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CHFR negatively regulates SIRT1 activity upon oxidative stress

View Article: PubMed Central - PubMed

ABSTRACT

SIRT1, the NAD+-dependent protein deacetylase, controls cell-cycle progression and apoptosis by suppressing p53 tumour suppressor. Although SIRT1 is known to be phosphorylated by JNK1 upon oxidative stress and subsequently down-regulated, it still remains elusive how SIRT1 stability and activity are controlled. Here, we have unveiled that CHFR functions as an E3 Ub-ligase of SIRT1, responsible for its proteasomal degradation under oxidative stress conditions. CHFR interacts with and destabilizes SIRT1 by ubiquitylation and subsequent proteolysis. Such CHFR-mediated SIRT1 inhibition leads to the increase of p53 acetylation and its target gene transcription. Notably, CHFR facilitates SIRT1 destabilization when SIRT1 is phosphorylated by JNK1 upon oxidative stress, followed by prominent apoptotic cell death. Meanwhile, JNK inhibitor prevents SIRT1 phosphorylation, leading to elevated SIRT1 protein levels even in the presence of H2O2. Taken together, our results indicate that CHFR plays a crucial role in the cellular stress response pathway by controlling the stability and function of SIRT1.

No MeSH data available.


CHFR negatively regulates SIRT1.(A) CHFR overexpression results in the decrease of endogenous SIRT1. HeLa cells were transfected with FLAG-CHFR (0, 0.5, or 2 μg) and treated with 2 μM MG132 for 12 h as indicated. (B) Both an E3 Ub-ligase activity and a binding capacity to SIRT1 are necessary for CHFR to down-regulate SIRT1. FLAG-CHFR DNA plasmids encoding WT, I306A (Ile306Ala), or ΔCR mutants were transfected into HeLa cells as indicated. Cell lysates were immunoblotted with anti-FLAG and anti-SIRT1 antibodies. (C) CHFR ubiquitylates SIRT1 in vivo. HEK293T cells were transiently transfected with indicated plasmids and treated with 2 μM MG132 for 12 h. Cell lysates were immunoprecipitated with anti-FLAG M2 resin and probed with anti-HA antibody. (D) SIRT1 is ubiquitylated by CHFR in vitro. Purified SIRT1 is incubated with either CHFR WT or I306A mutant in the presence of E1, UbcH5b, ATP, ubiquitin as indicated. After in vitro ubiquitylation reaction, samples were analysed by immunoblotting with anti-SIRT1 antibody.
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f2: CHFR negatively regulates SIRT1.(A) CHFR overexpression results in the decrease of endogenous SIRT1. HeLa cells were transfected with FLAG-CHFR (0, 0.5, or 2 μg) and treated with 2 μM MG132 for 12 h as indicated. (B) Both an E3 Ub-ligase activity and a binding capacity to SIRT1 are necessary for CHFR to down-regulate SIRT1. FLAG-CHFR DNA plasmids encoding WT, I306A (Ile306Ala), or ΔCR mutants were transfected into HeLa cells as indicated. Cell lysates were immunoblotted with anti-FLAG and anti-SIRT1 antibodies. (C) CHFR ubiquitylates SIRT1 in vivo. HEK293T cells were transiently transfected with indicated plasmids and treated with 2 μM MG132 for 12 h. Cell lysates were immunoprecipitated with anti-FLAG M2 resin and probed with anti-HA antibody. (D) SIRT1 is ubiquitylated by CHFR in vitro. Purified SIRT1 is incubated with either CHFR WT or I306A mutant in the presence of E1, UbcH5b, ATP, ubiquitin as indicated. After in vitro ubiquitylation reaction, samples were analysed by immunoblotting with anti-SIRT1 antibody.

Mentions: Given that SIRT1 is destabilized in the presence of CHFR as shown in Fig. 1A and they bind to each other, it is plausible that CHFR could act as a specific E3 Ub-ligase of SIRT1 to modulate its protein levels. In order to test this possibility, SIRT1 was transfected into HeLa cells, where CHFR is normally not expressed4, together with either mock- or CHFR-expression vector. SIRT1 protein levels were decreased in a CHFR dose-dependent manner and this reduction was blocked by the treatment of MG132 (Fig. 2A), indicating that CHFR leads to the proteasomal degradation of SIRT1. On the contrary, either E3 Ub-ligase-defective CHFR-I306A4 or SIRT1-binding-defective CHFR-ΔCR mutant failed to decrease SIRT1 protein levels compared to CHFR WT (Fig. 2B), suggesting that both an E3 Ub-ligase activity and a substrate binding ability of CHFR are necessary for SIRT1 destabilization.


CHFR negatively regulates SIRT1 activity upon oxidative stress
CHFR negatively regulates SIRT1.(A) CHFR overexpression results in the decrease of endogenous SIRT1. HeLa cells were transfected with FLAG-CHFR (0, 0.5, or 2 μg) and treated with 2 μM MG132 for 12 h as indicated. (B) Both an E3 Ub-ligase activity and a binding capacity to SIRT1 are necessary for CHFR to down-regulate SIRT1. FLAG-CHFR DNA plasmids encoding WT, I306A (Ile306Ala), or ΔCR mutants were transfected into HeLa cells as indicated. Cell lysates were immunoblotted with anti-FLAG and anti-SIRT1 antibodies. (C) CHFR ubiquitylates SIRT1 in vivo. HEK293T cells were transiently transfected with indicated plasmids and treated with 2 μM MG132 for 12 h. Cell lysates were immunoprecipitated with anti-FLAG M2 resin and probed with anti-HA antibody. (D) SIRT1 is ubiquitylated by CHFR in vitro. Purified SIRT1 is incubated with either CHFR WT or I306A mutant in the presence of E1, UbcH5b, ATP, ubiquitin as indicated. After in vitro ubiquitylation reaction, samples were analysed by immunoblotting with anti-SIRT1 antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: CHFR negatively regulates SIRT1.(A) CHFR overexpression results in the decrease of endogenous SIRT1. HeLa cells were transfected with FLAG-CHFR (0, 0.5, or 2 μg) and treated with 2 μM MG132 for 12 h as indicated. (B) Both an E3 Ub-ligase activity and a binding capacity to SIRT1 are necessary for CHFR to down-regulate SIRT1. FLAG-CHFR DNA plasmids encoding WT, I306A (Ile306Ala), or ΔCR mutants were transfected into HeLa cells as indicated. Cell lysates were immunoblotted with anti-FLAG and anti-SIRT1 antibodies. (C) CHFR ubiquitylates SIRT1 in vivo. HEK293T cells were transiently transfected with indicated plasmids and treated with 2 μM MG132 for 12 h. Cell lysates were immunoprecipitated with anti-FLAG M2 resin and probed with anti-HA antibody. (D) SIRT1 is ubiquitylated by CHFR in vitro. Purified SIRT1 is incubated with either CHFR WT or I306A mutant in the presence of E1, UbcH5b, ATP, ubiquitin as indicated. After in vitro ubiquitylation reaction, samples were analysed by immunoblotting with anti-SIRT1 antibody.
Mentions: Given that SIRT1 is destabilized in the presence of CHFR as shown in Fig. 1A and they bind to each other, it is plausible that CHFR could act as a specific E3 Ub-ligase of SIRT1 to modulate its protein levels. In order to test this possibility, SIRT1 was transfected into HeLa cells, where CHFR is normally not expressed4, together with either mock- or CHFR-expression vector. SIRT1 protein levels were decreased in a CHFR dose-dependent manner and this reduction was blocked by the treatment of MG132 (Fig. 2A), indicating that CHFR leads to the proteasomal degradation of SIRT1. On the contrary, either E3 Ub-ligase-defective CHFR-I306A4 or SIRT1-binding-defective CHFR-ΔCR mutant failed to decrease SIRT1 protein levels compared to CHFR WT (Fig. 2B), suggesting that both an E3 Ub-ligase activity and a substrate binding ability of CHFR are necessary for SIRT1 destabilization.

View Article: PubMed Central - PubMed

ABSTRACT

SIRT1, the NAD+-dependent protein deacetylase, controls cell-cycle progression and apoptosis by suppressing p53 tumour suppressor. Although SIRT1 is known to be phosphorylated by JNK1 upon oxidative stress and subsequently down-regulated, it still remains elusive how SIRT1 stability and activity are controlled. Here, we have unveiled that CHFR functions as an E3 Ub-ligase of SIRT1, responsible for its proteasomal degradation under oxidative stress conditions. CHFR interacts with and destabilizes SIRT1 by ubiquitylation and subsequent proteolysis. Such CHFR-mediated SIRT1 inhibition leads to the increase of p53 acetylation and its target gene transcription. Notably, CHFR facilitates SIRT1 destabilization when SIRT1 is phosphorylated by JNK1 upon oxidative stress, followed by prominent apoptotic cell death. Meanwhile, JNK inhibitor prevents SIRT1 phosphorylation, leading to elevated SIRT1 protein levels even in the presence of H2O2. Taken together, our results indicate that CHFR plays a crucial role in the cellular stress response pathway by controlling the stability and function of SIRT1.

No MeSH data available.