GO:0036054 protein-malonyllysine demalonylase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0036054 describes the enzymatic removal of a malonyl group from lysine residues on proteins, using NAD+ as a cofactor.
• SIRT5 is the primary enzyme known to catalyze this reaction, acting as a NAD-dependent demalonylase and desuccinylase.
• This activity regulates mitochondrial metabolism and protects against diabetic cardiomyopathy by modulating protein function [2,8].
• Dysregulation of demalonylation is linked to metabolic diseases, cancer, and cardiovascular disorders [2,4].
• Studying this activity requires tools like CRISPR knockout, point mutations, and proteomics to dissect its role in cellular pathways [1,5].
Description
Protein-malonyllysine demalonylase activity (GO:0036054) is a molecular function that catalyzes the removal of malonyl groups from lysine residues on proteins, a key post-translational modification involved in cellular metabolism. This activity is NAD+-dependent and produces 2''-O-malonyl-ADP-D-ribose, nicotinamide, and a de-malonylated protein. The discovery of this enzymatic activity expanded the understanding of sirtuin family functions beyond deacetylation, highlighting SIRT5 as a major demalonylase [3,4]. Researchers study this term to understand how mitochondrial proteins are regulated and how their dysfunction contributes to diseases such as diabetic cardiomyopathy and cancer [2,8]. The reaction is critical for maintaining metabolic homeostasis and responding to cellular stress [4,5].
protein-malonyllysine demalonylase activity At A Glance
| GO ID | GO:0036054 |
|---|---|
| GO term | protein-malonyllysine demalonylase activity |
| Ontology | molecular_function |
| Synonym | peptidyl-malonyllysine demalonylase activity; protein lysine demalonylation activity; protein malonyl lysine demalonylation activity |
| Major function | Removal of malonyl groups from lysine residues on proteins |
| Cofactor | NAD+ |
| Reaction products | 2''-O-malonyl-ADP-D-ribose, nicotinamide, L-lysyl-[protein] |
| Primary enzyme | SIRT5 |
| Related diseases | Diabetic cardiomyopathy, metabolic disorders, cancer |
What Is GO:0036054?
GO:0036054 describes the catalysis of the reaction: N(6)-malonyl-L-lysyl-[protein] + NAD+ + H2O = 2''-O-malonyl-ADP-D-ribose + nicotinamide + L-lysyl-[protein]. In simpler terms, it is an enzymatic activity that removes a malonyl modification from lysine residues on proteins, using NAD+ as a co-substrate and releasing nicotinamide and a malonyl-ADP-ribose byproduct.
Why Is protein-malonyllysine demalonylase activity Important in Cell Biology?
Protein-malonyllysine demalonylase activity is crucial for regulating protein function in response to metabolic cues, particularly in mitochondria where SIRT5 is localized [3,8]. This activity counteracts malonylation, a modification that can alter enzyme activity and protein interactions, thereby influencing pathways like glycolysis, fatty acid oxidation, and oxidative phosphorylation [4,8]. Dysregulation of demalonylation has been implicated in diabetic cardiomyopathy, where SIRT5-mediated demalonylation of GSTP1 suppresses cardiomyocyte pyroptosis. Understanding this activity provides insights into metabolic disease mechanisms and potential therapeutic targets [4,5].
• Regulates mitochondrial metabolism by reversing malonylation on key enzymes [3,8].
• Protects against diabetic cardiomyopathy through demalonylation of GSTP1.
• Influences cellular stress responses and apoptosis.
• Modulates insulin secretion and glucose homeostasis.
• Plays a role in cancer metabolism and tumor progression.
• Provides a mechanism for NAD+-dependent signaling.
• Potential target for treating metabolic disorders.
• Essential for maintaining protein stability and function.
• Involved in the regulation of reactive oxygen species.
• Contributes to the understanding of sirtuin biology beyond deacetylation [3,5].
What Happens During protein-malonyllysine demalonylase activity?
Substrate Recognition and Binding
In simple terms: The enzyme finds and grabs onto a protein that has a malonyl tag.
The demalonylase enzyme, such as SIRT5, recognizes target proteins with malonylated lysine residues. This recognition is mediated by specific structural features in the enzyme's active site that accommodate the malonyl group [3,6]. The binding is NAD+-dependent and involves conformational changes that position the substrate for catalysis.
Catalytic Mechanism and NAD+ Cleavage
In simple terms: The enzyme uses NAD+ to cut off the malonyl group.
Upon binding, the enzyme catalyzes the cleavage of NAD+, releasing nicotinamide and forming an ADP-ribose intermediate. This intermediate reacts with the malonyl-lysine, leading to the removal of the malonyl group and the formation of 2''-O-malonyl-ADP-D-ribose. The reaction is dependent on the presence of NAD+ and water.
Product Release and Protein Restoration
In simple terms: The malonyl group is removed, and the protein is restored to its unmodified state.
After catalysis, the de-malonylated protein is released, along with the byproducts 2''-O-malonyl-ADP-D-ribose and nicotinamide. The removal of the malonyl group can restore the protein's function, alter its interactions, or affect its stability [3,4]. This step is crucial for resetting the protein's modification status and allowing it to participate in subsequent cellular processes.
Regulation by Cellular NAD+ Levels
In simple terms: The reaction depends on how much NAD+ is available in the cell.
The activity of demalonylases is tightly linked to cellular NAD+ levels, which fluctuate with metabolic state. SIRT5, for example, senses NAD+ availability and adjusts its activity accordingly [3,8]. This connects demalonylation to energy metabolism and stress responses.
Key Genes Involved in GO:0036054 protein-malonyllysine demalonylase activity
The following genes and proteins are directly involved in or regulate protein-malonyllysine demalonylase activity.
| Gene | Major Role | Research Relevance |
|---|---|---|
| SIRT5 | Primary demalonylase enzyme | Central to studies of demalonylation in metabolism and disease |
| GSTP1 | Target of SIRT5 demalonylation | Demalonylation suppresses pyroptosis in diabetic cardiomyopathy |
| AGO2 | Indirectly linked to mitochondrial function | Protects against diabetic cardiomyopathy via mitochondrial translation |
| NAD+ | Essential cofactor | Required for demalonylase activity |
| SIRT1 | Related sirtuin with deacetylase activity | Provides comparative insights into sirtuin functions |
| SIRT3 | Mitochondrial deacetylase | Often studied alongside SIRT5 for mitochondrial regulation |
| SIRT4 | Mitochondrial sirtuin | Contributes to metabolic regulation |
| SIRT6 | Nuclear sirtuin | Involved in DNA repair and metabolism |
| SIRT7 | Nuclear sirtuin | Regulates transcription and stress responses |
| HSP60 | Mitochondrial chaperone | Potential target of demalonylation |
| SDHA | Succinate dehydrogenase | Malonylation affects TCA cycle enzymes |
| ATP5A1 | ATP synthase subunit | Malonylation impacts oxidative phosphorylation |
| CPT1A | Fatty acid oxidation enzyme | Regulated by malonylation/demalonylation |
| PDHA1 | Pyruvate dehydrogenase | Activity modulated by malonylation |
| GAPDH | Glycolytic enzyme | Malonylation affects glycolysis |
| LDHA | Lactate dehydrogenase | Linked to metabolic reprogramming |
| ACAT1 | Acetyl-CoA acetyltransferase | Mitochondrial enzyme subject to acylation |
How Is protein-malonyllysine demalonylase activity Regulated?
The activity of protein-malonyllysine demalonylases is primarily regulated by cellular NAD+ levels, which reflect the metabolic state of the cell [3,8]. SIRT5, the main enzyme, is also regulated at the transcriptional level and by post-translational modifications, though these mechanisms are less understood [4,5]. Additionally, the availability of malonylated substrates and the presence of inhibitors can modulate activity. Metabolic signals such as nutrient availability and stress can influence demalonylase function, integrating this activity into broader cellular signaling networks.
protein-malonyllysine demalonylase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| SIRT5 | Diabetic cardiomyopathy | Cardiomyocyte-specific knockout mice |
| GSTP1 | Cardiomyocyte pyroptosis | Point mutation at malonylated lysine |
| SIRT5 | Metabolic syndrome | Liver-specific knockout or overexpression |
| SIRT5 | Cancer | Xenograft models with SIRT5 knockdown |
| AGO2 | Diabetic cardiomyopathy | Cardiomyocyte overexpression |
Diabetic Cardiomyopathy
SIRT5-mediated demalonylation of GSTP1 suppresses cardiomyocyte pyroptosis, protecting against diabetic cardiomyopathy. Reduced SIRT5 activity leads to increased malonylation and exacerbated cardiac injury. This highlights the therapeutic potential of targeting demalonylation in cardiovascular complications of diabetes.
Metabolic Disorders
Dysregulation of demalonylation contributes to metabolic disorders such as obesity and insulin resistance. SIRT5 knockout mice exhibit altered mitochondrial metabolism and increased susceptibility to metabolic stress. The activity of SIRT5 is linked to fatty acid oxidation and glucose homeostasis.
Cancer
Altered expression of SIRT5 and other sirtuins has been observed in various cancers, where they can influence tumor metabolism and progression. Demalonylation of metabolic enzymes may support cancer cell proliferation under stress. Targeting SIRT5 is being explored as a potential anticancer strategy.
From protein-malonyllysine demalonylase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does SIRT5 demalonylase activity protect against cardiac injury? | Cardiomyocyte-specific SIRT5 knockout mice |
| What is the role of SIRT5 in metabolic regulation? | Liver-specific SIRT5 knockout or overexpression |
| How does malonylation affect enzyme activity? | Point mutations at specific lysine residues |
| Can demalonylation be monitored in live cells? | Knock-in of fluorescently tagged SIRT5 |
| What are the downstream targets of SIRT5? | Proteomics with SIRT5 overexpression |
| Is SIRT5 involved in cancer metabolism? | Cancer cell lines with SIRT5 knockout |
How to Study the protein-malonyllysine demalonylase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Mass spectrometry | Malonylated protein identification and quantification | Global malonylome profiling |
| Western blot | Specific protein malonylation levels | Validation of target demalonylation |
| Enzymatic assay | Demalonylase activity kinetics | Inhibitor screening |
| CRISPR knockout | Gene function in demalonylation | Identifying regulators |
| RNA-seq | Transcriptional changes upon SIRT5 modulation | Pathway analysis |
| Immunoprecipitation | Protein-protein interactions | Identifying SIRT5 substrates |
| Fluorescence microscopy | Subcellular localization | Live-cell imaging |
Proteomics and Malonylome Analysis
Mass spectrometry-based proteomics can identify malonylated proteins and quantify changes upon modulation of demalonylase activity. Enrichment of malonylated peptides using antibodies or chemical probes allows global profiling of this modification.
Enzymatic Activity Assays
In vitro assays using recombinant SIRT5 and malonylated substrates measure demalonylase activity by detecting the release of nicotinamide or the formation of de-malonylated products. These assays are essential for kinetic studies and inhibitor screening [3,7].
CRISPR-Based Genetic Screens
Genome-wide CRISPR knockout screens can identify genes that modulate demalonylase activity or sensitivity to metabolic stress. Such screens help uncover novel regulators and pathways [1,5].
Imaging and Cellular Localization
Fluorescence microscopy with tagged SIRT5 or malonylation-specific probes can visualize the subcellular localization and dynamics of demalonylation in response to metabolic cues.
How CRISPR Can Be Used to Study GO:0036054 protein-malonyllysine demalonylase activity
Knockout
CRISPR knockout of SIRT5 or other demalonylase genes allows researchers to study loss-of-function phenotypes, such as increased malonylation and metabolic dysfunction. Knockout models are valuable for validating the role of demalonylation in disease [2,8].
Point Mutation
Introducing point mutations at catalytic residues of SIRT5 or at specific malonylated lysine sites in target proteins can dissect the functional significance of individual modifications. This approach helps distinguish demalonylase activity from other sirtuin functions [3,6].
Knock-in
Knock-in of tagged or mutant SIRT5 allows for tracking endogenous protein levels and activity. Fluorescent tags enable live-cell imaging, while mutant knock-ins can mimic constitutive or inactive states.
Overexpression
Overexpression of SIRT5 or other demalonylases can enhance demalonylation, protecting against stress or altering metabolism. This approach is useful for gain-of-function studies and identifying downstream effects [2,4].
How EDITGENE Supports protein-malonyllysine demalonylase activity Research
Researchers studying protein-malonyllysine demalonylase activity-related genes often need to determine whether a candidate gene is causally involved in metabolic regulation, disease progression, or cellular stress responses. EDITGENE provides comprehensive CRISPR-based services to facilitate these investigations.
Contact EDITGENE today to design your custom CRISPR model for protein-malonyllysine demalonylase activity research.
Frequently Asked Questions About protein-malonyllysine demalonylase activity
What is protein-malonyllysine demalonylase activity?
It is an enzymatic activity that removes malonyl groups from lysine residues on proteins, using NAD+ as a cofactor, as defined by GO:0036054.
What genes are involved in protein-malonyllysine demalonylase activity?
The primary gene is SIRT5, which encodes a NAD-dependent demalonylase and desuccinylase.
Which diseases are associated with protein-malonyllysine demalonylase activity?
It is linked to diabetic cardiomyopathy, metabolic disorders, and cancer [2,4].
How can I study protein-malonyllysine demalonylase activity?
You can use CRISPR knockout, point mutations, proteomics, and enzymatic assays to investigate this activity [1,3].
What is the role of SIRT5 in demalonylation?
SIRT5 catalyzes the removal of malonyl groups from lysine residues, regulating mitochondrial metabolism and stress responses [3,8].
What are the substrates of protein-malonyllysine demalonylase activity?
Substrates include proteins with malonylated lysine residues, such as GSTP1 and various metabolic enzymes [2,4].
How is protein-malonyllysine demalonylase activity regulated?
It is regulated by cellular NAD+ levels and metabolic signals, integrating energy status with protein modification [3,8].
Can CRISPR be used to study protein-malonyllysine demalonylase activity?
Yes, CRISPR knockout, knock-in, and point mutation models are powerful tools to dissect the function of SIRT5 and related genes [1,2].
What methods measure demalonylase activity?
Enzymatic assays, mass spectrometry, and Western blotting are commonly used to measure demalonylase activity and malonylation levels [3,4].
Why is protein-malonyllysine demalonylase activity important for metabolism?
It reverses malonylation on metabolic enzymes, thereby regulating glycolysis, fatty acid oxidation, and oxidative phosphorylation [4,8].
Conclusion
Protein-malonyllysine demalonylase activity (GO:0036054) is a critical molecular function that regulates protein modification and metabolic pathways, with SIRT5 as the key enzyme. Its dysregulation contributes to diabetic cardiomyopathy, metabolic disorders, and cancer, making it a promising therapeutic target. Advances in CRISPR-based models and proteomic technologies will continue to unravel its complex roles in health and disease.
References
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- 2. Wei C et al.. 2024. SIRT5-related lysine demalonylation of GSTP1 contributes to cardiomyocyte pyroptosis suppression in diabetic cardiomyopathy.. Int J Biol Sci 20(2):585-605 PMID: 38169591
- 3. Du J et al.. 2011. Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase.. Science 334(6057):806-9 PMID: 22076378
- 4. Nahálková J. 2023. A new view on functions of the lysine demalonylase activity of SIRT5.. Life Sci 320:121572 PMID: 36921688
- 5. Sharma A et al.. 2023. Shedding light on structure, function and regulation of human sirtuins: a comprehensive review.. 3 Biotech 13(1):29 PMID: 36597461
- 6. Yuan H et al.. 2012. Structural basis for sirtuin activity and inhibition.. J Biol Chem 287(51):42428-35 PMID: 23086949
- 7. Yang L et al.. 2017. Sirtuin 5: a review of structure, known inhibitors and clues for developing new inhibitors.. Sci China Life Sci 60(3):249-256 PMID: 27858336
- 8. Newman JC et al.. 2012. Mitochondrial protein acylation and intermediary metabolism: regulation by sirtuins and implications for metabolic disease.. J Biol Chem 287(51):42436-43 PMID: 23086951
