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TYROSINE PHOSPHORYLATION MEDIATED REMODELING OF THE ERYTHROCYTE MEMBRANE IN SICKLE CELL DISEASE

thesis
posted on 04.11.2022, 12:23 authored by John M HausmanJohn M Hausman

The pathological hallmarks of sickle cell disease originate from a single mutation of the beta hemoglobin gene resulting in a valine at position 6 instead of the canonical glutamic acid. This small change perpetuates many factors, manifesting into chronic embolic processes in the microvasculature, causing painful vaso-occlusive episodes and eventual organ failure. There have been numerous therapies developed to reduce the mortality of sickle cell ranging from agents to induce production of fetal hemoglobin to chronic blood transfusions. Although each of these options are effective at improving the quality of life for sickle cell patients, they only treat one aspect of the disease and, for some, become ineffective over time. In the hope of producing a better therapy, a better understanding of the pathogenesis of vaso-occlusive episodes is needed. While many models have been offered to account for these vaso-occlusive events, one recently proposed mechanism stems from the elevated tyrosine phosphorylation of the cytoplasmic domain of the major erythrocyte membrane protein, Band 3. Band 3 serves as a hub for many critical proteins in the red cell. It binds ankyrin, which associates the spectrin cortical cytoskeleton to the red cell membrane, deoxygenated hemoglobin, the kinases Wnk1 and OSR1, which regulate cation transport, and a glycolytic enzyme metabolon that regulates the production of ATP and glutathione. When Band 3 is tyrosine phosphorylated, each of these proteins dissociate, causing significant changes to red cell homeostasis. These changes include an accumulation of reactive oxygen species, vesiculation and release of prothrombotic microvesicles, leakage of cell free hemoglobin, and a decrease in cell volume. Normally, Band 3 exists in a predominantly unphosphorylated state, however, in sickle cell disease, Band 3 is abundantly tyrosine phosphorylated. Reduction in the tyrosine phosphorylation of Band 3 has been documented to prevent the release of microvesicles and hemoglobin from sickle cell red blood cells. Because these microvesicles and cell free hemoglobin contribute to the vaso-occlusive episodes in sickle cell patients, inhibiting the mechanism for their release offers a potential therapeutic option. But to accomplish this, the molecular cause for the elevated tyrosine phosphorylation in sickle cell disease must be identified. Since tyrosine phosphorylation is performed by a tyrosine kinase and removed by a tyrosine phosphatase, the elevation in phosphorylation must be due to changes in both of these processes. Unfortunately, the identity and nature of these kinases and phosphatases are poorly understood. In this dissertation, I identified the tyrosine kinases Syk, Lyn, and Src attributed to Band 3

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phosphorylation that facilitates the release of microvesicles and hemoglobin in sickle cell red blood cells. Inhibition of Syk or one of the two Src family kinases is sufficient to prevent the destabilization of the red blood cell membrane. These kinases function in a hierarchy, where one of the three Src family kinase, Lyn phosphorylates Syk, activating it, and promoting the phosphorylation of Band 3 at tyrosines 8 and 21. Prevention of either phosphorylation event prevents the release of microvesicles and cell free hemoglobin. I also report the identification of PTP1B as the tyrosine phosphatase responsible for maintaining Band 3 in an unphosphorylated state. Interestingly, in sickle cell disease, this tyrosine phosphatase is proteolytically cleaved, resulting in a reduction in dephosphorylating potential. It has been reported previously that PTP1B is a substrate of the calcium dependent protease, calpain and that calpain inhibitors improve the cell morphology of sickle erythrocytes. Inhibition of this proteolytic process may offer an additional therapeutic option for the treatment of sickle cell disease.

Funding

NIH GM24417-40

History

Degree Type

Doctor of Philosophy

Department

Chemistry

Campus location

West Lafayette

Advisor/Supervisor/Committee Chair

Philip S. Low

Additional Committee Member 2

Angeline Lyon

Additional Committee Member 3

Nicolas Noinaj

Additional Committee Member 4

Shalini Low-Nam