In-cell and in vitro studies of disease related protein-protein interactions using NMR-spectroscopy
The receptor for advanced glycation end products (RAGE) is a multiligand cell surface macromolecule that plays a central role in the etiology of diabetes, inflammation, and neurodegeneration. The cytoplasmic domain of RAGE, ctRAGE, is critical for RAGE-dependent signal transduction. As the most membrane proximal event, mDia1 binds to ctRAGE and is essential for RAGE ligand-stimulated phosphorylation of AKT and cell proliferation/migration. We show that ctRAGE contains an unusual alpha-turn that mediates the mDia1-ctRAGE interaction and is required for RAGE dependent signaling. The results establish a novel mechanism through which an extracellular signal initiated by RAGE ligands regulates RAGE signaling in a manner requiring mDia1.
Interactions between biological macromolecules give rise to and regulate biological activity. This activity is manifest through structural dynamics and changes in the macromolecular structures that comprise these interactions. Until recently, mostly in vitro techniques have been used to study macromolecular interactions that govern biological processes under conditions remote from those existing in the cell. With the advent of in-cell Nuclear Magnetic Resonance (NMR) spectroscopy, these processes can now be studied within a cellular environment. Here we review the latest techniques in order to study proteins under more physiological relevant conditions.
Mycobacterium tuberculosis ability to resist nitric oxide stress, from the immune initiated response, relies on then bacteria's ability to target misfolded or damaged proteins. Mycobacterium tuberculosis uses (Prokaryotic Ubiquitin like protein) Pup to target proteins for proteasome mediated degradation. Proteins which have been tagged by Pup interact with mycobacterial ATPase (Mpa). Using STINT NMR we characterized the interactions between Pup and Mpa in-cell and show that Pup's C- terminus is engaged by Mpa. Pups N terminus was also shown to be engaged only in the presence of the Mpa-proteasome complex. We also demonstrate, through various biochemical assays that Pup which is not deamidated is not a substrate for proteasomal degradation.