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Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates
Published on: February 27, 2016
SIRT5 desuccinylates PRDX3 to promote its chaperone-mediated autophagy and mitigate mitochondrial dysfunction in MSU
Yaxin Deng1, Kailin Yu2, Lijun Ou2
1Institute of Rheumatology and Immunology, the Affiliated Hospital of North Sichuan Medical College, 1(#) South Maoyuan Road and Institute of Basic Medicine and Forensic Medicine, North Sichuan Medical College, 234(#) Fujiang Road, Nanchong, 637001, Sichuan province, China; North Sichuan Medical College Innovation Centre for Science and Technology, North Sichuan Medical College, 234# Fujiang Road, Nanchong 637001, China; Medical Imaging Key Laboratory of Sichuan Province, the Affiliated Hospital of North Sichuan Medical College, 1# South Maoyuan Road, Nanchong 637001, Sichuan, China.
Background:
Gout is triggered by monosodium urate (MSU) crystals and is closely linked to mitochondrial dysfunction and oxidative stress. However, the role of the desuccinylase SIRT5 in this process remains unclear. Here, we identify SIRT5 as a key negative regulator of MSU crystal-induced inflammation.
Methods:
Macrophages were challenged with MSU crystals to assess mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), and IL-1β secretion. The interaction between SIRT5 and PRDX3, as well as PRDX3 succinylation, was examined by co-immunoprecipitation (co-IP) and anti-succinyllysine immunoblotting. Co-IP coupled with mass spectrometry (MS) identified HSC70 as a PRDX3-interacting protein, and the interactions of PRDX3 with HSC70 and LAMP2A were validated by co-IP to assess chaperone-mediated autophagy (CMA). In vivo, inflammatory responses in MSU-induced peritonitis and arthritis models were evaluated in SIRT5-deficient mice.
Results:
SIRT5 preserved mitochondrial homeostasis and restrained NLRP3 inflammasome activation in macrophages in response to MSU crystals. Mechanistically, MSU crystal exposure reduced the SIRT5-PRDX3 interaction, increasing PRDX3 succinylation. This modification stabilized PRDX3 and impaired its chaperone-mediated autophagy (CMA)-dependent degradation, leading to PRDX3 accumulation and subsequent mitochondrial dysfunction. In vivo, SIRT5 deficiency exacerbated MSU crystal-induced inflammation and upregulated PRDX3 protein levels.
Conclusions:
Our findings reveal that SIRT5 desuccinylates PRDX3 to promote its CMA-dependent degradation, thereby preventing PRDX3 accumulation and mitochondrial dysfunction in MSU crystal-induced gouty inflammation.
Insights
SIRT5 prevents gout inflammation by regulating PRDX3 degradation. Loss of SIRT5 increases PRDX3, causing mitochondrial dysfunction and heightened inflammatory responses to MSU crystals.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Gout is an inflammatory condition caused by monosodium urate (MSU) crystals.
- Mitochondrial dysfunction and oxidative stress are linked to gout pathogenesis.
- The role of SIRT5 in MSU crystal-induced inflammation was previously unknown.
Purpose of the Study:
- To investigate the role of SIRT5 in MSU crystal-induced inflammation.
- To elucidate the molecular mechanisms by which SIRT5 regulates gouty inflammation.
Main Methods:
- Macrophages were treated with MSU crystals to analyze mitochondrial function and IL-1β secretion.
- Co-immunoprecipitation and mass spectrometry were used to identify protein interactions and modifications.
- Chaperone-mediated autophagy (CMA) was assessed.
- In vivo studies utilized SIRT5-deficient mice in models of gout.
Main Results:
- SIRT5 protects mitochondrial homeostasis and suppresses NLRP3 inflammasome activation in response to MSU crystals.
- MSU crystals decrease SIRT5-PRDX3 interaction, leading to PRDX3 succinylation and impaired CMA-dependent degradation.
- SIRT5 deficiency exacerbates MSU crystal-induced inflammation and increases PRDX3 levels in vivo.
Conclusions:
- SIRT5 desuccinylates PRDX3, promoting its degradation via CMA.
- This action prevents PRDX3 accumulation and subsequent mitochondrial dysfunction in gouty inflammation.
- SIRT5 is identified as a key negative regulator of MSU crystal-induced inflammation.
