Summary
From October 2014 the DFG is funding the Research Training Group 1947 Biochemical, Biophysical, and Biomedical Effects of Reactive Oxygen and Nitrogen Species on Biological Membranes - "BiOx" - at the University of Greifswald and the University Medicine Greifswald. Biological Membranes define living cells and determine their interaction with their environment. Reactive oxygen and nitrogen species (ROS/RNS) profoundly affect the physico-chemical properties and thereby the physiological functions of biological membranes.
Integrins are an important family of transmembrane adhesion receptors that mediate cell adhesion to the extracellular matrix proteins, or cell-cell interaction. AlphaIIbbeta3(αIIbβ3) is the most abundant integrin heterodimer on blood platelets with an average of 80,000 copies per cell.
Crystal structures revealed that low-affinity state integrins are in a bent conformation with their ligand binding sites placing in proximity to the membrane surface. Intermediate extended conformations with a closed head correspond to activated state integrin, where the conformational changes in the extracellular domain lead to increased ligand affinity. Binding of ligand induces further conformational changes, resulting in a high-affinity extended open conformation that fully activates integrin signaling downstream. αIIbβ3 plays a crucial role in haemostasis and is involved in the autoimmune disease Immune Thrombocytopenia (ITP) characterized by low platelet count and bleeding risks. Although it has been suggested that oxidants such as hydrogen peroxide may play a role in ITP, it is unknown whether such oxidants activate αIIbβ3.
We aim to study the influence of oxidants derived from platelets on the activation of αIIbβ3. For this purpose, αIIbβ3 will be reconstituted in a membrane environment (lipid bilayers) and will let to interact with exogenous oxidants and their products (e.g. hydrogen peroxide, H2O2; superoxides, O2-; nitric oxide, .NO).
Protein modification (activation) as a result of oxidative damage will be investigated using a combination of biophysical techniques. Liposome preparation and αIIbβ3 incorporation will be carried out and characterized by quartz crystal microbalance (QCM) and atomic force microscopy (AFM).
Förster resonance energy transfer (FRET) will be used to prove whether or not αIIbβ3 is activated. In addition, antibodies which recognize the activated αIIbβ3 will be used in an enzyme immunosorbent assay to validate protein activation by oxidants. The interaction of oxidants and their products with αIIbβ3 will be also investigated under various pH, ions and temperature conditions.
Publications
Karanth, S., Azinfar, A., Helm, C. A., Delcea, M., "Identification of a critical lipid ratio in raft-like phases exposed to nitric oxide: An AFM study" (2021), Biophysical Journal, doi: 10.1016/j.bpj.2021.06.009
Karanth, S., Meesaragandla, B., Delcea, M., "Changing surface properties of artificial lipid membranes at the interface with biopolymer coated gold nanoparticles under normal and redox conditions" (2020), Biophysical Chemistry, doi: 10.1016/j.bpc.2020.106465
Karanth, S., Delcea, M., "Nitrosative stress affects the interaction of integrin alphaIIbbeta3 with its ligands" (2020), Biochimica et Biophysica Acta (BBA) - Biomembranes, doi: 10.1016/j.bbamem.2020.183198
Collaborators
- Prof. Dr. Christiane A. Helm (Department Physics, University Greifswald, Germany) Link
- Dr. Christopher H. Lillig (University Medicine Greifswald, Germany) Link
Members
Sanjai Karanth