Researcher(s)
- Jholly Prell Burke, Applied Molecular Biology & Biotechnology, University of Delaware
Faculty Mentor(s)
- Arit Ghosh, DBI, University of Delaware
Abstract
Abstract:
In biological systems, the ubiquitin-proteasome system (UPS) has a fundamental role in selectively degrade mutated, misfolded or unwanted proteins. In this regard, the maturation of a red blood cell is a highly regulated process involving several proteostasis mechanisms that degrade and clear the maturing cell of excess proteins while upregulating hemoglobin levels. The UPS and related proteins are known to be critical towards the process of red blood cell maturation, however, there is not enough evidence on how proteasome inhibition directly induces ferroptosis, programmed cell death by manipulation of iron, in the erythroid cells. The proteasome inhibitor, MG132, is a molecule used to repress the UPS, leading to build-up of ubiquitin-conjugated proteins. In our study, the application of MG132 on the differentiated MELDS19 cells, in vitro, leads to build up of Heme Oxygenase 1 (Hmox1), a stress-inducible enzyme that breaks down heme and releases excess iron. A multiplexed assay was developed to detect transferrin receptor and lipid peroxidation of the MELDS19 erythroid cells. The results showed an increase of Hmox1 levels due to UPS inhibition directly correlates to increased oxidation of iron, which then induces ferroptosis. An eosin dye (EMA) was used to detect erythroid membrane molecule known as Band3. It was determined that there was dysregulation of membrane protein expression treated with MG132. Hence, we hypothesize that addition of MG132 will affect transferrin receptor biology, hemoglobin production, and the differentiation trajectory of erythroid cells. To visualize membrane destabilization, follow-up microscopy techniques such as FRAP (Fluorescence Recovery After Photobleaching) would help determine whether membrane integrity and cytoskeletal dynamics of differentiating erythroid cells are disrupted due to a malfunctioning UPS. Findings from this study can be used as a precursor for the upcoming studies using primary cells that could help better understand human blood diseases involving ferroptosis in human reticulocytes.