Sirtuin Substrate Identification
Sirtuin Substrate Identification - Related Videos
14:32
Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates
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Cited by 1 •
2016
This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.
11:13
Identification of Kinase-substrate Pairs Using High Throughput Screening
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Cited by 1 •
2015
Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.
11:11
Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach
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Cited by 6 •
2019
The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.
14:12
Rodent Identification II
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2015
Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN Animal records must be accurately maintained to ensure that data collection is correct. Records range from maintaining information on cage cards to having a detailed database with all of the relevant information on each animal. The primary component of recordkeeping is the individual identification of research animals. There are a variety of methods suitable for identifying mice and rats. This...
11:09
Rodent Identification I
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2015
Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN A fundamental requirement of biomedical research is the proper identification of research animals. It is essential that the right animal is utilized for procedures and data collection. Laboratory mice and rats can be identified with the following permanent methods: ear tags, ear punch codes, microchip implantation, tail tattoos for adult mice, and toe tattoos for neonates. Temporary methods of...
FAQs
The key recognition feature is an acylated lysine residue positioned for processing by a particular sirtuin. Substrate assignments therefore depend on both the lysine modification and the enzyme’s selectivity, rather than on protein identity alone. This chemical view helps explain why different sirtuins can connect NAD+-dependent deacylation to distinct cellular pathways.
NAD+ is not merely a required additive in this chemistry. During the reaction, its use is associated with formation of nicotinamide and an acyl-ADP-ribose product. Accounting for these products gives mechanistic support that an observed change in lysine acylation reflects sirtuin activity, helping distinguish enzymatic deacylation from an unrelated modification process.
Comparing acetylated, succinylated, and other related acylated lysines can reveal whether recognition is broad or chemically selective. The comparison matters because the acyl group changes the substrate’s chemical identity while the enzyme still uses NAD+. Such tests connect substrate preference with pathway-specific regulation and help prioritize which modification assignments require further validation.
A practical workflow combines a purified-enzyme assay with chemical and genetic checks. The enzyme is tested against a candidate substrate with a relevant acyl modification, mass spectrometry examines the modification, and mutational analysis tests whether altered substrate or enzyme features change the result. Agreement among these approaches makes the assignment more persuasive.
Mass spectrometry helps examine whether a candidate substrate carries the expected acylation state before and after exposure to a sirtuin. In this context, it links an enzyme assay to a chemically measurable substrate change. That evidence is especially useful when distinguishing acetyl, succinyl, or related acyl modifications during substrate assignment.
Mutational analysis tests whether selected substrate or enzyme features are important for the observed modification change. If altering those features changes the assay outcome, the result supports a specific recognition relationship rather than a nonspecific reaction. This approach complements purified-enzyme measurements and mass spectrometry by probing the molecular basis of substrate selectivity.
Mapping validated substrates can clarify how sirtuin activity connects chemical modifications with metabolic and signaling pathways. Those connections support investigations of gene regulation, cellular stress responses, aging, and therapeutic target development. The approach is therefore useful not only for cataloging modified proteins or metabolites, but also for interpreting the broader consequences of enzyme specificity.