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Role of the Hsp70 chaperone in erythropoiesis
Quantitative proteomics highlight vital protein quality control functions related to mature erythrocyte survival
Mature erythrocytes show a remarkable ability to sur- vive 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 main- taining 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 poten- tially cause eryptosis, a process by which mature erythro- cytes 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 quantita- tive analytical methods have identified over 2,600 pro- teins in the vestigial proteome of mature human red blood cells (2-5% of the progenitor proteome).94 We per- formed an enrichment analysis on these proteins or pro- tein classes focusing on function to uncover probable bio- logical 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 anhy- drase, and neither eliminated their transcription and translation machineries, nor lost their endoplasmic retic- ulum (ER), nuclei and mitochondrial compartments (Figure 5; Online Supplementary Tables S1-4).95 As expect- ed, 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 fac- tors, 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 erythro- cytes 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 pro-
teins 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 machin- ery 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 ery- throcyte 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 puz- zling 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 irre- versibly damaged proteins followed by transcription, trans- lation, 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 mol- ecules 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 vestig- ial proteome of mature red blood cells. These include the constitutively expressed HSPA8, a selective set of JDP cochaperones (DNAJA and DNAJB) that recognize misfold- ed/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 cochap- erones 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
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