ABSTRACT
Protein phosphatase 1 (PP1) is widely distributed among tissues and species and acts as a regulator of many important cellular processes. By targeting the catalytic part of PP1 (PP1C) toward particular loci and substrates, regulatory subunits constitute key elements conferring specificity to the holoenzyme. Here, we report the identification of an (alpha/beta)8-barrel-like structure within the N-ter stretch of the human PP1 regulatory subunit hGM, which is part of the family of diverse proteins associated with glycogen metabolism. Protein homology modeling gave rise to a three-dimensional (3D) model for the 381 N-ter residue stretch of hGM, based on sequence similarity with Streptomyces olivochromogenes xylose isomerase, identified by using FASTA. The alignment was subsequently extended by using hydrophobic cluster analysis. The homology-derived model includes the putative glycogen binding area located within the 142-230 domain of hGM as well as a structural characterization of the PP1C interacting domain (segment 51-67). Refinement of the latter by molecular dynamics afforded a topology that is in agreement with previous X-ray studies (Egloff et al., 1997). Finite difference Poisson-Boltzmann calculations performed on the interacting domains of PP1C and hGM confirm the complementarity of the local electrostatic potentials of the two partners. This work highlights the presence of a conserved fold among distant species (mammalian, Caenorhabditis elegans, yeast) and, thus, emphasizes the involvement of PP1 in crucial basic cellular functions.
Subject(s)
Glycogen/metabolism , Phosphoprotein Phosphatases/chemistry , Amino Acid Motifs , Amino Acid Sequence , Humans , Models, Molecular , Molecular Sequence Data , Protein Phosphatase 1 , Protein Structure, Tertiary , Sequence Alignment , Sequence Analysis, Protein , Static ElectricityABSTRACT
The vacuolar H+-ATPase (V-ATPase), located on the ruffled border of the osteoclast, is a proton pump which is responsible for secreting the massive amounts of protons that are required for the bone resorption process. With the aim to identify new agents which are able to prevent the excessive bone resorption associated with osteoporosis, we have designed a novel class of potent and selective inhibitors of the osteoclast proton pump, starting from the structure of the specific V-ATPase inhibitor bafilomycin A1. Compounds 3a-d potently inhibited the V-ATPase in chicken osteoclast membranes (IC50 = 60-180 nM) and were able to prevent bone resorption by human osteoclasts in vitro at low-nanomolar concentrations. Notably, the EC50 of compound 3c in this assay was 45-fold lower than the concentration required for half-maximal inhibition of the V-ATPase from human kidney cortex. These results support the validity of the osteoclast proton pump as a useful molecular target to produce novel inhibitors of bone resorption, potentially useful as antiosteporotic agents.