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Medical implications of understanding the functions of human small heat shock proteins Expert Rev. Proteomics 12(3), 295–308 (2015)

Evgeny V Mymrikov and Martin Haslbeck* Department Chemie, Technische Universita¨t Mu¨nchen, D-85747 Garching, Germany *Author for correspondence: Tel.: +49 089 289 13367 [email protected]

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that are implicated in a variety of diseases. Upon stress, they stabilize unfolding proteins and prevent them from aggregating. However, under physiological conditions without severe stress, some sHsps interact with other proteins. In a perspective view, their ability to bind specific client proteins might allow them to fine-tune the availability of the client for other, client-dependent cellular processes. Additionally, some sHsps seem to interact with specific co-chaperones. These co-chaperones are usually part of large protein machineries that are functionally modulated upon sHsps interaction. Finally, secreted human sHsps seem to interact with receptor proteins, potentially as signal molecules transmitting the stress status from one cell to another. This review focuses on the mechanistic description of these different binding modes for human sHsps and how this might help to understand and modulate the function of sHsps in the context of disease. KEYWORDS: molecular chaperone . protein aggregation . protein folding . protein homeostasis . protein–protein interaction

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small heat shock protein

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a-crystallin

Functional properties of human sHsps

Small heat shock proteins (sHsps) are ATPindependent molecular chaperones that form a first line of defense in protein homeostasis within the cell. They were among the first proteins detected as part of the cellular stress response, a network of proteins which evolved to protect cells against diverse harmful conditions (e.g., heat, cold and oxidative stress) [1,2]. Molecular chaperones are the most prominent group of these stress proteins. According to their molecular mass and evolutionary history, they comprise several families that cooperate in balancing protein homeostasis [1]. sHsps are present in all three domains of life and are the most omnipresent molecular chaperones besides proteins of the Hsp60 family [3]. However, among the molecular chaperone families, they present the least conserved one. Many additional functions besides the stabilization of other proteins have been subscribed to sHsps, for example, regulation of cytoskeleton network [4,5], involvement in regulation of informahealthcare.com

10.1586/14789450.2015.1039993

proteasomal degradation of proteins [6,7], inhibition of subcellular transport of proteins [8,9] or regulation of angiogenesis [10]. The most prominent and also well-studied members of the sHsp family are the two a-crystallins, aA- and aB-crystallin. They share 57% amino acid identity and account for over 30% of the protein content in the vertebrate eye lens where they maintain the visual properties of the lens by enhancing the solubility of aggregationprone lenticular proteins [11,12]. While aAcrystallin (HSPB4) is a lens specific protein, the importance of aB-crystallin (HSPB5) is highlighted by its additional expression in many other tissues [5,13–15]. It is implicated in a variety of diseases which are characterized by the deregulation of the physiological expression level of human sHsps (HSPBs) [16]. The respective diseases (or pathological conditions) range from different myopathies, ischemia, diabetes, multiple sclerosis to neurological disorders such as Alzheimer’s, Creutzfeldt–Jakob as well as diverse cancers and inflammatory disorders [14,17–19]. Besides these two a-crystallins, in

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Table 1. Standard and trivial nomenclature of human sHsps. Nomenclature name

Trivial names

HSPBs

Human small heat shock proteins

HSPB1

Hsp27, Hsp25, Hsp28

HSPB2

MKBP, myotonic dystrophy protein kinase binding protein

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HSPB3 HSPB4

aA-crystallin

HSPB5

aB-crystallin

HSPB6

Hsp20, p20

HSPB7

cvHsp, cardiovascular heat shock protein

HSPB8

Hsp22, H11 protein kinase, product of E2IG1 gene

HSPB9 HSPB10

ODF1, outer dense fiber protein

human eight additional sHsps are encoded (TABLE 1; also termed HSPB1–10 according to the proposed guidelines for the nomenclature of HSPBs [16,20]) and most of these have been correlated with the progression of diverse diseases as well [5,18]. Most currently available data strengthen the view that HSPBs protect against the proteotoxic damage induced by disease progression (or more generally: a stress situation; see below) [18,19]. Commonly, elevated levels of HSPBs seem to be able to protect cells and tissues against a multitude of stress situations. However, this is not always good for the organism, for example, enhanced levels of HSPBs also support the growth of tumor cells [18,19]. In the reversed view, reduced or insufficient levels of HSPBs (e.g., due to misregulation or mutations as well as too severe stress situations) can lead to diseases or cell death as well. Thus, in the context of diseases, HSPBs are presumably not causative, directly regulating or involved in disease progression. The observed effects are thought to be secondary and reflected by the changes of the overall health status of the cell [13,19]. Exceptions might be represented by diseases that are caused by mutations of a HSPB itself (e.g., cataract caused by the R120G mutation of HSPB5 or neuropathies caused by the K141N mutation in HSPB8 [17,21,22]). In terms of the underlying functional principle, sHsps are still the structurally and mechanistically least understood molecular chaperones. Nevertheless, the medical interest in HSPBs and their relevance as potential therapeutic targets might improve with the recently emerging high-resolution structural data, especially for HSPBs [23–29]. These structures are essential for the detailed analysis of their functional mechanism and especially for the understanding of their interaction with substrate proteins. Due to the still limited understanding of the 296

functional mechanism and its correlation with structural features, many seemingly contradictory results and, up to now, unexplained observations have been obtained. Especially, the link between descriptive in vivo data based on clinical or cellular systems and model-based in vitro mechanistic understanding is enigmatic in many respects and needs to be addressed in the future. Recent results increasingly indicate that HSPBs form a highly sensitive, balanced, protein-protective system that is closely integrated in many intra- and extra-cellular processes [5,13,18,19]. The triggers influencing the balance are diverse and the effects of changes in the balance seem to range between fine-tuning of single cellular processes and general stabilization of the proteome. Structural properties of sHsps

sHsps share a conserved organization of the primary structure that can be dissected in three major parts of functional importance [3,28,30–32]. A variable N-terminal sequence is followed by the conserved a-crystallin domain, which is the signature motif of the sHsps, and a short, variable C-terminal sequence (FIGURE 1A) [3,31,33]. Three-dimensionally, the a-crystallin domain represents a b-sheet sandwich composed of eight anti-parallel strands connected by an inter-domain loop (FIGURE 1B) [28]. Several highresolution structures of isolated a-crystallin domains of HSPBs demonstrate that these form dimers that represent the basic building blocks of higher oligomers (FIGURE 1C) [25,29,34,35]. All HSPBs studied so far show a b7-interface dimer where the b6-strand is fused with the b7-strand into an elongated b-strand connecting to its counterpart from the neighboring monomer in an anti-parallel orientation. The highly variable N- and C-terminal sequences of sHsps evolved independently [3] and are essential for the association of sHsps subunits with higher oligomers (FIGURE 1B). This potential to form higher order oligomers is one of the key features of sHsps. For example, in the case of the HSPB5, a 24-mer is formed where three dimers assemble into a hexamer via contacts formed by the conserved I-X-I/V motif in the C-terminal sequence [23,28]. Four such hexamers further associate into the 24-mer through contacts within the omnipresent and flexible N-terminal sequences [23,36]. Thus, the three domains contribute to the assembly process in a hierarchical way. However, some sHsp family members, such as HSPB6, lack the conserved I-X-I/V motif in the C-terminal sequence necessary for inter-dimer contacts [28,37,38]. These sHsps usually form only dimers and are unable to assemble into higher oligomers on their own. Interestingly, the hierarchical assembly system seems to be conserved among sHsps (from bacteria to human) and indicates that the total number of subunits in the oligomers can be modulated by respective variations in the C-terminal or especially in the N-terminal sequence [39]. Another key feature of sHsps is their tendency to populate different oligomeric states at equilibrium (FIGURE 1C). The oligomers are polydisperse and dynamic ensembles that Expert Rev. Proteomics 12(3), (2015)

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Understanding human sHsps

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constantly exchange subunits whereby the A B degree of heterogeneity varies for different members of the family [38,39]. This NTS S59 ACD ability of sHsps to generate oligomers of S19 different sizes from a single sequence is linked to the above-described modular architecture [28]. For example, in human S45 C HSPB5, heterogeneity is modulated N CTS through addition or subtraction of subunits from oligomeric complexes [23]. Such a seemingly simple mechanism of establishing a delicate balance between differC ent oligomer populations by adjusting the dissociation/association rates of building blocks would be unique to sHsps and seems to be tightly correlated with the regulation of their chaperone activity [40]. Figure 1. Structural features of HSPBs. (A) Domain organization of HSPBs, exempliThus, not only the amount of a HSPB fied by the monomer structure of full-length HSPB5 (human aB-crystallin) [23] (PDB: per se but also the presence of specific 2YGD). N-terminal sequence (NTS) (blue), a-crystallin domain (ACD) (green), C-terminal types of oligomers is important and indicsequence (CTS) (red). The motifs common to all sHsps and the three major phosphorylaative for the states of cells and tissues in tion sites of HSPB5 (S19, S45, S59) are indicated. (B) The hierarchy of the sHsp assembly, exemplified by the HSPB5 24-mer [23] (PDB: 2YGD). The dimeric building block the context of a variety of diseases [18] (see (green) assembles through the b7-interface of the adjacent protomers. A hexameric below). This distribution of oligomers is subassembly is formed by inter-dimer interactions, that is, through the binding of the tightly linked to post-translational modifiC-termini (red). The 24-mer assembles from the hexameric blocks through contacts cations of the sHsps [13,18]. Therefore a within the N-terminal sequence (blue). (C) Activation mechanism of HSPBs. HSPBs popudrug-mediated, targeted modulation of late at equilibrium a variety of inter-converting oligomers with different substrate affinities. The activation (transition grey to green; corresponding to an increase in chaperone the species distribution of HSPB ensemactivity) occurs through remodeling and shifting the ensemble composition to smaller bles might represent a valuable way to species. In some cases the first step throughout activation seems to be an expansion of influence the progression of the respective the oligomer (brackets) [40]. disease. However, as explained below, such approaches will be delicate, specific for certain diseases, and it remains to be seen if they can be sHsps–substrate complexes form a polydisperse ensemble of different species (FIGURE 2A) [40,46]. achieved. In vitro and in vivo experiments revealed that the non-native proteins trapped in sHsp-substrate complexes remain folding Implications of the chaperone activity: client versus competent and that in mammalian cells, the ATP-dependent substrate In the early 1990s, long time after their discovery, first Hsp70/Hsp40 system is required for the refolding of substrate in vitro studies demonstrated that sHsps bind denatured pro- proteins bound to HSPBs [47,48]. Thus, within the protein teins and prevent them from irreversible aggregation [41–43]. homeostasis network of the cell, sHsps function as a buffer sysMeanwhile, many sHsps from different species have been tem to bind and complex unfolding proteins, protecting them shown to bind a variety of non-native proteins in vitro and from irreversible aggregation (FIGURE 2A). This basic, highly promisin vivo leading to the general accepted view that they are cuous and effective (brute force) substrate-stabilizing mechanism promiscuous molecular chaperones acting fast when cells and and the energy-dependent release by other chaperone systems are organisms encounter stress conditions (FIGURE 2) [32]. They sta- conserved from bacteria to human and provide the organisms bilize early and/or late unfolding intermediates of with the capability of separating the prevention of aggregation aggregation-prone proteins. sHsps cannot rescue already from ATP-dependent refolding steps (FIGURE 2A) [47–51]. The substrate specificity of sHsps is still a matter of extensive aggregated substrates and must therefore be present during unfolding of the substrate [32]. The identity of the substrate, debate. First proteomic approaches in different organisms have the degree of unfolding and the specific properties of the shown that a significant number of cytosolic proteins interact respective sHsp determine the stability of the substrate inter- with sHsps under heat shock conditions [52–55]. Similarly, interaction. While some early unfolding intermediates of sub- action analysis of HSPB4 with human proteome microarrays strates may dissociate from the sHsp and refold indicated its promiscuity and revealed a preference for proteins spontaneously, most sHsp–substrate complexes appear to be from four functional clusters, DNA metabolic processes, prostable (FIGURE 2A) [30,38,44,45]. Similar to the sHsps itself, the tein transport and localization, organelle and nuclear lumen informahealthcare.com

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A

B

Substrate Stress

C Client

Ilate

D Co-chaperone

Cell 1

Signal Iearly +ATP

+ATP

Hsp40

P

Hsp40

Extra cellular space Hsp70

ot ei n Pr

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m ac hi

ne ry

Hsp70

HSPB-substrate complexes

HSPB-client complexes

HSPB-co-chaperone complexes

Receptor

Cell 2 HSPB-receptor complexes

Figure 2. Different modes of HSPB interaction. (A) Scheme of the HSPB-substrate interaction, representing the basic chaperone function of sHsps [32]. Upon stress conditions, the active HSPBs bind unfolding substrate proteins (I, unfolding intermediate) in an energyindependent manner and keep them in a folding-competent state in HSPB–substrate complexes. The substrates are subsequently refolded by ATP-dependent chaperone systems like Hsp70 and its co-chaperone Hsp40. (B) Scheme of the HSPB-client interaction. A partially unfolded client is captured and stabilized by a specific subspecies of the HSPB. Signals inducing changes in the phosphorylation status (purple P; unusual phosphorylation) of the HSPB commonly regulate the release of the client. The refolding might be enhanced by the presence of the Hsp70/40 system (in brackets). (C) Scheme of the HSPB-co-chaperone interaction. A co-chaperone binds to a specific subspecies of a HSPB (e.g., Bag3 to a HSPB8 dimer) facilitating its recruitment to a specific protein machinery (multi-protein complex). (D) Scheme of the extracellular HSPB-receptor interaction. Cell 1 secretes HSPBs, which travel to another cell (cell 2; e.g., macrophage) via body fluids and are recognized by receptor binding.

and cell cycle [56]. Intriguingly, in some organisms containing multiple cytosolic sHsps, the substrate spectra of individual members show substantial overlaps [54] and it remains enigmatic whether substrate specificity exists when multiple sHsps are present simultaneously. In higher eukaryotes, and especially in humans, where different subsets of HSPB are expressed in different tissues or developmental stages (see below), the respective substrate spectra might be optimized and adjusted to the proteome of the respective cells. For example, data describing the changes of the eye lens proteome in aA-R49C and aBR120G knock-in and aA/aB-knock-out mice indicate distinct variations in the interaction patterns [57,58]. Additionally, the available data on diverse interactions of HSPBs with cellular proteins have been summarized recently [13]. These data indicate that in humans, the largest fraction of proteins that interact with HSPBs seems to be linked to the cytoskeleton, cell adhesion and tissue integrity. Furthermore, most interactions described in this context have been discovered under physiological conditions and some interactions seem to be specific for single HSPBs. In a perspective view, this might indicate that many of the observed interactions of HSPBs with other proteins are not promiscuous ‘substrate’ interactions but more specific ‘client’ interactions that are rather specific and have a regulatory implication in the cell [59]. A ‘substrate’ then would be any type of non-native protein which is primarily recognized under general, (severe) stress conditions when the cell is in the need to stabilize its proteome by brute force and regulatory fine-tuning mechanisms are of secondary interest (FIGURE 3A, B). 298

A ‘client’ would be an at least partially unstable or intrinsically unfolded protein that is bound also under physiological conditions and where the binding and release of the client give the cell the opportunity to regulate and fine-tune cellular processes (FIGURES 2B & 3A, B) [13,15,59]. Thus, ‘clients’ would represent a subfraction of all ‘substrates’. A prominent example for such a client would be procaspase-3, which is bound and stabilized by HSPB1 and HSPB5 independent of general stress conditions [60–62]. Caspase-3 is an essential effector protein in apoptosis. For activation of caspase-3 the N-terminal prodomain has to be cleaved off. HSPB1 interacts with this prodomain and inhibits its cleavage, thus negatively regulating caspase-3 activity [60,61]. Interestingly, the recognition of procaspase-3 by HSPB1 is directly correlated with the phosphorylation state of HSPB1 and phosphorylation triggers the release of procaspase-3, and thus, its activation and induction of apoptosis [63]. On the other hand, enhanced phosphorylation of HSPB1 leads to more effective binding of eukaryotic translational initiation factor 4E (eIF4E), which protects eIF4E from proteasomal degradation and confers apoptosis resistance [64,65]. Hence, changes in the phosphorylation of HSPBs seem to represent a major regulatory tool to inhibit or favor recognition of specific clients to fine-tune cellular processes under physiological to low proteotoxic stress conditions (FIGURES 2B & 3A). Commonly, in these situations, independent of a severe general stress, anomalous phosphorylation results in deleterious effects for the cell, mainly by changes in the client-binding behavior [66]. In this context, it is not clear yet if a single or several Expert Rev. Proteomics 12(3), (2015)

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Understanding human sHsps

different HSPB subspecies can bind the respective client and whether they need to be activated or not. It also could be assumed that only partial activation of single or some subunits (leading to slight structural rearrangements) of the HSPB oligomer are sufficient to allow client interactions. Additionally, it is currently unclear if the Hsp70/40 system is involved in the release of clients from HSPBs as well, or if this energydependent release is only necessary for (unspecific) substrates. Further examples for clients seem to be p53 and Bax for HSPB5, where binding sequesters their translocation to mitochondria during apoptosis [13,59]. This shows that the decision on apoptosis induction seems to be dependent on several interactions of HSPBs with clients, indicating that in this case (and may be generally) the cell uses the availability of sHsps to bind clients as a fine-tuning system to monitor cell fate. Taken together, it might be hypothesized that whenever the sHsps are engaged in substrate binding upon severe stress conditions, the level of available sHsps for client binding is too low, and thus cell death via apoptosis is triggered by some of the clients. But also other apoptosis-independent clients seem to exist [13], for example, the dynein subunit TCTEL1 for the testis-specific human HSPB9 [67]. It has to be seen if and how this and other clients are integrated in regulatory processes of the respective cells. However, for many interacting proteins, the sorting into the substrate or client category might not be that easy. For example, the interactions of HSPB1 and other HSPBs with the cytoskeleton are somewhat in between or represent a mixture of the two interaction modes. While the modulation of the intermediate filament network by the interaction of HSPB1 with keratins might be more a ‘client type’ interaction, its binding to desmin, vimentin and actin on the other hand is dependent on stress-induced unfolding of the respective proteins, and thus, is more a ‘substrate type’ of interaction [5,13]. In addition, the modulation of the phosphorylation of HSPBs adjusts the appropriate substrate and client recognition of the respective HSPB involved in cytoskeleton organization [13]. Overall, client interactions of HSPBs with intermediate filament proteins seem to be directly regulating and modulating the cytoskeleton network [5,68–70]. The observed changes in the actin network on the other hand are secondary and mainly triggered by the decrease in available folded actin monomers due to the recognition of actin as HSPB substrate [5]. Similarly, the interaction of HSPBs with fibril-forming substrates (as causatives in many neurodegenerative disorders) [18,71,72] might represent a mixture of both binding modes, or a further, specific subtype of substrate interaction. Here, the transient binding of HSPBs to the unfolded substrate monomers seems to counteract the assembly of fibril nuclei [68]. Nevertheless, HSPBs are found attached to amyloid fibrils, indicating that a further type of interaction with the amyloid proteins is possible. For example, it has been shown in this context that the two yeast sHsps, Hsp26 and Hsp42, influence different steps in the assembly of amyloid fibrils of the yeast prion Sup35, seemingly by different but synergistic binding mechanisms that might informahealthcare.com

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be based on different recognition sites on Sup35 [73]. Hsp42 inhibits the formation of fibril nuclei, whereas Hsp26 inhibits the attachment of Sup35 monomers to already existing fibrils. The presence of the sHsps not only inhibits de novo fibril formation and self-templating but also promotes gradual depolymerization by the Hsp70/Hsp40 system (in this case assisted by Hsp110, a nucleotide exchange factor) [73]. It is tempting to speculate that in the human cell, an even more sophisticated and synergistic cooperation between different HSPBs exists, which interferes with fibril formation (and disassembly) in neurodegenerative diseases at different stages. Many observations showing differences between refolding (mainly observed for HSPB1 and HSPB5) and anti-aggregation properties (as observed for HSPB6, HSPB7, HSPB8 and HSPB9 in the context of poly-Q aggregation processes) hint in this direction [15,71,73]. However, we are far from understanding this multi-faced interactions of HSPBs with amyloids (monomers, oligomers and fibrils) and much more research using sophisticated, amyloidogenic disease-relevant model systems are necessary to understand and in the far end clinically address the role of HSPBs in the respective disease context. Up to now, it remains elusive how HSPBs might distinguish between a substrate and a client. Even worse, the recognition motifs of sHsps that are involved in substrate/client interaction are still largely unknown. The emerging picture is that there are several binding sites throughout the molecule which act together, presumably in a different manner for different substrates and/or clients. Studies utilizing the incorporation of hydrophobic dyes suggested that substrates might bind to short segments in the N-terminal sequence [51,74]. Recent evidence arising from crosslinking experiments and analyses by mass spectrometry or peptide libraries support this tendency [75–78]. But also substrate binding sites within the a-crystallin domain have been claimed [79], and a part of the a-crystallin domain (residues 73–92 called ‘mini-aB-crystallin’) of HSPB5 has been shown to prevent the aggregation of several denatured proteins [80,81]. Additionally, mutations of amino acid residues in the C-terminal sequence of HSPB5 affect chaperone activity and indicate that the flexibility of the C-terminus is necessary for substrate recognition [82]. Taken together, this scenario of seemingly several different recognition sites would be perfectly suited to adjust the interaction of substrates or clients in dependence of the combinatory engagement and availability of these different sites. Nevertheless, it seems most likely that substrate recognition is accomplished by the non-conserved variable sequences outside the a-crystallin domain that become exposed during the disassembly of the sHsp oligomers. Independent of the mode of interaction, it is also still enigmatic if this chaperoning function is directly needed in all cell types to stabilize the respective proteome. For example, in the static environment of the eye lens, irreversible binding of aging lens proteins by a-crystallin (a hetero-oligomer of HSPB4 and HSPB5) prevents aggregation and light scattering [83]. However, the b- and g-crystallins, which represent the most likely natural substrates in the eye lens, seem to be more stable than 299

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a-crystallin [12]. Thus, the key function of a-crystallin in the eye lens might probably not be the aggregation suppression by the chaperone function but rather the adjustment and regulation of the refractive index gradient [12]. Besides the two above-described modes of interaction (substrate vs client), a third intracellular interaction mode might exist in the form of the binding of co-chaperones that helps to target some HSPBs (or HSPB-substrate complexes) to other specific functional complexes or protein machineries of the cell (FIGURE 2C) [13,15]. The most prominent example for a cochaperone of HSPBs is Bag3, which binds to HSPB8 linking it to the Hsp70/ubiquitin ligation/proteasome machinery and the macroautophagy machinery [84]. The multichaperone complex HSPB8-Bag3-Hsp70 seems to act in the quality control deciding between proteasome-mediated degradation or autophagy [15,84,85]. Again, severe proteotoxic stress enhances macroautophagy, which enables the cell for fast and effective clearance of aggregated protein. A second example for a co-chaperone might be the 14-3-3 protein, which interacts specifically with phosphorylated HSPB6. It is assumed that this displaces phosphorylated cofilin and/or regulatory kinases and phosphatases (LIMK, TESK and SSH1L) from 14-3-3 inducing depolymerization of actin filaments, which results in muscle relaxation [5]. In summary, such a perspective view of different binding modes of HSPBs might provide a new basis to explain existing experimental data and address the interaction properties of HSPBs in a different manner. Especially, comparative in vivo proteome data of cells under different conditions are needed to verify or challenge this view. Overall, the described chaperone function of HSPBs and their incorporation into the protein homeostasis network is consistent with a general protective function in the dynamic environment of a living cell to combat proteotoxic stress. However, extracellular functions for HSPBs have been described as well. While HSPBs encode no secretion signals, a number of studies reported the extracellular presence of HSPBs [86–88]. While part of this extracellular HSPB fraction might be caused by leakage from dying cells, controlled secretion seems also to occur via non-conventional pathways involving exosomes [88]. Interestingly, the secreted HSPBs have been found to bind to specific surface receptors of other cells. Thus, this receptor binding represents a fourth mode of interaction of HSPBs with other proteins (FIGURE 2D). In this case, the HSPB was suggested to represent a signal molecule delivering information from one cell to another [88,89]. Since the secretion of HSPBs is enhanced in response to stress, this might be part of the cross-talk between different tissues and organs transmitting the stress status [90]. But also without stress, HSPB5 is constitutively secreted by retinal pigment cells at least in cell culture experiments [91,92] and also uptake of HSPB5 via photoreceptors was observed. Besides this potential signaling effect between tissue cells, it appears that the activation of macrophages or macrophage-like cells during inflammation is a general effect of extracellular HSPBs recognized by these cells [88,93,94]. This triggers innate immune response and in most cases seems to be 300

anti-inflammatory [88]. Additionally, the secretion of HSPBs seems to be especially high in the CNS, which was suggested to be the reason for their association with amyloid deposits [95]. This association shows that the extracellular HSPBs still seem to function as molecular chaperones and the amyloids are recognized basically as substrates [19,96,97]. However, it remains elusive if the extracellular HSPB fraction fulfills a general, protein protective function similar to other extracellular macromolecules of the body fluids, like clusterin, which shows quite similar mechanistic effects to sHsps in terms of chaperone activity and size distribution [98,99]. Regulation of the binding affinities of HSPBs

The above-described mechanistic features and seemingly different modes of interaction equip the cell with a variable toolkit to monitor and regulate protein fate. However, the binding affinity of the HSPBs towards substrates, clients and cochaperones must be strictly regulated to avoid dominant negative side effects. For other chaperones, a low- and high-affinity state for non-native proteins exists and ATP hydrolysis induces the transition between these two states [100]. In sHsps, the transition between high- and low-affinity states is regulated by intrinsic structural mechanisms and their ability to populate different oligomeric species [40]. Several lines of evidence indicate that various triggers, which involve conformational changes within the subunit and/or remodeling of the ensemble composition, regulate the transition between different binding states. A majority of sHsps requires a shift of the ensemble composition from high oligomers to smaller species (often dimers) for the efficient recognition and binding of substrate proteins (FIGURES 1C & 2A) [28,30,32,101]. In summary, these findings underline that the transition of sHsps between active and inactive states is not a simple, two-state mechanism, but a complex process involving several transitions. Some sHsps apparently make use of the full scale of possible transitions, while others show a predominance of specific species [24,40,102,103]. As triggers regulating the association/dissociation equilibria of sHsps lead to activation, four different stimuli have been identified: the presence of unfolded or partially folded substrates; changes in the environmental temperature; phosphorylation or more general post-translational modifications and the formation of hetero-oligomers [32,40]. Since in human cells, many HSPBs are already present under physiological conditions (and their expression is further induced under stress conditions), the first regulatory principle establishes HSPBs as first line of defense allowing the cell to preserve the stability of its proteome under physiological as well as upon stress conditions [32]. A dynamic assembly/disassembly behavior allows the substrate binding sites, which seem to be buried within the oligomeric complexes, to become exposed by dissociation [32,101]. Thus, in the ensemble, there will always be a fraction of active species with exposed substrate binding sites that would sense the presence of non-native proteins in the cellular environment. Similar to a titration process, this leads to a gradual shift of the ensemble to more active, substrate-binding Expert Rev. Proteomics 12(3), (2015)

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A

Review

B Physiological conditions

Stress conditions

Cellular process

Cellular process Client release (change of client availability) Efficient proteome stabilization

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Fine tuning regulating client availability

C

B4

B1

B6

B2

B5

B8

B3

B7

B9

B10

Figure 3. Cellular role of HSPBs. (A) Under physiological conditions, the HSPB-client interactions establish an equilibrium between free and HSPB-bound client, representing a regulatory tool for the cell to modulate cellular processes which depend on the availability of the respective client protein (FIGURE 2B). The HSPBs serve to fine-tune the cellular process by client binding and release in response to (slight) changes/fluctuations in the proteome balance (blue scheme) throughout the life of a cell. Unusual phosphorylation of the HSPBs (purple P) regulates client recognition. (B) Upon stress conditions where the proteome balance is severely affected, the HSPBs are preferentially engaged in efficient stabilization of the proteome. This affects the equilibrium between free and HSPB-bound client leading to changes in client availability, usually enhancing the client-dependent cellular process. (C) Scheme of the distribution and potential heterooligomer interactions of HSPBs. In a skeletal and cardiac muscle cell (symbolized by the square in antique pink square) seven different HSPBs exist in parallel under physiological conditions. In the eye lens (blue ellipse) and nerve cells (orange ellipse) the set of sHsps is adjusted and more restricted and specialized sets of HSPB are present. Some HSPBs like HspB9 and Hsp10 as well as HSPB4 (aA-crystallin) are only present in highly specific tissues (e.g., testis, purple ellipse; lens, blue ellipse). The most widely distributed members (represented by the class I members of HSPBs; green square) seemingly form one part of a hetero-oligomer network which is inter-connected with a second more specialized part via HSPB6 and HSPB8. Arrows indicate currently established hetero-oligomer formation and dashed arrows indicate controversially discussed hetero-oligomer formation [13,68].

species [32,101,104]. It is tempting to speculate that activation by temperature (and other physical, environmental stress situation) as a general trigger is most important in bacteria and lower eukaryotes [32]. In humans, phosphorylation and heterooligomer formation on the other hand might be the preferred and more sophisticated regulation mechanisms, especially to regulate client recognition. The regulation of the chaperone activity by phosphorylation or more general post-translational modifications is specific to eukaryotes and enables the abovementioned fine-tuning of the HSPBs activity (FIGURE 3A, B). The oligomeric state of HSPBs can be altered, in particular, by serine/threonine-specific phosphorylation by several kinases [66,105–107]. The informahealthcare.com

phosphorylation of HSPB1, HSPB4, HSPB5, HSPB6 and HSPB8 has been studied in some detail [5] and according to the UniProt database [108], for all other HSPBs phosphorylation was observed as well in accordance with the conservation of the respective phosphorylation sites [3,20]. The state of phosphorylation seems to be specific for a cell type and is regulated in response to stress, cytokines or growth factors [5,18,109]. Phosphatase-mediated dephosphorylation counter-regulates the phosphorylation [66,109]. Nevertheless, it should be kept in mind that stress situations are a major stimulus for the respective phosphorylation cascades and extensive phosphorylation enforces substrate binding via shifting the ensemble towards smaller 301

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species by dissociation of the larger oligomers [32]. In contrast to the other stimuli, phosphorylation commonly favors specific types of smaller species (often tetramers and hexamers), indicating that some of the equilibria within the disassembly process are affected stronger than others [24,47,110,111]. Such predominance of these species upon phosphorylation is based on the localization of the phosphorylation sites in the N-terminal sequence and is in accordance with the hierarchical assembly of the oligomers (FIGURE 1B) [23,24]. For HSPBs, several other post-translational modifications have been described to have regulatory influences on the chaperone activity as well. As described for a-crystallins, deamidation introduces a negative charge at the modification site that affects the oligomerization [112]. Similarly, modifications by glycation [113], oxidation [114,115], thiolation [116] and the attachment of methylglyoxal have been suggested to influence the oligomerization of HSPBs [117]. Being not really a posttranslational modification, the incorporation of bivalent metal ions, for example, Cu2+, represents nevertheless a modification that also changes the local charge pattern, and thus, a potential intra- or inter-molecular interaction site. This type of modification seems to be of special relevance in the context of amyloid [18,118] and lens protein [119] interaction. Additionally, and of special interest also for the extracellular HSPB fraction, redox-induced modifications were suggested as well [13,30,115,120]. HSPB1 and HSPB5 stabilize the cell against oxidative stress conditions and show anti-apoptotic activity [121,122] seemingly by influencing the glutathione pool of the cell [123]. Oxidation of HSPB1 freezes the ensemble in a more homogenous, high oligomer state because of the formation of disulfide bridges by the cysteine in the a-crystallin domain and results in a decrease of the overall chaperone activity [115]. Thus, the oxidized, extracellular fraction of HSPB1 might be estimated to be mainly inactive and in high oligomeric state. The fourth stimulus, hetero-oligomer formation of HSPBs of the same compartment, is very crucial, complicated and the effects are currently widely enigmatic in human cells. HSPB4 and HSPB5 in the human eye lens form the most prominent HSPB hetero-oligomer (a-crystallin) [14,124]. Hetero-oligomer formation was also shown for most other HSPBs (FIGURE 3C) [13]. Indeed, for example, in a human muscle cell, seven cytosolic HSPBs are present in moderate to high amounts in parallel. They can be divided roughly into two classes based on their expression and interactions between each other, HSPB1, HSPB5, HSPB6 and HSPB8 represent class I; they are found widely distributed in various tissues and are heat inducible. HSPB2, HSPB3, HSPB4 and HSPB7 represent class II. They show usually more tissue-restricted expression patterns and seemingly tissue-specific functions (i.e., substrates and clients) [13,18,125]. While most of the members of these two classes only form hetero-oligomers with HSPBs from the same class, HSPB6 and HSPB8 are inter-connecting the two classes by their ability to interact with members of both classes [13,126]. Intriguingly, the substrate spectra of the HSPBs become modified when they are incorporated into hetero-oligomers [13,127]. Furthermore, as the phosphorylation of the HSPBs additionally 302

influences their hetero-oligomer formation properties, the two stimuli (phosphorylation and hetero-oligomer formation) in combination allow a highly complex and variable adjustment of the binding properties for specific substrates or clients, for example, HSPB1 found in complex with HSPB5 is differently and less phosphorylated than HSPB1 found in homo-oligomers and the interactomes of hetero- and homo-oligomers vary significantly [68]. The situation in the human cytosol appears to be even more complex because only a fraction of HSPB1 is incorporated into hetero-oligomers and HSPB1 homo-oligomers coexist as well [13]. This fraction of HSPB1 homo-oligomers seems to be controlled by the phosphorylation status of HSPB1. Overall, these observations suggest that the formation of hetero-oligomers is tightly regulated and the coexistence of homo-oligomeric and hetero-oligomeric ensembles is possible. Nevertheless, a broad variety of different mixtures of heterooligomers is at least theoretically possible, which would allow a tremendous number of combinations of potentially slightly different binding sites and thus variability in substrate and client recognition. The expression of the respective sHsps represents the first level of modulation of the potential interaction patterns. For example, as already mentioned above, in the human skeletal and heart muscle seven different sHsps are present (FIGURE 3C). In nerve cells (e.g., sciatic nerve) especially the levels of HSPB5 and HSPB6 seem to be high [15]. Additionally, HSPB4 (lens), HSPB9 and HSPB10 (both testis) are highly tissue-specific and seem to be integrated in the hetero-oligomer network only in case of need [5,13,15]. However, it should be kept in mind that most of the currently available experimental data describe hetero-oligomer formation in vitro or in cellular lysates and real in vivo data are rare [127]. Much more work is needed to clarify the relevance of single hetero-oligomers as well as of the potential hetero-oligomer network in an intact cell, with subcellular compartmentalization. Expert commentary & five-year view

Taken together, the mechanistic correlation of the general chaperone activity of sHsps and their structural dynamics became clearer in recent years. For human HSPBs, however, the described different modes of interaction (FIGURE 2 & 3A, B) need to be evaluated in more detail. This is also extremely important because other ‘big’ Hsps, such as the Hsp90 system, seem to work in a similar mechanistic manner. Hsp90 also usually interacts with rather specific clients, in a co-chaperones regulated manner, but is additionally an effective general chaperone [128]. In terms of medical implications, the targeting of ‘client’ binding seems to be a highly promising way to trigger a specific cellular response. For example, in terms of sHsps, the targeted release of procaspase-3 would lead to the death of the respective cell. Apigenin, a natural plant flavonoid that emerged as a potentially promising compound for cancer prevention and therapy, is working in this direction by modulating the phosphorylation of HSPB1, triggering caspase-3 release [63]. However, a more detailed understanding of the respective Expert Rev. Proteomics 12(3), (2015)

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Understanding human sHsps

sHsp-client interactions is needed to find drugs with much more specificity, which would allow us to avoid side effects and control a specifically triggered cellular response. That the targeting of chaperone–substrate/client interaction (including HSPB interactions) is principally a valuable tool to address diseases at least in model systems has been demonstrated recently for Hsp70 [129,130]. Similarly, ‘peptide aptamers’, which bind to HSPBs and interfere with their function, have been demonstrated to be valuable [131]. To understand and specifically use emerging compounds that address these interactions, it will be crucial to determine the respective interaction sites of the HSPBs in detail and in dependence of diverse substrates and clients. Therefore, it will be highly important to increase the number of available high resolution 3D structures of full-length human HSPBs. Currently, we are still far from understanding the mechanistic details and differences of the various members of the sHsp family. It remains elusive how different assembly types can be achieved by variations in the N-terminal sequence or modulation of contacts sites. Understanding the different interaction modes in structural detail would be extremely exciting and should enable the design of highly specific drugs to target selected functional features. Besides the substrate and client recognition sites, especially the co-chaperone interaction site(s) is tantalizing. Recently, it has been shown that inhibitors of the Hsp70-Bag3 interaction might have promising potential in anti-cancer therapy [132]. It remains to be seen if inhibitors targeting the other side of the multi-chaperone complex, the HSPB8-Bag3 interaction, could be similarly effective. Generally, HSPBs might be highly promising targets for therapy of diverse diseases, especially because it seems possible to render the targeting highly specific for a single disease once the correlation of disease progression and HSPBs is understood in detail. While currently other molecular chaperones like Hsp70 or Hsp90 seem to be more in the focus of compound search to interfere with substrate and co-chaperone interaction, HSPBs might turn out to be the better target in the long run due to the expected smaller side effects on general cell fate (i.e., Hsp90 and Hsp70 are of higher central importance and incorporated in many more cellular processes than single HSPBs). However, such compound-based specific targeting of HSPBs seems to be a more futuristic vision. As evident by the abovedescribed diverse and fine-tuning effects of HSPBs, they have the potential to trigger both, health promoting and health endangering effects [19]. Thus, an in-depth understanding of the implication of the respective HSPB in a specific disease is needed to allow precise therapeutic targeting. What might be good in a context of one disease might make the situation worse in the context of another disease, or even promote additional diseases. And, for human HSPBs, we are far away from such a detailed comprehension. We still lack mechanistic, comparative data on most of the HSPB members. The understanding of the tissues specificity of HSPBs is also missing. Even the regulation of the expression of HSPBs is not clear in most cases [133]. The hetero-oligomerization network is still under discussion and its in vivo relevance remains widely enigmatic. informahealthcare.com

Review

The ensemble dynamics needs further, detailed analysis in a comparative manner. The inter-connection of HSPBs in the progression of the various diseases they have been assigned to is in most cases not clear [5,14,18,19]. Influencing the expression levels of HSPBs (or more generally the complete stress response) by drugs [114–137], in restricted tissues or body parts, was shown to be beneficial in the context of some disease models [18,19]. However, such strategies are discussed quite controversially and it remains to be seen if such treatments are effective and how adverse side effects can be omitted [18,19]. It also has to be taken into account that other chaperones might be additionally implicated in the respective targeted disease (or other diseases) and that a change in the level of one component (e.g., HSPBs) will influence the complete chaperone network [138]. Hence, it seems that, at least, highly specific drugs targeting not the general stress response are needed. One possibility in this context might be the use of short interfering RNAs to silence a specific gene [139,140]. Such strategies seem to be especially suited for cancer therapy. Cancer cells are usually in the need of high amounts of functional chaperones (e.g., including HSPB1 and other HSPBs). While the inhibition of a respective HSPB might not affect normal cells, it seems to sensitize (or even destroy) tumor cells [18,19,59,140]. Similarly, the specific, transgenic overexpression of HSPBs (e.g., HSPB1) in several non-cancerous disease models also showed a protective effect [18,141]. Gene therapy approaches delivering single HSPB expression cassettes (i.e., to compensate for functional losses due to mutations in the respective HSPB genes, which is also causative for several diseases [17]) have been discussed and went under trial in the past [17], but seem to have gotten out of focus in recent years. Besides targeting their expression or function, HSPBs themselves can be considered as potential therapeutic agents. Especially, approaches to inhibit acute or chronic inflammatory processes by intravenous administration of purified HSPB5 are highly promising and have been demonstrated to already work in mice [88,142] and a company (Delta Crystallon BV) aiming to exploit the therapeutic properties of HSPB5 (aB-crystallin) in immunological diseases and especially multiple sclerosis exists. Thus, exploiting the extracellular function of HSPBs for therapies seems to be on the horizon. Nevertheless, studies on functional activity of extracellular HSPBs and more work on the identification of receptors they are interacting with and how this interaction is mediated are necessary to rule out side reactions/adverse effects upon applying them into body fluids. Taken together HSPBs seem to be promising therapeutic targets. However, much care has to be taken because they have the potential to promote health or death of a cell. To be able to develop dedicated drugs to target HSPBs, the function of the HSPBs in context of the diverse diseases has to be analyzed specifically and in detail. Furthermore, a much more detailed understanding of the recognition of interaction partners by HSPBs is needed to address their respective function correctly. The perspective view presented here of four potential binding modes for HSPBs hopefully provides a new basis for 303

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experimental strategies to address and challenge the respective interaction modes in vitro and in vivo. Acknowledgements

Due to the journal’ s restrictions in the number of references, many original papers could not be cited. We apologize for that and would like to acknowledge all the colleagues in the field for their valuable contributions. We thank Sevil Weinkauf for critical reading the manuscript.

Financial & competing interests disclosure

The authors are supported by the Deutsche Forschungsgemeinschaft (SFB 1035), CIPSM and the Peter und Traudl Engelhorn Stiftung are acknowledged for financial support. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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Key issues .

Small heat shock proteins (HSPBs) are implicated in a variety of diseases.

.

They represent a first line of defense in protein and cell homeostasis.

.

They form dynamic ensembles of different, hierarchically organized oligomers.

.

Human HSPBs might show four different modes of interaction with other proteins. Upon severe stress conditions they are able to promiscuously bind many cellular proteins and prevent them from aggregation. Under physiological conditions they seem to interact primarily with specific client proteins, fine-tuning the availability of the client for other, client-dependent cellular processes. Several HSPBs interact with specific co-chaperones. These co-chaperones are usually part of larger protein machineries that are functionally modulated upon HSPBs interaction. Secreted HSPBs interact with receptors and extracellular protein deposits.

.

Post-translational modifications of HSPBs and hetero-oligomer formation seem to be major regulatory tools to modulate the interaction modes of HSPBs.

.

Client and co-chaperone interactions seem to represent a valuable target for the development of therapeutic strategies to influence the function of HSPBs in the context of diseases. However, care has to be taken because HSPBs can promote health as well as death of cells.

.

Extracellular HSPBs activate macrophages and show anti-inflammatory properties. Exploiting this function for therapies of immunological diseases seems to be on the horizon.

alpha 7. Bioch Biophys Acta 2001;1544: 311-19

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Expert Rev. Proteomics 12(3), (2015)

Medical implications of understanding the functions of human small heat shock proteins.

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that are implicated in a variety of diseases. Upon stress, they stabilize unfold...
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