8 research outputs found
Different Poses for Ligand and Chaperone in Inhibitor Bound Hsp90 and GRP94: Implications for Paralog-specific Drug Design
Hsp90 chaperones contain an N-terminal ATP binding site that has been effectively targeted by competitive inhibitors. Despite the myriad of inhibitors, none to date have been designed to bind specifically to just one of the four mammalian hsp90 paralogs, which are cytoplasmic Hsp90Ξ± and Ξ², ER GRP94, and mitochondrial Trap-1. Given that each of the hsp90 paralogs is responsible for chaperoning a distinct set of client proteins, specific targeting of one hsp90 paralog may result in higher efficacy and therapeutic control. Specific inhibitors may also help elucidate the biochemical roles of each hsp90 paralog. Here we present side by side comparisons of the structures of yeast Hsp90 and mammalian GRP94, bound to the pan-hsp90 inhibitors Geldanamycin and Radamide. These structures reveal paralog specific differences in the Hsp90 and GRP94 conformations in response to Geldanamycin binding. We also report significant variation in the pose and disparate binding affinities for the Geldanamycin-Radicicol chimera Radamide when bound to the two paralogs, which may be exploited in the design of paralog-specific inhibitors
Heterozygous Yeast Deletion Collection Screens Reveal Essential Targets of Hsp90
Hsp90 is an essential eukaryotic chaperone with a role in folding specific βclientβ proteins such as kinases and hormone receptors. Previously performed homozygous diploid yeast deletion collection screens uncovered broad requirements for Hsp90 in cellular transport and cell cycle progression. These screens also revealed that the requisite cellular functions of Hsp90 change with growth temperature. We present here for the first time the results of heterozygous deletion collection screens conducted at the hypothermic stress temperature of 15Β°C. Extensive bioinformatic analyses were performed on the resulting data in combination with data from homozygous and heterozygous screens previously conducted at normal (30Β°C) and hyperthermic stress (37Β°C) growth temperatures. Our resulting meta-analysis uncovered extensive connections between Hsp90 and (1) general transcription, (2) ribosome biogenesis and (3) GTP binding proteins. Predictions from bioinformatic analyses were tested experimentally, supporting a role for Hsp90 in ribosome stability. Importantly, the integrated analysis of the 15Β°C heterozygous deletion pool screen with previously conducted 30Β°C and 37Β°C screens allows for essential genetic targets of Hsp90 to emerge. Altogether, these novel contributions enable a more complete picture of essential Hsp90 functions
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A structural basis for lithium and substrate binding of an inositide phosphatase
Inositol polyphosphate 1-phosphatase (INPP1) is a prototype member of metal-dependent/lithium-inhibited phosphomonoesterase protein family defined by a conserved three-dimensional core structure. Enzymes within this family function in distinct pathways including inositide signaling, gluconeogenesis, and sulfur assimilation. Using structural and biochemical studies, we report the effect of substrate and lithium on a network of metal binding sites within the catalytic center of INPP1. We find that lithium preferentially occupies a key site involved in metal-activation only when substrate or product is added. Mutation of a conserved residue that selectively coordinates the putative lithium-binding site results in a dramatic 100-fold reduction in the inhibitory constant as compared with wild-type. Furthermore, we report the INPP1/inositol 1,4-bisphosphate complex which illuminates key features of the enzyme active site. Our results provide insights into a structural basis for uncompetitive lithium inhibition and substrate recognition and define a sequence motif for metal binding within this family of regulatory phosphatases
Structures of GRP94-Nucleotide Complexes Reveal Mechanistic Differences between the hsp90 Chaperones
Different Poses for Ligand and Chaperone in Inhibitor-Bound Hsp90 and GRP94: Implications for Paralog-Specific Drug Design
Characterization of a Selective Inhibitor of Inositol Hexakisphosphate Kinases: USE IN DEFINING BIOLOGICAL ROLES AND METABOLIC RELATIONSHIPS OF INOSITOL PYROPHOSPHATES*Sβ
Inositol hexakisphosphate kinases (IP6Ks) phosphorylate inositol
hexakisphosphate (InsP6) to yield 5-diphosphoinositol
pentakisphosphate (5-[PP]-InsP5 or InsP7). In this
study, we report the characterization of a selective inhibitor,
N2-(m-(trifluoromethy)lbenzyl)
N6-(p-nitrobenzyl)purine (TNP), for these
enzymes. TNP dose-dependently and selectively inhibited the activity of IP6K
in vitro and inhibited InsP7 and InsP8
synthesis in vivo without affecting levels of other inositol
phosphates. TNP did not inhibit either human or yeast Vip/PPIP5K, a newly
described InsP6/InsP7 1/3-kinase. Overexpression of
IP6K1, -2, or -3 in cells rescued TNP inhibition of InsP7
synthesis. TNP had no effect on the activity of a large number of protein
kinases, suggesting that it is selective for IP6Ks. TNP reversibly reduced
InsP7/InsP8 levels. TNP in combination with genetic
studies was used to implicate the involvement of two pathways for synthesis of
InsP8 in yeast. TNP induced a fragmented vacuole phenotype in
yeast, consistent with inhibition of Kcs1, a Saccharomyces cerevisiae
IP6K. In addition, it also inhibited insulin release from Min6 cells in a
dose-dependent manner further implicating InsP7 in this process.
TNP thus provides a means of selectively and rapidly modulating cellular
InsP7 levels, providing a new and versatile tool to study the
biological function and metabolic relationships of inositol
pyrophosphates