Pathological Role of Halted Proteasome Machinery in Sickle Cell Disease

Although proteasome machinery is a conserved cellular component to maintain their normal function, its function in erythrocyte under stress conditions is largely unknown, especially in sickle cell disease (SCD). To determine whether proteasome machinery is altered in SCD erythrocyte, we conducted western blot to detect total ubiquitinated proteins on the erythrocyte membrane in both mice and humans with or without SCD. We found that ubiquitinated proteins were significantly accumulated in SCD mice and humans compared to WT mice and normal controls, indicating that proteasome machinery is halted in SCD. Next, to determine which specific proteins are ubiquitinated and accumulated in SCD, we conducted robust and nonbiased proteomic profiling by immunoprecipitation ubiquitinated proteins followed by proteomics analysis. We found significant accumulation of several categories of ubiquitinated proteins on the erythrocyte membrane in SCD, including cytoskeleton proteins (Spectrin, Actin, Ankryin), glycolytic enzymes (GAPDH, 2,3-BPG mutase, Pyruvate Kinase, G6PD), transporters (Band3, large neutral AA transporter, calcium transporter, ENT1), hemoglobin, components of proteasome machinery [E2, E3 ligases, and valosin-containing protein (p97)]. Finally, to determine the effect of halted proteasome machinery in SCD functionally, we conducted in vitro hypoxia induced red blood cell (RBC) sickling assay. We found that inhibition of RBC proteasome machinery by targeting p97 using CB-5083 or targeting proteasome using MG132 increases SCD RBC sickling. Overall, our findings reveal a novel role of halted proteasome machinery in the pathophysiology of SCD and open up new therapies for the disease.