Profiling the E3 Ubiquitin Ligase Landscape in Normal and Sickle Red Blood Cells

Abstract

Sickle cell disease (SCD) is the most common genetic disease affecting about 100,000 Americans and millions of people in developing countries. In sub-Saharan Africa, about 50% of children with SCD die before age five. The pathophysiology of SCD is associated with the formation of sickle hemoglobin (HbS) polymers during hypoxia which can be pharmacologically antagonized. Our goal is to exploit targeted protein degradation (TPD) as a novel modality for preventing or delaying sickling. As a first step, we utilized a label-free quantitative proteomic workflow to qualitatively and quantitatively analyze differential protein expression between red blood cells (RBC) from healthy and SCD patients. Since mature RBCs have a simple but active cellular proteome responsible for degrading proteins damaged by oxidative stress, which is significantly increased in SCD (D'Alessandro et al.,Blood Transfus, 2019), we focused on assessing the differential expression of proteins associated with the ubiquitin-proteasome pathway. We identified a total of 2090 proteins, including 41 proteins in the ubiquitin proteasome pathway as well as 27 and 35 E2 and E3 ligases, respectively. Identified proteins and their abundances correlated with previous literature findings in healthy samples (Bryk and Wiśniewski, J Proteome Res, 2017; Gautier et al., Blood Adv, 2018). We also found E3 ligases that demonstrated selective expression in RBC samples from SCD patients. These ligases have been implicated in various signaling activities including cell regulation pathway, metabolic control, heme synthesis, xenophagy and anti-tumor immunity. Based on this work, we will further explore the feasibility of developing innovative TPD based medicines for SCD and other RBC disorders.