ABSTRACT
Tim23, the central subunit of the TIM23 protein-translocation complex, forms a voltage-gated channel in the mitochondrial inner membrane (MIM), an energy-conserving membrane that generates a proton-motive force to drive vital processes. Using high-resolution fluorescence mapping of a channel-facing transmembrane segment (TMS2) of Tim23 from Saccharomyces cerevisiae, we demonstrate that changes in the energized state of the MIM cause marked structural alterations in the channel region. In an energized membrane, TMS2 forms a continuous α-helix that is inaccessible to the aqueous intermembrane space (IMS). Upon depolarization, the helical periodicity of TMS2 is disrupted, and the channel becomes exposed to the IMS. Kinetic measurements confirm that changes in TMS2 conformation coincide with depolarization. These results reveal how the energized state of the membrane drives functionally relevant structural dynamics in membrane proteins coupled to processes such as channel gating.
Subject(s)
Membrane Transport Proteins/chemistry , Mitochondrial Membranes/metabolism , Models, Molecular , Multiprotein Complexes/chemistry , Proton-Motive Force/physiology , Saccharomyces cerevisiae Proteins/chemistry , Amino Acid Sequence , Cloning, Molecular , Kinetics , Membrane Transport Proteins/metabolism , Microscopy, Fluorescence , Mitochondrial Precursor Protein Import Complex Proteins , Molecular Sequence Data , Multiprotein Complexes/metabolism , Protein Structure, Secondary , Saccharomyces cerevisiae Proteins/metabolism , Sequence AlignmentABSTRACT
A major goal of treatment strategies for cancer is the development of agents which can block primary tumor growth and development as well as the progression of tumor metastasis without any treatment associated side effects. Using mini peptide display (MPD) technology, we generated peptides that can bind to the human vascular endothelial growth factor (VEGF) receptor KDR. These peptides were evaluated for their ability to block angiogenesis, tumor growth and metastasis in vitro and in vivo. A D-amino acid peptide with high serum stability (ST100,059) was found to have the most potent activity in vitro as indicated by inhibition of VEGF stimulation of endothelial cells. It was also found to be the most active of the series in blocking VEGF-mediated activity in vivo, as measured in Matrigel-filled angioreactors implanted in mice. ST100,059 blocks VEGF-induced MAPK phosphorylation, as well as inhibits VEGF-induced changes in gene expression in HUVEC cells. In in vivo studies, treatment of female C57BL/6 mice inoculated with B16 mouse melanoma cells with ST100,059 resulted in a dose-dependent decrease in tumor volume and lung metastasis as compared to control groups of animals receiving vehicle alone. These studies demonstrate that by using MPD, peptides can be identified with enhanced affinity relative to those discovered using phage display. Based on these studies we have identified one such peptide ST100,059 which can effectively block tumor growth and metastasis due to its anti-angiogenic effects and ability to block intracellular signaling pathways involved in tumor progression.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Melanoma, Experimental/pathology , Neovascularization, Pathologic/metabolism , Peptides/pharmacology , Vascular Endothelial Growth Factor Receptor-2/metabolism , Amino Acid Sequence , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/metabolism , Animals , Cattle , Cell Line , Cell Survival/drug effects , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Growth Factors/pharmacology , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Melanoma, Experimental/metabolism , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Peptide Library , Peptides/chemistry , Peptides/metabolism , Protein Binding , Signal Transduction/drug effects , Signal Transduction/genetics , Xenograft Model Antitumor AssaysABSTRACT
The structural changes that accompany activation of a G-protein coupled receptor (GPCR) are not well understood. To better understand the activation of rhodopsin, the GPCR responsible for visual transduction, we report studies on the three-dimensional structure for the activated state of this receptor, metarhodopsin II. Differences between the three-dimensional structure of ground state rhodopsin and metarhodopsin II, particularly in the cytoplasmic face of the receptor, suggest how the receptor is activated to couple with transducin. In particular, activation opens a groove on the surface of the receptor that could bind the N-terminal helix of the G protein, transducin alpha.
