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Role of the Hsp70 chaperone in erythropoiesis
mitochondria localized apoptosis-inducing factor (AIF, AIFM1).59-62 The AIF released into the cytosol enters the nucleus via a nuclear localization signal63 and initiates cas- pase-independent chromatin condensation, DNA frag- mentation and nuclear shrinkage to fully commit cells to apoptosis.62 In healthy erythroblasts, however, Hsp70 chaperone appears to play an important role in neutraliz- ing this pro-apoptotic signaling pathway (Figure 4A). Cytosol-localized Hsp70 (primarily the stress induced HSPA1A) directly interacts with AIF and prevents its translocation into the nucleus.59,63,64 On the contrary, in “unhealthy” erythroblasts (e.g., cells experiencing acute oxidative stress), Hsp70 is largely sequestered away by the accumulating misfolded/aggregated proteins. This sequestration prevents the neutralization of AIF signaling, thus leading to fitness checkpoint failure and rapid elimi- nation of unhealthy cells (Figure 4B).
The second checkpoint appears to monitor the capacity of Hsp70 to protect GATA-1 from caspase-3 mediated proteolytic cleavage. EPO stimulation triggers the translo- cation of HSPA1A from the cytosol to the nucleus.65 In healthy erythroblasts, the nuclear translocated HSPA1A binds directly to GATA-1 and prevents the transcription factor from being cleaved, which allows the initiation of erythropoiesis (Figure 4A).65 However, if there is a defi- ciency in Hsp70 levels, activity and/or the chaperone is insufficiently translocated to the nucleus due to the sequestration away by cytosolic protein aggregates,66 the unprotected GATA-1 becomes targeted by caspase-3 (Figure 4B). Reduction in the level of GATA-1 inhibits both the terminal differentiation program and anti-cell death signaling via Bcl-xL67 that ultimately trigger clear- ance of unhealthy cells via apoptosis. This fitness check- point seems to function throughout the early stages of erythropoiesis. During the latter stage of erythropoiesis, a small heat shock protein (sHSP) named Hsp27 (also know as HSPB1) translocates to the nucleus (triggered by post- translational modifications) and appears to outcompete Hsp70 from binding to GATA-1, which results in the degradation of the transcription factor.68 In a nutshell, i) adequate nuclear and cytosolic levels of “free” Hsp70 chaperones and ii) favorable inputs from both of the pro- teostasis fitness checkpoints seem to be required to selec- tively initiate the terminal differentiation of healthy ery- throblasts.
Hsp70’s role in hemoglobin folding and assembly
Despite decades of research, the mechanistic under- standing of the folding and assembly of Hb (Figure 1B) remains incomplete. In particular, little is known about how heme moieties are inserted into nascent globin chains during de novo folding. Several chaperones includ- ing Hsp70, Hsp90, and AHSP have been recognized to assist in globin folding and assembly in erythrocytes.66,69,70 Whether Hsp70 directly assists in Hb biogenesis is still an open question. Early studies have identified a role for Hsp70 in stabilizing α-globin and preventing its aggrega- tion during erythropoiesis.66 However, this could also result indirectly through the regulation of heme regulated inhibitor of translation (HRI) by Hsp70, which affects Hb assembly. HRI is the main kinase, which fine-tunes the cellular levels of heme and globin proteins to facilitate efficient Hb assembly during erythropoiesis.71-73 In healthy erythroblasts with sufficient levels of heme, HRI is kept inactive by an autoregulatory mechanism involv-
ing complex formation with heme and HSPA8.74,75 When cellular heme levels decrease, the inhibition is released and the activated kinase rapidly phosphorylates the eukaryotic translation initiation factor 2α (eIF2α). This halts protein synthesis and prevents the overproduction of aggregate-prone globin chains.72,73,76 Under proteotoxic stress conditions, HRI is similarly activated to block Hb production, but now as a result of HSPA8 being seques- trated away by misfolded/aggregated proteins.77 This activation appears to be independent of heme levels in erythroblasts.75,77 Apart from inhibiting protein transla- tion, HRI initiates an integrated stress response in ery- throid precursors by selectively switching-on the tran- scriptional factor ATF-4 signaling pathway to induce mul- tiple antioxidants that help mitigate oxidative stress.78 The triggering of this mechanism can be clearly observed in heat shocked erythroblasts.73,77 The activity of HRI is critical for the viability of stressed erythroid progenitors since induction of Hsp70 and other chaperones alone is insufficient to mitigate proteotoxicity in these cells.72,79 Additionally, Hsp70 may in part help fold HRI adding another level of complexity to this regulation.75 In essence, Hsp70 directly and/or indirectly facilitates the efficient biogenesis of Hb during erythropoiesis.
Clearance of aberrant proteins by the Hsp70 system duringerythropoiesis
Triggering of ineffective erythropoiesis as a result of increased levels of protein aggregation has been observed in disease conditions such as b-thalassemia (see section on Hsp70 associated blood disorders).66 Interestingly, the induction of Hsp70, but not other major stress chaper- ones, has been detected in heat shocked erythroblasts.4,80 This demonstrates the existence of a somewhat special- ized stress response in erythroblasts to perhaps selective- ly induce Hsp70-based PQC activities. Previous work has shown that rabbit reticulocyte lysates have the capacity to resolve in vitro generated protein aggregates81 suggest- ing that differentiating erythroblasts possess strong pro- tein disaggregation/refolding activity. This activity is most likely generated via the recently discovered Hsp70- based protein disaggregases in human cells.82-87 These dis- aggregases could potentially co-operate with cellular pro- tein degradation systems82 to rapidly clear aggregated pro- teins and reduce associated toxicities to facilitate ery- thopoiesis
Suppression of apoptosis in differentiating erythroblasts by Hsp70
HSPA1A, which is upregulated in response to proteosta- sis insults has been demonstrated to block pro-apoptotic pathways that lead to caspase activation in cells.88 Erythroblasts appear to rely on the same Hsp70 homolog to prevent premature apoptosis during terminal differenti- ation.89-91 This necessity may partially explain why there is an unusually high level of HSPA1A present even in early erythroid progenitors primed to undergo erythropoiesis (Figure 2B). Additionally, the EPO signaling induced mito- chondrial HSPA9 could also inhibit apoptosis in part by suppressing the production of ROS.59 These Hsp70 mediat- ed anti-apoptotic signals together with the induction of anti-apoptotic protein Bcl-xL by GATA-167 appears to help prevent erythroblasts from undergoing premature death despite the considerable proteostasis challenges associated with normal erythropoiesis.
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