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Y. Mathangasinghe et al.
ubiquitin proteasome system (UPS) and the autophagy pathway.26,27 This drastic proteome remodeling expected- ly depletes components of chaperone and proteolytic machineries and decreases the ability of these cells to induce global PQC pathways that help buffer against pro- teotoxic stresses.28 For example, unlike other cell types, reticulocytes show inability to fully recover critical cellu- lar 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 con- served molecular chaperone systems critical for maintain- ing cellular proteostasis. This highly versatile chaperone system supports a plethora of housekeeping and stress- related cellular repair processes that protect cells against proteotoxic stresses (for a review, see Rosenzweig et al.30). The key housekeeping activities include facilitating fold- ing of newly synthesized proteins, transport of polypep- tides 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 regulat- ed via the cooperation of dedicated cochaperones from the J-domain protein (JDP) family and nucleotide exchange factors (NEF) that fine-tune Hsp70’s ATP- dependent allosteric control of substrate binding and release (Figure 2A). JDP form the largest and the most diverse family of cochaperones in humans (over 42 mem- bers) and provide specificity to the Hsp70 family (13 homologs in humans) by selecting substrates.30,31 Concomitant interaction of Hsp70 with a JDP and sub- strate 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 misfold- ed 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 stat- ed, 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 pro- tein folding chaperone systems (e.g., Hsp60 and Hsp90) promote the buildup and maintenance of relatively high levels of native protein conformers under non-equilibri- um 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 sys- tem: the energy from the sun is harnessed by photosyn- thesis 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 mole- cules. 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 pro- tein 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 chap- erone could target different clientele and switch between functions, which allows this highly versatile chaperone system to rapidly respond to different cell growth and dif- ferentiation conditions.38,39 The main roles of Hsp70 dur- ing erythropoiesis include: (i) aiding in maintaining ery- throid progenitors (ii) assessing fitness of progenitors prior to initiating lineage specific terminal cell differentia- tion (iii) supporting Hb biogenesis (iv) counteracting pro- teotoxicities and preventing premature apoptosis of dif- ferentiating erythroblasts and (v) conceivably promoting viability of differentiated (mature) erythrocytes via pro- tein repair.
Hsp70 regulates dormancy and cell cycle quiescence of erythroid precursor cells
Continuous proliferation and differentiation of hematopoietic stem cells into committed erythroid pro- genitor 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 dur- ing 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 consti- tutively 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 unstruc- tured (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 triske- lions, 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
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haematologica | 2021; 106(6)