Heat shock proteins (Hsps) were first described by Tissières in 1974 in the salivary glands of Drosophila third instar larvae exposed to sub-lethal temperatures (temperature shift of 20–35 °C). Hsps were then discovered in all living organisms, from bacteria to human including plants (Lindquist and Craig 1988). Five families of heat shock proteins have been described: the HspH (large Hsps), 90 kDa (HspC-Hsp90), 70 kDa (HspA-Hsp70), 60 kDa (HspD-Hsp60), and the 20–30 kDa small heat shock proteins (HspB-small Hsps, sHsps). The interest in these proteins was stimulated by discovering that their expression can be triggered by many environmental stress conditions as well as by toxins known to alter the folding of proteins.This lead to the finding that Hsps are molecular chaperones whose function is to attenuate stress-induced damages in protein folding and participate in the refolding or elimination of aberrantly folded polypeptides. Cells expressing Hsps have therefore an improved ability to withstand stress and transiently develop resistance. Hsps, which are also often referred as stress proteins, are now considered as important actors of the so-called cellular stress response. The human family of small heat shock proteins is represented by ten members (HspB1–HspB10) which share a C-terminal domain (the crystallin domain), a less conserved N-terminal domain containing an hydrophobic WD/PF motif, and a flexible C-terminal tail with a IXI/V motif. In addition, the N-terminal domain of several small Hsps, including HspB1, contains serine sites that are phosphorylated under specific conditions(Kappe et al. 2003). Small Hsps are oligomeric polypeptides that display very large and heterodispersed native sizes (50–800 kDa). Three of them are stress inducible (HspB1, HspB5, and HspB8) and only four have a conserved ATP-independent chaperone activity (HspB1, HspB4, HspB5, and HspB8). HspB1, one of the most studied small Hsp, was first purified and characterized in the late 1980s (Arrigo and Welch 1987). In addition to its upregulated expression and major role in stress conditions, HspB1 is also an efficient antiapoptotic and antioxidant polypeptide and a crucial modulator of F-actin dynamics. Moreover, it is constitutively expressed in many normal and pathological tissues where it acts as a specialized chaperone (Arrigo 2012).
HspB1 Phosphorylation and Oligomerization
Human HspB1 phosphorylation occurs at the level of three serine sites (ser15, ser78, and ser82) localized in the N-terminal part of the protein while murine HspB1 (also called Hsp25) has only two phosphorylated serine sites (ser15 and ser86). The kinases responsive of HspB1 phosphorylation are the mitogen-activated protein kinases associated protein kinases (MAPK/MK2,3) that are activated by phosphorylation by MAP p38 protein kinase. In some particular conditions, HspB1 can also be phosphorylated by MK5-PRAK, PKCγ, PKD, and cGMP-dependent protein kinase. Hence, HspB1 has rather complex patterns of phosphorylation due to the many transduction pathways that activate its kinases.The oligomerization of HspB1 is a fascinating dynamic phenomenon directly linked to the physiology of the cell and to its environment. For example, in growing HeLa cancer cells where it is constitutively expressed, HspB1 displays a typical pattern of phosphorylation-oligomerization characterized by three subpopulations with different native sizes (<150 kDa, 150–400 kDa, and >400 kDa) and phosphorylated serines. Phosphoserine 15 is present only in <150 kDa oligomers while phosphoserine 78 is recovered only in the medium sized oligomers. In contrast, phosphoserine 78 is distributed between the small and large oligomers (Paul et al. 2010). However, this typical organization is very dynamic and can be transiently modified by changes in the environment, growth conditions, or differentiation. It is believed that HspB1 alpha-crystallin domain, which contains a unique cysteine residue and a beta-7 strand, is essential for the formation of dimers that can multimerize under the control of the phosphorylation-sensitive N-terminal domain (Mymrikov et al. 2010). Is phosphorylation a key factor regulating HspB1 oligomerization? It is difficult to give a straight answer to this question. Studies performed using nonphosphorylatable as well as phosphomimicry mutants revealed that in some conditions phosphorylation plays a role. For example, an intense phosphorylation usually induces the dissociation of HspB1 large oligomers but this not the case when cells are starved, confluent, exposed to reducing conditions, or growing in tumors (Bruey et al. 2000). The relation phosphorylation-oligomerization of HspB1 appears mainly regulated by the contacts between cells, such as those that exist in tissues and tumors. Hence, whatever the status of phosphorylatable serines could be, cell to cell contacts induce the formation of large oligomeric structures and small oligomers are induced by starvation. This clearly points to the role played by HspB1 as a crucial modulator of F-actin dynamics through its association with the plasma membrane cytoskeleton compartment where nucleation occurs (Pichon et al. 2004).
HspB1 in Heat Shock Conditions
HspB1 in Oxidative Conditions
HspB1 in Apoptotic Conditions
The expression of HspB1 is not upregulated by apoptotic programs. Therefore, as in oxidative conditions, a putative activity of this protein can only be detected if it is constitutively expressed, as for example in many human cells, particularly when they are of cancer origin. In that respect, we showed that HspB1 efficiently counteracts apoptosis triggered by many inducers through drastic changes in its structural organization (Mehlen et al. 1996; Paul et al. 2010). To interfere with apoptotic pathways, HspB1 targets several key polypeptides both upstream and downstream of mitochondria (Fig. 2). Major targeted upstream pathways are Bid or Daxx dependent or induced by alterations in F-actin or nucleus architecture integrity. Downstream pathways include the interaction with cytochrome c once it is released from mitochondria, impairment of the apoptosome-associated procaspase-9 activation, and interaction with effector caspase-3. The dynamic oligomerization and phosphorylation of HspB1 in cells undergoing apoptosis depends on the pathway triggered by the inducer (Paul et al. 2010). Two classes of apoptotic inducers could be defined. The first class is represented by etoposide and Fas which transiently shift HspB1 toward large oligomeric structures. The second class contains staurosporine and cytochalasin D, drugs known to induce a rapid disruption of F-actin architecture, which rapidly shift HspB1 in small oligomers before large ones are re-formed. The upstream effects, particularly at the level of F-actin, may first require small oligomers while the downstream effects require larger oligomeric structures (Bruey et al. 2000). These transient changes in oligomerization/phosphorylation, which correlate with HspB1 ability to transiently counteract the inducers effectiveness to activate caspases, suggest that this chaperone has multiple and complex strategies to negatively modulate apoptotic pathways. In addition, HspB1 is probably also efficient towards other death programs, as shown recently in the case of ferroptosis.
HspB1 in Differentiating Cells
HspB1 expression is usually transiently upregulated during the early phase of many differentiation programs where it is essential to avoid apoptosis. HspB1 probably acts against the drastic changes in protein and cytoskeletal organization occurring in differentiating cells which could sponteanously induce apoptosis (Arrigo 2005). This protecting effect is associated with drastic changes in HspB1 oligomerization and phosphorylation: from small phosphorylated oligomers to large unphosphorylated ones and then later to unphosphorylated small oligomers (Mehlen et al. 1997).
The Client Chaperone Machinery of HspB1
HspB1 in Tumor Survival and Dissemination
In cancerous pathologies, a high level of expression of HspB1 is often observed which promotes deleterious resistance and aggressive behavior of tumor cells and consequently poor clinical outcome (Ciocca et al. 2013). In addition of being able to protect cancer cells against the apoptotic signals mediated by the immune system, HspB1 is also deeply involved in the survival of tumor cells (Fig. 2) and their dissemination into surrounding tissues as well as in the formation of metastatic colonies. It also counteracts cancer treatments, such as chemotherapy, hyperthermia, and radiation. At the molecular level, HspB1 modulates the activity of many client proteins sharing potent prooncogenic activity (Ciocca et al. 2013; Arrigo and Gibert 2014), such as: the cell survival kinase Akt, the tumor suppressor PTEN whose degradation is stimulated by HspB1, the PEA-15 molecular switch of Fas-induced apoptosis, HDAC6 which contributes to oncogenic pathways activation, Her-2/neu oncogene, β-catenin which plays a key role in in tumor cell survival, and Snail which regulates the maintenance of cancer stem cells as well as polypeptides involved in the control of genetic imbalances (haploinsufficiency). The oncogene-induced senescence pathway, a host anticancer response, as well as the cellular matrix are other important targets of HspB1. Indeed, HspB1 acts at the level of HDM2, which serves as ubiquitin ligase to target p53 for degradation, matrix metalloproteinases, and SPARC (secreted protein, acidic and rich in cysteine), a polypeptide playing an important role in cell adhesion and migration. Among several other mechanisms that are not yet elucidated an important one is the promotion of the metastatic phenotype. In that regard, HspB1 modulates the expression of prometastatic genes dependent of the STAT3/Twist signaling by playing a role in the binding of STAT3 to the Twist promoter, hence promoting epithelial-mesenchymal transition (EMT) and metastasis. Of interest, HspB1 elimination suppresses EMT signatures and induces long-term dormancy. GATA1, another transcription factor modulated by HspB1, is essential for erythroid differentiation and inactive in almost all megakaryoblastic leukemias in patients with Down syndrome. Unfortunately, the structural active forms of HspB1 that interact with these procancerous polypeptides targets are still not known. However, experiments performed in syngeneic rats bearing colon cancer cells revealed that large oligomeric structures are responsible of the tumorigenic activity of human HspB1 (Garrido et al. 1998). HspB1 phosphorylation or even an aberrant phosphorylation of HspB1 has also been suggested to be associated with cancer development and progression.
The small heat shock protein HspB1 plays an essential role in the cellular response to heat shock. This phospho-oligomeric ATP-independent molecular chaperone, whose expression is upregulated by heat shock, acts as a storage compartment for misfolded polypeptides before they are refolded by ATP-dependent chaperones. These storage compartments result of specific changes in HspB1 dynamic oligomerization and phosphorylation. Being also constitutively expressed during differentiation as well as in many human cell types, it can induce a protection against different stresses, such as apoptosis and oxidative stress. In unstressed cells, it regulates a large panel of cellular processes and plays major roles in many human pathological diseases. The large number of apparently unrelated functions assigned to constitutively expressed HspB1 results of its interactions with crucial client polypeptides that are subsequently modified in their half-life and/or activity. The dynamic changes in HspB1 oligomerization and phosphorylation that are observed in response to modifications in the cellular physiology are key factors to promote the formation of structural platforms aimed at recognizing specific client polypeptides. Hence, HspB1 is a molecular sensor that allows cells to better adapt or respond to changes in their physiology or environment.
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