A Novel C-Terminal Homologue of Aha1 Co-Chaperone Binds to Heat Shock Protein 90 and Stimulates Its ATPase Activity in Entamoeba histolytica

Singh, Meetali; Shah, Varun; Tatu, Utpal

doi:10.1016/j.jmb.2014.01.008

Cited by 18 publications

(26 citation statements)

References 34 publications

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“…Unfortunately, a co-staining of Aha1 and Hsp90 and the visualisation by immunofluorescence microscopy was not possible due to unspecific binding of the anti-Aha1 antibody. The results confirm that Leishmania Aha1 behaves similarly to other known Aha1 orthologues (Chua et al 2012;Panaretou et al 2002;Singh et al 2014). Whether Leishmania Aha1 has interaction partners other than Hsp90 or chaperone activity by itself as described for human Aha1 (Tripathi et al 2014) remains to be investigated.…”

Section: Aha1supporting

confidence: 66%

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Hsp90 inhibitors radicicol and geldanamycin have opposing effects on Leishmania Aha1-dependent proliferation

Bartsch

Hombach-Barrigah

Clos

2017

Cell Stress and Chaperones

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Hsp90 and its co-chaperones are essential for the medically important parasite Leishmania donovani, facilitating life cycle control and intracellular survival. Activity of Hsp90 is regulated by co-chaperones of the Aha1 and P23 families. In this paper, we studied the expression of L. donovani Aha1 in two life cycle stages, its interaction with Hsp90 and the phenotype of Aha1 null mutants during the insect stage and inside infected macrophages. This study provides a detailed in vitro analysis of the function of Aha1 in Leishmania parasites and the first instance of a reverse genetic analysis of Aha1 in a protozoan parasite. While Aha1 is non-essential under standard growth conditions and at elevated temperature, Aha1 protects against ethanol stress. However, both overexpression and lack of Aha1 affected parasite growth in the presence of the Hsp90 inhibitors radicicol (RAD) and geldanamycin (GA). Under RAD pressure, P23 and Aha1 act in an antagonistic way. By contrast, expression levels of both co-chaperones have similar effects under GA treatment, indicating different inhibition mechanisms by the two compounds. Aha1 is also secreted in virulenceenhancing exosomes. This may explain why the loss of Aha1 reduces the infectivity of L. donovani in ex vivo mouse macrophages, indicating a role during the intracellular mammalian stage.

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Section: Aha1supporting

confidence: 66%

“…The architecture of Aha1 in other protozoan parasites, e.g. Giardia lamblia and Entamoeba histolytica, is reduced to either of the two domains without losing ATPase activation activity (Rehn and Buchner 2015;Singh et al 2014). The Aha1 gene is also conserved in the protozoan parasite Leishmania spp.…”

Section: Introductionmentioning

confidence: 99%

Hsp90 inhibitors radicicol and geldanamycin have opposing effects on Leishmania Aha1-dependent proliferation

Bartsch

Hombach-Barrigah

Clos

2017

Cell Stress and Chaperones

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“…Binding to its cochaperone, Aha1 (activator of Hsp90 ATPase), at a ratio of one Aha1 per Hsp90 dimer stimulates the ATPase activity of Hsp90 by threefold (54). Human Hsp90 ATPase has a K m value of 324 μM for ATP and a k cat /K m of 46 min −1 ·M −1 (55), which is about ninefold less than that of human p97. With such a low basal ATPase rate, Hsp90 not only needs an activating cochaperone but also requires inhibiting cochaperones to reduce activity (56).…”

Section: Discussionmentioning

confidence: 99%

Altered cofactor regulation with disease-associated p97/VCP mutations

Zhang

Gui

Zhang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

147

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Dominant mutations in p97/VCP (valosin-containing protein) cause a rare multisystem degenerative disease with varied phenotypes that include inclusion body myopathy, Paget's disease of bone, frontotemporal dementia, and amyotrophic lateral sclerosis. p97 disease mutants have altered N-domain conformations, elevated ATPase activity, and altered cofactor association. We have now discovered a previously unidentified disease-relevant functional property of p97 by identifying how the cofactors p37 and p47 regulate p97 ATPase activity. We define p37 as, to our knowledge, the first known p97-activating cofactor, which enhances the catalytic efficiency (k cat /K m ) of p97 by 11-fold. Whereas both p37 and p47 decrease the K m of ATP in p97, p37 increases the k cat of p97. In contrast, regulation by p47 is biphasic, with decreased k cat at low levels but increased k cat at higher levels. By deleting a region of p47 that lacks homology to p37 (amino acids 69-92), we changed p47 from an inhibitory cofactor to an activating cofactor, similar to p37. Our data suggest that cofactors regulate p97 ATPase activity by binding to the N domain. Induced conformation changes affect ADP/ATP binding at the D1 domain, which in turn controls ATPase cycling. Most importantly, we found that the D2 domain of disease mutants failed to be activated by p37 or p47. Our results show that cofactors play a critical role in controlling p97 ATPase activity, and suggest that lack of cofactorregulated communication may contribute to p97-associated disease pathogenesis.AAA ATPase | p97/VCP | MSP1 | p47 | steady-state kinetics

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“…Hsp90 works in concert with a number of chaperones and co-chaperones that modulate the activity of Hsp90 and the binding and conformation of its client proteins [65, 66]. We have previously found that Hsp70 is a negative regulator of Nox5[28], however, the roles of other important co-chaperones like HOP, Hsp40 and p23 have not been studied.…”

Section: Discussionmentioning

confidence: 99%

Nox5 stability and superoxide production is regulated by C-terminal binding of Hsp90 and CO-chaperones

Chen

Haigh

et al. 2015

Free Radical Biology and Medicine

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Heat shock protein 90 (Hsp90) is a molecular chaperone that orchestrates the folding and stability of proteins that regulate cellular signaling, proliferation and inflammation. We have previously shown that Hsp90 controls the production of reactive oxygen species by modulating the activity of Noxes1-3 and 5, but not Nox4. The goal of the current study was to define the regions on Nox5 that bind Hsp90 and determine how Hsp90 regulates enzyme activity. In isolated enzyme activity assays, we found that Hsp90 inhibitors selectively decrease superoxide, but not hydrogen peroxide, production. The addition of Hsp90 alone only modestly increases Nox5 enzyme activity but in combination with the co-chaperones, Hsp70, HOP, Hsp40, and p23 it robustly stimulated superoxide, but not hydrogen peroxide, production. Proximity ligation assays reveal that Nox5 and Hsp90 interact in intact cells. In cell lysates using a co-IP approach, Hsp90 binds to Nox5 but not Nox4, and the degree of binding can be influenced by calcium-dependent stimuli. Inhibition of Hsp90 induced the degradation of full length, catalytically inactive and a C-terminal fragment (aa398–719) of Nox5. In contrast, inhibition of Hsp90 did not affect the expression levels of N-terminal fragments (aa1–550) suggesting that Hsp90 binding maintains the stability of C-terminal regions. In Co-IP assays, Hsp90 was bound only to the C-terminal region of Nox5. Further refinement using deletion analysis revealed that the region between aa490–550 mediates Hsp90 binding. Converse mapping experiments show that the C-terminal region of Nox5 bound to the M domain of Hsp90 (aa310–529). In addition to Hsp90, Nox5 bound other components of the foldosome including co-chaperones Hsp70, HOP, p23 and Hsp40. Silencing of HOP, Hsp40 and p23 reduced Nox5-dependent superoxide. In contrast, increased expression of Hsp70 decreased Nox5 activity whereas a mutant of Hsp70 failed to do so. Inhibition of Hsp90 results in the loss of higher molecular weight complexes of Nox5 and decreased interaction between monomers. Collectively these results show that the C-terminal region of Nox5 binds to the M domain of Hsp90 and that the binding of Hsp90 and select co-chaperones facilitate oligomerization and the efficient production of superoxide.

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A Novel C-Terminal Homologue of Aha1 Co-Chaperone Binds to Heat Shock Protein 90 and Stimulates Its ATPase Activity in Entamoeba histolytica

Cited by 18 publications

References 34 publications

Hsp90 inhibitors radicicol and geldanamycin have opposing effects on Leishmania Aha1-dependent proliferation

Hsp90 inhibitors radicicol and geldanamycin have opposing effects on Leishmania Aha1-dependent proliferation

Altered cofactor regulation with disease-associated p97/VCP mutations

Nox5 stability and superoxide production is regulated by C-terminal binding of Hsp90 and CO-chaperones

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