Threonine-408 Regulates the Stability of Human Pregnane X Receptor through Its Phosphorylation and the CHIP/Chaperone-Autophagy Pathway

Sugatani, Junko; Noguchi, Yuji; Hattori, Yoshiki; Yamaguchi, Masahiko; Yamazaki, Yoji; Ikari, Akira

doi:10.1124/dmd.115.066308

Cited by 17 publications

(14 citation statements)

References 59 publications

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“…Client proteins of CHIP can be targeted either to lysosomes or to proteasomes for degradation [50, 54]. While short-chain ubiquitylation increases AQP2 endocytosis and lysosomal degradation [22], polyubiquitylation targets AQP2 to proteasomes, in fact MG132 treatment increased the stability of AQP2 by impairing its proteasomal degradation [23].…”

Section: Discussionmentioning

confidence: 99%

AQP2 Abundance is Regulated by the E3-Ligase CHIP Via HSP70

Centrone

Ranieri

Mise

et al. 2017

Cell Physiol Biochem

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Background/Aims: AQP2 expression is mainly controlled by vasopressin-dependent changes in protein abundance which is in turn regulated by AQP2 ubiquitylation and degradation, however the proteins involved in these processes are largely unknown. Here, we investigated the potential role of the CHIP E3 ligase in AQP2 regulation. Methods: MCD4 cells and kidney slices were used to study the involvement of the E3 ligase CHIP on AQP2 protein abundance by cell homogenization and immunoprecipitation followed by immunoblotting. Results: We found that AQP2 complexes with CHIP in renal tissue. Expression of CHIP increased proteasomal degradation of AQP2 and HSP70 abundance, a molecular signature of HSP90 inhibition. Increased HSP70 level, secondary to CHIP expression, promoted ERK signaling resulting in increased AQP2 phosphorylation at S261. Phosphorylation of AQP2 at S256 and T269 were instead downregulated. Next, we investigated HSP70 interaction with AQP2, which is important for endocytosis. Compared with AQP2-wt, HSP70 binding decreased in AQP2-S256D and AQP2-S256D-S261D, while increased in AQP2-S256D-S261A. Surprisingly, expression of CHIP-delUbox, displaying a loss of E3 ligase activity, still induced AQP2 degradation, indicating that CHIP does not ubiquitylate and degrade AQP2 itself. Conversely, the AQP2 half-life was increased upon the expression of CHIP-delTPR a domain which binds Hsc70/HSP70 and HSP90. HSP70 has been reported to bind other E3 ligases such as MDM2. Notably, we found that co-expression of CHIP and MDM2 increased AQP2 degradation, whereas co-expression of CHIP with MDM2-delRING, an inactive form of MDM2, impaired AQP2 degradation. Conclusion: Our findings indicate CHIP as a master regulator of AQP2 degradation via HSP70 that has dual functions: (1) as chaperone for AQP2 and (2) as an anchoring protein for MDM2 E3 ligase, which is likely to be involved in AQP2 degradation.

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Section: Discussionmentioning

confidence: 99%

AQP2 Abundance is Regulated by the E3-Ligase CHIP Via HSP70

Centrone

Ranieri

Mise

et al. 2017

Cell Physiol Biochem

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“…Our recent efforts combined with that of others indicate that phosphorylation, ubiquitylation, and SUMOylation of PXR play a pivotal and likely interactive role in regulating the biological function of this nuclear receptor protein [11-18]. However, where the PTMs occur on the protein and how they might interact with each other in live cells to regulate its complex biology in liver and intestine is only beginning to be understood.…”

Section: Introductionmentioning

confidence: 99%

A SUMO-acetyl switch in PXR biology

Cui

Sun

Zhang

et al. 2016

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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Post-translational modification (PTM) of nuclear receptor superfamily members regulates various aspects of their biology to include sub-cellular localization, the repertoire of protein-binding partners, as well as their stability and mode of degradation. The nuclear receptor pregnane X receptor (PXR, NR1I2) is a master-regulator of the drug-inducible gene expression in liver and intestine. The PXR-mediated gene activation program is primarily recognized to increase drug metabolism, drug transport, and drug efflux pathways in these tissues. The activation of PXR also has important implications in significant human diseases including inflammatory bowel disease and cancer. Our recent investigations reveal that PXR is modified by multiple PTMs to include phosphorylation, SUMOylation, and ubiquitination. Using both primary cultures of hepatocytes and cell-based assays, we show here that PXR is modified through acetylation on lysine residues. Further, we show that increased acetylation of PXR stimulates its increased SUMO-modification to support active transcriptional suppression. Pharmacologic inhibition of lysine de-acetylation using trichostatin A (TSA) alters the sub-cellular localization of PXR in cultured hepatocytes, and also has a profound impact upon PXR transactivation capacity. Both the acetylation and SUMOylation status of PXR is affected by its ability to associate with the lysine de-acetylating enzyme histone de-acetylase (HDAC)3 in a complex with silencing mediator of retinoic acid and thyroid hormone receptor (SMRT). Taken together, our data support a model in which a SUMO-acetyl ‘switch’ occurs such that acetylation of PXR likely stimulates SUMO-modification of PXR to promote the active repression of PXR-target gene expression.

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“…These pathways in the heart are activated by several cardio-protective compounds [ 42 ] including fenofibrate [ 43 , 44 , 45 ]. Moreover, CHIP is known to play an important role in autophagy [ 12 , 46 , 47 , 48 , 49 , 50 ]. To explore the mechanism behind the phenotype observed in CHIP −/− mice, we used qPCR analysis to determine if fenofibrate had differential effects on metabolic or autophagy/mitophagy gene expression in hearts isolated from CHIP −/− versus wild-type mice.…”

Section: Resultsmentioning

confidence: 99%

Adverse Effects of Fenofibrate in Mice Deficient in the Protein Quality Control Regulator, CHIP

Ravi

Parry

Willis

et al. 2018

JCDD

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We previously reported how the loss of CHIP expression (Carboxyl terminus of Hsc70-Interacting Protein) during pressure overload resulted in robust cardiac dysfunction, which was accompanied by a failure to maintain ATP levels in the face of increased energy demand. In this study, we analyzed the cardiac metabolome after seven days of pressure overload and found an increase in long-chain and medium-chain fatty acid metabolites in wild-type hearts. This response was attenuated in mice that lack expression of CHIP (CHIP−/−). These findings suggest that CHIP may play an essential role in regulating oxidative metabolism pathways that are regulated, in part, by the nuclear receptor PPARα (Peroxisome Proliferator-Activated Receptor alpha). Next, we challenged CHIP−/− mice with the PPARα agonist called fenofibrate. We found that treating CHIP−/− mice with fenofibrate for five weeks under non-pressure overload conditions resulted in decreased skeletal muscle mass, compared to wild-type mice, and a marked increase in cardiac fibrosis accompanied by a decrease in cardiac function. Fenofibrate resulted in decreased mitochondrial cristae density in CHIP−/− hearts as well as decreased expression of genes involved in the initiation of autophagy and mitophagy, which suggests that a metabolic challenge, in the absence of CHIP expression, impacts pathways that contribute to mitochondrial quality control. In conclusion, in the absence of functional CHIP expression, fenofibrate results in unexpected skeletal muscle and cardiac pathologies. These findings are particularly relevant to patients harboring loss-of-function mutations in CHIP and are consistent with a prominent role for CHIP in regulating cardiac metabolism.

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Threonine-408 Regulates the Stability of Human Pregnane X Receptor through Its Phosphorylation and the CHIP/Chaperone-Autophagy Pathway

Cited by 17 publications

References 59 publications

AQP2 Abundance is Regulated by the E3-Ligase CHIP Via HSP70

AQP2 Abundance is Regulated by the E3-Ligase CHIP Via HSP70

A SUMO-acetyl switch in PXR biology

Adverse Effects of Fenofibrate in Mice Deficient in the Protein Quality Control Regulator, CHIP

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