Rescue of mutant rhodopsin traffic by metformin-induced AMPK activation accelerates photoreceptor degeneration

Protein misfolding caused by inherited mutations leads to loss of protein function and potentially toxic 'gain of function', such as the dominant P23H rhodopsin mutation that causes retinitis pigmentosa (RP). Here, we tested whether the AMPK activator metformin could affect the P23H rhodopsin synthesis and folding. In cell models, metformin treatment improved P23H rhodopsin folding and traffic. In animal models of P23H RP, metformin treatment successfully enhanced P23H traffic to the rod outer segment, but this led to reduced photoreceptor function and increased photoreceptor cell death. The metformin-rescued P23H rhodopsin was still intrinsically unstable and led to increased structural instability of the rod outer segments. These data suggest that improving the traffic of misfolding rhodopsin mutants is unlikely to be a practical therapy, because of their intrinsic instability and long half-life in the outer segment, but also highlights the potential of altering translation through AMPK to improve protein function in other protein misfolding diseases

Magic angle spinning nuclear magnetic resonance spectroscopy of G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the largest family of membrane receptors and mediate a diversity of cellular processes. These receptors have a common seven-transmembrane helix structure, yet have evolved to respond to literally thousands of different ligands. In this chapter, we describe the use of magic angle spinning solid-state NMR spectroscopy for characterizing the structure and dynamics of GPCRs. Solid-state NMR spectroscopy is well suited for structural measurements in both detergent micelles and membrane bilayer environments. We first outline the methods for large-scale production of stable, functional receptors containing 13 C- and 15 N-labeled amino acids. The expression methods make use of eukaryotic HEK293S cell lines that produce correctly folded, fully functional receptors. We subsequently describe the basic methods used for magic angle spinning solid-state NMR measurements of chemical shifts and dipolar couplings, which reveal detailed information on GPCR structure and dynamics. © 2013 Elsevier Inc