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Updated: May 5, 2026
Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates
Published on: February 27, 2016
The NAD-dependent deacetylase SIRT2 is required for programmed necrosis
Nisha Narayan1, In Hye Lee, Ronen Borenstein
1Center for Molecular Medicine, National Heart, Lung and Blood Institute, NIH, Bethesda, Maryland 20892, USA.
Abstract:
Although initially viewed as unregulated, increasing evidence suggests that cellular necrosis often proceeds through a specific molecular program. In particular, death ligands such as tumour necrosis factor (TNF)-α activate necrosis by stimulating the formation of a complex containing receptor-interacting protein 1 (RIP1) and receptor-interacting protein 3 (RIP3). Relatively little is known regarding how this complex formation is regulated. Here, we show that the NAD-dependent deacetylase SIRT2 binds constitutively to RIP3 and that deletion or knockdown of SIRT2 prevents formation of the RIP1-RIP3 complex in mice. Furthermore, genetic or pharmacological inhibition of SIRT2 blocks cellular necrosis induced by TNF-α. We further demonstrate that RIP1 is a critical target of SIRT2-dependent deacetylation. Using gain- and loss-of-function mutants, we demonstrate that acetylation of RIP1 lysine 530 modulates RIP1-RIP3 complex formation and TNF-α-stimulated necrosis. In the setting of ischaemia-reperfusion injury, RIP1 is deacetylated in a SIRT2-dependent fashion. Furthermore, the hearts of Sirt2(-/-) mice, or wild-type mice treated with a specific pharmacological inhibitor of SIRT2, show marked protection from ischaemic injury. Taken together, these results implicate SIRT2 as an important regulator of programmed necrosis and indicate that inhibitors of this deacetylase may constitute a novel approach to protect against necrotic injuries, including ischaemic stroke and myocardial infarction.
Insights
SIRT2 regulates programmed necrosis by deacetylating RIP1, preventing RIP1-RIP3 complex formation. Inhibiting SIRT2 protects against injuries like ischaemia-reperfusion, suggesting therapeutic potential for necrosis.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- Cellular necrosis, once considered unregulated, is now understood to follow specific molecular pathways.
- Tumour necrosis factor-alpha (TNF-α) triggers necrosis via the formation of a receptor-interacting protein 1 (RIP1) and receptor-interacting protein 3 (RIP3) complex.
- The regulation of this RIP1-RIP3 complex formation remains largely unknown.
Purpose of the Study:
- To investigate the role of SIRT2 in the regulation of programmed necrosis.
- To determine if SIRT2 modulates the formation of the RIP1-RIP3 complex.
- To evaluate the therapeutic potential of SIRT2 inhibition in conditions involving necrosis.
Main Methods:
- Constitutive binding of SIRT2 to RIP3 was assessed.
- SIRT2 deletion or knockdown was performed in mice to evaluate its effect on RIP1-RIP3 complex formation.
- TNF-α-induced necrosis was measured in the presence of genetic or pharmacological SIRT2 inhibition.
- RIP1 deacetylation and its effect on RIP1-RIP3 complex formation and necrosis were analyzed using gain- and loss-of-function mutants.
- Ischaemia-reperfusion injury models were used to assess the in vivo role of SIRT2.
Main Results:
- SIRT2 constitutively binds to RIP3, and its absence prevents RIP1-RIP3 complex formation.
- Inhibition of SIRT2 blocks TNF-α-induced cellular necrosis.
- RIP1 is a key target of SIRT2-dependent deacetylation, with acetylation of RIP1 lysine 530 modulating complex formation and necrosis.
- SIRT2 inhibition protects against ischaemia-reperfusion injury in vivo.
Conclusions:
- SIRT2 is a critical regulator of programmed necrosis.
- Targeting SIRT2 with inhibitors may offer a novel therapeutic strategy for necrotic injuries, including ischaemic stroke and myocardial infarction.
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