Abstract
Succinylation is a novel post-translational modification identified on many proteins and is involved in multiple biological processes. Succinylation levels are dynamically regulated, balanced by succinylation and desuccinylation processes, and are closely connected to metabolic state in vivo. Sirtuins have been shown to possess NAD+-dependent desuccinylation activity in vitro and in vivo, among which the desuccinylation activity of SIRT5 is most extensively studied. Our understanding of the response of succinylation levels to different metabolic conditions, is hampered by the lack of a fast NAD+-dependent desuccinylation assay in a physiological context. In the present study, we therefore optimized and validated a fluorescence-based assay for measuring NAD+-dependent desuccinylation activity in cell lysates. Our results demonstrated that shorter and stricter reaction time was critical to approach the initial rate of NAD+-dependent desuccinylation activity in crude cell lysate systems, as compared to the desuccinylation reaction of purified His-SIRT5. Analysis of desuccinylation activity in SIRT5 knockout HEK293T cells confirmed the relevance of SIRT5 in cellular desuccinylation activity, as well as the presence of other NAD+-dependent desuccinylase activities. In addition, we were able to analyse desuccinylation and deacetylation activity in multiple cell lines using this assay. We showed a remarkably higher desuccinylase activity, but not deacetylase activity, in proliferative cultured muscle and adipose cells in comparison with their differentiated counterparts. Our results reveal an alteration in NAD+-dependent desuccinylation activity under different metabolic states.
Highlights
We examined whether the assay set-up for pure His-SIRT5 could be applied to measure desuccinylase activity in a crude cell lysate
The desuccinylase activity was lower in HEK293T SIRT5 knockout cell lysates as compared to wild type cell lysates at all tested reaction time, which ranged from 2.5 to 60 min (Fig. 2d)
This result demonstrates that SIRT5 contributes to the desuccinylation activity of the crude cell lysate
Summary
A fluorescence based, homogenous, NAD+-dependent desuccinylation activity assay to study desuccinylase activity in crude cell lysate directly and rapidly is lacking. Its availability would allow for studying desuccinylation in a physiological context. Fluorogenic assays have been used for identifying activators and inhibitors of recombinant SIRT5 in vitro[32,33,34]. A limited number of studies analysed desuccinylase activity in the more physiological context of a cell lysate[35,36]. We optimize and validate a fluorescence-based assay for detecting N AD+-dependent cellular desuccinylase activity. We use this assay to gain insights into NAD+-dependent desuccinylase activity in different cellular contexts, as well as its relation to global protein succinylation levels
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