Geldanamycin-associated Inhibition of Intracellular Trafficking Is Attributed to a Co-purified Activity

Abstract

Geldanamycin, an ansamycin antibiotic that specifically inhibits heat-shock protein-90 (HSP90) and its endoplasmic reticulum homologue, glucose-regulated protein-94 (GRP94), accelerates the degradation of selected cellular proteins. We showed previously that geldanamycin inhibits maturation and transport of the epidermal growth factor receptor in addition to accelerating its degradation ( Chromatography of S. hygroscopicus var. geldanus extracts on a silica-gel column allowed separation between the inhibition of intracellular trafficking and geldanamycin-mediated degradation. One fraction that was devoid of geldanamycin blocked secretion of a soluble form of the erythropoietin receptor, retarded maturation of the epidermal growth factor receptor without enhancing its degradation, and blocked anterograde transport of a temperature-sensitive mutant of the vesicular stomatitis virus G protein (VSVGtsO45) from the early Golgi cisternae. This fraction was enriched (>95%) in 17-demethylgeldanamycin. However, as synthetically derived 17-demethylgeldanamycin did not inhibit intracellular trafficking, we concluded that 17-demethylgeldanamycin is not the active component. We thus propose that a compound(s) that co-purifies with benzoquinone ansamycins inhibits intracellular transport. Taken together, our data demonstrate that the inhibitory effects on protein maturation and intracellular trafficking, previously attributed to geldanamycin, are mediated by another distinct moiety.

Natalamycin A, an ansamycin from a termite-associated Streptomyces sp.

“…However, additional compounds were observed in these fractions. Through a careful series of semi-preparative reverse-phase HPLC experiments with a phenyl-hexyl column, reblastatin ( 3 ), 5 17- O -demethyl-geldanamycin ( 4 ), 6 19- S -methylgeldanamycin ( 5 ), 7 17-amino-17-demethoxy-geldanamycin ( 6 ), 8 methyl geldanamycinate derivative 7, 9 and recently reported geldanamycin analog 9 were resolved and isolated. 10 There were also two previously unreported metabolites.…”

Non-Invasive PET Imaging of EGFR Degradation Induced by a Heat Shock Protein 90 Inhibitor

“…The activity of Hsp90 promotes the attainment and maintenance of proper conformation of its clients, including EGFR, HER-2, Akt, and wild type or mutated androgen receptor, which are of potential importance in mediating prostate cancer progression [17][18][19]. An Hsp90 inhibitor, geldanamycin (GM), has been shown to have two distinct effects on EGFR: promoting its degradation and mediating its intracellular retention [20]. It has also been reported that 17-allyamino-17-demethoxygeldanamycin (17-AAG), a GM-derived Hsp90 inhibitor, could induce the degradation of both wild-type and mutant form EGFR [21].…”

An extracellular region of the erythropoietin receptor of the subterranean blind mole rat Spalax enhances receptor maturation

“…Sol EPO-R Secretion. BOSC-23T cells expressing sol EPO-R cDNA (41,61) were cultured in 6-cm plates. Confluent cell cultures were incubated at 37°C for 3 h in 1 ml of DMEM containing 0.2% FCS.…”

“…Cells were lysed in 0.5 ml of sample buffer X2 and boiled for 15 min. Aliquots of media (40 l) and cell lysates (20 l) were subjected to Western blot analysis (61).…”