Phosphorylation Dependence of Hsp27 Multimeric Size and Molecular Chaperone Function

Abstract

The molecular chaperone Hsp27 exists as a distribution of large oligomers that are disassembled by phosphorylation at Ser-15, -78, and -82. It is controversial whether the unphosphorylated Hsp27 or the widely used triple Ser-to-Asp phosphomimic mutant is the more active molecular chaperone in vitro. This question was investigated here by correlating chaperone activity, as measured by the aggregation of reduced insulin or ␣-lactalbumin, with Hsp27 self-association as monitored by analytical ultracentrifugation. Furthermore, because the phospho-mimic is generally assumed to reproduce the phosphorylated molecule, the size and chaperone activity of phosphorylated Hsp27 were compared with that of the phospho-mimic. Hsp27 was triply phosphorylated by MAPKAP-2 kinase, and phosphorylation was tracked by urea-PAGE. An increasing degree of suppression of insulin or ␣-lactalbumin aggregation correlated with a decreasing Hsp27 self-association, which was the least for phosphorylated Hsp27 followed by the mimic followed by the unphosphorylated protein. It was also found that Hsp27 added to pre-aggregated insulin did not reverse aggregation but did inhibit these aggregates from assembling into even larger aggregates. This chaperone activity appears to be independent of Hsp27 phosphorylation. In conclusion, the most active chaperone of insulin and ␣-lactalbumin was the Hsp27 (elongated) dimer, the smallest Hsp27 subunit observed under physiological conditions. Next, the Hsp27 phospho-mimic is only a partial mimic of phosphorylated Hsp27, both in self-association and in chaperone function. Finally, the efficient inhibition of insulin aggregation by Hsp27 dimer led to the proposal of two models for this chaperone activity.

Small heat-shock proteins: important players in regulating cellular proteostasis

“…Phosphorylation reduces the average oligomer size, and increases oligomeric polydispersity and rate of subunit exchange of αBc [98,[132][133][134], whereas it leads to a dramatic decrease in the size of Hsp27 oligomers, such that the triply phosphorylated isoform is predominately dimeric in solution [135,136]. Thus, under stress conditions, Hsp27 is phosphorylated, triggering dissociation of the high molecular mass Hsp27 oligomers [117,135,137,138] and an increase in the amount of exposed hydrophobicity on the newly formed Hsp27 dimers [139].…”

“…Significantly, phosphomimics of Bc and Hsp27 have similar attributes to the phosphorylated forms of the protein with regards to their oligomeric distribution, chaperone activity, subcellular localisation and cellular trafficking [136,143,150,151]. For example, the translocation of αBc to the nucleus and its association with nuclear speckles during mitosis is phosphorylation-dependent and this is replicated by the S19D/S45D/S59D αBc phosphomimic [143] and both in vitro phosphorylated Hsp27 and the S15D/S78D/S82D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Hsp27 phosphomimic have comparable abilities to prevent the aggregation of target proteins in vitro [136]. Both S59 phosphorylated and S59E phosphomimicking forms of Bc bind to the anti-apoptotic regulator Bcl-2, promoting apoptosis of breast cancer MCF7 cells and therefore making them more susceptible to chemotherapeutic agents [152].…”

“…For example, some studies have concluded that Hsp27 phosphorylation increases chaperone activity (e.g. [136,138]), others have reported no difference (e.g. [153]) whilst others have reported that phosphorylation decreases activity (e.g.…”

“…Various factors may account for these apparent differences including the target protein used to assess the chaperone activity, the final concentration of sHsp in the assays (wild-type Hsp27 dissociates at low concentrations and therefore may be predominately dimeric, like the phosphorylated form, in some assays [136,138]), and buffer and temperature conditions of the assay, all of which have been shown to influence sHsp chaperone activity [133]. Overall, the consensus emerging from work with Bc and Hsp27 concerning the impact of phosphorylation on sHsp chaperone function is that phosphorylation boosts chaperone activity by (i) promoting dissociation of large oligomers into smaller species [98,132,133,135,136] and (ii) enhancing their affinity to bind destabilised target proteins [103,138]. Based on structural modelling of a full-length Bc into a 24-mer oligomer [154].…”

pH-dependent structural modulation is conserved in the human small heat shock protein HSBP1

“…WT-HSPB1 forms large (∼450 kDa), polydisperse oligomers that are highly sensitive to factors such as temperature (Hayes et al 2009;Lelj-Garolla and Mauk 2006), phosphorylation (Shashidharamurthy et al 2005), and disease-associated missense mutations (Almeida-Souza et al 2010;Muranova et al 2015;Nefedova et al 2013a, b). Earlier elution of HSPB1 oligomers at low pH in size-exclusion chromatography (SEC) has been previously reported (Chernik et al 2004).…”