The Hsp70 chaperone system: distinct roles in erythrocyte formation and maintenance

Abstract

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.

Figure 1.

Biogenesis of hemoglobin during erythropoiesis. (A) A simplified schematic diagram showing the key cell stages of erythropoiesis. Hematopoietic stem cells (HSC) differentiate into a common myeloid progenitor, which further transition into a committed erythroid lineage. The proerythroblast is the earliest morphologically identifiable erythroid precursor cell in the bone marrow. Erythropoietin (EPO) signaling initiates terminal differentiation of erythroblasts to generate mature erythrocytes. During terminal differentiation, cells reduce in size and undergo major changes including chromatin condensation, proteome remodeling and ultimately the elimination of cellular organelles to provide room for hemoglobin (Hb). Hb expression levels in differentiating cells are indicated by intensity of the red color. (B) Chaperone assisted folding and assembly of Hb. The folding of nascent globin chains is assisted by chaperones such as the α-Hb–stabilizing protein (AHSP), heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90). Errors in Hb subunit synthesis and assembly, iron/heme imbalances, deficiencies in protein quality control activities and exposure to reactive oxygen species (ROS), however, could trigger the misfolding and aggregation of globin proteins. In particular, misfolded and unassembled α-globin chains are highly prone to form cytotoxic aggregates leading to ineffective erythropoiesis. c: cytosol; n: nucleus; UPS: ubiquitin proteasome system.

Figure 2.

Roles of the Hsp70 chaperone system in differentiating erythroblasts and mature erythrocytes. (A) Domain organization of the heat shock protein 70 (Hsp70) chaperone (top); the Hsp70 chaperoning cycle (bottom). Substrate binding is dictated in Hsp70 by the allosteric coupling of ATP binding and hydrolysis at the N-terminal nucleotide binding domain (NBD), which results in conformational changes at the substrate binding domain (SBD).147 The conformational cycle linked to substrate capture is defined by ATP hydrolysis driven large scale movements in the α-helical-lid domain (SBDα) that closes over the b-sandwich substrate binding subdomain (SBDb) in the ADP state, resulting in low substrate off-rates (i.e., high affinity towards bound substrates).30 J-domain proteins (JDP) select substrates for Hsp70. Concomitant interactions of the Hsp70 (in ATP state) with JDP and substrate result in increased stimulation of ATP hydrolysis trapping the substrate in Hsp70. Subsequently, nucleotide exchange factors (NEF) induce ADP dissociation from Hsp70 allowing ATP rebinding, which triggers substrate release to complete Hsp70 cycle. Substrate unfolding and refolding is facilitated by multiple cycles of substrate binding and release. Hsp70 recognizes a highly degenerative and frequently occurring peptide motif enriched with five hydrophobic amino acids, flanked by preferentially positively charged amino acids (statistically occurring in every 30-40 residues in polypeptide chains).148 Such hydrophobic motifs are typically buried inside a natively folded protein, but become exposed in unfolded or misfolded conformers, which allow the Hsp70 machinery to discriminate between natively folded and unfolded/misfolded/aggregated substrates.30 (B) Expression profiles of selected chaperone systems in different stages of erythropoiesis from quantitative proteomics data.3,5 The black dotted line represents the median relative abundance of non-hemoglobin (Hb) proteins in each cell type. The cytosolic Hsp70/110, Hsp60, and Hsp90 initially represent about 1% of the total proteome in erythroid progenitor stages. Their abundance, however, gradually decreases as the proportion of Hb increases during erythropoiesis, but much less so than ribosomes and histones. Below, multifaceted functions of the Hsp70 chaperone system at major steps of red blood cell generation. Ability to synthesize proteins is lost in mature erythrocytes. Protein degradation capacity is largely reduced in mature erythrocytes. A selective Hsp70 system seems to drive protein repair in terminally differentiated erythrocytes. PQC: protein quality control.

Figure 3.

Hsp70 chaperone system modulates the maintenance of dormancy and cell cycle quiescence of stem cell progenitors of erythrocytes. Heat shock protein 70 (Hsp70) binds and shuttles key cyclins that control proliferation and differentiation of erythroid precursor cells. Growth factor regulated cyclin-dependent kinase inhibitors (CDKi) appear to modulate these activities by interacting (directly or indirectly) with Hsp70-cyclin complexes. EPO: erythropoietin; HSC: hematopoietic stem cell; SCF: stem cell factor.

Figure 4.

Hsp70 facilitates proper initiation of erythropoiesis. Heat shock protein 70 (Hsp70) is utilized as a stress sensor molecule in two fitness checkpoints to assess proteostasis deficiencies in erythroid progenitors. (A) Checkpoint 1, in essence, “gauges” and tests whether erythroblasts contain sufficient Hsp70 levels in the cytosol to block the nuclear translocation of apoptosis-inducing factor (AIF), a pro-apoptotic factor released from transiently depolarized mitochondria as a result of erythropoietin (EPO) signaling. In unhealthy cells, Hsp70 is sequestered away by protein aggregates and consequently AIF translocates into the nucleus to initiate cell death. Conversely, checkpoint 2 evaluates the ability of Hsp70 to protect GATA-1, the master regulator of erythropoiesis, from caspase 3 cleavage. This too indirectly evaluates the Hsp70 chaperoning capacity in early erythroblasts. Satisfaction from both tests (green) appears to be required to initiate a robust terminal differentiation process. (B) Both fitness checkpoints fail (red) in “unhealthy” cells (e.g., due to increased oxidative stress) containing high levels of misfolded and aggregated proteins that sequester Hsp70.

Figure 5.

Hsp70 mediates protein repair function in mature erythrocytes. (A) Mass-fraction abundance of proteins in mature erythrocytes. Black bars indicate masswise enrichment or depletion of selected proteins and protein categories in total erythrocytes in comparison to unstressed Jurkat cells (control). Red bars indicate adjusted fold changes in the enrichment or depletion of selected proteins and protein categories normalized to the average abundance of all non-hemoglobin (Hb) proteins in total mature erythrocytes over Jurkat cells. Proteins linked to oxidative stress, cytoskeleton, ubiquitin/proteasome, glycolysis/tricarboxylic acid (TCA) cycle/pentose phosphate (PP), mitochondria, endoplasmic reticulum (ER) lumen and ribosomes were defined according to gene ontology annotations; the remaining categories (Hb, carbonic anhydrase, HSPA1A, Hsp70 system, Hsp60, Hsp90) were custom-defined. (B-C) Mass fractions of members of the Hsp70 system comprising of Hsp70, J-domain proteins (JDP) and nucleotide exchange factors (NEF) in unstressed Jurkat cells (B) and in mature erythrocytes (C). Values within brackets indicate mass fraction of Hsp70 chaperones and cochaperones as percent of the total proteome. NEF types include HSPBP1 and BAG-family proteins. Hsp110-type NEF vital for protein disaggregation (HSPH1-3) are indicated separately. A complete list of mass fractions of individual chaperones and cochaperones are provided in the Online Supplementary Tables S1-2. (D) Surface representation of the human 26S proteasome (PDB entry 5L4G). Core particle (CP) in green; regulatory particle (RP) in orange. (E-F) Mass fractions of members of the ubiquitin proteasome system (UPS) in unstressed Jurkat cells (E), and in mature erythrocytes (F). E1, E2 and E3 ubiquitin ligase enzymes indicated in different shades of blue. Ubiquitin (Ubi), CP, RP proteins are shown in yellow, green and orange, respectively. Values within brackets indicate mass fraction of the protein/protein groups as percent of the total proteome. Complete list of mass fractions of individual chaperones and cochaperones are provided in the Online Supplementary Tables S3-4. The analyzed proteomics datasets were obtained from Label-Free Quantification (LFQ) experiments.5,94,95,149 For each quantified protein/protein group, LFQ values were obtained from the MaxQuant software150 and were proportional to the mass-wise abundance of the corresponding polypeptides. LFQ values were normalized by the sum of all LFQ values from a given sample and indicated as the relative mass-wise abundance of a protein/ protein group and termed as mass fractions. (G) Putative Hsp70-based protein repair-only functions in mature erythrocytes.