1 Introduction

In 1972, Christian B. Anfinsen, along with Stanford Moore and William H. Stein, received the Nobel Prize in Chemistry for research on the folding (i.e., formation of the spatial structure) of ribonuclease A, an enzyme responsible for the degradation of RNA, one of the nucleic acids. This research concerned the relationship between the activity of a protein and its amino acid sequence and conformation (spatial structure). The outstanding American biochemist proved that a denatured ribonuclease, a ribonuclease with an altered spatial structure, may return to its appropriate conformation and maintain its enzymatic activity. Anfinsen’s studies have led to what is referred to as the thermodynamic hypothesis, also known as Anfinsen’s dogma, which states that the amino acid sequence of a protein defines its spatial structure. Anfinsen’s dogma postulates that in particular conditions needed for proteins to fold, depending, among other requirements, on temperature, solvent composition, and solvent concentration, the native structure of a protein (i.e., the spatial structure that allows the protein to perform its biological functions) is unique and stable. It, however, may be lost due to protein denaturation (Anfinsen, 1973).

Studies focusing on protein folding that have been carried out over many years have confirmed this dogma. However, many exceptions have been discovered that do not meet the criteria of the dogma, e.g., prions and other proteins involved in the formation of pathogenic aggregates (precipitated ‘agglomerations’ of abnormal proteins) that are related to the development of various neurodegenerative disorders, such as, Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. Furthermore, it has been discovered that, during folding, many polypeptides require the presence of other assisting proteins, called chaperones, to reach their native structure. One of the tasks that chaperones carry out is preventing proteins from aggregating (‘sticking together’) before reaching their native state (Cox et al., 2020; Hetz & Mollereau, 2014; Higuchi-Sanabria et al., 2018).

How does a protein ‘know’ how to change a linear sequence of amino acids into a functional structure with a specific spatial orientation? How long does it take for a protein to attain native conformation? These questions have already given biochemists many sleepless nights over the past several decades. The rising interest in protein folding inspired a researcher named Cyrus Levinthal to carry out a thought experiment, now commonly known as Levinthal’s paradox. The paradox relates to a considerable discrepancy between the theoretical time needed for a protein to be folded correctly (determined using a mathematical model) and the real-time for a protein to reach its native conformation (Levinthal, 1969). Assuming that the process takes place through a random sampling of all possible conformations and estimating it using a mathematical model, we can calculate the theoretical amount of time that a protein requires to attain its stable native conformation. The total theoretical time is longer than the supposed age of the Universe! The real-time of folding ranges from milliseconds to seconds. This is because folding is not a linear process; it results from a progressing stabilization of transition forms (Rooman et al., 2002). Protein folding is described with a physical model depicting the concept of minimizing free energy, a theory known as the folding funnel (Dill & Chan, 1997; Sali et al., 1994).

2 Protein: Structure and Functions

Proteins are key components of cells. Alongside nucleic acids, carbohydrates and lipids, they belong to one of the main four classes of macromolecules that constitute the biochemical basis of life. Structurally speaking, proteins are also the most complex and diverse macromolecules. They can be thought of as the ‘driving force’ of each organism. Their importance is proven by the fact that they directly or indirectly participate in all biological processes (Berg et al., 2002).

The spatial structure of a protein, i.e., protein conformation, depends on the arrangement of amino acids in a polypeptide chain and the chain’s manner of folding. A few organization levels can be distinguished in the structure of a protein: primary, secondary, tertiary and quaternary (Figure 5.1). It should be emphasized that despite a strictly determined spatial structure, proteins maintain relative structural flexibility. Thanks to this, they can perform biological functions requiring, for example, local deformations that are necessary for the binding of another molecule (Berg et al., 2002).

3 Proteome: The Protein Pool of a Cell

The set of proteins inside every cell, the proteome, is constantly changing. Maintaining biological equilibrium (homeostasis) requires precise control of the processes that take part in the protein cycle, e.g., the synthesis and degradation of proteins. The state of this equilibrium can be regarded as dynamic. The total pool of proteins at the disposal of a cell may change, for instance, in response to different external signals or due to an illness. These changes cause a new equilibrium to be established (Hinkson & Elias, 2011).

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The types of protein structure. Proteins have four levels of organization: primary, secondary, tertiary, and quaternary structures. The primary structure is a linear sequence of amino acids. The secondary structure refers to three-dimensional forms like alpha-helices or beta-sheets, shaped by weak interactions like hydrogen bonds. The tertiary structure describes the folding of secondary elements into a spatial arrangement through interactions such as hydrogen bonds and disulfide bridges. Quaternary structure arises when multiple folded polypeptide chains combine, though not all proteins have this level

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Proteostasis, i.e., protein homeostasis, is the state of equilibrium of the proteome. It encompasses mechanisms responsible for both stabilizing proteins in physiological and stress conditions (mostly involving the participation of chaperone proteins) as well as the mechanisms responsible for the degradation of proteins (involving specialized structures, such as proteasomes and lysosomes, which also engage various chaperone proteins). The protein quality control system (PQC) is responsible for maintaining the integrity of the proteome. The inability to maintain proteostasis causes disorders involving an excessive production, degradation, and accumulation of proteins with an incorrect conformation (Powers & Gierasch, 2021).

4 HSP s: The Basic Elements of the Protein Quality Control System

In 1962, an Italian geneticist, Ferruccio Ritossa, discovered that raising the body temperature of a common fruit fly (Drosophila melanogaster) increases the synthesis of numerous proteins. The result of this observation was the very first identification of heat shock proteins (HSP s, in 1974), the name of which refers to the circumstances surrounding their discovery (Ritossa, 1996). Because of their function, they are called assisting proteins or chaperones, and they are ‘molecular guardians’ for the cell. HSP s are a family of proteins produced by cells in response to exposure to stress conditions. In non-stress conditions, these proteins take part in the regulation of fundamental cellular functions, namely housekeeping. HSP s were first described in the context of the thermal shock that increased their synthesis. However, as it was soon discovered, they may be synthesized through other stress factors, such as free radicals, UV radiation, infections, inflammation and hypoxia (Dubrez et al., 2020; Kregel, 2002; Niforou et al., 2014).

The most popular classification of the family of HSP s was conducted based on their mass, expressed in kilodaltons (kDa). According to this criterion, the family can be divided into six main classes: small heat shock proteins (sHSP s), with a mass between 15 and 30 kDa (Jagla et al., 2018), and HSP40, HSP60, HSP70, HSP90 and HSP100, the names of which denote that the mass of the proteins from these classes equals 40, 60, 70, 90 and 100 kDa, respectively. Cells are usually equipped with many representatives of each class of chaperones (Kampinga et al., 2009).

Chaperones usually work in cycles that involve binding and releasing a substrate, i.e., a protein requiring ‘guardianship’. They are regulated by various cofactors, i.e., chemical substances supporting the functioning of proteins. The characteristic feature of most representatives of the HSP family is their activity involving the hydrolysis of high-energy bonds present in adenosine triphosphate (ATP), the substance that ‘fuels’ most life processes. Thanks to this activity, HSP s obtain the energy necessary to perform their main function, namely, taking part in the folding of other proteins (Martin Vabulas et al., 2010). For example, HSP70 (Figure 5.2) is switched between two states in its cycle: a closed state, with a high affinity for a protein ‘client’, and an open state, with a low affinity for a protein substrate. The ‘client’ may be an unfolded or partially folded polypeptide, and the nucleotide exchange factor (NEF) is a cofactor. The NEF takes part in the exchange of adenosine diphosphate (ADP), i.e., ATP after it has been used to obtain energy (Rosenzweig et al., 2019). The HSP70 cycle aims to form the correct structure of proteins belonging to the fast-folding proteins. Polypeptides, which require more time to attain native conformation, use an additional folding system based on HSP60 or HSP90 (Figure 5.3). For them, the HSP70 cycle is a safe transition state, which ensures conditions that prevent potentially toxic aggregation (Saibil, 2013).

Systems based on proteins from the HSP family do not act independently. Instead, they are interconnected and assisted by other proteins that also belong to the family of chaperone proteins. For example, a protein substrate is delivered to the HSP70 system through a protein belonging to the HSP40 class (Rosenzweig et al., 2019). In turn, the repair of damaged polypeptides requires collaboration between the HSP70 and HSP100 or sHSP s systems, depending on the type of damage (Barends et al., 2010; Dubińska-Magiera et al., 2014).

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A simplified diagram of the HSP70 protein’s reaction cycle. A. HSP70 binds the substracte, an unfolded or misfolded polypeptide, delivered by an HSP40 chaperone. HSP70 remins in an open state. B. After binding the substrate, HSP40 is released, and ATP is hydrolyzed, providing energy for HSP70 to transition to a closed state. C. The HSP70-ADP-substrate complex binds to a nucleotide exchange factor (NEF), which facilitates the release of ADP and its replacement with ATP, causing HSP70 to return to an open state. D. The folded substrate and NEF are released, allowing the cycle to begin again

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5 The Life Cycle of a Protein

The life cycle of a protein begins with the expression of the gene that encodes the protein, while the degradation of the protein marks the endpoint of its cycle (Figure 5.4). Gene expression is understood as reading the information contained in a gene and transcribing it first into mRNA (messenger RNA) and then translating it into a protein. Other processes connected with this cycle also encompass folding and transport to cellular compartments and the modification of a protein involving, for example, the addition of a chemical group or the addition of another protein. The appropriate time coordination of these processes is carried out by the elements of the PQC system, primarily the proteins of the HSP family. It is of fundamental importance for the functioning of a cell (Berg et al., 2002).

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A simplified diagram of the network of HSP family proteins, the main component of the protein quality control system (PQC). Heat shock proteins (HSP s) are produced by all organisms to maintain proteostasis, with expression levels increased by stress factors. Their main function is to assist in the folding of newly synthesized and denatured polypeptides. HSP70, the primary protein in folding system, collaborates with other chaperone systems like HSP60 and HSP90, mediated by specialized factors not shown in the diagram. Repair systems, such as HSP100, remove protein aggregates and help refold damaged proteins. Small heat shock proteins (s HSP s) prevent irreversible aggregation, directing proteins to repair pathways. Black arrows show protein folding, while red arrows indicate pathways for unfolding, repair, and aggregation triggered by stress factors

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Translation involves the synthesis of a protein on a ribosome, which decodes information hidden in mRNA and, based on this information, adds specific amino acids, one after another, creating a linear, unbranched polypeptide chain. Folding begins during translation and primarily concerns the formation of secondary structures (Berg et al., 2002; Buchan & Stansfield, 2007). The majority of chaperone proteins that assist other polypeptides in assembling or disassembling their chains mainly function during the post-translational stages of the protein life cycle. However, specific chaperones begin their work immediately after a sufficiently long fragment of a new polypeptide chain; usually, longer than 60 amino acids, leaves a ribosome. Their involvement mainly concerns promoting interactions between amino acids and between the atoms of a peptide bond. The task of these and many other chaperones also involves binding to, and shielding, hydrophobic amino acids exposed on the surface of the produced protein (Martin Vabulas et al., 2010).

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A simplified diagram of a protein life cycle. During its life cycle, a protein undergoes various processes encompassing the translation of mRNA on a ribosome, folding assisted by chaperones, transport to cellular compartments (such as e.g., a mitochondrion), post-translational modifications and degradation (solid lines). However, under stress conditions (e.g., increased temperature), additional scenarios are possible, e.g., denaturation (the loss of native conformation) and then either repair by re-folding, aggregation, or degradation (dashed lines). The diagram does not include all the aspects of the functioning of chaperone systems and possible degradation pathways. For the sake of clarity, this diagram does not include all PQC systems and the other possible pathways of protein degradation

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It should be emphasized that the duration of the life cycles of various proteins may be different. Some proteins are physiologically short-lived molecules, and their half-life is measured in minutes. They include regulatory proteins, e.g., p53, which is a transcription factor with the properties of a tumour suppressor. These proteins are rapidly released and rapidly degraded, which is significant for the regulation of key processes, including transcription, translation and cell growth. In turn, other proteins are characterized by a much longer half-life, expressed in days, for instance, the structural proteins of muscles, including myosin and actin. Others, such as collagen and elastin, the proteins responsible for skin elasticity or crystallin, a protein present in the lens of the eye, have their half-lives expressed in years (Glickman & Ciechanover, 2002; Toyama & Hetzer, 2012).

6 Folding

Protein folding is a physical process that involves the folding of a polypeptide chain. As a result of this process, the protein gains a highly organized, stable spatial structure. The correct spatial structure of each protein determines its functions (Berg et al., 2002). It should be emphasized that folding is a complex process prone to errors. Disturbances during the adaptation of the correct spatial structure by a protein usually lead to the synthesis of a molecule with altered properties. These, in extreme cases, might be toxic for the organism and cause, for example, neurodegenerative diseases resulting from the accumulation of incorrect polypeptides (Berg et al., 2002).

Despite the fact that folding is determined mainly by the primary structure of a protein, the process depends on many different factors, such as the presence of water in the environment where folding occurs (cytosol in a cell). Water causes hydrophobic amino acids to display a tendency to ‘hide’ inside the synthesized protein; in turn, hydrophilic amino acids are located on the protein’s surface, where they can interact with surface molecules. As a result, the polypeptide produced is very unstable and requires additional help to arrive at and maintain its native conformation. For many proteins, the requirement for the correct folding of a polypeptide chain is the presence of chaperone proteins, which assist in this process. The folding times of different proteins vary. Usually, small proteins attain their three-dimensional structure very quickly. However, larger molecules require more time and additional assistance from chaperones to reach their native conformation (Hartl & Hayer-Hartl, 2009).

7 Aggregation: Disastrous Consequences for the Cell

The relative structural flexibility of proteins in performing their biological functions also causes their main weakness. Due to various stress factors (e.g., oxidative stress, high temperatures, chemical substances), proteins can easily lose their native conformation (the polypeptide chain gets unfolded). Therefore, proteins also lose their physical, chemical and biological properties. As a result, this leads to the formation of protein aggregates in the cell. An essential and simultaneously dangerous (when speaking of the maintenance of proteostasis) feature of incorrectly folded proteins is their ability to disturb polypeptides with correct conformation. This may lead to the elimination of theoretically functional proteins from the total pool of the proteome by ‘trapping’ them in toxic aggregates (Ross & Poirier, 2004). Considering the fact that the cellular environment is very ‘crowded’, it seems justified that a cell decidedly tries to avoid the accumulation of incorrectly folded proteins with a tendency for aggregation. An excess of protein aggregates in the cell is toxic and may lead to cell death and even the death of the entire organism. In humans, over 30 diseases associated with protein aggregation have now been described (Ciechanover & Kwon, 2017). These include neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease, familial amyloid polyneuropathy and amyotrophic lateral sclerosis. Aggregates created by incorrect proteins may be deposited both inside and outside the cell. The latter is often observed in patients with advanced neurodegenerative disorders (Ulrich Hartl, 2017).

The first and most straightforward method of defence against the formation of toxic deposits is the prevention of undesirable aggregation. This is why organisms have developed a complex network of molecular chaperones. Such a network can ‘catch’ the aggregation-prone transition forms that are created during protein folding and unfolding. Chaperones, including the HSP70 protein can recognize proteins, which due to the unfolding of the polypeptide chain, expose previously ‘hidden’ hydrophobic amino acids. In the case of failure, organisms have another line of defence that, apart from proteolytic degradation, uses a system based on HSP100 and allows aggregates to be reactivated without the destruction of proteins (Figure 5.3). The HSP100 system temporarily dissolves deactivated proteins through collaboration with the system based on HSP70 (Barends et al., 2010).

Even though cells have developed effective mechanisms that are responsible for both the repair and the removal of damaged proteins, these mechanisms may sometimes fail, e.g., during prolonged stress conditions. In such cases, an option to protect the integrity of the proteome is the sequestration (i.e., capturing and isolating) of inappropriately folded proteins in the spatially separated structures. ‘Capturing’ incorrectly folded proteins prevents their harmful interference in the biogenesis of newly synthesized polypeptides, which are susceptible to disturbances, thus ‘maintaining the order’ in the cellular environment (Escusa-Toret et al., 2013; Sontag et al., 2014).

8 Machinery Responsible for the Degradation of Proteins

The machinery responsible for the degradation of proteins encompasses a few different components. The elimination of proteins often results from a cell’s response to stress stimuli and leads to reduced protein content in a cell and the release of amino acids. These amino acids may be used again by cells, e.g., during translation (Figure 5.4). Thus, another protein’s life cycle, administered by chaperones, may begin. Substrates intended for degradation include, for example, unnecessary proteins that have already fulfilled their biological functions, non-functional proteins, inappropriately folded or unfolded proteins, and protein aggregates. Their degradation occurs with the use of specialized cellular structures, such as proteasomes and lysosomes, which perform the function of ‘molecular shredders’. Degradation may occur in several ways, including proteasomal degradation, ER-associated degradation and lysosomal degradation (Ciechanover & Kwon, 2017; Lemberg & Strisovsky, 2021; Soto & Estrada, 2008).

The ubiquitin-proteasome system (UPS) plays a key role in regulating the protein cycle in eukaryotic organisms and thus supports intracellular homeostasis. Its main role involves eliminating incorrectly folded and damaged polypeptides (Figure 5.4) (Toyama & Hetzer, 2012). It is the main system for the degradation of most proteins, particularly proteins with a short half-life (Lilienbaum, 2013). For the protein to be degraded via this system, the polypeptide must be ubiquitylated, which involves attaching ubiquitin molecules to this protein to be recognized by a proteasome (a protein complex with a cylindrical structure comprising many subunits) (Hartl & Hayer-Hartl, 2009). Many structurally related enzymes are involved in the ubiquitylation process, which are specific to various cellular compartments, for instance, the cell nucleus. These enzymes recognize and selectively ubiquitylate particular proteins, the so-called substrates (Glickman & Ciechanover, 2002; Natoli & Chiocca, 2008; Varshavsky, 2012).

Lysosomal degradation encompasses various pathways and involves the degradation of macromolecules in lysosomes, i.e., specialized structures containing numerous hydrolytic enzymes. One such pathway is chaperone-mediated autophagy (CMA) (Figure 5.4) (Kaushik & Cuervo, 2018; Kettern et al., 2010). The CMA is mostly activated as a result of oxidative stress. This pathway contributes to the degradation of soluble proteins, which are equipped with a specific protein motif. This motif is recognized by the heat shock cognate protein 70 (Hsc70), a chaperone that, along with its partners, transports the protein ‘client’ to the surface of a lysosome. The complex binds to a lysosomal membrane receptor, which allows the complex to be transported inside the lysosome, where the protein substrate is degraded (Bandyopadhyay et al., 2008).

9 PQC System: Collaboration between Components

Eukaryotic cells are divided into various regions that specialize in particular cellular processes. These regions include the cytoplasm, cell nucleus, endoplasmic reticulum, mitochondria and others. For instance, translation, i.e., the process of translating the information contained in a genome into a protein sequence, takes place in the cytoplasm, while other processes related to storing this information and making it available to the cell at the appropriate time occur in the nucleus.

Each cellular compartment also has specific and individual elements of the PQC system (Goldberg, 2003). However, it should be emphasized that despite spatial separation, these systems collaborate closely with one another (Haynes & Ron, 2010; Shibata & Morimoto, 2014). This phenomenon can be easily illustrated using the cell nucleus as an example. The cell nucleus constitutes the isolated but not hermetically closed, ‘command centre’ of a cell. Due to its physical separation, the nuclear proteome is to a certain extent protected from changes resulting from the conformation disorders of cytoplasmic proteins. The machinery responsible for the protection of the cell’s nucleus comprises the components of nuclear PQC (Foresti et al., 2014; Khmelinskii et al., 2014), which form this permanent equipment of this compartment, and also contain some elements of PQC systems that are characteristic of other cellular compartments (Shibata & Morimoto, 2014).

Protein folding disorders are identified by ‘detectors’ that are specific to the cellular compartment in which they occur. Apart from functioning autonomously, these systems can extend their influence outside the cell, protecting the entire organism from the harmful consequences of proteotoxic stress. The signals that warn against stress may be sent by the cells of other tissues, which can thus participate in protecting other cells located in remote regions of the organism. For instance, stress induced in neurons can protect muscle cells from the consequences of chronic proteotoxicity (Taylor et al., 2014).

The aging of an organism leads to the insufficient functioning of the PQC systems and, consequently, the inappropriately folded or entirely unfolded proteins and the formation of intracellular and extracellular toxic deposits containing ‘clustered’ proteins and aggregates (Koga et al., 2011). These deposits are manifested in many age-related disorders, such as Alzheimer’s disease, Parkinson’s disease and cataracts (Ciechanover & Kwon, 2017; Moreau & King, 2012).

10 Conclusions

The Greek origin of the word proteostasis contains the notion of stasis. However, ‘stasis’ is not used here in the sense of invariability. The term can be considered to mean the dynamic maintenance of equilibrium, i.e., harmony. A cell can maintain its integrity (appearing to remain unchanged) through the numerous perfectly collaborating elements that react to various stimuli from the constantly changing environment. Under the guise of stasis, a fierce fight for the maintenance of equilibrium takes place. To deal with this challenge, cells have to be flexible and systematically verify the quantity and quality of their components, including proteins. This is why keeping the proteome’s composition of correctly folded proteins, which are present at a precise time and place inside the cell, is so important for maintaining proteostasis. Broadly defined, stress involves a disturbance to the equilibrium and shifts the internal conditions away from the optimal state. In other words, it causes the loss of harmony. The maintenance of proteostasis is ensured by a complex system of biological pathways where components such as heat shock proteins play the leading role. These proteins supervise the quality and circulation of all polypeptides present in the cell. Thanks to the fact that all of these ‘instruments’ are perfectly tuned up, the melody of life can be heard in this ‘orchestra’.