolded proteins. The use of specific small molecules is doing this. These small molecules are called pharmacological chaperones; they help to rescue the misfolded proteins by shifting the equilibrium toward the folded state, regaining its function, minimizing misfolding, enhancing the degradation of misfolded proteins, and even facilitating protein folding or the proteostasis regulators (PRs) that enhance protein folding and reduce the rate of misfolding [172]. For the treatment of cystic fibrosis, CFTR modulators, including ivacaftor, lumacaftor etc., are being used in the clinics as per the mutation in the CFTR gene [173]. Similarly for Parkinson's disease, celastrol is being used [172].

Moreover, a very recent study by Malhotra et al. (2021) has shown that curcumin, which is being used in clinical trials for various cancers, helps stabilize the inactive and misfolded mutant of p53(Y220C) and restores its function in human pancreatic cancer leading to p53 mediated apoptosis [174]. Similarly, several small molecules are under trial or being used to treat protein-misfolding diseases. Targeted gene therapies are also implied to cure genetic disorders caused due to mutations and misfolded proteins. For instance, Wang et al. (2021) have employed targeted gene therapy based on lentiviral vectors that carry out the insertional mutagenesis and code for normal beta globin chain [175,176].

8. Conclusion

Proteins are the major drivers of the plethora of cellular functions. Various biochemical and biophysical factors, such as numerous interactions, pH, temperature, molecular crowding effect, PTMs, etc., influence the stability of proteins. Under the optimum conditions, the proteins attain the suitable confirmation required for their ideal functions. The epigenetic modifications also contribute to controlling the functions of various proteins such as p53, Histones, etc. The interplay between the epigenetic players and PTMs is crucial in modulating protein stability. However, the protein molecules that are misfolded and unstable undergo a series of events ranging from Hsp70-mediated refolding to ubiquitin-specific proteasomal degradation and autophagy. This highlights that a cell has devised a beautiful system that takes care of each and every aspect of protein synthesis, folding, functioning and degradation. The impairment in the protein's stability due to mutations, PTMs, etc, might result in aggregation or dysregulation of various cellular cascades or pathways associated with that protein, hence ultimately leading to diseases. A missense mutation is known to be the major cause affecting more than 60% of monogenic disease mutations affecting protein structure, leading to the impairment of protein stability. Therefore, it is essential to understand the protein evolution and cellular and epigenetic perspective of protein stability to decode the mechanism behind this 60% of diseases occurring due to poor folding of proteins under missense mutations.

9. Future perspective

Protein stability predictions are becoming indispensable in medicine for the discovery of novel immunotherapeutic agents and for drug development. Variations in the genome play a crucial role in altering protein function by enhancing or decreasing the stability of many proteins. Although the mutation occurring in the protein domain is often neutral, it may hamper protein-protein interaction, whereas the mutation occurring in the core region affects protein folding. Understanding these mutational events is paramount, which can help drug sensitivity or resistance in a particular patient for better therapeutic intervention. Such a scenario is imperative in designing novel proteins by site-directed or random mutagenesis for a better precision medicine approach. Therefore, identifying potential drivers or passenger mutation requires understanding missense mutation and mapping the genetic variations to 3D structures [177]. It has been shown that oncogenic driver mutation can stabilize an active or inactive conformation of proteins. For instance, the Leu858 driver mutation in EGFR is oncogenic in lung cancer and stabilizes the αC-helix in the active conformation, whereas the T790M mutation stabilizes the hydrophobic R-spine, destabilizing the inactive state.

Furthermore, engineering the protein that will provide more stable proteins or peptide candidates is amenable for better immunotherapeutic intervention; however, such effective candidates are lacking in clinical settings due to high cost and time consumption. Such scenarios are significant for the adoptive transfer of autologous T cells, chimeric antigen receptor (CAR), or T cell receptor (TCR) based immunotherapy approach. Therefore, a practical computational approach and logarithm need to be implemented to predict a booming immunotherapeutic target. Currently, NetMHCstab and NetMHCstabpan methods have shown promising capability in predicting the immunogenicity of peptide-MHC-I complexes [178]. In addition, stability-optimized vaccines comprising of proteins serving as antigens are instrumental in immunogenicity [179]. Exploring the thermodynamics impact on the several proteins resulting from different variations must be addressed. In the recent past, several investigations have taken the challenge in predicting the influence of amino acid variants on protein stability and assessment of free energy of its variants [180,181]. Numerous computational methods have been deployed for the prediction of protein stability of a mutant protein, such as ELASPIC, FOLDX, MAESTROweb and many more [182]. These methods compute the change in Gibbs energy upon mutation to predict protein stability. Unfortunately, the correlation between the experimental and predicted Gibbs energy change is very low. Prediction accuracy is the biggest limitation in utilizing computational approaches in predicting protein stability, which must be addressed. These approaches can serve as a guiding light for designing proteins with specified levels of stability and enzymatic activity and can be very beneficial for drug design [183]. Also, certain issues must be addressed, and effective methods should be developed for novel, precise medical approaches for genetic disorders and neurodegenerative diseases [181]. Furthermore, future studies should focus on the polymer or nano-conjugated resistant delivery methods or mutagenesis to develop novel functional enzymes with specified stability to withstand the thermal denaturation, proteolysis, and acidic or alkaline environment in the cellular compartments or body. Lastly, developing antigens with specific stability that can illicit immunogenicity and serve as vaccines is the dire need of the hour.

Funding Statement

The work was supported by CSIR Fellowship (File No. 09/006(0487)/2019-EMR-I) and grant number. 5/3/8/31/ITR-F/2022-ITR, Indian Council of Medical Research (ICMR), Govt of India.

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17501911.2024.2351788

Author contributions

N Manav searched the literature and reviewed and wrote the manuscript. BP Jit and N Manav drew the diagrams and tables. B Kataria corrected the manuscript. A Sharma formulated the idea and planned and corrected the manuscript.

Financial disclosure

The work was supported by CSIR Fellowship (File No. 09/006(0487)/2019-EMR-I) and grant number. 5/3/8/31/ITR-F/2022-ITR, Indian Council of Medical Research (ICMR), Govt of India. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.