Hui Cang

SENP3 is responsible for HIF-1 transactivation under mild oxidative stress via p300 de-SUMOylation

The physiological function of Sentrin/SUMO‐specific proteases (SENPs) remains largely unexplored, and little is known about the regulation of SENPs themselves. Here, we show that a modest increase of reactive oxygen species (ROS) regulates SENP3 stability and localization. We found that SENP3 is continuously degraded through the ubiquitin‐proteasome pathway under basal condition and that ROS inhibit this degradation. Furthermore, ROS causes SENP3 to redistribute from the nucleoli to the nucleoplasm, allowing it to regulate nuclear events. The stabilization and redistribution of SENP3 correlate with an increase in the transcriptional activity of the hypoxia‐inducing factor‐1 (HIF‐1) under mild oxidative stress. ROS‐enhanced HIF‐1 transactivation is blocked by SENP3 knockdown. The de‐SUMOylating activity of SENP3 is required for ROS‐induced increase of HIF‐1 transactivation, but the true substrate of SENP3 is the co‐activator of HIF‐1α, p300, rather than HIF‐1α itself. Removing SUMO2/3 from p300 enhances its binding to HIF‐1α. In vivo nude mouse xenografts overexpressing SENP3 are more angiogenic. Taken together, our results identify SENP3 as a redox sensor that regulates HIF‐1 transcriptional activity under oxidative stress through the de‐SUMOylation of p300.

Redox regulation of the stability of the SUMO protease SENP3 via interactions with CHIP and Hsp90

The molecular chaperone heat shock protein 90 (Hsp90) and the co‐chaperone/ubiquitin ligase carboxyl terminus of Hsc70‐interacting protein (CHIP) control the turnover of client proteins. How this system decides to stabilize or degrade the client proteins under particular physiological or pathological conditions is unclear. We report here a novel client protein, the SUMO2/3 protease SENP3, that is sophisticatedly regulated by CHIP and Hsp90. SENP3 is maintained at a low basal level under non‐stress condition due to Hsp90‐independent CHIP‐mediated ubiquitination. Upon mild oxidative stress, SENP3 undergoes thiol modification, which recruits Hsp90. Hsp90/SENP3 association protects SENP3 from CHIP‐mediated ubiquitination and subsequent degradation, but this effect of Hsp90 requires the presence of CHIP. Our data demonstrate for the first time that CHIP and Hsp90 interplay with a client alternately under non‐stress and stress conditions, and the choice between stabilization and degradation is made by the redox state of the client. In addition, enhanced SENP3/Hsp90 association is found in cancer. These findings provide new mechanistic insight into how cells regulate the SUMO protease in response to oxidative stress.

Oxidative modification of caspase-9 facilitates its activation via disulfide-mediated interaction with Apaf-1

Intracellular reactive oxygen species (ROS) are known to regulate apoptosis. Activation of caspase-9, the initial caspase in the mitochondrial apoptotic cascade, is closely associated with ROS, but it is unclear whether ROS regulate caspase-9 via direct oxidative modification. The present study aims to elucidate the molecular mechanisms by which ROS mediate caspase-9 activation. Our results show that the cellular oxidative state facilitates caspase-9 activation. Hydrogen peroxide treatment causes the activation of caspase-9 and apoptosis, and promotes an interaction between caspase-9 and apoptotic protease-activating factor 1 (Apaf-1) via disulfide formation. In addition, in an in vitro mitochondria-free system, the thiol-oxidant diamide promotes auto-cleavage of caspase-9 and the caspase-9/Apaf-1 interaction by facilitating the formation of disulfide-linked complexes. Finally, a point mutation at C403 of caspase-9 impairs both H2O2-promoted caspase-9 activation and interaction with Apaf-1 through the abolition of disulfide formation. The association between cytochrome c and the C403S mutant is significantly weaker than that between cytochrome c and wild-type caspase-9, indicating that oxidative modification of caspase-9 contributes to apoptosome formation under oxidative stress. Taken together, oxidative modification of caspase-9 by ROS can mediate its interaction with Apaf-1, and can thus promote its auto-cleavage and activation. This mechanism may facilitate apoptosome formation and caspase-9 activation under oxidative stress.

SENP3-mediated De-conjugation of SUMO2/3 from Promyelocytic Leukemia Is Correlated with Accelerated Cell Proliferation under Mild Oxidative Stress

Small ubiquitin-like modifier (SUMO) 2/3 is known to conjugate to substrates in response to a variety of cellular stresses. However, whether and how SUMO2/3-specific proteases are involved in de-conjugation under cell stress is unclear. Here, we show that low doses of hydrogen peroxide (H2O2) induce an increase of the SENP3 protein, which removes SUMO2/3 from promyelocytic leukemia (PML). Low dose H2O2 causes SENP3 to co-localize with PML bodies and reduces the number of PML bodies in a SENP3-dependent manner. Furthermore, de-conjugation of SUMO2/3 from PML is responsible for the accelerated cell proliferation caused by low dose H2O2. Knocking down PML promotes basal cell proliferation as expected. This can be reversed by reconstitution with wild-type PML but not its mutant lacking SUMOylation, indicating that only the SUMOylated PML can play an inhibitory role for cell proliferation. Thus, SENP3 appears to be a key mediator in mild oxidative stress-induced cell proliferation via regulation of the SUMOylation status of PML. Furthermore, SENP3 is over-accumulated in a variety of primary human cancers including colon adenocarcinoma in which PML is hypo-SUMOylated. These results reveal an important role of SENP3 and the SUMOylation status of PML in the regulation of cell proliferation under oxidative stress.

SUMO2 and SUMO3 transcription is differentially regulated by oxidative stress in an Sp1-dependent manner

Protein SUMOylation (SUMO is small ubiquitin-related modifier) is a dynamic process that is strictly regulated under physiological and pathological conditions. However, little is known about how various intra- or extra-cellular stimuli regulate expression levels of components in the SUMO system. SUMO isoforms SUMO2 and SUMO3 can rapidly convert to be conjugated in response to a variety of cellular stresses. Owing to the limitations of sequence homology, SUMO2 and SUMO3 cannot be differentiated between and are thus referred to as SUMO2/3. Whether these two isoforms are regulated in distinct manners has never been addressed. In the present paper we report that the expression of SUMO3, but not SUMO2, can be down-regulated at the transcription level by cellular oxidative stress. In the present study, we checked SUMO2 and SUMO3 mRNA levels in cells exposed to various doses of H2O2 and in cells bearing different levels of ROS (reactive oxygen species). We found an inverse relationship between SUMO3 transcription and ROS levels. We characterized a promoter region specific for the mouse Sumo3 gene that is bound by the redox-sensitive transcription factor Sp1 (specificity protein 1) and demonstrated oxidation of Sp1, as well as suppression of Sp1-DNA binding upon oxidative stress. This revealed for the first time that the expression of SUMO2 and SUMO3 is regulated differently by ROS. These findings may enhance our understanding about the regulation of SUMOylation and also shed light on the functions of Sp1.

The endogenous reactive oxygen species promote NF-κB activation by targeting on activation of NF-κB-inducing kinase in oral squamous carcinoma cells

Reactive oxygen species (ROS) could stimulate or inhibit NF-κB pathways. However, most results have been obtained on the basis of the exogenous ROS and the molecular target of ROS in NF-κB signalling pathways has remained unclear. Here, the oral squamous carcinoma (OSC) cells, with a mild difference in the endogenous ROS level, were used to investigate how slight fluctuation of the endogenous ROS regulates NF-κB activation. This study demonstrates that NF-κB-inducing kinase (NIK) is a critical target of the endogenous ROS in NF-κB pathways. The results indicate that ROS may function as a physiological signalling modulator on NF-κB signalling cascades through its ability to facilitate the activity of NIK and subsequent NF-κB transactivation. In addition, the data are useful to explain why the altered intracellular microenvironment related to redox state may influence biological behaviours of cancer cells.

The biphasic redox sensing of SENP3 accounts for the HIF-1 transcriptional activity shift by oxidative stress

Aim:To investigate the mechanisms underlying the biphasic redox regulation of hypoxia-inducible factor-1 (HIF-1) transcriptional activity under different levels of oxidative stress caused by reactive oxidative species (ROS).Methods:HeLa cells were exposed to different concentrations of H2O2 as a simple model for mild and severe oxidative stress. Luciferase reporter assay and/or quantitative real-time PCR were used to investigate the transcriptional activity. Immunoblot was used to detect protein expression. Chromatin immunoprecipitation assay was used to detect HIF-1/DNA binding. The interaction of p300 with HIF-1α or with SENP3, and the SUMO2/3 conjugation states of p300 were examined by coimmunoprecipitation.Results:HIF-1 transcriptional activity in HeLa cells was enhanced by low doses (0.05–0.5 mmol/L) of H2O2, but suppressed by high doses (0.75–8.0 mmol/L) of H2O2. The amount of co-activator p300 bound to HIF-1α in HeLa cells was increased under mild oxidative stress, but decreased under severe oxidative stress. The ROS levels differentially modified cysteines 243 and 532 in the cysteine protease SENP3, regulating the interaction of SENP3 with p300 to cause different SUMOylation of p300, thus shifting HIF-1 transcriptional activity.Conclusion:The shift of HIF-1 transactivation by ROS is correlated with and dependent on the biphasic redox sensing of SENP3 that leads to the differential SENP3/p300 interaction and the consequent fluctuation in the p300 SUMOylation status.