Geldanamycin Leads to Superoxide Formation by Enzymatic and Non-enzymatic Redox Cycling

Dikalov, Sergey; Landmesser, Ulf; Harrison, David G.

doi:10.1074/jbc.m203271200

Cited by 116 publications

(49 citation statements)

References 27 publications

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“…The role of Hsp90 as a "switch" from superoxide to NO production was also demonstrated in neural NO synthase (48). However, later studies extended these findings and showed that geldanamycin is able to increase superoxide production independently of endothelial NO synthase activation both in in vivo and in vitro systems, possibly by its quinone group, which may participate in redox cycling (21). Similar data were reported on neural NO synthase (20).…”

Section: Discussionsupporting

confidence: 67%

“…2B), which suggested that the two effects are not independent from each other. Reaching this point in our experiments the first results of geldanamycininduced superoxide generation appeared (20,21). What if shaking provided an additional oxygen inducing a larger amount of superoxides?…”

Section: Geldanamycin Induces An Accelerated Lysis Of Jurkatmentioning

confidence: 89%

“…Accelerated superoxide production is a recently observed feature of geldanamycin, the first Hsp90-independent function of this highly specific Hsp90 inhibitor (20,21). Superoxide production has been first implicated in geldanamycin action as an alternative route of endothelial NO synthase function leading to an uncoupled superoxide production parallel with a decreased NO synthesis (47).…”

Section: Discussionmentioning

confidence: 99%

“…However, later experiments demonstrated that the extent of geldanamycin-induced enhancement of cell lysis was dependent on the experimental conditions, namely, if cells were shaken during the experiment or not. At this time the first results of geldanamycin-induced superoxide generation appeared (20,21). These results turned our attention to examine the contribution of superoxide-related versus Hsp-related events to diminished cellular integrity after Hsp90 inhibition.…”

mentioning

confidence: 99%

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Hsp90 Inhibition Accelerates Cell Lysis

Sreedhar¹,

Kálmán²,

Pato³

et al. 2003

Journal of Biological Chemistry

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The 90 kDa heat shock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells. Here we analyzed the involvement of Hsp90 in the maintenance of cellular integrity using partial cell lysis as a measure. Inhibition of Hsp90 by geldanamycin, radicicol, cisplatin, and novobiocin induced a significant acceleration of detergent-and hypotonic shock-induced cell lysis. The concentration and time dependence of cell lysis acceleration was in agreement with the Hsp90 inhibition characteristics of the N-terminal inhibitors, geldanamycin and radicicol. Glutathione and other reducing agents partially blocked geldanamycin-induced acceleration of cell lysis but were largely ineffective with other inhibitors. Indeed, geldanamycin treatment led to superoxide production and a change in membrane fluidity. When Hsp90 content was diminished using anti-Hsp90 hammerhead ribozymes, an accelerated cell lysis was also observed. Hsp90 inhibition-induced cell lysis was more pronounced in eukaryotic (yeast, mouse red blood, and human T-lymphoma) cells than in bacteria. Our results indicate that besides the geldanamycin-induced superoxide production, and a consequent increase in cell lysis, inhibition or lack of Hsp90 alone can also compromise cellular integrity. Moreover, cell lysis after hypoxia and complement attack was also enhanced by any type of Hsp90 inhibition used, which shows that the maintenance of cellular integrity by Hsp90 is important in physiologically relevant lytic conditions of tumor cells.The 90 kDa heat shock protein (Hsp90) 1 is a central part of a chaperone meshwork chaperoning a large number of substrate proteins (1-5). Besides being a partner of a large number of co-chaperones and substrates, Hsp90 binds to filamentous actin and tubulin (6 -8) and the involvement of the cytoskeleton in the traffic of Hsp90 substrates has also been demonstrated (5, 9). Together with other chaperones, like Hsp27 and Hsp70, Hsp90 is involved in cytoprotection (10 -12).Cell lysis is one of the most commonly used methods to test cellular integrity. Moreover, lysis rate anomalies (13,14) together with diffusional anomalies (15, 16) were used as important arguments for the organization of the cytoplasm. Since cellular integrity is preserved after a partial cell lysis to a large extent (13,14), partial lysis provides a highly sensitized, but still somewhat organized cellular system, where the contribution of various components to both the cytoplasmic organization and cellular stability can be studied.The original aim of the present study was to examine whether Hsp90 inhibition induces any change in the rate of cell lysis induced by mild detergent treatment or hypotonic shock. The rationale behind these experiments was to test, whether Hsp90, a cytoprotective chaperone, binding to "thousand-andone" substrates and other proteins is involved in the maintenance of cellular integrity (17), and whether its inhibition renders cells more "lysis-prone." The first experiments were very promising: geldan...

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Section: Discussionsupporting

confidence: 67%

Section: Geldanamycin Induces An Accelerated Lysis Of Jurkatmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Hsp90 Inhibition Accelerates Cell Lysis

Sreedhar¹,

Kálmán²,

Pato³

et al. 2003

Journal of Biological Chemistry

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“…anion (8). Superoxide could interact with NO to form peroxynitrite and reduce the amounts of NO that are available to interact with sGC independent of the actions of geldanamycin on HSP90.…”

Section: Fig 7 Fluorescent Colocalization Of Hsp90 and Sgc In Baecsmentioning

confidence: 99%

Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase

Venema

et al. 2003

American Journal of Physiology-Heart and Circulatory Physiology

112

120

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. Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase. Am J Physiol Heart Circ Physiol 285: H669-H678, 2003. First published April 3, 2003 10.1152/ajpheart.01025.2002 is an important downstream intracellular target of nitric oxide (NO) that is produced by endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). In this study, we demonstrate that sGC exists in a complex with eNOS and heat shock protein 90 (HSP90) in aortic endothelial cells. In addition, we show that in aortic smooth muscle cells, sGC forms a complex with HSP90. Formation of the sGC/eNOS/HSP90 complex is increased in response to eNOS-activating agonists in a manner that depends on HSP90 activity. In vitro binding assays with glutathione S-transferase fusion proteins that contain the ␣-or ␤-subunit of sGC show that the sGC ␤-subunit interacts directly with HSP90 and indirectly with eNOS. Confocal immunofluorescent studies confirm the subcellular colocalization of sGC and HSP90 in both endothelial and smooth muscle cells. Complex formation of sGC with HSP90 facilitates responses to NO donors in cultured cells (cGMP accumulation) as well as in anesthetized rats (hypotension). These complexes likely function to stabilize sGC as well as to provide directed intracellular transfer of NO from NOS to sGC, thus preventing inactivation of NO by superoxide anion and formation of peroxynitrite, which is a toxic molecule that has been implicated in the pathology of several vascular diseases.smooth muscle cells; endothelium; vascular endothelial growth factor; bradykinin; cGMP accumulation SOLUBLE GUANYLATE CYCLASE (sGC), an ␣/␤-heterodimeric heme protein, catalyzes the conversion of GTP to cGMP in many cells including vascular endothelial cells (ECs) and vascular smooth muscle cells (SMCs). Activation of sGC is by direct binding of nitric oxide (NO) to the sGC heme prosthetic group. Formation of the nitrosyl heme adduct induces a conformational change in sGC that results in an increase in its enzymatic activity (21). The NO that activates sGC in various cells is the product of a reaction that is catalyzed by one of three distinct NO synthase (NOS) molecules, which catalyze the oxidation of L-arginine to produce L-citrulline and NO (1). In ECs, NO production is mediated by the constitutively expressed endothelial NOS (eNOS). Activation of eNOS is by Ca 2ϩ -calmodulin (CaM) after agonist-stimulated elevations in intracellular Ca 2ϩ concentrations. Two signaling pathways exist that involve eNOS and sGC. The first is an intercellular pathway whereby NO, which is produced by eNOS in ECs, diffuses to the underlying SMCs and promotes blood vessel relaxation (16). The second is an intracellular eNOS-sGC pathway that is essential for vascular endothelial growth factor (VEGF)-induced increases in EC permeability and proliferation (24,25,30).Initially, eNOS was thought to function as an isolated homodimer. It is now known, however, that eNOS exists in multiprotein complexes in which it interacts with other proteins. These pr...

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Hsp 90 Inhibitors

Blagg

Hadden

2010

Burger's Medicinal Chemistry and Drug Discovery

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The Hsp90 molecular chaperones represent a promising target for the development of anticancer and neuroprotective agents as they are responsible for the conformational maturation of numerous signaling proteins and the refolding of aggregated proteins, respectively. In this chapter, a summary of recent developments toward the treatment of these diseases by small‐molecule modulators of Hsp90 is provided.

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Geldanamycin Leads to Superoxide Formation by Enzymatic and Non-enzymatic Redox Cycling

Cited by 116 publications

References 27 publications

Hsp90 Inhibition Accelerates Cell Lysis

Hsp90 Inhibition Accelerates Cell Lysis

Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase

Hsp 90 Inhibitors

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