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- Publisher Website: 10.1021/jacs.9b06913
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- PMID: 31644285
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Article: Chemical Probes Reveal Sirt2’s New Function as a Robust “Eraser” of Lysine Lipoylation
Title | Chemical Probes Reveal Sirt2’s New Function as a Robust “Eraser” of Lysine Lipoylation |
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Authors | |
Keywords | Catalytic efficiencies Cellular environment Fluorogenic probes Kinetic experiment Physiological substrate |
Issue Date | 2019 |
Publisher | American Chemical Society. The Journal's web site is located at http://pubs.acs.org/journals/jacsat/index.html |
Citation | Journal of the American Chemical Society, 2019, v. 141 n. 46, p. 18428-18436 How to Cite? |
Abstract | Lysine lipoylation, a highly conserved lysine post-translational modification, plays a critical role in regulating cell metabolism. The catalytic activity of a number of vital metabolic proteins, such as pyruvate dehydrogenase (PDH), depends on lysine lipoylation. Despite its important roles, the detailed biological regulatory mechanism of lysine lipoylation remains largely unexplored. Herein we designed a powerful affinity-based probe, KPlip, to interrogate the interactions of lipoylated peptide/proteins under native cellular environment. Large-scale chemical proteomics analysis revealed a number of binding proteins of KPlip, including sirtuin 2 (Sirt2), an NAD+-dependent protein deacylase. To explore the potential activity of Sirt2 toward lysine lipoylation, we designed a single-step fluorogenic probe, KTlip, which reports delipoylation activity in a continuous manner. The results showed that Sirt2 led to significant delipoylation of KTlip, displaying up to a 60-fold fluorescence increase in the assay. Further kinetic experiments with different peptide substrates revealed that Sirt2 can catalyze the delipoylation of peptide (DLAT-PDH, K259) with a remarkable catalytic efficiency (kcat/Km) of 3.26 × 103 s–1 M–1. The activity is about 400-fold higher than that of Sirt4, the only mammalian enzyme with known delipoylation activity. Furthermore, overexpression and silencing experiments demonstrated that Sirt2 regulates the lipoylation level and the activity of endogenous PDH, thus unequivocally confirming that PDH is a genuine physiological substrate of Sirt2. Using our chemical probes, we have successfully established the relationship between Sirt2 and lysine lipoylation in living cells for the first time. We envision that such chemical probes will serve as useful tools for delineating the roles of lysine lipoylation in biology and diseases. |
Persistent Identifier | http://hdl.handle.net/10722/288395 |
ISSN | 2023 Impact Factor: 14.4 2023 SCImago Journal Rankings: 5.489 |
ISI Accession Number ID |
DC Field | Value | Language |
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dc.contributor.author | Xie, Y | - |
dc.contributor.author | Chen, L | - |
dc.contributor.author | Wang, R | - |
dc.contributor.author | Wang, J | - |
dc.contributor.author | Li, J | - |
dc.contributor.author | Xu, W | - |
dc.contributor.author | Li, Y | - |
dc.contributor.author | Yao, SQ | - |
dc.contributor.author | Zhang, L | - |
dc.contributor.author | Hao, Q | - |
dc.contributor.author | Sun, H | - |
dc.date.accessioned | 2020-10-05T12:12:15Z | - |
dc.date.available | 2020-10-05T12:12:15Z | - |
dc.date.issued | 2019 | - |
dc.identifier.citation | Journal of the American Chemical Society, 2019, v. 141 n. 46, p. 18428-18436 | - |
dc.identifier.issn | 0002-7863 | - |
dc.identifier.uri | http://hdl.handle.net/10722/288395 | - |
dc.description.abstract | Lysine lipoylation, a highly conserved lysine post-translational modification, plays a critical role in regulating cell metabolism. The catalytic activity of a number of vital metabolic proteins, such as pyruvate dehydrogenase (PDH), depends on lysine lipoylation. Despite its important roles, the detailed biological regulatory mechanism of lysine lipoylation remains largely unexplored. Herein we designed a powerful affinity-based probe, KPlip, to interrogate the interactions of lipoylated peptide/proteins under native cellular environment. Large-scale chemical proteomics analysis revealed a number of binding proteins of KPlip, including sirtuin 2 (Sirt2), an NAD+-dependent protein deacylase. To explore the potential activity of Sirt2 toward lysine lipoylation, we designed a single-step fluorogenic probe, KTlip, which reports delipoylation activity in a continuous manner. The results showed that Sirt2 led to significant delipoylation of KTlip, displaying up to a 60-fold fluorescence increase in the assay. Further kinetic experiments with different peptide substrates revealed that Sirt2 can catalyze the delipoylation of peptide (DLAT-PDH, K259) with a remarkable catalytic efficiency (kcat/Km) of 3.26 × 103 s–1 M–1. The activity is about 400-fold higher than that of Sirt4, the only mammalian enzyme with known delipoylation activity. Furthermore, overexpression and silencing experiments demonstrated that Sirt2 regulates the lipoylation level and the activity of endogenous PDH, thus unequivocally confirming that PDH is a genuine physiological substrate of Sirt2. Using our chemical probes, we have successfully established the relationship between Sirt2 and lysine lipoylation in living cells for the first time. We envision that such chemical probes will serve as useful tools for delineating the roles of lysine lipoylation in biology and diseases. | - |
dc.language | eng | - |
dc.publisher | American Chemical Society. The Journal's web site is located at http://pubs.acs.org/journals/jacsat/index.html | - |
dc.relation.ispartof | Journal of the American Chemical Society | - |
dc.rights | This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://pubs.acs.org/doi/abs/10.1021/jacs.9b06913# | - |
dc.subject | Catalytic efficiencies | - |
dc.subject | Cellular environment | - |
dc.subject | Fluorogenic probes | - |
dc.subject | Kinetic experiment | - |
dc.subject | Physiological substrate | - |
dc.title | Chemical Probes Reveal Sirt2’s New Function as a Robust “Eraser” of Lysine Lipoylation | - |
dc.type | Article | - |
dc.identifier.email | Hao, Q: qhao@hku.hk | - |
dc.identifier.authority | Hao, Q=rp01332 | - |
dc.description.nature | postprint | - |
dc.identifier.doi | 10.1021/jacs.9b06913 | - |
dc.identifier.pmid | 31644285 | - |
dc.identifier.scopus | eid_2-s2.0-85074875986 | - |
dc.identifier.hkuros | 315238 | - |
dc.identifier.volume | 141 | - |
dc.identifier.issue | 46 | - |
dc.identifier.spage | 18428 | - |
dc.identifier.epage | 18436 | - |
dc.identifier.isi | WOS:000499738700013 | - |
dc.publisher.place | United States | - |
dc.identifier.issnl | 0002-7863 | - |