Abstract Background: The ubiquitin-proteasome system (UPS)is essential for maintaining red blood cell (RBC) membrane homeostasis by degrading damaged and misfolded proteins, thus preventing the accumulation of potentially harmful protein aggregates. Ubiquitin, a small polypeptide, contains seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) that can form various types of ubiquitin chains. Among these, K48-linked chains are the most abundant and are responsible for mediating the proteasomal degradation. Valosin-containing protein (VCP/p97) is crucial in preserving RBC membrane integrity by removing and transporting proteins polyubiquitinated with K48-linked ubiquitin from the membrane to the UPS. In sickle RBCs, oxidative stress and insufficient bioenergetics (ATP) can impair p97 function, leading to the accumulation of dysfunctional p97 on the RBC membrane, elevated levels of K48-polyubiquitinated (K48-Ub) proteins, and compromised RBC membrane integrity. Mitapivat, a pyruvate kinase activator of both PKR and PKM2, is under clinical development for treating sickle cell disease (SCD). A key mechanism by which mitapivat treatment increases hemoglobin levels is through the enhancement of RBC integrity, achieved by boosting ATP production and reducing oxidative stress in patients with SCD. Here, we demonstrate that, in addition to the reduction in tyrosine-phosphorylation of RBC band 3, another underlying mechanism that contributes to improved RBC integrity is the restoration of p97 function and a decrease in the accumulation of ubiquitinated proteins. Methods: RBC ghosts (membranes) were isolated from frozen whole blood samples from 3 individuals each with HbAA, HbAS, and HbSS enrolled under protocol NCT00047996. The levels of K48-Ub and p97 were analyzed by Western blotting and densitometry analysis. To investigate the effect of mitapivat on membrane accumulation of p97 and K48-Ub, RBCs from HbSS patients were treated with varying concentrations (1 µM, 3 µM, 10 µM, and 30 µM) of mitapivat for 1h, 2 h, 4 h, 8 h, and 16 h ex vivo. The levels of ATP and K48-Ub and p97 were measured using a Luminescent ATP Detection Assay Kit (abcam) and Western blotting, respectively. We also utilized frozen whole blood samples from patients in the Phase 1 dose-ascending study (NCT04000165), of which 7 of 16 were accessible. RBC ghosts at baseline, after 2 weeks of 5, 20, 50, and 100 mg twice daily mitapivat, at the end of drug taper, and at the end of study were isolated. The level of membrane K48-Ub was analyzed by Western blotting and quantified by densitometry analysis; p97 was not measured due to shortage of material. The percentage change of K48-Ub from baseline was calculated for each sample at different timepoints, and a one-sample, two-sided t-test was used to assess the significance of the mean change of the 7 patients' samples. Statistical significance was annotated as follows: p 0.05 (*), p 0.01 (**), and p ≥ 0.05 (ns). To evaluate the association between K48-Ub and ATP, a Spearman rank correlation analysis was performed using the relative change of K48-Ub and ATP value corrected for hematocrit (ATP/Hct). Results: Significantly higher levels of K48-Ub (p 0.05) and p97 (p 0.05) were observed on the RBC membranes of HbSS RBCs compared to HbAA and HbAS RBCs, consistent with previous studies. HbSS RBCs treated withmitapivat ex-vivo showed a dose- and time-dependent increase in ATP production accompanied by a decrease in membrane-associated p97 and K48-Ub levels. ATP/Hct level of the 7 patients in the Phase 1 study was significantly increased after 5-, 20-, 50- and 100-mg mitapivat treatment (p 0.01, p 0.05, p 0.01, and p 0.05, respectively), while K48-Ub levels of RBC membranes were decreased, suggesting a negative correlation between K48-Ub and ATP/Hct levels, which was confirmed by Spearman rank correlation analysis. The Spearman correlation coefficient was R = –0.38, with a p-value of 0.017, indicating a moderate and statistically significant negative association between K48-Ub and ATP/Hct. Conclusion: The function of p97 was impaired in HbSS RBCs, leading to the accumulation of p97 and K48-Ub on the cell membrane. Mitapivat as a PKR and PKM2 activator, significantly increased ATP production, restoring p97 function and reducing K48-Ub accumulation, further improving RBC membrane integrity.
Gao et al. (Mon,) studied this question.