Oxidative stress occurs as a consequence of an imbalance between the production and scavenging of ROS. Severe oxidative stress leads to oxidation and aggregation of vital proteins and DNA. The expression of Hsps may increase dramatically following such stress (Swindell et al, 2007
). Some Hsps, mainly members of the Hsp70 family and its co-chaperones, select and direct aberrant proteins to the proteasome or lysosome for degradation (Whitesell and Lindquist, 2005
). In some cases when proteins can be rescued, the same system can refold damaged proteins (Broadley and Hartl, 2009
). The Hsp and co-chaperone system thus has a crucial function as a cleanser under these circumstances. However, when mild oxidative stress occurs, cells may modify their signalling and gene expression profiles to adjust their behaviour. Diverse post-translational modifications on proteins may be involved in these processes, during which ROS lead to oxidative modification, rather than typical oxidative damage, of proteins (Kumsta and Jakob, 2009
). Whether and how the Hsp and co-chaperone system participate in these adaptive responses remain largely unknown. The present study provides an example of the role of Hsp90 and its co-chaperone CHIP in the stabilization of a normal protein under mild oxidative stress induced by low doses of H2
We found that the binding of Hsp90 to SENP3, a SUMO2/3-specific protease, leads to SENP3 stabilization under mild oxidative stress and that this is achieved via blockage of the CHIP-mediated ubiquitination that otherwise constantly occurs under non-stressed conditions. We previously demonstrated that following nuclear accumulation of SENP3, it re-localizes from the nucleoli to all over the nucleoplasm, which provides this SUMO protease with a brand-new spectrum of substrates. The important transcription co-activator p300 and the nuclear body component PML, among others, become substrates of SENP3 under mild oxidative stress. De-conjugation of SUMO2/3 from p300 and PML leads to changes in their functions; the transcriptional activity of hypoxia-inducible factor-1 is enhanced, and the role of negative regulator of PML for cell proliferation is impaired (Huang et al, 2009
; Han et al, 2010
), which all contribute to cellular adaptation to mild oxidative stress. Therefore, Hsp90 and CHIP cooperate as critical modulators that ensure the function of a protein in this process.
Hsp90 is an abundant and essential protein in eukaryotic cells as well as in bacteria. As one of the most important chaperones, Hsp90 stabilizes, refolds, and activates its client proteins (Young et al, 2001
). Hsp90 stabilizes mutant oncogenic proteins that are prone to misfolding, thereby enabling malignant transformation (Cowen, 2009
). Compromising Hsp90 function can reverse oncogenic cellular phenotypes (Neckers, 2007
). Hsp90 also stabilizes unmutated regulators of signalling in fungal cells, conferring drug resistance (Cowen, 2009
). In neuronal cells, phosphorylated tau is a client of the Hsp90 chaperone network. In addition, abnormal tau (hyperphosphorylated) displays enhanced binding to Hsp90 (Dickey et al, 2007
). Although an increasing body of research has revealed many in vivo
Hsp90 client proteins and proven that the association of client proteins with Hsp90 complexes is important for their activity, the interaction of Hsp90 with client proteins is currently still poorly understood. Among the most intriguing puzzles are the specific mechanism of recognition of non-native substrates by Hsp90 and the coordination of Hsp90 with the CHIP degradation machinery (Richter and Buchner, 2001
Being in a complex together with the client, CHIP and Hsp90 can have cooperative or antagonistic effects on the client (Qian et al, 2006
; Xia et al, 2007
). For instance, dephosphorylation and refolding of p-tau is initially facilitated by an Hsp90-containing complex that prevents degradation; however, when refolding is subverted by Hsp90 inhibition, p-tau is transferred to the Hsp70/CHIP complex and degraded via polyubiquitination (Dickey et al, 2007
). Our present study revealed that CHIP mediates constitutive ubiquitination and degradation of SENP3 under non-stress condition, whereas by contrast, Hsp90 binds to SENP3 under mild oxidative stress and mediates its stabilization. This is the first demonstration to our knowledge that CHIP and Hsp90 are responsible, respectively, for controlling the levels of a protein under non-stressed and stressed conditions. Moreover, our data provide new insight into the mutual relationship of these two molecules. CHIP is believed to mediate degradation of the clients in a chaperone-dependent manner, until recently it is noticed that CHIP can be independent of Hsp90 or other chaperones (Parsons et al, 2008
; Shang et al, 2009
) Our findings here highlight this novel concept by confirming that Hsp90 is dispensable for CHIP-mediated SENP3 degradation under non-stressed condition. On the contrary, CHIP is known to be indispensable for the chaperones under various circumstances (McDonough and Patterson, 2003
; Rosser et al, 2007
), although the precise contributions of CHIP as a co-chaperone to Hsp90 remain unclear. Our study demonstrates that Hsp90 abrogates the ubiquitin ligase function of CHIP, but Hsp90's action requires the presence of CHIP and the CHIP–Hsp90 interaction. This previously unperceived contribution suggests an alternative co-chaperone function of CHIP: a molecule supporting the complex for the Hsp90-mediated client stabilization.
Our present data show that, upon oxidative stress, Hsp90 binds to a region of SENP3 comprising the redox-sensing domain and the ubiquitin-modified domain. More interestingly, a prerequisite for this Hsp90–SENP3 association is that Hsp90 recognizes specific oxidative modifications on cysteines 243 and/or 274 in the redox-sensing domain of SENP3. This study is the first demonstration that oxidation of a protein diverts its fate from degradation to stabilization. A study of structure biology in the future may provide better elucidation to whether the site for Hsp90 binding is spatially close to the site for CHIP binding, and how the Hsp90–SENP3 binding affects the function of CHIP but simultaneously requires the presence of CHIP.
Reversibility, a crucial aspect of all regulatory mechanisms, is one of the most important characteristics of thiol oxidative modification (Kumsta and Jakob, 2009
). We showed that stabilization of SENP3 is reversed by an anti-oxidant thiol-reducing agent as well as by an Hsp90 inhibitor. This result implies that Hsp90 binding to SENP3 and the consequent SENP3 stabilization are subject to physiological modulation along with environmental ROS fluctuations.
Redox regulation of chaperones has been illustrated in two molecules, prokaryotic Hsp33 and eukaryotic 2-Cys peroxiredoxin, which use ROS to activate their chaperone function (Jang et al, 2004
; Winter et al, 2008
; Kumsta and Jakob, 2009
). However, the client selectivity of the chaperones under these circumstances has not been explained. By contrast, our report unveils an intrinsic signal formed by oxidative modification of the client protein instead of the chaperone. This unique mechanism may better explain the high specificity of SENP3 stabilization, as we have determined that only this SUMO protease in the SENP family is immediately stabilized by the low concentrations of H2
we used (Han et al, 2010
The role of Hsp90 in cancer has received much attention in the past decade, because it is upregulated in various primary cancers and functions as an integral part of the machinery that allows cancer cells to escape normal regulation (Pick et al, 2007
). Recently, CHIP was found to suppress tumour progression in human breast cancer by inhibiting oncogenic pathways, and CHIP levels are negatively correlated with the malignancy of human breast tumour tissues (Kajiro et al, 2009
). Our previous and current data demonstrate an accumulation of SENP3 in a series of primary cancers. The present data have further revealed that binding between Hsp90 and SENP3 is enhanced in cancerous hepatocytes in which ubiquitination of SENP3 is attenuated. Therefore, this study, in line with the ideas that Hsp90 is oncogenic whereas CHIP is a tumour suppressor, provides a novel redox-sensitive client protein that is aberrantly regulated by the CHIP–Hsp90 machinery in cancer.