Erythropoiesis is a tightly regulated cell differentiation process in which specialized oxygen- and carbon dioxide-carrying red blood cells are generated in vertebrates. Extensive reorganization and depletion of the erythroblast proteome leading to the deterioration of general cellular protein quality control pathways and rapid hemoglobin biogenesis rates could generate misfolded/aggregated proteins and trigger proteotoxic stresses during erythropoiesis. Such cytotoxic conditions could prevent proper cell differentiation resulting in premature apoptosis of erythroblasts (ineffective erythropoiesis). The heat shock protein 70 (Hsp70) molecular chaperone system supports a plethora of functions that help maintain cellular protein homeostasis (proteostasis) and promote red blood cell differentiation and survival. Recent findings show that abnormalities in the expression, localization and function of the members of this chaperone system are linked to ineffective erythropoiesis in multiple hematological diseases in humans. In this review, we present latest advances in our understanding of the distinct functions of this chaperone system in differentiating erythroblasts and terminally differentiated mature erythrocytes. We present new insights into the protein repair-only function(s) of the Hsp70 system, perhaps to minimize protein degradation in mature erythrocytes to warrant their optimal function and survival in the vasculature under healthy conditions. The work also discusses the modulatory roles of this chaperone system in a wide range of hematological diseases and the therapeutic gain of targeting Hsp70.
Cells are highly vulnerable to proteotoxic stresses during widespread remodeling of proteomes that typically accompany cell differentiation. Under such challenging conditions, molecular chaperones that constitute an essential part of cellular protein quality control (PQC) pathways, help maintain proteostasis by decreasing protein misfolding and aggregation, and promote cell viability.1,2 In response to cell differentiation, considerable rearrangements in cellular chaperomes have been detected,2,3 but the functional consequences of such changes largely remain enigmatic. In particular, the heat shock protein 70 (Hsp70) chaperone system is maintained at high levels during red blood cell differentiation.3-5 Emerging data demonstrate that Hsp70 machineries have distinct functions ranging from modulating cell signaling to PQC activities at different stages of erythropoiesis. These multifaceted roles of the Hsp70 chaperone include maintaining erythroid progenitors, assessing fitness of progenitors prior to initiating lineage specific terminal cell differentiation, supporting hemoglobin (Hb) biogenesis, counteracting proteotoxicities and preventing premature apoptosis of differentiating erythroblasts, and promoting viability of terminally differentiated erythrocytes via protein repair. Hence, the dysfunction of this chaperone system is invariably associated with ineffective erythropoiesis, which leads to chronic anemia in several hematological diseases in humans.
Formation of red blood cells
Erythropoiesis is a vital process throughout vertebrate life, which helps maintain adequate tissue oxygenation under physiological and nonphysiological states (e.g., hypoxia, hemorrhage or other anemic conditions). This cell differentiation event leads to the generation of highly specialized erythrocytes that function as dedicated oxygen and carbon dioxide transporting cells across the body. Erythrocytes have a finite lifespan (approximately 120 days in humans) in the circulatory system before they are recycled mainly in the spleen by macrophages.6 These cells, therefore, must be continuously and rapidly replaced in vertebrates. About two million new erythrocytes per second are generated in adult humans7 via proliferation and differentiation of a self-renewing population of pluripotent hematopoietic stem cells (HSC) located in the yolk sac, liver, spleen (antenatal) or bone marrow (postnatal) that give rise to early erythroid progenitors. 6 During erythropoiesis, these progenitors undergo a red cell lineage specific terminal differentiation program to generate mature erythrocytes.
Erythrocyte production is tightly regulated by a set of hormones. For example, glucocorticoids regulate both the proliferation and differentiation of early erythroid progenitors known as early burst forming unit-erythroid (BFU-E) cells.8 The proliferation and differentiation of subsequent erythroid progenitors including late stage BFU-E and colony forming unit-erythroid (CFU-E) cells occur after the stimulation by erythropoietin (EPO), a glycoprotein cytokine secreted by the kidneys.9 EPO stimulation is vital for the induction of GATA-binding factor 1 (GATA-1) transcription factor, the master regulator of erythropoiesis. GATA-1 together with the transcription factor STAT5, promote further erythroblast proliferation10 and turn on the gene activation and repression program, which drives the multistep terminal differentiation process of these cells.11 In mammals, erythropoiesis can be resolved into six morphologically distinct cell stages that result from a series of mitotic cell divisions (Figure 1A). These stages include: (i) proerythroblast, (ii) basophilic erythroblast, (iii) polychromatophilic erythroblast, (IV) orthochromatophilic erythroblast, (V) reticulocyte, and (VI) mature erythrocyte. During terminal differentiation, the erythroblasts decrease in cell size, condense chromatin, reorganize and reduce cellular proteome and membranes, and eliminate organelles, thereby making space for the rapidly increasing levels of Hb, the oxygen-trafficking protein complex. The most striking morphological change occurs in orthochromatophilic erythroblasts that eject nuclei to form reticulocytes in the bone marrow (Figure 1A). These reticulocytes loose ribosomes and the bulk of RNA molecules, and develop into Hb packed mature erythrocytes that have a characteristic biconcave disk-like shape with a flattened center.
The mature erythrocytes contain a remarkably high concentration of Hb molecules (approximately 29.5 pg/cell),12 which represents approximately 98% of the proteome.13 Hb is a tetrameric globular protein made up of four globin protein subunits/chains, each containing a heme group, which reversibly binds to oxygen and carbon dioxide. The adult human hemoglobin (HbA) is made from two 141 amino acid-long α-globin chains and two 146 amino acid-long b-globin chains (α2b2) (Figure 1B). Under physiological conditions, HbA makes up >97% of the Hb constituent in adult humans. The remaining Hb contains fetal Hb (HbF; α2γ2) and HbA2 (α2d2), two isotypes generated by switching the b globin chain with either γ or d globin chains.
Proteotoxic stresses associated with erythropoiesis
Proteostasis is maintained by balancing the cellular pathways that facilitate protein synthesis, folding, assembly, trafficking, and degradation under varying environmental and metabolic conditions.1 Even under normal growth conditions, cells experience a continuous influx of misfolded proteins generated from various protein biogenesis mistakes such as errors in transcription, translation and folding. Additionally, most proteins are at risk of misfolding due to the marginal stability of their native conformations.14 Cells have evolved a set of intricate PQC pathways comprising of molecular chaperones and protein degradation systems that operate constantly to decrease the levels of misfolded proteins that otherwise would easily form aggregates in crowded cellular environments. Protein aggregates typically show poor solubility in aqueous cellular environments, have no physiological function per se and could instead elicit cytotoxicity.1 By untangling and unfolding such aberrant protein species, the ATP fueled chaperone machineries are able to “repair” and rescue proteins, which leads to a considerable reduction in the risk of proteotoxicities in cells. Protein degradation pathways also represent an important line of defense by clearing misfolded proteins and preventing their accumulation.1 However, in aging and/or stressed cells, such defense mechanisms could become overwhelmed leading to the buildup of potentially toxic protein aggregate species. Many disease-linked mutant proteins also form such aggregates that are refractory to PQC systems, including degradation pathways.15
Due to various protein biosynthetic errors and considerable attenuation of basic PQC pathways, erythroid maturation is highly exposed to protein misfolding/aggregation. The first PQC challenge during erythropoiesis involves the folding and assembly of the α-globin chains that show a high degree of instability. In the absence of the partnering b-globin, the free α-chains with heme/iron could readily misfold to form protein aggregate deposits called Heinz bodies (for a review, see Voon et al.16). These highly toxic protein aggregates, when accumulated, could trigger the generation of reactive oxygen species (ROS)17 that damage cellular proteins, nucleic acids and lipids and induce oxidative stress in erythroblasts leading to premature cell death (Figure 1B).18 This is circumvented to a certain degree with the assistance of a dedicated chaperone named alpha hemoglobin stabilizing protein (AHSP). AHSP mimics the α-helix-loop-α-helix motif of b-globin and assists in the folding of α-chains in a “template” directed manner19,20 (for a review, see Weiss et al.)21. Apart from the α-chain instability, the generally high synthesis rates of globin proteins (300 mg of Hb per hour in healthy adult humans22) during this atypical state could also proportionally increase the level of intrinsic errors in folding and assembly23 of Hb. In particular, heme/iron imbalances could result in globin misfolding, which could induce severe oxidative stress in erythroblasts. A tightly regulated supply of iron to support the production of heme is required for efficient Hb biogenesis. Heme synthesis is mediated by conjugating iron and protoporphyrin in a series of enzymatic reactions occurring in mitochondria and cytosol.24 The hydroxyl radicals produced by elevated levels of free heme/iron undergoing the Fenton reaction in erythroblast cytosol could damage and induce the aggregation of both Hb and other critical biomolecules.25
The next PQC challenge in red blood cell maturation occurs from the reduction/disruption of crucial PQC pathways that typically protect cells against protein aggregation. In order to make space for the increasing levels of Hb, the proteome of the terminally differentiating erythroblasts is rapidly reduced to 2-5% via bulk degradation of many cellular proteins and organelles3 by the ubiquitin proteasome system (UPS) and the autophagy pathway.26,27 This drastic proteome remodeling expectedly depletes components of chaperone and proteolytic machineries and decreases the ability of these cells to induce global PQC pathways that help buffer against proteotoxic stresses.28 For example, unlike other cell types, reticulocytes show inability to fully recover critical cellular functions such as protein synthesis after heat shock.4,29 On the whole, terminally differentiating erythroblasts seem particularly vulnerable to stresses associated with protein misfolding and aggregation.
The Hsp70 chaperone system
Hsp70 forms one of the most abundant and highly conserved molecular chaperone systems critical for maintaining cellular proteostasis. This highly versatile chaperone system supports a plethora of housekeeping and stressrelated cellular repair processes that protect cells against proteotoxic stresses (for a review, see Rosenzweig et al.30). The key housekeeping activities include facilitating folding of newly synthesized proteins, transport of polypeptides across cellular membranes, assembly/disassembly of protein complexes and regulation of protein activity. In stressed cells, the Hsp70 chaperone system functions to prevent aggregation of aberrant proteins, refold misfolded proteins, solubilize aggregated proteins, and cooperate with cellular degradation machineries to clear terminally damaged proteins (for a review, see Rosenzweig et al.30).
The chaperoning functions of Hsp70 are tightly regulated via the cooperation of dedicated cochaperones from the J-domain protein (JDP) family and nucleotide exchange factors (NEF) that fine-tune Hsp70’s ATPdependent allosteric control of substrate binding and release (Figure 2A). JDP form the largest and the most diverse family of cochaperones in humans (over 42 members) and provide specificity to the Hsp70 family (13 homologs in humans) by selecting substrates.30,31 Concomitant interaction of Hsp70 with a JDP and substrate boosts ATP hydrolysis in Hsp70. This dual trigger allows Hsp70 to efficiently trap and unfold substrates (Figure 2A).32 The timely release of substrates is mediated by nucleotide exchange factors that release ADP and allow subsequent rebinding of ATP, thus resetting the Hsp70 chaperone to its open, low substrate affinity state to receive a new client.30,33
Hsp70 generally shows a high affinity towards misfolded and aggregated substrates, and a low affinity for native proteins, which may, thus be considered as the products of such polypeptide unfolding enzymes.34 As earlier stated, the energy from ATP hydrolysis drives the iterative protein unfolding cycles of Hsp70 that allow the refolding of misfolded proteins (Figure 2A). Hsp70 and other protein folding chaperone systems (e.g., Hsp60 and Hsp90) promote the buildup and maintenance of relatively high levels of native protein conformers under non-equilibrium stress conditions where without chaperones or ATP, the denatured protein conformers would readily seek equilibrium and turn into stable inactive misfolded species.35 Conceptually, this aligns with Erwin Schrödinger’s view, which states that living matter evades the decay to equilibrium.36 The term “evades” implies that living cells must constantly consume energy in order to avoid spontaneous entropy-driven decomposition of their macromolecules (e.g., proteins), leading to cell death. This is possible because the biosphere is not a closed system: the energy from the sun is harnessed by photosynthesis to produce ATP for all organisms to fuel their repair (and replace) mechanisms. The chaperone-based protein repair mechanisms constantly counteract the natural entropic tendency of proteins to misfold and further decay by hydrolysis and oxidation into simpler molecules. In other words, when acting as ATP-fueled iterative unfolding nanomachines, chaperones such as Hsp70 can correct or “repair” structurally damaged proteins as they are formed under stressful non-equilibrium conditions.37 Erythropoiesis is a prime example of how Hsp70’s protein repair and regulatory functions are fully deployed to support cell differentiation and viability.
Multifaceted roles of the Hsp70 chaperone system in erythropoiesis
During red blood cell generation, the Hsp70 chaperone system functions in a number of regulatory and PQC activities. By changing its cochaperones, the Hsp70 chaperone could target different clientele and switch between functions, which allows this highly versatile chaperone system to rapidly respond to different cell growth and differentiation conditions.38,39 The main roles of Hsp70 during erythropoiesis include: (i) aiding in maintaining erythroid progenitors (ii) assessing fitness of progenitors prior to initiating lineage specific terminal cell differentiation (iii) supporting Hb biogenesis (iv) counteracting proteotoxicities and preventing premature apoptosis of differentiating erythroblasts and (v) conceivably promoting viability of differentiated (mature) erythrocytes via protein repair.
Hsp70 regulates dormancy and cell cycle quiescence of erythroid precursor cells
Continuous proliferation and differentiation of hematopoietic stem cells into committed erythroid progenitor cells6 is required for maintaining healthy levels of mature erythrocytes in the peripheral vasculature. The cyclin dependent cell cycle entry from G1 to S phase during proliferation of hematopoietic stem cells is modulated largely by the opposing actions of cyclin dependent kinases (CDK) and cyclin-dependent kinase inhibitors (CDKi).40 In order to terminate the dormancy of hematopoietic stem cells and initiate cell cycle entry, cyclin D1, the regulatory subunit of CDK4 and CDK6, has to translocate from the cytosol to the nucleus.41,42 This key step in HSC proliferation is mediated by the constitutively expressed heat shock cognate protein 70 (Hsc70/HSPA8), which binds to cyclin D1 and shuttles it across the nuclear membrane (Figure 3).41 Here, HSPA8 appears to recognize a peptide segment in an unstructured (likely a looped or terminal) region exposed on the surface of folded cyclin D1. Similar types of interactions between Hsp70 and native proteins leading to regulatory activities have been demonstrated with clathrin triskelions, immunoglobulin heavy chain, Escherichia coli heat shock transcription factor σ32 and plasmid replication protein RepE.43-45 The cyclin D1-HSPA8 complex is retained in the cytosol by forming additional interaction(s) with p57KIP2 and p27KIP1, two critical CDKi that are known to prevent the cell cycle progression of hematopoietic stem cells (Figure 3).41 It is tempting to speculate that through complexing, the CDKi mask the nuclear localization related signal of HSPA841,42 until the CDKi are degraded via stem cell factor signaling coupled to the initiation of erythropoiesis. ERK signaling, which plays a modulatory role in erythropoiesis46 also plays a role in the nuclear shuttling of Hsp70,47 but little is known about how these signals are integrated, if at all. The rodent mammalian relative of DnaJ (MRJ), an ortholog of human JDP DNAJB6, was identified from recent stem cell work48 to play a role in promoting cell quiescence by binding to a cyclin D1 inhibitor.49 The same JDP was implicated in playing a role in stem cell self-renewal.50 Whether MRJ/DNAJB6 or another JDP directs the selection of clients in the CDKi-HSPA8-cyclin D1-mediated HSC proliferation pathway remains to be determined. The terminal differentiation of erythroblasts also requires cell cycle regulating cyclins. Cyclins A2 and D3 are required to control cytokinesis, erythrocyte size and number.51,52 Here too, the Hsp70 system facilitates the shuttling of cyclins A2 and D3 and co-operate with CDKi to regulate terminal differentiation of erythroblasts. An analysis of erythroblasts obtained from differentiating human cord blood CD34+ cells shows that only three out of the seven CDKi (p18INK4c, p19INK4d and p27KIP1) are expressed to significant levels during early and late terminal differentiation steps of red blood cells. In contrast to the functions of p57KIP2 and p27KIP1 in HSC, p19INK4d appears to be promoting erythroblast differentiation by facilitating nuclear localization of the stress inducible Hsp70 (HSPA1A) through the activation of the ERK, but not AKT, signal transduction pathway (Figure 3).47 p19INK4d may even play a role in the proteostasis-based fitness checkpoints in human erythroblasts (see below). How EPO stimulation leads to the induction and ERK-mediated nuclear translocation of Hsp70 and the role of CDKi in modulating this process during erythropoiesis remains to be dissected.53,54
The mammalian mitochondrial Hsp70 (mortalin/ HSPA9) is also implicated in the proliferation/maintenance of early progenitors of erythrocytes.55 HSPA9 cooperates with the inner mitochondrial translocase (TIM) complex to facilitate the translocation of mitochondrial matrix proteins that are essential for mitochondrial function and cell viability.56 Therefore, it is conceivable that any depletion of HSPA9 levels leads to increased mitochondrial dysfunction and activation of pro-apoptotic factors that induce hematopoietic progenitor cell death.57 Intriguingly, however, compared to progenitors of other lineages of hematopoiesis, a greater reduction of BFU-E progenitors was observed when HSPA9 was knocked down in rodents, suggesting that this Hsp70 paralog possibly plays an additional role(s) in maintaining the erythroid progenitor cell niche.58
Hsp70 checks the fitness of erythroblasts at the initiation of erythropoiesis
The continuous generation of large amounts of red blood cells to traffic O2/CO2 in vertebrates comes with a heavy energy cost (ATP-wise). By allowing only healthy erythroid progenitors to undergo cell differentiation in part increases the fidelity of this process. In order to select healthy progenitors, two fitness checkpoints seem to have evolved around the Hsp70 chaperone system. In both checkpoints, the Hsp70 chaperone appears to monitor proteostasis deficiencies in EPO stimulated erythroid progenitors that are primed to undergo differentiation. By acting as a sensor of global folding status, Hsp70 is able to gauge the levels of misfolded/aggregated proteins in these cells. The first fitness checkpoint seems to be initiated by momentarily triggering a pro-apoptotic insult during EPO stimulation, which induces mitochondria to undergo transient depolarization.59 This prompts the activation of several pro-apoptotic signals including the release of the 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 caspase- independent chromatin condensation, DNA fragmentation and nuclear shrinkage to fully commit cells to apoptosis.62 In healthy erythroblasts, however, Hsp70 chaperone appears to play an important role in neutralizing 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 elimination 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 translocation 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 deficiency 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 clearance of unhealthy cells via apoptosis. This fitness checkpoint 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 posttranslational 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 proteostasis fitness checkpoints seem to be required to selectively initiate the terminal differentiation of healthy erythroblasts.
Hsp70’s role in hemoglobin folding and assembly
Despite decades of research, the mechanistic understanding 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 including 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 aggregation 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 involving 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 sequestrated away by misfolded/aggregated proteins.77 This activation appears to be independent of heme levels in erythroblasts.75,77 Apart from inhibiting protein translation, HRI initiates an integrated stress response in erythroid precursors by selectively switching-on the transcriptional factor ATF-4 signaling pathway to induce multiple 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 during erythropoiesis
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 chaperones, has been detected in heat shocked erythroblasts.4,80 This demonstrates the existence of a somewhat specialized stress response in erythroblasts to perhaps selectively induce Hsp70-based PQC activities. Previous work has shown that rabbit reticulocyte lysates have the capacity to resolve in vitro generated protein aggregates81 suggesting that differentiating erythroblasts possess strong protein disaggregation/refolding activity. This activity is most likely generated via the recently discovered Hsp70- based protein disaggregases in human cells.82-87 These disaggregases could potentially co-operate with cellular protein degradation systems82 to rapidly clear aggregated proteins and reduce associated toxicities to facilitate erythropoiesis
Suppression of apoptosis in differentiating erythroblasts by Hsp70
HSPA1A, which is upregulated in response to proteostasis 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 differentiation. 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 mitochondrial HSPA9 could also inhibit apoptosis in part by suppressing the production of ROS.59 These Hsp70 mediated 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.
Quantitative proteomics highlight vital protein quality control functions related to mature erythrocyte survival
Mature erythrocytes show a remarkable ability to survive up to 120 days in the circulation6 while supporting a plethora of enzymatic reactions required for preserving the cytoskeletal ultrastructure, biomolecule trafficking across membranes and signal transduction (e.g., for maintaining lipid homeostasis92). Apart from the mechanical stress insults that induce shape changes while navigating through the vasculature, these cells could be subjected to a range of environmental (e.g., chronic hypoxia at high altitudes, hyperosmotic shock, and energy depletion) or chemical (e.g., oxidative stress and high intracellular Ca2+ levels) stresses93 that could trigger protein misfolding, damage and/or aggregation. Such conditions could potentially cause eryptosis, a process by which mature erythrocytes undergo apoptosis-like cell death. Unlike other cell types that contain stress-sensing and signaling pathways (e.g., HSF-1 transcription factor-mediated heat shock response) to produce large quantities of new chaperones to help buffer against such proteostasis insults,1 mature erythrocytes that lack ribosomes have to utilize already existing PQC elements present in their vestigial proteome to counteract protein misfolding/aggregation.
Advancements in mass spectrometry-based quantitative analytical methods have identified over 2,600 proteins in the vestigial proteome of mature human red blood cells (2-5% of the progenitor proteome).94 We performed an enrichment analysis on these proteins or protein classes focusing on function to uncover probable biological processes important for erythrocyte survival. We compared published label-free quantitative proteomics data obtained from complete cytosolic extracts of mature erythrocytes (originating from age-matched, healthy donors),94 to human cells (unstressed Jurkat cells) that have not massively accumulated Hb and carbonic anhydrase, and neither eliminated their transcription and translation machineries, nor lost their endoplasmic reticulum (ER), nuclei and mitochondrial compartments (Figure 5; Online Supplementary Tables S1-4).95 As expected, our analysis showed that mature erythrocytes (likely containing ~1% reticulocytes with ribosomes and ER),94 are markedly enriched with Hb, carbonic anhydrase and antioxidant enzymes, such as catalases (Figure 5A). Our analysis also confirmed that mature erythrocytes are severely depleted in DNA polymerases, transcription factors, RNA polymerases, ribosomes, as well as nuclear, ER and mitochondrial proteins compared to Jurkat cells (Figure 5A). On average, the replacement of the proteome with high levels of Hb and carbonic anhydrases in mature erythrocytes should theoretically reduce the levels of any other given protein by ~280-fold in abundance compared to Jurkat cells.94,95 Thus, it is reasonable to assume that proteins or protein classes that are significantly less depleted than ~280-fold have been purposefully retained to sustain functions associated with the maintenance and survival of mature erythrocytes. For example, compared to Jurkat cells, the cytoskeletal proteins are 44-fold more abundant than the average non-Hb protein in erythrocytes thus supporting this view (Figure 5A). We also detected a high level of Hsp60 chaperones that facilitate in the folding and assembly of cytoskeletal proteins. The above observations highlight the critical role of these proteins in maintaining cell shape and cytoskeletal integrity of red blood cells.
The role of Hsp70 in terminally differentiated erythrocytes
Molecular chaperones make up approximately 5% of the total protein mass of naïve mammalian cells, and up to 10% in stress-resistant cancer cells, attesting to their central role in maintaining cell viability. The Hsp70-JDP-NEF machinery alone contributes to ~1% of the total protein mass of most mammalian cells.96 Comparatively, the chaperome of mature erythrocytes is 0.28% of the total protein mass (including Hb) (Online Supplementary Tables S1-2). The erythrocyte Hsp70-JDP-NEF machinery, however, accounts for about a third of the mature erythrocyte chaperome (0.1% of the total protein mass; Figure 5; Online Supplementary Table S2). From a proteostasis angle, it is somewhat puzzling as to why this particular chaperone system, which is conventionally thought to function in de novo folding of newly synthesized proteins,97 is maintained at relatively high levels in terminally differentiated erythrocytes that lack protein-synthesizing capability.
Up to a certain point, protein repair is less costly than protein replacement
At the cellular level, life may evade protein decay by maintaining a subtle balance between ATP-fueled protein repair by unfolding chaperones and ATP-fueled protein replacement mediated by controlled degradation of irreversibly damaged proteins followed by transcription, translation, folding and assembly of new functional proteins. Noticeably, in terms of ATP cost, it is energetically cheaper to repair structurally damaged proteins with unfolding chaperones than to degrade them by the proteasome and synthetize replacements at the cost of at least two ATP molecules per peptide bond.37
Mature erythrocytes are an extreme case of living cells that completely lack protein replacement mechanisms. This leaves protein repair as the sole mechanism to counteract the time-dependent entropic decay of labile proteins into aggregates and support erythrocyte survival in circulation. Intriguingly, we detected all the components of the Hsp70 chaperone system necessary for protein repair in the vestigial proteome of mature red blood cells. These include the constitutively expressed HSPA8, a selective set of JDP cochaperones (DNAJA and DNAJB) that recognize misfolded/ aggregated proteins, and Hsp110-type nucleotide exchange factors that support protein disaggregation/ refolding in human cells (Figure 5C; Online Supplementary Tables S1-2).82-84,86,98,99 When combined, the identified Hsp70 chaperone system components were 14-fold more enriched in mature erythrocytes with respect to Jurkat cells (Figure 5A, red bars). Interestingly, the DNAJA and DNAJB cochaperones showed significant qualitative rearrangements in mature erythrocytes, compared to Jurkat cells (Figures 5B and C). The JDP composition shifted from a DNAJA:DNAJB ratio of ~2:1 in Jurkat cells (Figure 5B) to a DNAJB class-dominated ratio (approximately DNAJA:DNAJB = 1:9) in erythrocytes (Figure 5C). Hsp110 cochaperones were also re-arranged in erythrocytes; HYOU1 (the ER-resident HSP110) and HSPH3 (APG-1, cytosolic) were considerably depleted, while the proportion of HSPH2 (APG-2, cytosolic) was only slightly decreased from ~9% (of the Hsp70/110 chaperome) in Jurkat cells to ~7% in total erythrocyte cytosol (Figures 5B and C). In 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 erythrocytes (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 erythrocytes (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 differentiation (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 primarily solubilize and repair misfolded/aggregated proteins. Functional studies are now required to deconvolute from the alternative, whereby some of the more abundant chaperones 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 erythrocyte 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 postmitotic terminally differentiated erythrocytes, given that their functions are mainly associated with gene transcription, 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 erythrocytes. Importantly, we also detected an enrichment of metabolic enzymes required to support glycolysis and the pentose phosphate cycle. These catabolic and metabolic 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) UPSmediated 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 premature apoptosis of differentiating erythroblasts in the bone marrow and the rapid destruction of circulating erythrocytes 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 severity of the condition.105 Apart from the mutation driven bglobin dosage effect, it was also found that the co-inheritance 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-globin 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 impeded 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 triggers ineffective erythropoiesis leading to anemia. In parallel, sequestration of cytosolic Hsp70 by protein aggregates could also i) compromise the overall chaperoning capacity of early erythroblasts and ii) affect protein synthesis due to HRI activation.76 Although, a reduction in protein synthesis may temporally help prevent further accumulation of aggregate-prone proteins, such as α-globin 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 microcytosis 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 produced in Hbbth3/+ genetic background. The Hbbth3/+ Ube2o erythrocytes also showed a dramatic decrease in the levels of aggregated Hb. The authors speculated that the observed decrease in α-globin aggregation resulted from an increase in eIF2α phosphorylation, which reduced the production of globin chains (approximately 20% and 40% reduction in α- and b-globin, respectively, compared to control animals).27 However, the steady-state α-globin: b-globin ratio in the soluble protein fraction, in which the aggregate prone α-globin was still largely in excess, did not change between Hbbth3/+ and Hbbth3/+ Ube2o−/− cells. A closer look at the chaperone levels in the Hbbth3/+ Ube2o−/− erythrocytes suggests an alternative explanation. The study shows that the deletion of UBE2O resulted in elevated levels of AHSP. Similarly, the Hsp70 and Hsp90 chaperone systems were also induced in these cells.27 Such chaperone inductions could boost the protein repair capacity consequently leading to the observed decrease in α-globin precipitation in Hbbth3/+ Ube2o−/− erythroblasts despite defects in degrading excess α-globin. The moderate decrease in α-globin synthesis may also help reduce the burden on PQC machineries contributing to the remarkable rescue of erythroblasts in the Hbbth3/+ Ube2o−/− animals. Based on the findings from our proteomics data analysis (Figure 5), we speculate that the degradation of a certain amount of damaged proteins, despite the inability to replace them, might be tolerated and perhaps advantageous for the long-term survival of mature erythrocytes under healthy conditions. However, in unhealthy erythroblasts (e.g., early b-thalassemic erythroblasts108), a strong induction of the UPS due to stress could generate an aberrant PQC condition where even foldable conformers of proteins might be partitioned towards rapid degradation. 101 Such a condition could disrupt the fine balance between protein repair and clearance in these cells (Figure 5G). A careful study of the potential misregulation of PQC pathways in erythroblasts is required to fully comprehend the underlining mechanism of pathology in bthalassemia. It is tempting to speculate that even slight increases in the levels of certain Hsp70 machineries at pre- or very early stages of erythropoiesis may favorably tilt the folding equilibrium of globin chains and minimize the formation of cytotoxic Hb aggregates. Such chaperone manipulations at clinical level could result in reducing the symptoms of b-thalassemia.
Hsp70, a key modulator of inflammation in sickle cell disease?
Sickle cell disease (SCD) is another autosomal recessive genetic disorder associated with chronic anemia. SCD results from massive cyclic-polymerization of a structurally aberrant variant of adult Hb S (HbS) under hypoxic conditions. The resulting HbS fibers deform mature erythrocytes into rigid “sickle” shaped cells that aggregate and readily undergo premature destruction in the vasculature. Whether these conditionally formed reversible protein fibers (distinct from amyloid-type fibers formed e.g., in neurodegenerative disorders) trigger any proteostasis insult involving the Hsp70 system is unknown. The Hsp70 chaperone, however, may play an important role in activating the inflammatory response in SCD.109,110 Stressed cells have been observed to secrete Hsp70 into the extracellular matrix.109 Immune cells generate specific peptides from secreted Hsp70 that act as key mediators of stress-induced inflammation.111-113 A considerable buildup of circulating Hsp70 levels have been detected in patients with SCD109 suggesting that there is an active secretion of Hsp70 by blood cells. Under hypoxic conditions, sickled erythrocytes show differential recruitment of the stress-inducible HSPA1A to the cell membrane,114 possibly representing an early step in this secretion process. Interestingly, a similar observation was recently noted in b-thalassemia intermedia. 115 Together, the observations suggest that the high levels of extracellularly circulating Hsp70 may serve as an important immune modulator that trigger inflammation in these hemoglobinopathies, leading to increased red blood cell destruction by macrophages.
Haploinsufficiency of Hsp70 associated with myelodysplastic syndromes
Myelodysplastic syndromes (MDS) are a heterogeneous, but closely related group of hematopoietic malignancies characterized by ineffective erythropoiesis leading to peripheral blood cytopenias.116 The mitochondria localized HSPA9 is strongly implicated as a protein that contributes to MDS. The HSPA9 locus (5q31.2) is frequently deleted in patients with MDS, leading to a haploinsufficiency of this chaperone.117 Recent work showed that mutating HSPA9 causes an MDS-like phenotype in zebrafish118 and a knockdown in rodents results in considerable delay in erythroid progenitor maturation.58 HSPA9 is implicated in the pathogenesis of MDS at two levels. First, the haploinsufficiency of the chaperone may contribute to the phenotype as a result of altered mitochondrial import and refolding of heme-synthesis enzymes required for heme biogenesis during erythropoiesis. 55 Second, a decrease in HSPA9 could activate p53, a nuclear transcription factor, resulting in cell cycle arrest and premature apoptosis of hematopoietic progenitor cells.57
Further, a recent study demonstrated that defects in EPO induced nuclear translocation of Hsp70 in erythroblasts could also be an important driver of these disorders.119 The ineffective erythropoiesis observed in MDS could largely be reversed by protecting GATA-1 from caspase 3 cleavage using an Hsp70 variant (lacking the nuclear export signal) that accumulates in the nucleus. 119 Protein aggregates containing aberrant p53 (as in cancer cells)120,121 have been detected in erythroblasts in some forms of MDS.122 These aggregates could conceivably sequester Hsp70, thus triggering ineffective erythropoiesis in a similar mechanism to that in b-thalassemia. Alternatively, defective EPO signaling or/and other mechanisms affecting nuclear transportation of proteins, could lead to the Hsp70 trafficking defect observed in MDS.119
Hsp70 is a modifier of polycythemia vera
Polycythemia vera is a hyperproliferative disorder characterized by increased synthesis of red blood cells resulting in hyperviscosity of whole-blood. Recent proteomic studies on polycythemia vera have shown that increased levels of Hsp70 along with Hsp90 stabilize JAK2 kinase. This triggers a prolonged aberrant activation of the kinase, which results in massive proliferation of erythroid progenitors and abnormal stimulation of erythropoiesis. 123-126 Inhibition of either Hsp70 or Hsp90 has been demonstrated to promote the apoptosis of the abnormally proliferating erythroid progenitors and is currently being investigated as a potential therapeutic approach to delay the progression of this disease in humans.123,126
Haploinsufficiency of Hsp70 modulates Diamond Blackfan anemia
Diamond Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome resulting from ineffective erythropoiesis.127 In this disease, erythroid differentiation arrests between BFU-E and CFU-E stages.128 More than 70% of the cases of DBA occur due to haploinsufficiency of genes that encode for the small and large ribosomal subunit proteins.129-131 The defective ribosome biogenesis triggers ineffective erythropoiesis in part due to the decreased production of GATA-1.132 This is further promoted by imbalances in globin chain and heme synthesis leading to α-globin aggregation and induction of oxidative stress in erythroblasts133 similar to b-thalassemia. Additionally, in DBA associated with RPL11, but not RPL19 haploinsufficiency, the Hsp70 chaperone is considerably degraded in the erythroblasts via the UPS.133,134 The reason for the rapid degradation of Hsp70 in some permutations of this disorder remains unclear. Recent work shows that aberrations in chromatin organization resulting from low levels of the global chromatin organizer SATB1 prevents the induction of Hsp70 in early erythroblasts in DBA.135 Together, these observations suggest that the differential Hsp70 expression and degradation rates may have considerable effect on red blood cell viability, differentiation and Hb biogenesis, thus partly explaining the variability in the observed phenotypes of DBA. Remarkably, the restoration of Hsp70 levels in affected erythroblasts inhibits premature apoptosis and substantially restores erythropoiesis in DBA,133,134 thus providing an important therapeutic avenue for the treatment of this blood disorder.
A predicted pathological role for Hsp70 in congenital sideroblastic anemias
Congenital sideroblastic anemias (CSA) are inherited rare blood disorders characterized by erythroblasts displaying ring sideroblasts formed by the pathological depositions of iron in mitochondria. Patients with CSA show a significant reduction in the regeneration of erythrocytes leading to anemic conditions. The ineffective erythropoiesis in CSA is caused by defects in iron-sulfur cluster biogenesis essential for a broad range of cellular functions. Patients with CSA show mutations in genes directly (e.g., GLRX5, ABCB7)136,137 and indirectly (e.g., HSPA9)138,139 associated with this biosynthetic pathway. It is important to note that the ring sideroblasts are uncommon in MDS cases that are also associated with HSPA9 deletion mutations (see above), but whether this is due to an epistatic suppression by another modifier remains to be investigated.138 As in CSA, HSPA9 may also play an indirect role in some forms of dyserythropoietic anemias that are also characterized by pathological iron-loading defects and ineffective erythropoiesis140 and warrants further investigation.
Aging is attributed to a decline in hematopoiesis with high incidents of anemia.141 The viability and self-renewal of HSC/early erythroid progenitors depend on maintaining robust PQC activity and high levels of Hsp70.142,143,144 These abilities decline in stem cells during aging.145,146 As a consequence, Hsp70-mediated functions such as maintenance of erythroid dormancy, cell cycle quiescence and cell cycle entry may breakdown leading to an age-related exhaustion of HSC.
Recent findings have considerably broadened our understanding of the multifaceted roles of Hsp70 in erythrocyte differentiation and how deficiencies in its activity modify several blood disorders in humans. The tuning of the Hsp70 chaperone system to cater to different PQC needs during erythropoiesis sheds extremely valuable insight on cell repair and viability and provides a conceptual framework for investigating chaperone-based therapeutic avenues for a wide spectrum of blood disorders.
- Received July 29, 2019
- Accepted September 25, 2020
No conflicts of interest to disclose.
NBN conceptualized the work. BF, PG and NBN analyzed published proteomics data. YM, BF, SJ, PG and NBN wrote and edited the manuscript.
- Labbadia J, Morimoto RI. The biology of proteostasis in aging and disease. Annu Rev Biochem. 2015; 84:435-464. https://doi.org/10.1146/annurev-biochem-060614-033955PubMedPubMed CentralGoogle Scholar
- Vonk WI, Rainbolt TK, Dolan PT, Webb AE, Brunet A, Frydman J.. Differentiation drives widespread rewiring of the neural stem cell chaperone network. Mol Cell. 2020; 78:329-345. https://doi.org/10.1016/j.molcel.2020.03.009PubMedPubMed CentralGoogle Scholar
- Gautier E-F, Ducamp S, Leduc M. Comprehensive proteomic analysis of human erythropoiesis. Cell Rep. 2016; 16(5):1470-1484. https://doi.org/10.1016/j.celrep.2016.06.085PubMedPubMed CentralGoogle Scholar
- Banerji SS, Theodorakis N, Morimoto RI. Heat shock-induced translational control of HSP70 and globin synthesis in chicken reticulocytes. Mol Cell Biol. 1984; 4(11):2437-2448. https://doi.org/10.1128/MCB.4.11.2437PubMedPubMed CentralGoogle Scholar
- Gautier EF, Leduc M, Cochet S. Absolute proteome quantification of highly purified populations of circulating reticulocytes and mature erythrocytes. Blood Adv. 2018; 2(20):2646-2657. https://doi.org/10.1182/bloodadvances.2018023515PubMedPubMed CentralGoogle Scholar
- Hall JE. Red blood cells, anemia and polycythemia. Guyton and Hall Textbook of Medical Physiology 13 ed. 2015;445-454. Google Scholar
- Higgins JM. Red blood cell population dynamics. Clin Lab Med. 2015; 35(1):43-57. https://doi.org/10.1016/j.cll.2014.10.002PubMedPubMed CentralGoogle Scholar
- Li H, Natarajan A, Ezike J. Single cell resolution of glucocorticoid effects on erythroid progenitor cells. Blood. 2018; 132(Suppl 1):S751. https://doi.org/10.1182/blood-2018-99-112445PubMedGoogle Scholar
- Peslak SA, Wenger J, Bemis JC. EPOmediated expansion of late-stage erythroid progenitors in the bone marrow initiates recovery from sublethal radiation stress. Blood. 2012; 120(12):2501-2511. https://doi.org/10.1182/blood-2011-11-394304PubMedPubMed CentralGoogle Scholar
- Wierenga AT, Vellenga E, Schuringa JJ. Down-regulation of GATA1 uncouples STAT5-induced erythroid differentiation from stem/progenitor cell proliferation. Blood. 2010; 115(22):4367-4376. https://doi.org/10.1182/blood-2009-10-250894PubMedGoogle Scholar
- Chiba T, Ikawa Y, Todokoro K.. GATA-1 transactivates erythropoietin receptor gene, and erythropoietin receptor-mediated signals enhance GATA-1 gene expression. Nucleic Acids Res. 1991; 19(14):3843-3848. https://doi.org/10.1093/nar/19.14.3843PubMedPubMed CentralGoogle Scholar
- Bain BJ, Bates I, Laffan MA. Reference ranges and normal values. 2017;2-17. https://doi.org/10.1016/B978-0-7020-6696-2.00002-3Google Scholar
- Roux-Dalvai F, Gonzalez de Peredo A, Simo C. Extensive analysis of the cytoplasmic proteome of human erythrocytes using the peptide ligand library technology and advanced mass spectrometry. Mol Cell Protiomics. 2008; 7(11):2254-2269. https://doi.org/10.1074/mcp.M800037-MCP200PubMedGoogle Scholar
- Goloubinoff P, Sassi AS, Fauvet B, Barducci A, De Los Rios P. Chaperones convert the energy from ATP into the nonequilibrium stabilization of native proteins. Nat Chem Biol. 2018; 14(4):388-395. https://doi.org/10.1038/s41589-018-0013-8PubMedGoogle Scholar
- Stefani M. Protein misfolding and aggregation: new examples in medicine and biology of the dark side of the protein world. Biochim Biophys Acta. 2004; 1739(1):5-25. https://doi.org/10.1016/j.bbadis.2004.08.004PubMedGoogle Scholar
- Voon HPJ, Vadolas J.. Controlling α-globin: a review of α-globin expression and its impact on b-thalassemia. Haematologica. 2008; 93(12):1868-1876. https://doi.org/10.3324/haematol.13490PubMedGoogle Scholar
- Bank A. Hemoglobin synthesis in β-thalassemia: the properties of the free α-chains. J Clin Invest. 1968; 47(4):860-866. https://doi.org/10.1172/JCI105779PubMedPubMed CentralGoogle Scholar
- Fibach E, Dana M.. Oxidative stress in betathalassemia. Mol Diagn Ther. 2019; 23(2):245-261. https://doi.org/10.1007/s40291-018-0373-5PubMedGoogle Scholar
- Feng L, Gell DA, Zhou S. Molecular mechanism of AHSP-mediated stabilization of α-hemoglobin. Cell. 2004; 119(5):629-640. https://doi.org/10.1016/j.cell.2004.11.025PubMedGoogle Scholar
- Mollan TL, Khandros E, Weiss MJ, Olson JS. Kinetics of α-globin binding to α-hemoglobin stabilizing protein (AHSP) indicate preferential stabilization of hemichrome folding intermediate. J Biol Chem. 2012; 287(14):11338-11350. https://doi.org/10.1074/jbc.M111.313247PubMedPubMed CentralGoogle Scholar
- Weiss MJ, Zhou S, Feng L. Role of alpha hemoglobin stabilizing protein in normal erythropoiesis and β-thalassemia. Ann NY Acad Sci. 2005; 1054(1):103-117. https://doi.org/10.1196/annals.1345.013PubMedGoogle Scholar
- Barrett KE, Barman SM, Brooks H, Yuan J.. Ganong’s review of medical physiology. 26 ed. 2019;543-551. Google Scholar
- Drummond DA, Wilke CO. The evolutionary consequences of erroneous protein synthesis. Nat Rev Genet. 2009; 10(10):715. https://doi.org/10.1038/nrg2662PubMedPubMed CentralGoogle Scholar
- Camaschella C, Hoffbrand AV, Hershko C.. Iron metabolism, iron deficiency and disorders of haem synthesis. Postgrad Haematol. 2015;21-39. https://doi.org/10.1002/9781118853771.ch3PubMedGoogle Scholar
- Kruszewski M. Labile iron pool: the main determinant of cellular response to oxidative stress. Mutat Res. 2003; 531(1-2):81-92. https://doi.org/10.1016/j.mrfmmm.2003.08.004PubMedGoogle Scholar
- Yanagitani K, Juszkiewicz S, Hegde RS. UBE2O is a quality control factor for orphans of multiprotein complexes. Science. 2017; 357(6350):472-475. https://doi.org/10.1126/science.aan0178PubMedPubMed CentralGoogle Scholar
- Nguyen AT, Prado MA, Schmidt PJ. UBE2O remodels the proteome during terminal erythroid differentiation. Science. 2017; 357(6350):471. https://doi.org/10.1126/science.aan0218PubMedPubMed CentralGoogle Scholar
- Pilla E, Schneider K, Bertolotti A.. Coping with protein quality control failure. Annu Rev Cell Dev Biol. 2017; 33:439-465. https://doi.org/10.1146/annurev-cellbio-111315-125334PubMedGoogle Scholar
- Mizuno S. Temperature sensitivity of protein synthesis initiation: inactivation of a ribosomal factor by an inhibitor formed at elevated temperatures. Arch Biochem Biophys. 1977; 179(1):289-301. https://doi.org/10.1016/0003-9861(77)90114-XGoogle Scholar
- Rosenzweig R, Nillegoda NB, Mayer MP, Bukau B.. The Hsp70 chaperone network. Nat Rev Mol Cell Biol. 2019; 11:665-680. https://doi.org/10.1038/s41580-019-0133-3PubMedGoogle Scholar
- Kampinga HH, Craig EA. The HSP70 chaperone machinery: J-proteins as drivers of functional specificity. Nat Rev Mol Cell Biol. 2010; 11(8):579-592. https://doi.org/10.1038/nrm2941PubMedPubMed CentralGoogle Scholar
- Kityk R, Kopp J, Mayer MP. Molecular mechanism of J-domain-triggered ATP hydrolysis by Hsp70 chaperones. Mol Cell. 2018; 69(2):227-237. https://doi.org/10.1016/j.molcel.2017.12.003PubMedGoogle Scholar
- Cyr DM. Swapping nucleotides, tuning Hsp70. Cell. 2008; 133(6):945-947. https://doi.org/10.1016/j.cell.2008.05.036PubMedPubMed CentralGoogle Scholar
- Finka A, Mattoo RU, Goloubinoff P.. Experimental milestones in the discovery of molecular chaperones as polypeptide unfolding enzymes. Annu Rev Biochem. 2016; 85:715-742. https://doi.org/10.1146/annurev-biochem-060815-014124PubMedGoogle Scholar
- De Los Rios P, Barducci A.. Hsp70 chaperones are non-equilibrium machines that achieve ultra-affinity by energy consumption. Elife. 2014; 3:e02218. https://doi.org/10.7554/eLife.02218PubMedPubMed CentralGoogle Scholar
- Schrödinger E. What is life? The physical aspect of the living cell. 1944. Google Scholar
- Sharma SK, De los Rios P, Christen P, Lustig A, Goloubinoff P.. The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase. Nat Chem Biol. 2010; 6(12):914-920. https://doi.org/10.1038/nchembio.455PubMedGoogle Scholar
- Trinklein ND, Chen WC, Kingston RE, Myers RM. Transcriptional regulation and binding of heat shock factor 1 and heat shock factor 2 to 32 human heat shock genes during thermal stress and differentiation. Cell Stress Chaperon. 2004; 9(1):21. https://doi.org/10.1379/1466-1268(2004)009<0021:TRABOH>2.0.CO;2Google Scholar
- Saretzki G, Armstrong L, Leake A, Lako M, von Zglinicki T. Stress defense in murine embryonic stem cells is superior to that of various differentiated murine cells. Stem Cells. 2004; 22(6):962-971. https://doi.org/10.1634/stemcells.22-6-962PubMedGoogle Scholar
- Matsumoto A, Takeishi S, Kanie T. p57 is required for quiescence and maintenance of adult hematopoietic stem cells. Cell Stem Cell. 2011; 9(3):262-271. https://doi.org/10.1016/j.stem.2011.06.014PubMedGoogle Scholar
- Zou P, Yoshihara H, Hosokawa K. p57Kip2 and p27Kip1 cooperate to maintain hematopoietic stem cell quiescence through interactions with Hsc70. Cell Stem Cell. 2011; 9(3):247-261. https://doi.org/10.1016/j.stem.2011.07.003PubMedGoogle Scholar
- Tesio M, Trumpp A.. Breaking the cell cycle of HSCs by p57 and friends. Cell Stem Cell. 2011; 9(3):187-192. https://doi.org/10.1016/j.stem.2011.08.005PubMedGoogle Scholar
- Böcking T, Aguet F, Harrison SC, Kirchhausen T.. Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating. Nat Struct Mol. 2011; 18(3):295. https://doi.org/10.1038/nsmb.1985PubMedPubMed CentralGoogle Scholar
- Chakraborty A, Mukherjee S, Chattopadhyay R, Roy S, Chakrabarti S.. Conformational adaptation in the E. coli sigma 32 protein in response to heat shock. J Phys Chem. 2014; 118(18):4793-4802. https://doi.org/10.1021/jp501272nPubMedGoogle Scholar
- Marcinowski M, Höller M, Feige MJ, Baerend D, Lamb DC, Buchner J.. Substrate discrimination of the chaperone BiP by autonomous and cochaperone-regulated conformational transitions. Nat Struct Cell Biol. 2011; 18(2):150. https://doi.org/10.1038/nsmb.1970PubMedGoogle Scholar
- Zhang J, Socolovsky M, Gross AW, Lodish HF. Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry–based novel culture system. Blood. 2003; 102(12):3938-3946. https://doi.org/10.1182/blood-2003-05-1479PubMedGoogle Scholar
- Han X, Zhang J, Peng Y. Unexpected role for p19INK4d in posttranscriptional regulation of GATA1 and modulation of human terminal erythropoiesis. Blood. 2017; 129(2):226-237. https://doi.org/10.1182/blood-2016-09-739268PubMedPubMed CentralGoogle Scholar
- Sterrenberg JN, Blatch GL, Edkins AL. Human DNAJ in cancer and stem cells. Cancer Lett. 2011; 312(2):129-142. https://doi.org/10.1016/j.canlet.2011.08.019PubMedGoogle Scholar
- Zhang Y, Yang Z, Cao Y. The Hsp40 family chaperone protein DnaJB6 enhances Schlafen1 nuclear localization which is critical for promotion of cell-cycle arrest in Tcells. Biochem J. 2008; 413(2):239-250. https://doi.org/10.1042/BJ20071510PubMedGoogle Scholar
- Watson ED, Mattar P, Schuurmans C, Cross JC. Neural stem cell self-renewal requires the Mrj co-chaperone. Dev Dynam. 2009; 238(10):2564-2574. https://doi.org/10.1002/dvdy.22088PubMedGoogle Scholar
- Ludwig LS, Cho H, Wakabayashi A. Genome-wide association study follow-up identifies cyclin A2 as a regulator of the transition through cytokinesis during terminal erythropoiesis. Am J Hematol. 2015; 90(5):386-391. https://doi.org/10.1002/ajh.23952PubMedPubMed CentralGoogle Scholar
- Sankaran VG, Ludwig LS, Sicinska E. Cyclin D3 coordinates the cell cycle during differentiation to regulate erythrocyte size and number. Genes Dev. 2012; 26(18):2075-2087. https://doi.org/10.1101/gad.197020.112PubMedPubMed CentralGoogle Scholar
- Arai A, Kanda E, Miura O.. Rac is activated by erythropoietin or interleukin-3 and is involved in activation of the Erk signaling pathway. Oncogene. 2002; 21(17):2641. https://doi.org/10.1038/sj.onc.1205346PubMedGoogle Scholar
- Song H, Kim W, Kim S-H, Kim K-T. VRK3- mediated nuclear localization of HSP70 prevents glutamate excitotoxicity-induced apoptosis and Aβ accumulation via enhancement of ERK phosphatase VHR activity. Sci Rep. 2016; 6:38452. https://doi.org/10.1038/srep38452PubMedPubMed CentralGoogle Scholar
- Shan Y, Cortopassi G.. Mitochondrial Hspa9/Mortalin regulates erythroid differentiation via iron-sulfur cluster assembly. Mitochondrion. 2016; 26:94-103. https://doi.org/10.1016/j.mito.2015.12.005PubMedGoogle Scholar
- Yamamoto H, Momose T, Yatsukawa Y-i. Identification of a novel member of yeast mitochondrial Hsp70-associated motor and chaperone proteins that facilitates protein translocation across the inner membrane. FEBS Lett. 2005; 579(2):507-511. https://doi.org/10.1016/j.febslet.2004.12.018PubMedGoogle Scholar
- Liu T, Krysiak K, Shirai CL. Knockdown of HSPA9 induces TP53-dependent apoptosis in human hematopoietic progenitor cells. PLoS One. 2017; 12(2):e0170470. https://doi.org/10.1371/journal.pone.0170470PubMedPubMed CentralGoogle Scholar
- Chen TH-P, Kambal A, Krysiak K. Knockdown of Hspa9, a del (5q31. 2) gene, results in a decrease in hematopoietic progenitors in mice. Blood. 2011; 117(5):1530-1539. https://doi.org/10.1182/blood-2010-06-293167PubMedPubMed CentralGoogle Scholar
- Weiss MJ, dos Santos CO. Chaperoning erythropoiesis. Blood. 2009; 113(10):2136-2144. https://doi.org/10.1182/blood-2008-09-115238PubMedPubMed CentralGoogle Scholar
- Zermati Y, Garrido C, Amsellem S. Caspase activation is required for terminal erythroid differentiation. J Exp Med. 2001; 193(2):247-254. https://doi.org/10.1084/jem.193.2.247PubMedPubMed CentralGoogle Scholar
- Kolbus A, Pilat S, Husak Z. Raf-1 antagonizes erythroid differentiation by restraining caspase activation. J Exp Med. 2002; 196(10):1347-1353. https://doi.org/10.1084/jem.20020562PubMedPubMed CentralGoogle Scholar
- Cande C, Vahsen N, Garrido C, Kroemer G.. Apoptosis-inducing factor (AIF): caspaseindependent after all. Cell Death Differ. 2004; 11(6)https://doi.org/10.1038/sj.cdd.4401400PubMedGoogle Scholar
- Gurbuxani S, Schmitt E, Cande C. Heat shock protein 70 binding inhibits the nuclear import of apoptosis-inducing factor. Oncogene. 2003; 22(43):6669. https://doi.org/10.1038/sj.onc.1206794PubMedGoogle Scholar
- Lui JC-K, Kong S-K. Heat shock protein 70 inhibits the nuclear import of apoptosisinducing factor to avoid DNA fragmentation in TF-1 cells during erythropoiesis. FEBS Lett. 2007; 581(1):109-117. https://doi.org/10.1016/j.febslet.2006.11.082PubMedGoogle Scholar
- Ribeil JA, Zermati Y, Vandekerckhove J. Hsp70 regulates erythropoiesis by preventing caspase-3-mediated cleavage of GATA-1. Nature. 2007; 445(7123):102-105. https://doi.org/10.1038/nature05378PubMedGoogle Scholar
- Arlet JB, Ribeil JA, Guillem F. HSP70 sequestration by free alpha-globin promotes ineffective erythropoiesis in beta-thalassaemia. Nature. 2014; 514(7521):242-246. https://doi.org/10.1038/nature13614PubMedGoogle Scholar
- Gregory T, Yu C, Ma A, Orkin SH, Blobel GA, Weiss MJ. GATA-1 and erythropoietin cooperate to promote erythroid cell survival by regulating bcl-xL expression. Blood. 1999; 94(1):87-96. https://doi.org/10.1182/blood.V188.8.131.523k41_87_96PubMedGoogle Scholar
- de Thonel A, Vandekerckhove J, Lanneau D. HSP27 controls GATA-1 protein level during erythroid cell differentiation. Blood. 2010; 116(1):85-96. https://doi.org/10.1182/blood-2009-09-241778PubMedGoogle Scholar
- Ghosh A, Garee G, Sweeny EA, Nakamura Y, Stuehr DJ. Hsp90 chaperones hemoglobin maturation in erythroid and nonerythroid cells. Proc Natl Acad Sci U S A. 2018; 115(6):E1117-E1126. https://doi.org/10.1073/pnas.1717993115PubMedPubMed CentralGoogle Scholar
- Kihm AJ, Kong Y, Hong W. An abundant erythroid protein that stabilizes free α- haemoglobin. Nature. 2002; 417(6890):758. https://doi.org/10.1038/nature00803PubMedGoogle Scholar
- Liu S, Bhattacharya S, Han A. Haemregulated eIF2α kinase is necessary for adaptive gene expression in erythroid precursors under the stress of iron deficiency. Br J Haematol. 2008; 143(1):129-137. https://doi.org/10.1111/j.1365-2141.2008.07293.xPubMedPubMed CentralGoogle Scholar
- Zhang S, Macias-Garcia A, Ulirsch JC. HRI coordinates translation necessary for protein homeostasis and mitochondrial function in erythropoiesis. eLife. 2019; 8:e46976. https://doi.org/10.7554/eLife.46976PubMedPubMed CentralGoogle Scholar
- Han AP, Yu C, Lu L. Heme-regulated eIF2α kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency. EMBO J. 2001; 20(23):6909-6918. https://doi.org/10.1093/emboj/20.23.6909PubMedPubMed CentralGoogle Scholar
- Thulasiraman V, Xu Z, Uma S, Gu Y, Chen JJ, Matts RL. Evidence that Hsc70 negatively modulates the activation of the heme-regulated eIF-2α kinase in rabbit reticulocyte lysate. Eur J Biochem. 1998; 255(3):552-562. https://doi.org/10.1046/j.1432-1327.1998.2550552.xPubMedGoogle Scholar
- Uma S, Thulasiraman V, Matts RL. Dual role for Hsc70 in the biogenesis and regulation of the heme-regulated kinase of the alpha subunit of eukaryotic translation initiation factor 2. Mol Cell Biol. 1999; 9:5861-5871. https://doi.org/10.1128/MCB.19.9.5861PubMedPubMed CentralGoogle Scholar
- Han A-P, Fleming MD, Chen J-J. Heme-regulated eIF2α kinase modifies the phenotypic severity of murine models of erythropoietic protoporphyria and β-thalassemia. J Clin Invest. 2005; 115(6):1562-1570. https://doi.org/10.1172/JCI24141PubMedPubMed CentralGoogle Scholar
- Lu L, Han A-P, Chen J-J. Translation initiation control by heme-regulated eukaryotic initiation factor 2α kinase in erythroid cells under cytoplasmic stresses. Mol Cell Biol. 2001; 21(23):7971-7980. https://doi.org/10.1128/MCB.21.23.7971-7980.2001PubMedPubMed CentralGoogle Scholar
- Suragani RN, Zachariah RS, Velazquez JG. Heme-regulated eIF2α kinase activated Atf4 signaling pathway in oxidative stress and erythropoiesis. Blood. 2012; 119(22):5276-5284. https://doi.org/10.1182/blood-2011-10-388132PubMedPubMed CentralGoogle Scholar
- Zhang S, Macias-Garcia A, Velazquez J, Paltrinieri E, Kaufman RJ, Chen J-J. HRI coordinates translation by eIF2αP and mTORC1 to mitigate ineffective erythropoiesis in mice during iron deficiency. Blood. 2018; 131(4):450-461. https://doi.org/10.1182/blood-2017-08-799908PubMedPubMed CentralGoogle Scholar
- Morimoto R, Fodor E.. Cell-specific expression of heat shock proteins in chicken reticulocytes and lymphocytes. J Cell Biol. 1984; 99(4):1316-1323. https://doi.org/10.1083/jcb.99.4.1316PubMedPubMed CentralGoogle Scholar
- Lee GJ, Roseman AM, Saibil HR, Vierling E.. A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state. EMBO J. 1997; 16(3):659-671. https://doi.org/10.1093/emboj/16.3.659PubMedPubMed CentralGoogle Scholar
- Nillegoda NB, Wentink AS, Bukau B.. Protein disaggregation in multicellular organisms. Trends Biochem Sci. 2018; 43(4):285-300. https://doi.org/10.1016/j.tibs.2018.02.003PubMedGoogle Scholar
- Rampelt H, Kirstein-Miles J, Nillegoda NB. Metazoan Hsp70 machines use Hsp110 to power protein disaggregation. EMBO J. 2012; 31(21):4221-4235. https://doi.org/10.1038/emboj.2012.264PubMedPubMed CentralGoogle Scholar
- Mattoo RU, Sharma SK, Priya S, Finka A, Goloubinoff P.. Hsp110 is a bona fide chaperone using ATP to unfold stable misfolded polypeptides and reciprocally collaborate with Hsp70 to solubilize protein aggregates. J Biol Chem. 2013; 288(29):21399-21411. https://doi.org/10.1074/jbc.M113.479253PubMedPubMed CentralGoogle Scholar
- Nillegoda NB, Kirstein J, Szlachcic A. Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation. Nature. 2015; 524(7564):247. https://doi.org/10.1038/nature14884PubMedPubMed CentralGoogle Scholar
- Nillegoda NB, Stank A, Malinverni D. Evolution of an intricate J-protein network driving protein disaggregation in eukaryotes. Elife. 2017; 6:e24560. https://doi.org/10.7554/eLife.24560PubMedPubMed CentralGoogle Scholar
- Kirstein J, Arnsburg K, Scior A. In vivo properties of the disaggregase function of Jproteins and Hsc70 in Caenorhabditis elegans stress and aging. Aging Cell. 2017; 16(6):1414-1424. https://doi.org/10.1111/acel.12686PubMedPubMed CentralGoogle Scholar
- Beere HM, Green DR. Stress management– heat shock protein-70 and the regulation of apoptosis. Trends Cell Biol. 2001; 11(1):6-10. https://doi.org/10.1016/S0962-8924(00)01874-2Google Scholar
- Li C-Y, Lee J-S, Ko Y-G, Kim J-I, Seo J-S. Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation. J Biol Chem. 2000; 275(33):25665-25671. https://doi.org/10.1074/jbc.M906383199PubMedGoogle Scholar
- Bivik C, Rosdahl I, Öllinger K.. Hsp70 protects against UVB induced apoptosis by preventing release of cathepsins and cytochrome c in human melanocytes. Carcinogenesis. 2007; 28(3):537-544. https://doi.org/10.1093/carcin/bgl152PubMedGoogle Scholar
- Gao T, Newton AC. The turn motif is a phosphorylation switch that regulates the binding of Hsp70 to protein kinase C. J Biol Chem. 2002; 277(35):31585-31592. https://doi.org/10.1074/jbc.M204335200PubMedGoogle Scholar
- Mohandas N, Gallagher PG. Red cell membrane: past, present, and future. Blood. 2008; 112(10):3939-3948. https://doi.org/10.1182/blood-2008-07-161166PubMedPubMed CentralGoogle Scholar
- Pretorius E, du Plooy JN, Bester J.. A comprehensive review on eryptosis. Cell Physiol Biochem. 2016; 39(5):1977-2000. https://doi.org/10.1159/000447895PubMedGoogle Scholar
- Bryk AH, Wiśniewski JR. Quantitative analysis of human red blood cell proteome. J Proteome Res. 2017; 16(8):2752-2761. https://doi.org/10.1021/acs.jproteome.7b00025PubMedGoogle Scholar
- Finka A, Sood V, Quadroni M, De Los Rios P, Goloubinoff P.. Quantitative proteomics of heat-treated human cells show an acrossthe- board mild depletion of housekeeping proteins to massively accumulate few HSPs. Cell Stress Chaperon. 2015; 20(4):605-620. https://doi.org/10.1007/s12192-015-0583-2PubMedPubMed CentralGoogle Scholar
- Finka A, Goloubinoff P.. Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis. Cell Stress Chaperon. 2013; 18(5):591-605. https://doi.org/10.1007/s12192-013-0413-3PubMedPubMed CentralGoogle Scholar
- Balchin D, Hayer-Hartl M, Hartl FU. In vivo aspects of protein folding and quality control. Science. 2016; 353(6294):aac4354. https://doi.org/10.1126/science.aac4354PubMedGoogle Scholar
- Nillegoda NB, Bukau B.. Metazoan Hsp70- based protein disaggregases: emergence and mechanisms. Front Mol Biosci. 2015; 2(57)https://doi.org/10.3389/fmolb.2015.00057PubMedPubMed CentralGoogle Scholar
- Shorter J. The mammalian disaggregase machinery: Hsp110 synergizes with Hsp70 and Hsp40 to catalyze protein disaggregation and reactivation in a cell-free system. PLoS One. 2011; 6(10):e26319. https://doi.org/10.1371/journal.pone.0026319PubMedPubMed CentralGoogle Scholar
- Sarikas A, Hartmann T, Pan Z-Q. The cullin protein family. Genome Biol. 2011; 12(4):220. https://doi.org/10.1186/gb-2011-12-4-220PubMedPubMed CentralGoogle Scholar
- Nillegoda NB, Theodoraki MA, Mandal AK. Ubr1 and Ubr2 function in a quality control pathway for degradation of unfolded cytosolic proteins. Mol Biol Cell. 2010; 21(13):2102-2116. https://doi.org/10.1091/mbc.e10-02-0098PubMedPubMed CentralGoogle Scholar
- Theodoraki MA, Nillegoda NB, Saini J, Caplan AJ. A network of ubiquitin ligases is important for the dynamics of misfolded protein aggregates in yeast. J Biol Chem. 2012; 287(28):23911-23922. https://doi.org/10.1074/jbc.M112.341164PubMedPubMed CentralGoogle Scholar
- Sultana R, Theodoraki MA, Caplan AJ. UBR1 promotes protein kinase quality control and sensitizes cells to Hsp90 inhibition. Exp Cell Res. 2012; 318(1):53-60. https://doi.org/10.1016/j.yexcr.2011.09.010PubMedPubMed CentralGoogle Scholar
- McDonough H, Patterson C.. CHIP: a link between the chaperone and proteasome systems. Cell Stress Chaperon. 2003; 8(4):303. https://doi.org/10.1379/1466-1268(2003)008<0303:CALBTC>2.0.CO;2Google Scholar
- Taher AT, Weatherall DJ, Cappellini MD. Thalassaemia. Lancet. 2018; 391(10116):155-167. https://doi.org/10.1016/S0140-6736(17)31822-6Google Scholar
- Gringras P, Wonke B, Old J. Effect of alpha thalassaemia trait and enhanced gamma chain production on disease severity in beta thalassaemia major and intermedia. Arch Dis Child. 1994; 70(1):30-34. https://doi.org/10.1136/adc.70.1.30PubMedPubMed CentralGoogle Scholar
- dos Santos CO, Costa FF. AHSP and betathalassemia: a possible genetic modifier. Hematology. 2005; 10(2):157-161. https://doi.org/10.1080/10245330500067280PubMedGoogle Scholar
- Khandros E, Thom CS, D'Souza J, Weiss MJ. Integrated protein quality-control pathways regulate free α-globin in murine b-thalassemia. Blood. 2012; 119(22):5265-5275. https://doi.org/10.1182/blood-2011-12-397729PubMedPubMed CentralGoogle Scholar
- Adewoye AH, Klings ES, Farber HW. Sickle cell vaso-occlusive crisis induces the release of circulating serum heat shock protein-70. Am J Hematol. 2005; 78(3):240-242. https://doi.org/10.1002/ajh.20292PubMedPubMed CentralGoogle Scholar
- Sundd P, Gladwin MT, Novelli EM. Pathophysiology of sickle cell disease. Annu Rev Pathol Mech. 2019; 14:263-292. https://doi.org/10.1146/annurev-pathmechdis-012418-012838PubMedPubMed CentralGoogle Scholar
- Elsner L, Flugge PF, Lozano J. The endogenous danger signals HSP70 and MICA cooperate in the activation of cytotoxic effector functions of NK cells. J Cell Mol Med. 2010; 14(4):992-1002. https://doi.org/10.1111/j.1582-4934.2008.00677.xPubMedPubMed CentralGoogle Scholar
- De Maio A, Vazquez D.. Extracellular heat shock proteins: a new location, a new function. Shock. 2013; 40(4):239-246. https://doi.org/10.1097/SHK.0b013e3182a185abPubMedPubMed CentralGoogle Scholar
- Multhoff G, Pfister K, Gehrmann M. A 14-mer Hsp70 peptide stimulates natural killer (NK) cell activity. Cell Stress Chaperon. 2001; 6(4):337-344. https://doi.org/10.1379/1466-1268(2001)006<0337:AMHPSN>2.0.CO;2Google Scholar
- Biondani A, Turrini F, Carta F. Heatshock protein-27, -70 and peroxiredoxin-II show molecular chaperone function in sickle red cells: Evidence from transgenic sickle cell mouse model. Proteomics Clin Appl. 2008; 2(5):706-719. https://doi.org/10.1002/prca.200780058PubMedGoogle Scholar
- Levin C, Koren A, Rebibo-Sabbah A, Koifman N, Brenner B, Aharon A.. Extracellular vesicle characteristics in betathalassemia as potential biomarkers for spleen functional status and ineffective erythropoiesis. Front Physiol. 2018; 9:1214. https://doi.org/10.3389/fphys.2018.01214PubMedPubMed CentralGoogle Scholar
- Ogawa S. Genetics of MDS. Blood. 2019; 133(10):1049-1059. https://doi.org/10.1182/blood-2018-10-844621PubMedPubMed CentralGoogle Scholar
- Horrigan SK, Arbieva ZH, Xie HY. Delineation of a minimal interval and identification of 9 candidates for a tumor suppressor gene in malignant myeloid disorders on 5q31. Blood. 2000; 95(7):2372-2377. https://doi.org/10.1182/blood.V95.7.2372.007k20_2372_2377PubMedGoogle Scholar
- Craven SE, French D, Ye W, de Sauvage F, Rosenthal A.. Loss of Hspa9b in zebrafish recapitulates the ineffective hematopoiesis of the myelodysplastic syndrome. Blood. 2005; 105(9):3528-3534. https://doi.org/10.1182/blood-2004-03-1089PubMedGoogle Scholar
- Frisan E, Vandekerckhove J, de Thonel A. Defective nuclear localization of Hsp70 is associated with dyserythropoiesis and GATA-1 cleavage in myelodysplastic syndromes. Blood. 2012; 119(6):1532-1542. https://doi.org/10.1182/blood-2011-03-343475PubMedGoogle Scholar
- Xu J, Reumers J, Couceiro JR. Gain of function of mutant p53 by coaggregation with multiple tumor suppressors. Nat Chem Biol. 2011; 7(5):285-295. https://doi.org/10.1038/nchembio.546PubMedGoogle Scholar
- de Oliveira GA, Rangel LP, Costa DC, Silva JL. Misfolding, aggregation, and disordered segments in c-Abl and p53 in human cancer. Front Oncol. 2015; 5:97. https://doi.org/10.3389/fonc.2015.00097PubMedPubMed CentralGoogle Scholar
- Malcovati L, Karimi M, Papaemmanuil E. SF3B1 mutation identifies a distinct subset of myelodysplastic syndrome with ring sideroblasts. Blood. 2015; 126(2):233-241. https://doi.org/10.1182/blood-2015-03-633537PubMedPubMed CentralGoogle Scholar
- Gallardo M, Barrio S, Fernandez M. Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera. Mol Cancer. 2013; 12(1):142. https://doi.org/10.1186/1476-4598-12-142PubMedPubMed CentralGoogle Scholar
- Sevin M, Girodon F, Garrido C, de Thonel A.. HSP90 and HSP70: implication in inflammation processes and therapeutic approaches for myeloproliferative neoplasms. Mediators Inflamm. 2015; 2015:970242. https://doi.org/10.1155/2015/970242PubMedPubMed CentralGoogle Scholar
- Vainchenker W, Kralovics R.. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017; 129(6):667-679. https://doi.org/10.1182/blood-2016-10-695940PubMedGoogle Scholar
- Marubayashi S, Koppikar P, Taldone T. HSP90 is a therapeutic target in JAK2-depen- dent myeloproliferative neoplasms in mice and humans. J Clin Invest. 2010; 120(10):3578-3593. https://doi.org/10.1172/JCI42442PubMedPubMed CentralGoogle Scholar
- Nathan DG, Clarke BJ, Hillman DG, Alter BP, Housman DE. Erythroid precursors in congenital hypoplastic (Diamond-Blackfan) anemia. J Clin Invest. 1978; 61(2):489-498. https://doi.org/10.1172/JCI108960PubMedPubMed CentralGoogle Scholar
- Ohene-Abuakwa Y, Orfali KA, Marius C, Ball SE. Two-phase culture in Diamond Blackfan anemia: localization of erythroid defect. Blood. 2005; 105(2):838-846. https://doi.org/10.1182/blood-2004-03-1016PubMedGoogle Scholar
- Choesmel V, Fribourg S, Aguissa-Touré A-H. Mutation of ribosomal protein RPS24 in Diamond-Blackfan anemia results in a ribosome biogenesis disorder. Hum Mol Genet. 2008; 17(9):1253-1263. https://doi.org/10.1093/hmg/ddn015PubMedGoogle Scholar
- Farrar JE, Nater M, Caywood E. Abnormalities of the large ribosomal subunit protein, Rpl35a, in Diamond-Blackfan anemia. Blood. 2008; 112(5):1582-1592. https://doi.org/10.1182/blood-2008-02-140012PubMedPubMed CentralGoogle Scholar
- Quarello P, Garelli E, Brusco A. High frequency of ribosomal protein gene deletions in Italian Diamond-Blackfan anemia patients detected by multiplex ligationdependent probe amplification assay. Haematologica. 2012; 97(12):1813-1817. https://doi.org/10.3324/haematol.2012.062281PubMedPubMed CentralGoogle Scholar
- Ludwig LS, Gazda HT, Eng JC. Altered translation of GATA1 in Diamond-Blackfan anemia. Nat Med. 2014; 20(7):748-753. https://doi.org/10.1038/nm.3557PubMedPubMed CentralGoogle Scholar
- Rio S, Gastou M, Karboul N. Regulation of globin-heme balance in Diamond-Blackfan anemia by HSP70/GATA1. Blood. 2019; 133(12):1358-1370. https://doi.org/10.1182/blood-2018-09-875674PubMedPubMed CentralGoogle Scholar
- Gastou M, Rio S, Dussiot M. The severe phenotype of Diamond-Blackfan anemia is modulated by heat shock protein 70. Blood Adv. 2017; 1(22):1959-1976. https://doi.org/10.1182/bloodadvances.2017008078PubMedPubMed CentralGoogle Scholar
- Wilkes MC, Takasaki K, Youn M, Chae HD, Narla A, Sakamoto KM. Chromatin Organization By SATB1 Regulates HSP70 Induction in Early Erythropoiesis and Lost in Diamond Blackfan Anemia. Blood. 2018; 132(Suppl 1):S2591. https://doi.org/10.1182/blood-2018-99-119729PubMedGoogle Scholar
- Allikmets R, Raskind WH, Hutchinson A, Schueck ND, Dean M, Koeller DM. Mutation of a putative mitochondrial iron transporter gene (ABC7) in X-linked sideroblastic anemia and ataxia (XLSA/A). Hum Mol Genet. 1999; 8(5):743-749. https://doi.org/10.1093/hmg/8.5.743PubMedGoogle Scholar
- Liu G, Guo S, Anderson GJ, Camaschella C, Han B, Nie G.. Heterozygous missense mutations in the GLRX5 gene cause sideroblastic anemia in a Chinese patient. Blood. 2014; 124(17):2750-2751. https://doi.org/10.1182/blood-2014-08-598508PubMedGoogle Scholar
- Schmitz-Abe K, Ciesielski SJ, Schmidt PJ. Congenital sideroblastic anemia due to mutations in the mitochondrial HSP70 homologue HSPA9. Blood. 2015; 126(25):2734-2738. https://doi.org/10.1182/blood-2015-09-659854PubMedPubMed CentralGoogle Scholar
- Furuyama K, Kaneko K.. Iron metabolism in erythroid cells and patients with congenital sideroblastic anemia. Int J Hematol. 2018; 107(1):44-54. https://doi.org/10.1007/s12185-017-2368-0PubMedGoogle Scholar
- Lefèvre C, Bondu S, Le Goff S, Kosmider O, Fontenay M.. Dyserythropoiesis of myelodysplastic syndromes. Curr Opin Hematol. 2017; 24(3):191-197. https://doi.org/10.1097/MOH.0000000000000325PubMedGoogle Scholar
- Gazit R, Weissman IL, Rossi DJ. Hematopoietic stem cells and the aging hematopoietic system. Semin Hematol. 2008; 45(4):218-224. https://doi.org/10.1053/j.seminhematol.2008.07.010PubMedGoogle Scholar
- Pang Q, Keeble W, Christianson TA, Faulkner GR, Bagby GC. FANCC interacts with Hsp70 to protect hematopoietic cells from IFN-γ/TNF-α-mediated cytotoxicity. EMBO J. 2001; 20(16):4478-4489. https://doi.org/10.1093/emboj/20.16.4478PubMedPubMed CentralGoogle Scholar
- Mortensen M, Soilleux EJ, Djordjevic G. The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance. J Exp Med. 2011; 208(3):455-467. https://doi.org/10.1084/jem.20101145PubMedPubMed CentralGoogle Scholar
- De Franceschi L, Bertoldi M, De Falco L. Oxidative stress modulates heme synthesis and induces peroxiredoxin-2 as a novel cytoprotective response in b-thalassemic erythropoiesis. Haematologica. 2011; 96(11):1595-1604. https://doi.org/10.3324/haematol.2011.043612PubMedPubMed CentralGoogle Scholar
- Higuchi-Sanabria R, Frankino PA, Paul JW, Tronnes SU, Dillin A.. A futile battle? Protein quality control and the stress of aging. Dev Cell. 2018; 44(2):139-163. https://doi.org/10.1016/j.devcel.2017.12.020PubMedPubMed CentralGoogle Scholar
- Vilchez D, Saez I, Dillin A.. The role of protein clearance mechanisms in organismal ageing and age-related diseases. Nat Commun. 2014; 5:5659. https://doi.org/10.1038/ncomms6659PubMedGoogle Scholar
- Zhuravleva A, Clerico EM, Gierasch LM. An interdomain energetic tug-of-war creates the allosterically active state in Hsp70 molecular chaperones. Cell. 2012; 151(6):1296-1307. https://doi.org/10.1016/j.cell.2012.11.002PubMedPubMed CentralGoogle Scholar
- Rudiger S, Germeroth L, Schneider-Mergener J, Bukau B.. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J. 1997; 16(7):1501-1507. https://doi.org/10.1093/emboj/16.7.1501PubMedPubMed CentralGoogle Scholar
- Pesciotta EN, Lam H-S, Kossenkov A. In-depth, label-free analysis of the erythrocyte cytoplasmic proteome in diamond blackfan anemia identifies a unique inflammatory signature. PLoS One. 2015; 10(10):e0140036. https://doi.org/10.1371/journal.pone.0140036PubMedPubMed CentralGoogle Scholar
- Cox J, Mann M.. MaxQuant enables high peptide identification rates, individualized ppb-range mass accuracies and proteomewide protein quantification. Nat Biotechnol. 2008; 26(12):1367. https://doi.org/10.1038/nbt.1511PubMedGoogle Scholar
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