Regulation of protein turnover by heat shock proteins.

Free Radical Biology and Medicine 77 (2014) 195–209

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Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed

Review Article

Regulation of protein turnover by heat shock proteins Perinur Bozaykut, Nesrin Kartal Ozer, Betul Karademir n Genetic and Metabolic Diseases Research and Investigation Center, Department of Biochemistry, Faculty of Medicine, Marmara University, 34854 Maltepe, Istanbul, Turkey

art ic l e i nf o

a b s t r a c t

Article history: Received 25 February 2014 Received in revised form 11 August 2014 Accepted 11 August 2014 Available online 16 September 2014

Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin–proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system. & 2014 Elsevier Inc. All rights reserved.

Keywords: Heat shock proteins Protein turnover Proteasome Autophagy Protein quality system Posttranslational modifications Free radicals

Contents Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The main structure and function of heat shock proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HSP90 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HSP70 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HSP40 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HSP100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Small HSPs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heat shock proteins in protein turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ERAD of misfolded proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lysosomal degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial protein maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proteasomal degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of protein modifications on protein turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PTMs of heat shock proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PTMs of the ubiquitin–proteasome system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PTMs of autophagy-related proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Redox regulation of heat shock proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

196 196 196 197 197 197 197 198 199 200 200 201 202 203 203 204 204

Abbreviations: 4-HNE, 4-hydroxy-2-nonenal; AMPK, AMP-dependent protein kinase; BAG-1, Bcl-2-associated athanogene-1; Bcl-2, B cell lymphoma; CaMKII, Ca2 þ / calmodulin-dependent protein kinase II; CFTR, cystic fibrosis transmembrane conductance regulator; CK-II, casein kinase II; CHIP, carboxyl terminus of HSP70-interacting protein; CMA, chaperone-mediated autophagy; c-Src, proto-oncogene tyrosine-protein kinase; eNOS, endothelial nitric oxide synthase; ER, endoplasmic reticulum; ERAD, ER-associated degradation; Hip, heat shock cognate 70-interacting protein; HO, heme oxygenase; Hop, HSP70/HSP90-organizing protein; Hsc, heat shock cognate; HSP, heat shock protein; IκBα, nuclear factor κ-light-chain polypeptide gene enhancer in B cells inhibitor α; IKK, IκB kinase; LAMP-2A, lysosomal-associated membrane protein type 2A; NAC, nascent polypeptide-associated complex; NAD, nicotinamide adenine dinucleotide; NF-κB, nuclear factor κ-light-chain enhancer of activated B cells; PARP, poly (ADP-ribose) polymerase; PDI, protein disulfide isomerase; PKA, protein kinase A; PP2A, protein phosphatase 2; PTM, posttranslational modification; PQC, protein quality control; Rpn, regulatory particle non-ATPase subunit; ROS, reactive oxygen species; SBD, substrate-binding domain; TPR, tetratricopeptide repeat; UGT, UDP-glucose: glycoprotein glucosyltransferase; UPS, ubiquitin–proteasome system. n Corresponding author. Fax: þ 90 216 421 44 30. E-mail address: [email protected] (B. Karademir). http://dx.doi.org/10.1016/j.freeradbiomed.2014.08.012 0891-5849/& 2014 Elsevier Inc. All rights reserved.

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Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

Introduction Proteins have major functions in cells such as signaling, transport, catalysis, membrane fusion, cell protection, and regulation [1]. After ribosomal synthesis, proteins have to gain their three-dimensional structure to be functional [2]. But during life, the cell is exposed to stress conditions such as heat, oxidative stress, inflammation, irradiation, and heavy metals or other toxic compounds, which can cause protein unfolding, nonspecific aggregation, and imbalance in protein homeostasis. Proteins are the molecules most sensitive to cellular stress conditions [3]. The balance between the synthesis, folding, and degradation of proteins controls the proteome function [4] and there are two main pathways to keep this balance: (i) the cellular degradation machinery, which targets proteins for proteolysis, and (ii) molecular chaperones, which prevent aggregation and ensure the folding of proteins to their native state [5]. Heat shock proteins (HSPs)1 as molecular chaperones exist in high concentrations in the cell and play crucial roles in sustaining protein homeostasis during cellular stress (Table 1) [3]. There are four large and ubiquitous, ATP-dependent families classified as HSP100, HSP90, HSP70, and HSP60. There are also ATP-independent chaperones, including small HSPs [4]. The HSP60 family members function as chaperonins to prevent aggregation by providing the correct folding of mainly mitochondrial proteins [6]. HSP70 and HSP90 bind to unfolded polypeptide sequences in the cytoplasm and induce correct folding [7]. HSP27 mediates client holding and protein folding in an ATP-independent manner [8]. The coordinated work of these chaperones is important for the efficient folding of proteins and for the balance of protein homeostasis [9]. The protein quality control system also maintains intracellular homeostasis of cellular proteins that proceed through the degradation machinery under stress conditions [3]. This quality control system includes the action of degradation mechanisms such as endoplasmic reticulum (ER)-associated degradation (ERAD), the ubiquitin–proteasome system (UPS), and autophagy-driven vacuola (lysosomal) proteolysis. In the process of 26S proteasomal degradation, small ubiquitin proteins are covalently attached to proteins, providing a degradation signal. This process requires a ubiquitinactivating enzyme (E1), a ubiquitin-conjugating enzyme (E2), a ubiquitin–protein ligase (E3), and, for some substrates, a ubiquitin chain-elongation factor (E4) [1]. The proteasomal system and molecular chaperones are closely connected and the interaction of these two systems is well regulated. Whether a protein is stabilized and refolded or is ubiquitinated and degraded by the proteasome depends on the binding of different cochaperones and also the degree of the damage [3]. Furthermore, the interaction of chaperones and proteasome is also crucial for the folding machinery of the ER. In the ER, the initiation of the ERAD process depends on whether a

Table 1 Heat shock protein (HSP) families and their functions. HSP family

Function

Small HSPs HSP40

Prevent the accumulation of aggregated proteins Substrate delivery to HSP70, targets nonnative proteins to ERAD Protein quality control and turnover Mainly responsible for cell viability, keeps proteins in folded state Solubilize aggregated proteins

HSP70 HSP90 HSP100

newly synthesized protein is functional and correctly folded [10]. In eukaryotic cells, another mechanism for protein degradation is autophagy, which is responsible for the degradation of long-lived proteins and organelles, whereas short-lived proteins are especially degraded by the ubiquitin–proteasome system [11]. In the case of a nonfunctional and misfolded protein, the binding of a molecular chaperone, such as HSP70 and HSP90, determines whether the protein can be repaired or, if not repairable, be degraded by the proteasomal system. The switch between refolding and degradation is mainly mediated by the help of cochaperones including CHIP (carboxyl terminus of HSP70-interacting protein), BAG-1 (Bcl-2-associated athanogene-1), Hip/p48 (heat shock cognate 70-interacting protein), and Hop/Sti1 (HSP-organizing protein/ stress-inducible protein), all of which influence the binding affinity of HSP70 and HSP90 [3]. In this review, chaperones and their modulators, degradation mechanisms, and their interactions in protein turnover are discussed.

The main structure and function of heat shock proteins HSPs are a highly conserved protein family that accounts for 1–2% of the total protein pool. Their expression is upregulated after exposure to stress conditions such as heat shock, excessive reactive oxygen species (ROS), drugs, or inflammation [12]. Although all the functions of HSPs are still not fully clarified, most of the HSPs act as molecular chaperones and perform essential functions in the proper folding of proteins and repair protein damage [13]. HSPs are mainly classified into two groups according to their molecular weights [3,14]. High-molecular-weight HSPs are ATPdependent chaperones, and cochaperones stabilize their ATP- or ADP-binding state, modulating their chaperone function. Different from the high-molecular-weight HSPs, small HSPs are ATP-independent. The well-studied members of this family are HSP27 and αB-crystallin [15]. HSP90 HSP90 is a highly conserved molecular chaperone family, which is expressed in many organisms from prokaryotes to eukaryotes [16]. It has been indicated that HSP90 proteins are found in the cytosol, nucleoplasm, ER, mitochondria, and chloroplast. The ubiquitous protein HSP90 is required for the viability of eukaryotes [17]. HSP90 is already present under normal conditions and its expression is further increased by 10-fold under stress conditions [18]. HSP90 contains three domains, which are the C-terminal domain, including binding sites for other cochaperones and a dimerization motif; the N-terminal domain, containing drug, nucleotide, and cochaperone binding sites; and the middle domain, which provides binding sites for client proteins and other cochaperones [19,20]. Both N and C domains have ATP-binding sites [21]. Additionally, the C-terminal domain has a calmodulin-binding site [22,23]. The middle domain coordinates proper substrate activation, because it presents a high affinity for cochaperones and client proteins, which results in discriminating between substrate types [24,25]. In contrast to other HSPs, HSP90 is responsible for keeping client proteins in a functional folded state instead of binding unfolded proteins and preventing aggregation. The client proteins consist of many protein families such as protein kinases, nuclear hormone receptors, cell surface receptors, or transcription factors [3]. HSP90


also plays a role in RNA and DNA metabolism, such as RNA transcription, RNA processing, DNA replication, DNA recombination, and DNA repair [26]. Various cochaperones affect the activity and function of HSP90 by exerting a special binding preference for different HSP90 conformations. The EEVD motifs at the C terminus of HSP90 function as binding sites for small tetratricopeptide repeat (TPR) domains. Important TPR domain-containing cochaperones are Hop/Sti1, CHIP (ubiquitin ligase), and Cyp40 (protein maturation). Hop/Sti1 serves to maintain the interaction of HSP90 and HSP70/HSP40, which is required for steroid hormone receptor stabilization, protein kinase binding, or the transfer of a client protein from HSP90 to HSP70. CHIP is another cochaperone, which has a ubiquitin E3 ligase activity on proteins that are bound on HSP90/HSP70 and thus provides for their degradation. On the other hand, Sti1 upregulation leads to the inhibition of HSP90 ATPase activity, thus keeping HSP90 in a conformation that could facilitate its interaction with its substrate. This results in the prevention of protein aggregation [15,26–28].

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to HSP70 [37]. HSP40 binds to HSP70 when it is in the ATP-bound state. It is suggested that the association of HSP40 with HSP70 results in an allosteric shift in the structure of the HSP40 protein during the delivery of a substrate from HSP40 to HSP70, which reduces the binding affinity of HSP40 to the substrate. ATP hydrolysis occurs after the binding of the J-domain of HSP70, which brings the substrate very close to the HSP70 SBD. ATP hydrolysis results in an increase in HSP70 substrate affinity, leading to the release of HSP40 from the substrate and from HSP70 [37]. In addition to the HSP70 substrate delivery, HSP40s have diverse functions, which are related to “ERdj5,” an ER-located type 3 HSP40. This type of HSP40 plays an important role in ERAD and is involved in substrate recognition and retranslocation into the cytosol. ERdj5 targets nonnative proteins for ERAD via the cleavage of disulfide bonds and thereby facilitates the retranslocation process [37]. HSP100

HSP70 Heat shock protein 70 has an active role in the assembly of newly synthesized proteins, membrane translocation of organellar and secretory proteins, refolding of misfolded and aggregated proteins, and control of the activities of regulatory proteins [29– 31]. Thus, HSP70 is crucial for protein quality control and turnover during both normal and stress conditions [32]. Regarding its various functions, HSP70 proteins are expressed in two different forms: a constitutive form (Hsc70), which maintains the normal cell function, and a stress-inducible form (HSP70/72), which mainly prevents protein damage or protein aggregation under stress conditions [33]. HSP70 proteins have an N-terminal ATPase domain and a C-terminal substrate-binding domain (SBD), which can be a β-sandwich domain or an α-helical subdomain [33]. The ATPase domain is responsible for its chaperone and folding activity. HSP70 exhibits a low affinity for the bound substrate in the ATP-bound form and thus facilitates the binding and release of the substrate. Meanwhile, the ADP-bound form has more affinity for the bound substrate; therefore, the process of binding and releasing of proteins is slower [34]. The J-domain protein HSP40 (prokaryotes: DnaJ) is responsible for the transformation of ATP to ADP. Moreover, HSP40 also maintains HSP70 substrate specificity. HSP40 functions by recognizing aromatic and aliphatic side chains and marks them as substrates for HSP70 on its SBD [33,35]. Like HSP90, HSP70 contains an EEVD domain at the C terminus that enables its interaction with HSP90, in addition to TPR domaincontaining cochaperones such as Hop/Sti1 and CHIP [36]. Two other cochaperones for HSP70, Hip (human homolog: p48) and BAG-1, interact with the N-terminal ATPase domain. Hip/p48 binding is known to stabilize the ADP state of the chaperone and thus promotes the folding activity of HSP70 by increasing its affinity to the substrate [34]. On the other hand, Hip/p48 competes with BAG-1 for binding to the N terminus. BAG-1 belongs to the BAG protein family (BAGs 1, 2, 3, 4, 5, and 6), all of which possess at least one BAG domain, which promotes HSP70 binding. The effect of BAG-1 on HSP70 is different from the effect of Hip/p48, in that the interaction of BAG-1 with the ATPase domain of HSP70 leads to the faster release of ADP and ensuing faster return of HSP70 to the ATP-bound state [34]. HSP40 HSP40 proteins are intermediates between molecular chaperones and small chaperones [37]. HSP40s are homologous to the Escherichia coli cochaperone DnaJ, and the conserved J-domain is the most important domain of HSP40, because it is responsible for recognizing/binding unfolded proteins and transferring them

The HSP100 protein family can be found in bacteria, yeast, plants, and mammals and therefore its nomenclature depends on the species: HSPH1, HSP101, HSP105, or HSP104 (eukaryotes) and CIpA, CIpB, CIpC, CIpD, CIpE, CIpX, or CIpY (bacteria) [38]. They all include an N-terminal domain that is responsible for substrate binding, two AAA nucleotide-binding domains for the 19S regulator of the 26S proteasome, a wing domain, and a small Cterminal domain [38,39]. These six subunits generate the functional hexameric HSP100 cylinder structure [39]. HSP100 is highly induced after cellular stress and thus mainly serves to solubilize aggregated proteins. Additionally, the HSP100 family interacts with HSP70 to dissolve aggregated proteins, thus translocating bound proteins after ATP hydrolysis [40]. Small HSPs These HSP family proteins differ from other families in that they (i) are ATP independent, (ii) have much smaller molecular masses (12–42 kDa), (iii) can build large oligomers, and (iv) contain a conserved α-crystallin domain [41,42]. Small HSPs have a less conserved structure than high-molecular-weight HSPs except for the α-crystallin domain, and they are expressed in many different organisms. Humans possess 10 representatives of the small HSP family [42]. HSP27 and αB-crystallin are ubiquitous proteins induced in response to various stress conditions and their main function is to prevent the accumulation of aggregated proteins [15]. Their activity is controlled by the dynamic organization of HSP27 and αBcrystallin oligomers and the oligomerization state depends on the phosphorylation of proteins [15]. These large oligomers are responsible for the storage of unfolded or misfolded proteins. In addition, the catalytic activity of 26S proteasome is increased by HSP27, thus inducing the degradation of ubiquitinated proteins under stress stimuli [15]. Different from HSP70, HSP27 possesses higher affinity for long ubiquitin chains than for monoubiquitin and directly interacts with the 19S proteasome subunit [43]. HSP27 induces the ubiquitination of proteins such as p27kip1 and the transcription factor GATA-1 [44]. HSP27 might act as an E4 factor, which cooperates with E3 ubiquitin ligase for the ubiquitination of proteins [15]. HSP27 could indirectly affect apoptosis by the degradation of IκBα and the consequent stimulation of the death regulatory protein NF-κB [15]. A study showed that the cytoprotective effect of HSP27 was decreased in dominant-negative NF-κB mutants, which demonstrates the crucial role of HSP27 in the NF-κB-related apoptotic pathway [45]. On the other hand, the ubiquitin–proteasome system itself plays a major role in the modulation of apoptosis, which was correlated with HSP27. The proteasome inhibitor bortezomib was shown to induce apoptosis via the expression of the HSP27 protein

198


mechanism that maintains protein homeostasis, which is also known as proteostasis [1]. Recently it has been shown that HSPs not only facilitate the folding process of newly synthesized polypeptides and the refolding process of functional proteins that are damaged in cellular stresses, but they also cooperate with the degradation mechanism of misfolded proteins [55]. In addition, whether a protein will be folded or degraded is determined by the protein quality control mechanism, which is a network of HSPs and degradation systems. The involvement of heat shock proteins in protein quality control is summarized in Fig. 1. Different classes of HSPs are involved in protein quality control with several functions. HSP70 targets proteins for degradation when the protein cannot be properly renatured, in addition to folding nascent proteins and refolding denatured proteins [14]. This role of HSP70/Hsc70 in protein degradation is cooperative with the cochaperones CHIP and BAG-1 [14] (Fig. 2). Because CHIP possesses a ubiquitin ligase E3 activity, it captures unfolded proteins and catalyzes the ubiquitination [56,57]. CHIP's structure ensures the cooperation of HSPs with the proteasome. On one hand, its functional domain TPR interacts with other TPR-containing proteins, such as HSP70 or HSP90. On the other hand, its “U-box” domain interacts with the UBCH5 family of E2 ubiquitin-conjugating enzymes, which is responsible for CHIP's ability to ubiquitinate and target proteins to the proteasome system [58–60]. CHIP also cooperates with the S5a component of the 19S proteasome subunit during the delivery of ubiquitinated proteins [56]. Several proteins were shown to be ubiquitinated with the assistance of CHIP, among

[46]. HSP27 phosphorylation leads to the disaggregation of HSP27 multimers, which may affect the cellular localization of the chaperone and its targets. Therefore, phosphorylated HSP27 can be found in both the nucleus and the cytosol, inducing the degradation of both cytosolic and nuclear proteins [15].

Heat shock proteins in protein turnover The final conformation and folding of the polypeptide chain are related to the protein amino acid sequence, which has already started during protein synthesis in the ribosome [2]. Protein folding is a thermodynamically favored process that requires high energy from the action of heat shock proteins. ATP hydrolysis is coupled with HSP-induced protein folding. HSPs engage in folding by recognizing hydrophobic amino acids of unfolded and incompletely folded proteins and thus preventing protein aggregation [47–49]. Even though folding is assisted by the action of HSPs, several mistakes may occur in the process [1]. Specifically, stress conditions can disrupt protein folding and result in protein misfolding. Because misfolded proteins do not have the proper structure, aggregation is inevitable for them [50,51]. In humans protein aggregation is important because it leads to several diseases such as Alzheimer disease, Parkinson disease, Huntington disease, Creutzfeldt–Jakob disease, or type 2 diabetes. In addition, aging and cancer are connected to protein misfolding and aggregate formation [51–54]. For these reasons, the cell has a strong protein quality control

Folded protein Hip Hsp 70

Hsp 40

Hop Hsp 104

Hsp 90

Hsp 70

Resolubilization

Hsp 70 Hsp 40

Aggregates

BAG-1

Hsp 70

Ub

CHIP

Ub

E2

E2

26S proteasome

ER

Cell stress Oligo peptides Misfolded protein

CMA Lamp-2a

Ub

E2 KFERQ CHIP

BAG-1 Hsc 70 Hsp 40

E3

Oxidative stress

Hip Hop Hsp 90

Lysosome

mtHsps Mitochondria

Fig. 1. The involvement of heat shock proteins in the protein quality system. Whether nonfunctional and misfolded proteins will be repaired or degraded is determined by the binding of molecular chaperones HSP70 and HSP90. The switch between refolding and degradation is mainly mediated by the help of cochaperones including CHIP, BAG-1, Hip/p48, and Hop/Sti1. Intracellular homeostasis depends on the removal of misfolded proteins through degradation, which is also regulated by heat shock proteins. These quality control systems include degradation mechanisms such as ERAD, the UPS, CMA, and mitochondrial protein maintenance.


Hsp 40

Hsp 40

Hsp 70

Hip Promotes folding

Hop

199

Hsp 70

BAG-1

CHIP Recruits the proteasome

Recruits Hsp90

Protein folding

Mediates ubiquitination

Protein degradation

Fig. 2. HSP70 chaperone system for the folding and degradation of proteins. The binding of different cochaperones determines the fate of the unfolded/misfolded protein. Hip and BAG-1 bind to the ATPase domain and Hop and CHIP bind to the C terminus of HSP70. (A) In the folding machinery, Hop recruits HSP90 and Hip promotes folding. (B) CHIP mediates ubiquitination and BAG-1 recruits the proteasome in the degradation machinery.

which are the cystic fibrosis transmembrane conductance regulator (CFTR) and the glucocorticoid receptor [56,61]. Furthermore, CHIP has a role in the lysosomal degradation of target proteins [62]. There are two pathways related to lysosomal degradation known as CMA (chaperone-mediated autophagy) and CASA (chaperone-assisted selective autophagy) [63]. CMA is activated in response to nutritional and cellular stress conditions under which specific cytosolic proteins are directed to lysosomal degradation. In this pathway, the Hsc70associated cochaperone CHIP leads proteins to the lysosome by recognizing a motif related to the pentapeptide KFERQ [64]. Meanwhile, CASA is another lysosomal degradation machinery mostly involved in the maintenance of a protein assembly called the Z disk. The Z disk is required for actin anchoring in striated muscles and Hsc70 forms a complex with the small HSP22 (HSPB8) and CHIP to assist in the degradation of Z-disk components [65]. On the other hand, BAG-1 has the ability to interact with the antiapoptotic protein B cell lymphoma 2 (Bcl-2) to bind the ATPase domain of HSP70 [66]. Moreover, BAG-1 also possesses a ubiquitin-like domain interacting with the 19S subunit of the proteasome and the 20S core proteasome [67]. BAG-1 and CHIP also interact with each other, and their coexpression enhances the degradation of client proteins [68]. The nascent polypeptide-associated complex (NAC) is responsible for cotranslational protein folding [69,70]. The interaction of this heterodimeric complex with nascent polypeptides results in the prevention of incorrect interactions. Moreover, NAC also interacts with the ribosome-associated HSP70/HSP40-chaperone system comprising a RAC complex (HSP70 chaperone Ssz1, HSP40 chaperone Zuo1) and the HSP70 chaperone Ssb1 [70–72]. Recently, it has been shown that the accumulation of aggregation-prone proteins referred to as poly-Q proteins is prevented by the colocalization of these folding mediators [73]. On the other hand, misfolded cytosolic proteins are stabilized after the loss of HSP70 [74,75], leading to their sequestration into insoluble inclusions [76]. The HSP70–HSP40 system can dissolve small aggregates by itself but larger aggregates need the cooperation of HSP104, an AAA-ATPase chaperone of the HSP100 family [77,78]. Different from the HSP70 mechanism, HSP104 is responsible only for the disaggregation of denatured proteins [39,78,79] and therefore directly functions in the protein turnover process. In concert with HSP40 and HSP70, HSP104 can reactivate proteins that have been denatured and are thus prone to aggregation where other chaperones are ineffective [39]. In fact, HSP104 is crucial to preventing the accumulation of aggregates when the amount of misfolded proteins exceeds the folding capacity of other chaperones [39]. HSP90 has various roles in the conformational regulation of many signaling proteins [80–82]. Studies have shown that HSP90 inhibition resulted in the reversal of transformation and

differentiation and of apoptosis in cancer cell lines [83–85]. The role of HSP90 in the cancer process is related to HSP90 client proteins including Bcr-Abl, FLT3, and ErbB2 [86–88]. HSP90 inhibition induced apoptotic cell death correlated with mitochondrial pathways accompanied by the cytosolic accumulation of cytochrome c and SMAC/Diablo [89]. Because HSP90 is responsible for the stability of many HSP90 client proteins, an impairment of HSP90 chaperoning or treatment with HSP90 inhibitors causes inefficient folding of these client proteins. Furthermore, these unfolded/misfolded client proteins are ubiquitinated by an E3 ligase, leading to their degradation by proteasome [90], which can be ensured through CHIP and also HSP70 [91,92]. Another mechanism is accepted to be an indirect pathway that includes the HSP90induced transfer of client proteins to HSP70 and CHIP-related ubiquitination and proteasomal degradation [61,93]. However, CHIP is not the only mediator for ubiquitination of HSP90 client proteins, and other E3 ligases are also involved [94]. Geldanamycin administration for HSP90 inhibition resulted in a complex formation consisting of the E3 ubiquitin–protein ligase Triad3A, receptorinteracting protein 1, and HSP90 [95]. ERAD of misfolded proteins In a cell, 30% of the total proteins are involved in the secretory pathway composed of the ER, Golgi apparatus, lysosome, plasma membrane, and exterior of the cell [1]. These proteins are imported to the secretory pathway mainly from the ER by an aqueous channel named Sec61, which is part of a large multiprotein complex. Protein import consists of many different pathways that are cotranslational and posttranslational [96–98]. The proteins have to be folded in the ER to be functional before they are imported. The ER has proteins that are responsible for folding, including chaperones, cochaperones, oxidoreductases, glycan-modifying enzymes, and lectins [1]. The basic steps of the ERAD process are substrate recognition, retrotranslocation across the ER membrane to the cytoplasm, polyubiquitination, recognition, and degradation by the proteasome [99]. Several protein modifications occur, such as cleavage of the signal sequences, glycosylation, disulfide bond formation, or lipid conjugation, during entry into the ER and the folding process [100,101]. The first action of a cell against stress-induced accumulation of unfolded/misfolded proteins in the ER is the unfolded protein response (UPR). The UPR functions by decreasing the translation and import of secretory proteins into the ER and increasing the activities of chaperones and folding supportive proteins. In addition, increase in the ER volume by transcriptional upregulation of the ERAD machinery is crucial for the degradation of misfolded proteins [102–104]. The ERAD pathway prevents the aggregation of unfolded proteins, which results in

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protein precipitation, because the aforesaid may bind to other proteins and disturb their action. These altered protein conformations may also be secreted to form extracellular amyloids. As a result, the ERAD pathway is very important because it rescues cells from various diseases linked with protein aggregates [105,106]. The ER is not responsible for the degradation of misfolded proteins, however, which are instead degraded by the cytoplasmic UPS [107,108]. It was astonishing to find out first in yeast that an ER-imported, fully glycosylated mutant protein, which was thought to be transferred to the vacuole, was retrotranslocated out of the ER back into the cytoplasm, polyubiquitinated, and degraded by the proteasome [109]. Other studies have also revealed that retrograde transport and degradation in the cytoplasm are a general mechanism to eliminate unfolded proteins of ER [110,111]. Folding in the ER is accompanied by HSP70, HSP40, and chaperone-like proteins as well as lectins [112–114]. Glucoseregulated protein of 78 kDa (GRP78), the ER homolog of HSP70, recognizes the hydrophobic patches of nascent proteins and determines the folding status of proteins in the ER [115]. GRP78 includes a KDEL ER-retention signal in addition to ATPase and peptide binding domains of HSP70 [116]. Under normal conditions, GRP78 is inactive when bound to the activating transcription factor-6 the inositol-requiring transmembrane kinase/endoribonuclease 1, and the double-stranded RNA-activated protein kinaselike eukaryotic initiation 2 kinase [117]. GRP78 performs its folding activity through the N-linked carbohydrate on the proteins consisting of triple-branched trees of Glc3–Man9–GlcNAc2 named the “glyco-code” [118]. The proteins gain their final and proper folding conformation with the assistance of the HSP70 system during the cleavage of three glucose residues by glucosidases I and II. If proper folding still fails in mammalian cells, calnexin and calreticulin-associated oxidoreductase ERp57 binds to the polypeptide and a folding sensor, the UDP-glucose:glycoprotein glucosyltransferase (UGT), reglucosylates the Man9 residue, leading to another round of folding. All together, calnexin and calreticulin, UGT1, and glucosidases ensure the proper folding of proteins in the ER [101,118]. GRP94, the ER homolog of HSP90, is accepted as involved in ERAD by its interaction with other chaperones but its clear role has still not been elucidated [119]. Lysosomal degradation Under normal conditions, autophagy is responsible for the removal of damaged and/or nonfunctional organelles and proteins, and it provides amino acids and essential constituents for the synthesis of new functional intracellular components [120,121]. Under stress conditions such as nutritional deprivation, oxidative stress, toxic compounds, and host invasion by pathogen agents, autophagy is induced to remove damaged intracellular components [122]. Autophagy is required during life because it is essential for cell differentiation and tissue remodeling, and it also has a role in cellular survival and the cell death process [123]. Autophagy is carried out by various mechanisms that occur in lysosomes [122]. Although different autophagy forms have common features, in mammals three main forms of autophagy have been identified: macroautophagy, microautophagy, and CMA [122]. Different from other autophagy forms, CMA exhibits selective degradation of soluble proteins in lysosomes and does not require vesicle formation or major changes in the lysosomal membrane. In this process, target proteins are able to cross the lysosomal membrane directly to reach the lumen, where they are degraded [122]. A pentapeptide protein motif on the target proteins named KFERQ is first recognized by a complex including Hsc70 and its cochaperones in the cytoplasm. And then, the target protein interacts with a lysosomal receptor on the lysosomal membrane named lysosomal-associated membrane protein type 2A (LAMP-2A) and is translocated across the membrane into the

lysosomal lumen with the help of the lysosomal chaperone [122]. Because the substrates are transported across the lysosomal membrane and not engulfed into a vesicle, CMA is a proper mechanism for the degradation of soluble proteins but not of organelles. The selectivity and direct substrate translocation properties of CMA determine its roles under various physiological and pathological conditions [122]. According to the selectivity of the lysosomal system, formerly it was not considered to function for the removal of oxidized proteins in contrast to the proteasomal system. For this reason, early studies of oxidative stress and the lysosomal system focused on the contribution of this organelle to oxidation-induced damage, because the lysosomal compartment is destabilized during exposure to severe oxidative conditions [124,125]. Later on, several studies suggested that CMA has a role in the removal of oxidized proteins, because it was shown that (1) CMA activity decreased in aged cells, which led to enhancement of oxidized proteins [126], (2) CMA was activated during toxic exposure, which resulted in selective degradation [127], and (3) the CMA degradation of IκB was decreased after treatment with antioxidants [128,129]. Although the mechanism is not fully elucidated, it is considered to be related to the recognition of substrates by chaperones following oxidative stress-related conformational changes. Moreover, increased de novo synthesis of LAMP-2A and increased levels of Hsc70 and HSP90 were found to be related to the removal of oxidized proteins via CMA [129]. Hsc70 is involved in the unfolding of the substrate protein required for its transport into the lysosome [122]. As already mentioned, ATP hydrolysis promotes binding to the substrate and these ATP/ADP binding cycles are modulated by various cochaperones such as HSP40, Hip, Hop, and BAG-1 [130]. HSP40 assists the hydrolysis of ATP into ADP to facilitate substrate protein and Hsc70 binding, and Hip is responsible for the stabilization of the substrate/Hsc70 complex by avoiding the binding of the new ATP molecule. In contrast, Hop and BAG-1 promote the release of the substrate by providing ATP binding. In this process, HSP90 binds to the unfolded region of a substrate to prevent the aggregation of the substrate protein during binding/release cycles. Hop promotes the interaction of HSP90 and Hsc70 by acting as a bridge. In summary, for the transport of the substrate protein to the lysosome in the CMA process, it is important to ensure that the interaction site of Hsc70 with the substrates is free, the nucleotides ATP and ADP are adequate for binding/release cycles, and there are chaperone/cochaperone interactions [130]. Mitochondrial protein maintenance The mitochondrion has diverse functions such as ATP production, iron/sulfur cluster biogenesis, calcium buffering, or the regulation of apoptosis. On the other hand, the mitochondrion is an important source of ROS, which cause DNA damage and the production of nonfunctional proteins. Therefore cells should have an effective mitochondrial protein quality control system to overcome oxidative stress-related mistakes [1]. Mitochondrial protein quality control mechanisms (PQCs) consist of chaperones and proteases that facilitate the repair and/or removal of damaged proteins [131]. In fact, chaperones are truly significant, because a nascent polypeptide chain in the cytosol to be transported into the mitochondrion has to bind a cytosolic chaperone to keep its unfolded conformation. Furthermore, the mitochondrion is surrounded by a double membrane; thus, the matrix proteins are not directly transferred to the cytoplasm or to the UPS. The translocation of the proteins between the outer and the inner mitochondrial membranes is mediated by protein complexes that cooperate with mitochondrial chaperones [132]. HSP70 and HSP90 chaperones (mtHsp70, Ssc1 in yeast) with their cochaperones are responsible for the translocation and folding of newly


synthesized mitochondrial proteins and for the prevention of protein aggregation in the cytosol [133]. These chaperones recognize and bind to the hydrophobic segments of polypeptides to keep them in an unfolded conformation and protect them from aggregation. They also provide the translocation of polypeptides by interacting with TPR motifs of mitochondrial surface receptors [133,134]. Both matrix and the intermembrane space have their own chaperones to assist in the folding of translocated precursor proteins. Newly arrived nascent polypeptides and also stress-induced denatured proteins interact first with mtHsp70 to get their functional conformation. HSP70 binds to the hydrophobic segments of the substrate protein for the correct folding of nascent polypeptides [135]. In addition, the chaperonin HSP60 and the cochaperonin HSP10 are responsible for the folding of substrates [136]. HSP60 interacts with the substrate protein after it is released from mtHsp70 [137] and it has been shown that an impairment of HSP60 function results in the accumulation of protein aggregates in the matrix [138]. In addition to HSP60 and HSP10, HSP104 also stimulates disaggregase activity. Meanwhile, the LON protease is responsible for the final degradation of misfolded and damaged mitochondrial matrix proteins [139,140]. Severe stress conditions may cause the denaturation of mitochondrial proteins that exceed the capacity of chaperone repair systems. Because inner mitochondrial membrane proteins are close to respiratory chain complexes, which are responsible for ROS generation, they are more likely to be damaged by oxidative stress and to form aggregates [141]. For this reason, many cell types contain special quality control systems located in the inner membrane consisting of specialized chaperones. These chaperones are in the heat shock protein 100/caseinolytic peptidase subfamily of the AAA þ (ATPases associated with various cellular activities) protein family and they are responsible for the reactivation of the aggregates and are essential for thermotolerance. They are known to cooperate with HSP70 in catalyzing protein unfolding, disassembly, and disaggregation in bacteria, plants, and fungi [39,142]. On the other hand, it has been indicated that the UPS plays a role in mitochondrial protein quality control with two different mechanisms. The first mechanism is related to a mitochondrial adaptor protein complex, which extracts and retrotranslocates damaged mitochondrial proteins to the cytosol for proteasomal degradation under oxidative stress conditions [143]. The second mechanism is a direct action of proteasome: the recruitment of the proteasome to the mitochondria degrades previously ubiquitinated substrates [144]. In a study, proteasome inhibition resulted in the stabilization of the F1F0-ATPase subunit oligomycin sensitivity-conferring protein in a ubiquitinated form in the outer mitochondrial membrane, which shows similarity to the retrotranslocatian system in ERAD [145]. Furthermore, the UPS also controls mitochondrial maintenance by a form of autophagosomal degradation called mitophagy, which mediates the degradation of the whole organelle [146,147]. A mammalian ubiquitin ligase, parkin, cooperating with another kinase called PINK, is recruited to the damaged mitochondria, where it ubiquitinates proteins of the outer mitochondrial membrane, which facilitates fusion/fission events leading to mitophagy [148,149]. In addition, another mammalian E3 ligase, mitol, which is located in the outer mitochondrial membrane, ubiquitinates the proteins and therefore leads to mitochondrial fission events [150]. Early studies on HSP90 inhibitors indicated that HSP90 inhibition resulted in increased mitochondrial protein expression and mitochondrial dysfunction [151]. Furthermore, it was also related to defective protein folding in the endoplasmic reticulum, which showed that a mitochondrial unfolded protein response may play a role in the apoptotic effects of HSP90 and proteasome inhibitors [152]. HSP90 inhibitors also induce mitochondrial proliferation, which results in the accumulation of mitochondrial proteins as an early apoptotic event in cells [151,153].

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Proteasomal degradation The ubiquitin-related proteasomal system consists of the 26S multicatalytic protease, which is made of two 19S regulatory particles and the 20S core particle. The 19S regulatory particle has various functions, such as binding polyubiquitinated proteins, providing an entrance into the core particle for substrates, and unfolding the substrates, whereas the 20S core particle is a barrel-shaped complex that possesses all the proteolytic activity and selectively degrades proteins [154]. The proteolytic activities of the proteasome result in the generation of small-sized peptides ranging from 3 to 22 residues, and 19S particles liberate intact ubiquitins for the subsequent rounds of degradation [155]. The 19S regulators with proteases, unfoldases, and ATPases, which introduce the denatured protein to the 20S proteasome for degradation, are responsible for the ubiquitination and unfolding of the bound protein substrate [156]. The proteasome exists in multiple forms and also degrades other substrates [157] in an ATP/ubiquitin-independent manner and its activity is modulated by multiple regulators such as PA28αβ in the cytoplasm [158] and poly (ADP-ribose) polymerase (PARP) [159] and/or PA28γ [160] in the nucleus. The tagging of substrates with the 76-amino-acid polypeptide ubiquitin is achieved by the action of E1, E2, and E3 ubiquitinactivating, -conjugating, and -targeting enzymes [161] at the expense of energy in the form of ATP. Ubiquitin-activating enzymes (E1) have cysteine residues in their active sites, which allow the formation of an energy-rich thioester bond with the glycine residue of ubiquitin. Afterward, ubiquitin ligases (E3) mediate the transfer of the ubiquitin residue to a ubiquitin-conjugating enzyme, and ubiquitin is linked to the lysine side chains of the protein to generate an isopeptide bond. With this process, the proteasome recognizes its substrates and degrades ubiquitin-labeled proteins [162,163] (Fig. 3). In addition to lysine, ubiquitination also occurs on cysteine, serine, and threonine residues as a signal for proteasomal degradation [162]. Various in vivo and in vitro studies have shown that the proteasomal system is responsible for the degradation of oxidized proteins [164–166], whereby the oxidized proteins are degraded by the 20S proteasome without ubiquitination, via the recognition of hydrophobic patches directly by the proteasome [167]. However, a study of iron-mediated oxidation indicated that oxidized proteins are targeted for the proteasomal system after ubiquitination by a ubiquitin ligase (E3) [168]. Studies with mutated proteasome have indicated the main role of the ubiquitin–proteasome system in the removal of misfolded proteins: degradation rates and the accumulation of ubiquitinated proteins were considerably decreased in response to the induction of protein misfolding by canavanine [169]. Grune and co-workers [170,171] provided evidence that not the 26S proteasome but the 20S core proteasome, which dissociated from the 26S proteasome, is mostly responsible for the degradation of oxidized intracellular proteins. The activation of the nuclear 20S proteasome by PARP [171] leads to a significant increase in the proteolytic capacity, preventing the aggregation and accumulation of oxidized proteins [172]. In contrast, 26S proteasome impairment would cause serious problems in cellular metabolism [173] because the ubiquitin–26S proteasome system affects many processes such as transcription, translation, and turnover of perhaps 100 shock/ stress proteins [156]. Eventually, it becomes clear that when the 26S proteasome is reconstituted, the 20S proteasome is actively synthesized de novo to maintain the removal of oxidized proteins [156]. Like the 26S proteasome, HSP90 requires ATP for its in vivo functions [174]. Both HSP90 and proteasome systems are known to play roles in the degradation of proteins. On one hand, HSP90 may inhibit proteasome degradation: early studies showed that HSP90 acts as an inhibitor of the Z-Leu-Leu-Leu-MCA degrading activity of the proteasome [175]. On the other hand, several studies have shown that HSP90 inhibition results in increased proteasomal degradation [3]. The reason for increased ubiquitin-dependent

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Hsp 40

Substrate

Hsp 70

Native folded proteins

Hsp 70

Hsp 40

RNA Unfolded / Misfolded protein

Ribosome

Substrate

E3 Polyubiquitin

α subunit

19 S Ubiquitination

26S proteasome

Β subunit

20 S Β subunit α subunit E2 Ubiquitin E1

Peptides

Fig. 3. The roles of HSP40 and HSP70 in the degradation process of unfolded/misfolded proteins by 26S proteasome. (A) After synthesis, native folded proteins may be damaged by several factors and therefore denatured. These denatured proteins take unfolded or misfolded forms, which bind HSP40 and HSP70 onto their structure. Further binding of Hip and Hop proteins (Fig. 2) targets the substrate to the proteasome. The degradation of substrate protein by the 26S proteasome requires the ubiquitin chain attachment. This ubiquitination process is catalyzed by E1, E2, and E3 enzymes, which activate and ligate the ubiquitin. (B) 26S proteasome is composed of a 20S core particle and 19S regulators, which are responsible for the catalytic activities and entrance of ubiquitinated substrates, respectively.

proteasomal degradation after HSP90 inhibition may be explained by the fact that HSP90 facilitates the stabilization of client proteins. Therefore, HSP90 inhibition by geldanamycin and other derivatives results in destabilization, leading to the ubiquitination and proteasomal degradation of the client proteins [176]. This process was also demonstrated by a study on the CFTR protein [177]. Meanwhile, Imai et al. [178] showed that nearly complete dissociation of 26S proteasome into its components is caused by in vivo inactivation of HSP90 in yeast. This dissociation was demonstrated to be reversed in an HSP90-dependent manner. This process requires ATP hydrolysis. In addition, information about the genetic link between HSP90 and several proteasomal Rpn (regulatory particle non-ATPase subunit) genes confirms the importance of HSP90 in the integrity of the 26S proteasome [178]. Other studies have shown that the inhibition of HSP90 resulted in an increase in HSP70 expression in most cancer cells. It is also known that inhibition of CHIP results in decreased HSP90 client protein degradation, whereas CHIP overexpression leads to an enhancement in their degradation [179], which indicates that the degradation of HSP90 client proteins may be mediated by the HSP70–CHIP system [3]. This was proven by the experiments performed with the leucine-rich repeat kinase-2 protein, which is the most common cause of the development of late-onset Parkinson disease [180]. In contrast to HSP90 inhibition, HSP70 impairment results in a decrease in proteasomal degradation, leading to protein aggregation [179]: in the case of a decrease in the activity of HSP90, client proteins are stabilized by HSP70, ubiquitinated by CHIP, and degraded by the proteasome. Thus, HSP70 inhibition results in the accumulation of client protein

aggregates. In addition, HSP70 inhibition leads to a decrease in the interaction between CHIP and HSP90, which affects the folding or the stabilization of HSP90 client proteins [179,181]. Finally, whether a misfolded protein will be degraded by the proteasomal or autophagic system is determined through the ubiquitination process [1]. Some E3 ligases are responsible for the ubiquitination of nascent polypeptides. On the other hand, the E3 ligase Not4, as component of the CCR4/Not complex, is essential for mRNA integrity and has a role in the cotranslational protein quality control process [182]. Proteasomal or autophagic degradation may be determined also by the E2 ubiquitin ligases CHIP or parkin and E3 ligases, which can attach either to K48 or to K63 ubiquitin chains, respectively. Substrates that have K48 chains are targeted for proteasomal degradation, whereas substrates with K63 chains are degraded through autophagy [183,184]. As a conclusion, it is clear that all parts of the protein quality system work in collaboration and one part of the protein quality control system affects the other parts in a direct or indirect manner.

Effects of protein modifications on protein turnover Posttranslational modifications (PTMs), including phosphorylation, ubiquitination, nitrosylation, and oxidation, are the specific chemical modifications of proteins after their synthesis and they regulate protein stability, distribution, and function [185]. PTMs have an important role in the activity of a protein or its molecular interaction network and constitute a tool for regulating protein function. PTMs regulate the PQC system by affecting either misfolded


proteins or the various components of the PQC machinery, including chaperones, the UPS, and autophagy. Proteomics studies have made it possible to detect PTMs on multiple subunits of the same protein complex under a given condition; however, molecular pathways that modulate these PTMs and the significance of the PTMs need to be studied in more detail [185]. PTMs of heat shock proteins As mentioned above, HSPs are involved in protein turnover via folding or unfolding processes and assembly or disassembly of larger macromolecular complexes, and they contribute to degradation systems. Advances in proteomics techniques have revealed that HSP70, HSP90, and HSP27 are modulated by several PTMs, including acetylation, phosphorylation, ubiquitination, methylation, and nitrosylation [185]. Acetylation is a posttranslational modification in which an acetyl group is transferred from donor acetyl-coenzyme A to a target protein, a process that is catalyzed by histone acetyltransferase. The acetylation of target proteins has been shown to control protein turnover and may have various effects. Cotranslational N-terminal acetylation targets the protein mostly for degradation, whereas posttranslational lysine acetylation may stabilize the substrate protein or, in contrast, may lead to degradation [186]. Various enzymes such as p300, CBP, PCAF, and TAF1 have both acetyltransferase and ubiquitin-activating/conjugating or ligase activities. Histone deacetylase complexes, which are coimmunoprecipitated with HSP70, induce the reversal of acetylation [187]. The HSP70 chaperone, which requires ATP hydrolysis for its activity [188], also interacts directly with histone deacetylase 2 and is necessary for histone deacetylasedependent hypertrophy [189]. The acetylation of HSP90 occurs mostly on lysine residues, which disturbs the interaction of cochaperone and client proteins. For example Lys294 acetylation inhibits the binding of the cochaperones and client proteins of HSP90 [190,191]. In addition, HSP90 acetylation is also known to decrease ATP binding, which affects the substrate protein binding [192]. Phosphorylation is another well-known PTM in the cell, in which a phosphate group is hydrolyzed from ATP and covalently linked to the hydroxyl group of serine, threonine, or tyrosine on the target protein. Several studies have implicated the significance of phosphorylation for the HSP70 chaperone family [185]. On the other hand, the phosphorylation of HSP90 has diverse effects on client interactions depending on the client protein and the phosphorylation site [193]. Many different phosphorylation sites have been recognized in HSP90. For example, phosphorylation at S225 and S254 in HSP90 results in reduced affinity for the aryl hydrocarbon receptor [194]; on the other hand, the binding of eNOS needs c-Src phosphorylation at Y300 [195]. It has been indicated that, when HSP90 is hyperphosphorylated, interaction with its client kinase p60v-src is decreased. In addition, dephosphorylation of HSP90 by its cochaperone PP5/Ppt1 modulates its chaperone activity in a positive manner in vivo [196]. The phosphorylation of HSP90 from tyrosine residue affects its interaction depending on the client proteins. For example, tyrosine phosphorylation in HSP90, which is induced by statins, enhances the interaction of eNOS with HSP90 [197]. These data indicate that the phosphorylation of specific residues in HSP90 regulates its interaction with client proteins and also enables a mechanism to modulate the chaperoning of distinct client proteins. HSP22, HSP27, and αB-crystallin are also regulated by phosphorylation. αB-crystallin, which functions as a chaperone for desmin and maybe for myofibrillar proteins [198], is phosphorylated at three different residues (Ser19, Ser45, and Ser59) under stress conditions. A phospho-mutant αB-crystallin S19A, S45A, S59E that mimics the phosphorylation of αB-crystallin [199] results in osmotic and ischemic stresses leading to the stabilization of mitochondrial membrane potential, in vitro [200]. Additionally, HSP27 was shown

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to be phosphorylated at serine residues in ischemia models, and phospho-mutant transgenic mice were found to be protected against ischemia–reperfusion injury [201]. Therefore, phosphorylation may affect both the chaperone function and the cardioprotective actions of αB-crystallin and HSP27. Ubiquitination is an essential modification for proteasomal degradation catalyzed by E1, E2, E3, and E4 enzymes and mostly occurs on the lysine residues of the substrate protein [202]. Oxidative stress has been demonstrated to affect the ubiquitination of HSP90. A study showed that tubocapsenolide A treatment causes the thiol oxidation of HSP90 [203], which is linked with the proteasome-dependent degradation of the HSP90 clients, including Cdk4, cyclin D1, Raf-1, Akt, and mutant p53. This study clearly indicated that the thiol oxidation of HSP90 inhibits its chaperone activity [203]. Oxidative stress also causes lipid peroxidation, which results in the accumulation of thiol-reactive aldehyde 4-hydroxy-2-nonenal (4-HNE). 4-HNE causes the oxidation of HSP90 on Cys572, inhibiting its chaperoning activity [204]. It was demonstrated that a photodynamic inhibitor, hypericin, increases the ubiquitination of HSP90, which leads to the inhibition of the HSP90 chaperone function [205]. Protein methylation is the transfer of a methyl group from a methyl donor such as S-adenosylmethionine to the substrate protein. This modification mostly occurs on lysine and arginine residues, and also on histidine, glutamine, asparagine, glutamic acid, aspartic acid, and cysteine residues [206]. It has been shown that HSP70 chaperones are methylated at both lysine and arginine residues [207], and the pattern of methylation can also change under several conditions such as cellular proliferation [208]. Studies have shown that lysine methyltransferases modulate the interaction of HSP90α with both TPR-containing domains [209]. The HSP90 chaperone was methylated at K209 and K615 in vitro, a process that was antagonized by the HSP90 cofactor Hop, and the methylation of K615 was further shown to be reversible by a demethylase [210]. It has been demonstrated that the methylation of K615 is high in striated muscle tissue, in which the TPR-containing domain and Hsp90 interact with the myofilament protein titin, a component of striated muscle tissue. This interaction depends on the methylation of HSP90 by its associated methyltransferase. Thus, HSP90 methylation is important in the formation of sarcomeric structures during muscle development [185]. S-nitrosylation is another modification that modulates chaperone function in which the thiol side of the cysteine residue is covalently linked to nitric oxide, forming a thionitrite group [211]. This modification seems not to be catalyzed by an enzyme, but rather occurs as a consequence of changes in the redox state of the cell. Among a subset of cellular proteins, HSP90α is nitrosylated spontaneously [193]. It was previously known that phosphorylation of HSP90 enhances eNOS function, and recently the S-nitrosylation of HSP90 at C597 has also been shown to affect eNOS activation. The nitrosylated site resides within a region that is important for interaction with eNOS, but this modification results in an impairment of HSP90 ATPase activity and inhibits eNOS function [185,193].

PTMs of the ubiquitin–proteasome system The proteasome activity is modulated by PTMs at several levels. As previously stated, ubiquitination is essential for the targeting of substrate proteins for proteasomal degradation. Moreover, for many substrate proteins, other PTMs are required to induce ubiquitination. PTMs also have an effect on the process of ubiquitination by acting on ubiquitin ligases, and finally PTMs modulate the assembly and activities of the proteasome [212]. Several types of PTMs such as phosphorylation, acetylation, sumoylation, ubiquitination, modification by hydroxy-2-nonenal, oxidation, glycosylation, poly(ADP

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ribosylation), O-GlcNAc modification, nitrosylation, and N-myristoylation have been identified [213]. Ubiquitination modulates the stability, localization, and function of a substrate protein, which depend on the length and topology of the ubiquitin moieties [212]. Many lysine residues (K6, K11, K27, K29, K33, K48, K63) may be used to form polyubiquitin chains but the most commonly used are K48 and K63 residues. Ubiquitination on different residues may have diverse roles. For example, K48-linked polyubiquitination leads to the proteasomal degradation of target proteins, whereas K63-linked polyubiquitination modulates the subcellular distribution, partner protein binding, and activity of the modified protein [188]. A specific example of the latter is the membrane translocation and activation of Akt protein induced by K63-linked polyubiquitination [214]. In contrast to polyubiquitination, monoubiquitination mostly does not trigger degradation but rather leads to nonproteolytic processes such as signal transduction and transcriptional regulation. The ubiquitination starts with a signal in a substrate protein named degron and the direct interaction of this substrate with an active specific E3 ligase. PTMs of substrates are required to trigger ubiquitination as well as E3 ligase. A misfolded or damaged protein may undergo ubiquitination as a consequence of hydrophobicity, a buried degron, or the generation of a new degron [215]. The ubiquitination of ubiquitin ligases such as CHIP promotes E3 ligase activity [216]. The ubiquitination of proteasome subunits also regulates proteasome activities. The polyubiquitination of Rpn10 results in the degradation of Rpn10, and the monoubiquitination of Rpn10 at K84 reduces the proteolytic function of the proteasome [217]. However, α1, α2, α4, and Rpt3 and Rpt5 subunits have been shown to be modulated by oxidative modification, which leads to the impairment of proteasome activities [218]. Ubiquitination can be regulated by other PTMs, mainly by phosphorylation, which operates in at least three different ways [212]. Phosphodegron formation in the substrate induces its recognition by the E3 ligase, which brings the substrate and the E3 ligase to the same subcellular location. Additionally, the phosphorylation of E3 modulates its ligase activity. Many different subunits of the 20S proteasome have been shown to be phosphorylated at specific amino acid residues [219]. The phosphorylation of 20S subunits is regulated by PKA/PP2A [220], and the Rpt6 phosphorylation at the S120 site is catalyzed by PKA/CaMKII. Such modification has been shown to increase proteasomal activities [221,222]. Additionally, the phosphorylation of the α7 subunit at S243 and S250, regulated by CK-II, has been shown to enhance the interaction of 19S and 20S [223]. UPS substrates are also modulated by phosphorylation. One such example is IκB phosphorylation by IKK, which forms a phosphodegron and regulates its proteasomal degradation [199]. Other examples are Chk1 phosphorylation by the serine/threonine-protein kinase ATR, which exposes the degron for ubiquitination [224], and p27Kip1 phosphorylation, which results in translocation to the cytoplasm for degradation [225]. PTMs of autophagy-related proteins Autophagy is also modulated by PTMs in addition to HSPs and the proteasomal system [212]. Phosphorylation by AMP-dependent protein kinase (AMPK), which activates macroautophagy, is stimulated by low ATP or cell stress. AMPK phosphorylates tuberous sclerosis complex-2 and raptor, leading to the inhibition of mTORC1 [226], which stimulates autophagosome synthesis. AMPK also phosphorylates ULK1 on serine residues [227,228], and ULK1 (ATG1) forms a kinase complex with ATG13, ATG101, and FIP200, which leads to the activation of macroautophagy under nutritional deficiency. In addition, AMPK phosphorylation stimulates the nuclear accumulation of FOXO3, which results in an enhancement of macroautophagic gene expression [229].

ATG5, which is an essential component of macroautophagy, conjugates to ATG12, participating in the maturation of the autophagic isolation membrane [212]. ATG7 is a key regulator of autophagosome formation; it has a role in both the conjugation of ATG12 with ATG5 and the addition of phosphatidylethanolamine to ATG8 (LC3) [230]. ATG7 has an important role in stimulating macroautophagy and thus prevents the accumulation of misfolded proteins and aggregates. ATG7 is acetylated by the p300 acetyltransferase under conditions of starvation, which results in the suppression of autophagy [231]. In addition, p300 acetylates ATG4, ATG8 (LC3), and ATG12, which is related to decreased autophagosome content [231]. Several studies have shown that NAD þ also mediates the acetylation of ATG7, ATG5, and ATG8 (LC3), which can be deacetylated by SIRT1 [232]. SIRT1 is an NAD-dependent deacetylase that is upregulated under starvation conditions [233]. SIRT1 also plays a role in the FOXO-dependent expression of macroautophagy genes by deacetylating FOXO proteins [234]. SIRT1 deacetylation of FOXO1 was shown to enhance autophagosome levels in glucose-deprived cardiomyocytes [235]. Beclin-1 is a member of the phosphoinositide 3-kinase complex of autophagosome nucleation, which promotes the formation of isolation membranes [236]. Several studies have been performed to define the role of beclin-1 dependent macroautophagy in PQC, and beclin-1 depression has been shown to result in an increase in ubiquitinated proteins [237]. Beclin-1 is also known to interact with Bcl-2 and Bcl inhibits beclin-1-dependent macroautophagy by sequestering beclin-1 away from the autophagy nucleation complex [238,239]. Bcl-2 is regulated by phosphorylation, and Bcl-2 phosphorylation on Thr69, Ser70, and Ser87 residues by c-Jun N-terminal kinase disrupts Bcl-2/Beclin-1 interaction, leading to the induction of autophagy [240]. High-mobility-group box 1 also promotes the phosphorylation of Bcl-2 by extracellular signal-regulated kinase, activating starvation-induced macroautophagy [241]. Another study indicated that beclin-1 phosphorylation at Thr119, which is regulated by death-associated protein kinase 1, resulted in the dislodging of Bcl-2 and thus the activation of autophagy [242].

Redox regulation of heat shock proteins In addition to PTMs, redox regulation of heat shock proteins may be an important factor for their role in protein turnover. Redox regulation is described as the cellular responses at the gene and protein levels induced by reactive oxygen/nitrogen species [243]. Regulation of molecular chaperones by oxidative stress is tightly controlled via transcription factors named heat shock factors (HSFs). Among HSFs, HSF1 is responsible for the control of cellular responses to stress conditions [244,245]. Under normal conditions, HSF1 is in its inactive form and it is bound to chaperones/cochaperones such as HSP90, HSP70, or HSP40 [246]. When oxidative stress increases, chaperones bind to the unfolded proteins, which results in the liberation of HSF1. The phosphorylated and trimerized active form of HSF1 translocates to the nucleus where it regulates the transcriptional activity of chaperones [246]. Redox regulation may affect the activity of HSF1 at the posttranslational level by oxidizing two cysteine residues within HSF1's DNA-binding domain [247]. Several studies showed that several oxidants may affect the HSP70 protein levels. Cytokine- and also sodium nitroprussideinduced nitrosative stress was shown to stimulate HSP70 protein synthesis. In addition, the oxidant and antioxidant balance, which is maintained through glutathione status, and the antioxidant enzymes regulate the molecular mechanisms related to the NOinduced activation of the heat shock response [248-250]. H2O2 was shown to activate the HSP70 promoter via enhanced binding of STATs to cognate binding sites in the promoter and to stimulate HSP70 expression in a time-dependent manner [251]. Flavonoids


as antioxidant compounds inhibited the induction of HSP70, suggesting the role of the redox system in HSP70 induction [252]. HSP90 is another target chaperone protein shown to be affected by redox status. In a study, tubocapsenolide A (TA) treatment caused an increase in ROS and a decrease in glutathione levels in MDA-MB-231 breast cancer cells. TA also inhibited the chaperone activity of HSP90 by oxidizing thiol residues in its structure, and also HSP90 client proteins including cyclin D1, Akt, Cdk4, Raf-1, and mutant p53 were degraded by the proteasome [253]. Accumulation of the thiol-reactive aldehyde 4-HNE as a lipid peroxidation product was shown to oxidize Cys572 of HSP90 and inhibit its chaperoning activity [204]. HSP32 or heme oxygenase (HO) plays a role in the production of bilirubin as a rate-limiting enzyme [254]. Although HSP32 is not mentioned in previous parts, here we focus on it regarding its highly redox-regulated situation. HSP32 contains four cysteine residues that coordinate zinc under reducing conditions and these cysteines are highly responsive to changes in intracellular redox potential [255]. In a study, H2O2 treatment was shown to oxidize cysteine sulfhydryls, which form two intramolecular disulfide bridges that cause the release of the coordinated zinc [255,256]. In addition, there are three isoforms of HO, which are HO-1, an inducible form; HO-2, a constitutive form; and the recently discovered HO-3 [257,258]. HO-1 has a specialty compared to other chaperone proteins because it contains an antioxidant response element in its promoter region. Oxidative/nitrosative stress as well as glutathione depletion upregulate HO-1 rapidly [259]. Additionally, metal-chelating compounds and antioxidant molecules modulate HO-1 expression [254]. Protein disulfide isomerase (PDI) has a critical role in oxidative folding of the endoplasmic reticulum and extracellular proteins. Redox regulation of this protein induces a release of the compact form of the molecule and exposes the shielded hydrophobic areas, facilitating its chaperoning activity [260]. Furthermore, cigarette smoke was shown to increase the complex formation between PDI and client proteins [261].

Concluding remarks Protein quality control, which is mainly obtained by the regulation of protein turnover, is an important process for organisms. Although the recognition, delivery, and degradation processes of proteins seem simple and well organized, diverse degradation pathways and regulators lead to complexity. When investigated in detail, heat shock proteins have been found to play crucial roles in the process in addition to other chaperones, cochaperones, ubiquitin-related proteins, and many others. On the other hand, stress-related misfolding/unfolding of proteins is controlled by different factors, which makes the degradation procedure more complicated. Therefore it is important to detail the interactions of heat shock proteins and related cofactors in protein turnover, which include protein modifications, and there is still need for further studies in this area. The detailed understanding of this mechanism is not only of scientific interest; it might lead to new therapeutic approaches for protein turnover-related diseases. References [1] Amm, I.; Sommer, T.; Wolf, D. H. Protein quality control and elimination of protein waste: the role of the ubiquitin–proteasome system. Biochim. Biophys. Acta 1843:182–196; 2014. [2] Bartlett, A. I.; Radford, S. E. An expanding arsenal of experimental methods yields an explosion of insights into protein folding mechanisms. Nat. Struct. Mol. Biol. 16:582–588; 2009. [3] Kastle, M.; Grune, T. Interactions of the proteasomal system with chaperones: protein triage and protein quality control. Prog. Mol. Biol. Transl. Sci 109:113–160; 2012.

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Regulation of apoptosis by heat shock proteins.

Heat shock increases turnover of 90 kDa heat shock protein phosphate groups in HeLa cells.

Modulation of Alloimmunity by Heat Shock Proteins.

Heat Shock Protein (HSP) Drug Discovery and Development: Targeting Heat Shock Proteins in Disease.

Regulation of heat shock protein synthesis in rat astrocytes.

Heat shock proteins bind calcitonin.

Transcriptional regulation of heat shock proteins and ascorbate peroxidase by CtHsfA2b from African bermudagrass conferring heat tolerance in Arabidopsis.

Heat shock gene regulation by nascent polypeptides and denatured proteins: hsp70 as a potential autoregulatory factor.

Induction of heat-shock proteins by glutamine. The 'feeding effect'.

Transcription regulation of HYPK by Heat Shock Factor 1.

Regulation of bacterial heat shock stimulons.

Unraveling regulation of the small heat shock proteins by the heat shock factor HvHsfB2c in barley: its implications in drought stress response and seed development.

Heat Shock Protein HSP101 Affects the Release of Ribosomal Protein mRNAs for Recovery after Heat Shock.

Bacterial Heat Shock Protein Activity.

Diabetes and heat shock protein.

Heat-shock proteins: immunity and autoimmunity.

Mycobacterial heat-shock proteins as carrier molecules.

Heat shock proteins and cell proliferation.

Heat shock, stress proteins, chaperones, and proteotoxicity.

Heat shock proteins in host-parasite interactions.

Effect of heat shock on expression of proteins not involved in the heat-shock regulon.

Heat shock factor 2 is associated with the occurrence of lung cancer by enhancing the expression of heat shock proteins.

Are heat shock proteins involved in autoimmunity?

Heat shock proteins: friend and foe?