Biochemical And Structural Characterization Of Intramembrane Proteases

In regulated intramembrane proteolysis membrane proteins are cleaved within their transmembrane region, resulting in soluble fragments that regulate cell physiology. The intramembrane proteases responsible for cleavage are widespread with important roles in human biology and disease. Rhomboids catalyze the activation of epidermic growth factor receptor ligands in D. melanogaster. How rhomboids recognize their substrates and select which peptide bond to cleave is not understood. We have studied the substrate specificity and peptide bond selectivity of purified rhomboids from several organisms using chimeric transmembrane substrates. Analysis of the proteolytic products by mass spectrometry reveals that cleavage occurs in the membrane/water interface at sites shared by both eukaryotic and prokaryotic rhomboids. Mutagenesis of the substrates reveals a helical amino acid motif that is crucial for substrate recognition and peptide bond selection. With insight from computational data a model for the substrate-enzyme complex will be presented. Gamma-Secretase catalyzes the production of amyloid beta-peptides involved in Alzheimer's disease. Using negative-stain single-particle electron microscopy we have determined the structure of a native-like 500kDa gamma-secretase complex comprising presenilin, nicastrin, APH-1, and PEN-2 that is fully catalytically active. Antibody labeling of the extracellular domain of nicastrin was employed to ascertain the topology of the reconstruction. Active site labeling with a gold-coupled transition state analog inhibitor demonstrates that gamma-secretase contains a single active site facing a large conical internal cavity. This cavity, surrounded by a ~35Å thick transmembrane protein wall, extends from the extracellular side of the membrane to past the membrane centre, where it narrows to finally close at the cytoplasmic side. Based on our structure we suggest a model for gamma-secretase function, in which a hydrophobic transmembrane helix substrate is hydrolyzed by catalytic aspartyl moieties at the interface of a water-accessible internal cavity away from the surrounding lipid environment.