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Role of the Hsp70 chaperone in erythropoiesis
essence, quantitative proteomic analysis revealed a strong bias towards maintaining a set of fully operational Hsp70 machineries conceivably functioning in specific protein repair activities important for the survival of mature ery- throcytes (Figure 5G). We also noticed a clear enrichment of the stress-induced form of Hsp70, HSPA1A (13-fold more enriched than in Jurkat cells, and >3,500 times more abundant than the average non-Hb protein) in mature ery- throcytes (Figures 5A and C). Analysis of the levels of this chaperone during red blood cell formation indicated that the HSPA1A is present 83-fold higher even in erythroblast progenitors compared to unstressed Jurkat cells, and then only decreases approximately 10-fold during terminal dif- ferentiation (Figure 2B; Online Supplementary Figure S1). The high levels of HSPA1A perhaps largely facilitate the blocking of erythroblasts from undergoing premature apoptosis during differentiation.89-91 However, in mature erythrocytes, HSPA1A may serve a different purpose where it could also form chaperone machines that prima- rily solubilize and repair misfolded/aggregated proteins. Functional studies are now required to deconvolute from the alternative, whereby some of the more abundant chap- erones such as the Hsp70 linger in mature erythrocytes simply because of an incomplete proteome reduction.
We also noticed the retention of a fully functional UPS, perhaps more fine-tuned to the needs of mature erythro- cyte maintenance (Figures 5A, D to F; Online Supplementary Tables S3-4). Although UPS proteins are 10- fold less abundant in erythrocytes than in Jurkat cells, they are 32-times less depleted than the average non-Hb protein (Figure 5A). A basal protein degradation system is likely needed to prevent the cytotoxic accumulation of terminally-damaged proteins in these cells (Figure 5G). As expected, the E2-E3 hybrid enzyme UBE2O was present in mature erythrocytes perhaps to more selectively target unpaired/damaged α-globin chains.27 We also, however, observed a considerable enrichment of Cullin-RING E3 ubiquitin ligase family members. It is somewhat puzzling as to why such E3 ubiquitin ligases are retained in post- mitotic terminally differentiated erythrocytes, given that their functions are mainly associated with gene transcrip- tion, cell cycle and development.100 In contrast, we observed the absence of E3 ubiquitin ligases that target misfolded proteins for degradation, such as members of the UBR family101-103 and STUB1/CHIP, which directly binds and ubiquitinate Hsp70 substrates104 (Online Supplementary Tables S3-4). This implies that protein degradation is considerably regulated and clearance of misfolded proteins might be kept to a minimal to further promote protein repair over proteolysis in mature ery- throcytes. Importantly, we also detected an enrichment of metabolic enzymes required to support glycolysis and the pentose phosphate cycle. These catabolic and meta- bolic pathways are vital for importing and breaking down glucose to produce ATP. The ATP generated from an active glycolysis reaction could supply the energy needed to support (i) chaperone-based protein repair (ii) UPS- mediated protein degradation and (iii) active ion pumps required for maintaining the steep ion gradients across plasma membrane in erythrocytes. Taking everything into account, it is intriguing to speculate that the retained Hsp70 chaperone system together with the UPS is cogged towards primarily repairing proteins in mature red blood cells. This is of particular interest given the wide array of hematological diseases associated with Hb aggregation.
Hsp70 associated blood disorders
The Hsp70 chaperone has been implicated in the pathophysiology of several prominent blood disorders in humans. Below we describe how this chaperone system may act as an important modifier which influences both the severity and progression of hematological disorders such as b-thalassemia, sickle cell disease, myelodysplastic syndromes, polycythemia vera and Diamond Blackfan anemia. Importantly, the ineffective erythropoiesis observed in these disorders is intimately linked to several key functions of the Hsp70 chaperone system in red blood cell differentiation.
Mislocalization of Hsp70 drives ineffective erythropoiesis in b-thalassemia
b-thalassemia is an autosomal recessive disease with three clinical phenotypes: b-thalassemia major (severe anemia), intermedia (mild to moderate anemia) and minor (clinically asymptomatic, patients act as “carriers” of the disease). One hallmark of this disease is the prema- ture apoptosis of differentiating erythroblasts in the bone marrow and the rapid destruction of circulating erythro- cytes by the reticuloendothelial system. b-thalassemia arises from a series of point mutations and deletions that reduce or prevent the production of functional b-globin. The decrease in b-globin levels correlates with the sever- ity of the condition.105 Apart from the mutation driven b- globin dosage effect, it was also found that the co-inheri- tance of the genetic variants of the globin genes has a modifying effect on the severity of the disease,105,106 which could be partly attributed to protein misfolding and aggregation. At a mechanistic level, the decrease in b-glo- bin levels leads to increased aggregation of unpaired α- globin chains,107 which triggers acute oxidative stress in erythroblasts leading to premature apoptosis.108
The Hsp70 chaperone system plays a pivotal role in the pathogenesis of b-thalassemia. During erythropoiesis, the stress-induced form of Hsp70 (HSPA1A) translocates in to the nucleus to protect GATA-1 from caspase-3 cleavage and initiate terminal differentiation (Figure 4).65 However, in b-thalassemia, this translocation step is largely imped- ed as a result of Hsp70 being sequestered into cytosolic aggregates formed by excess unpaired α-globin chains.66 This ultimately results in GATA-1 cleavage, which trig- gers ineffective erythropoiesis leading to anemia. In par- allel, sequestration of cytosolic Hsp70 by protein aggre- gates could also i) compromise the overall chaperoning capacity of early erythroblasts and ii) affect protein syn- thesis due to HRI activation.76 Although, a reduction in protein synthesis may temporally help prevent further accumulation of aggregate-prone proteins, such as α-glo- bin chains,76 it may also considerably affect the overall Hb biogenesis in these already compromised red blood cells.77 This mechanism may partly contribute to microcy- tosis that causes mild anemia in b-thalassemia minor.
A recent breakthrough study showed an unexpected suppression of the disease phenotype in a mouse model of b-thalassemia intermedia when UBE2O, which helps clear unpaired α-globin chains, was knocked out (Hbbth3/+ Ube2o−/−).27 The mitigation of anemia in these animals resulted from an increase in erythrocyte levels. Intriguingly, the erythrocytes generated in Hbbth3/+ Ube2o−/− animals were relatively healthier to those that were pro- duced in Hbbth3/+ genetic background. The Hbbth3/+ Ube2o−/−
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