Lysine malonylation

{{Short description|Post-translational modification}} thumb|300x300px|Protein with a lysine residue at physiological pH: unmodified (left) and malonylated (right). '''Lysine malonylation''' ('''Kmal''', '''maK'''), '''protein malonylation''' or '''malonylation''', is a reversible post-translational modification (PTM) in eukaryotic and prokaryotic cells, in which a malonyl group (–CO–CH2–COOH) is added to a lysine (K) residue of a protein.<ref name=":1" /><ref name=":5">{{Cite journal |last1=Du |first1=Yipeng |last2=Cai |first2=Tanxi |last3=Li |first3=Tingting |last4=Xue |first4=Peng |last5=Zhou |first5=Bo |last6=He |first6=Xiaolong |last7=Wei |first7=Peng |last8=Liu |first8=Pingsheng |last9=Yang |first9=Fuquan |last10=Wei |first10=Taotao |date=January 2015 |title=Lysine Malonylation Is Elevated in Type 2 Diabetic Mouse Models and Enriched in Metabolic Associated Proteins |journal=Molecular & Cellular Proteomics |language=en |volume=14 |issue=1 |pages=227–236 |doi=10.1074/mcp.M114.041947 |pmc=4288257 |pmid=25418362 |doi-access=free}}</ref><ref name=":0">{{Cite journal |last1=Galván-Peña |first1=Silvia |last2=Carroll |first2=Richard G. |last3=Newman |first3=Carla |last4=Hinchy |first4=Elizabeth C. |last5=Palsson-McDermott |first5=Eva |last6=Robinson |first6=Elektra K. |last7=Covarrubias |first7=Sergio |last8=Nadin |first8=Alan |last9=James |first9=Andrew M. |last10=Haneklaus |first10=Moritz |last11=Carpenter |first11=Susan |last12=Kelly |first12=Vincent P. |last13=Murphy |first13=Michael P. |last14=Modis |first14=Louise K. |last15=O'Neill |first15=Luke A. |date=2019-01-18 |title=Malonylation of GAPDH is an inflammatory signal in macrophages |journal=Nature Communications |language=en |volume=10 |issue=1 |article-number=338 |doi=10.1038/s41467-018-08187-6 |issn=2041-1723 |pmc=6338787 |pmid=30659183 |bibcode=2019NatCo..10..338G |doi-access=free}}</ref> It was first identified in 2011 by ''Peng et al.'' as an evolutionarily conserved modification and belongs to the acidic acyl modifications such as succinylation and glutarylation.<ref name=":11" /><ref name=":8">{{Cite journal |last1=Hirschey |first1=Matthew D. |last2=Zhao |first2=Yingming |date=September 2015 |title=Metabolic Regulation by Lysine Malonylation, Succinylation, and Glutarylation |journal=Molecular & Cellular Proteomics |language=en |volume=14 |issue=9 |pages=2308–2315 |doi=10.1074/mcp.R114.046664 |pmc=4563717 |pmid=25717114 |doi-access=free}}</ref> As a dynamically regulated modification, it responds to conditions such as stress responses, metabolic processes, and mutations, thereby influencing the charge, structure, and function of proteins.<ref name=":0" /><ref>{{Cite journal |last1=Xu |first1=Yan |last2=Ding |first2=Ya-Xin |last3=Ding |first3=Jun |last4=Wu |first4=Ling-Yun |last5=Xue |first5=Yu |date=2016-12-02 |title=Mal-Lys: prediction of lysine malonylation sites in proteins integrated sequence-based features with mRMR feature selection |journal=Scientific Reports |language=en |volume=6 |issue=1 |bibcode=2016NatSR...638318X |doi=10.1038/srep38318 |issn=2045-2322 |pmc=5133563 |pmid=27910954 |doi-access=free |article-number=38318}}</ref> This involves, among other things, the metabolic pathways of glucose and fatty acids as well as histone-mediated gene regulation, and is increasingly associated with immune regulation, angiogenesis, osteoarthritis, cancer and metabolic diseases such as obesity and type 2 diabetes.<ref name=":19">{{Cite journal |last1=Zou |first1=Lu |last2=Yang |first2=Yanyan |last3=Wang |first3=Zhibin |last4=Fu |first4=Xiuxiu |last5=He |first5=Xiangqin |last6=Song |first6=Jiayi |last7=Li |first7=Tianxiang |last8=Ma |first8=Huibo |last9=Yu |first9=Tao |date=2023 |title=Lysine Malonylation and Its Links to Metabolism and Diseases |journal=Aging and Disease |language=en |volume=14 |issue=1 |pages=84–98 |doi=10.14336/AD.2022.0711 |issn=2152-5250 |pmc=9937698 |pmid=36818560 |doi-access=free}}</ref><ref>{{Cite journal |last1=Guo |first1=Chenxu |last2=Zhang |first2=Mingliang |last3=Jin |first3=Xin |last4=Zhu |first4=Chao |last5=Xu |first5=Rui |last6=Sun |first6=Jiahe |last7=Qian |first7=Jun |date=2025-09-30 |title=Integrated proteome, phospho-proteome and malonyl-proteome revealed a molecular alteration of breast cancer |journal=Scientific Reports |language=en |volume=15 |issue=1 |article-number=34004 |doi=10.1038/s41598-025-11573-y |issn=2045-2322 |pmc=12484806 |pmid=41028821 |bibcode=2025NatSR..1534004G |doi-access=free}}</ref> Its biological significance is increasingly recognized, but many aspects of its regulation and function remain unresolved, so that its therapeutic potential is still unexplored.<ref name=":19" />

== Chemical properties == At physiological pH, the ε-amino group (–NH₂) of the lysine residue exists almost entirely in its protonated form (–NH₃⁺), whereas the carboxyl group (–COOH) of the malonyl group exists almost entirely in its deprotonated form (–COO^-).<ref name=":8" /> Through the covalent attachment of a malonyl group to the ε-amino group, the lysine residue loses its positive charge and assumes the negative charge of the malonyl group, resulting in a charge shift from +1 to −1.<ref name=":14">{{Cite journal |last1=Kulkarni |first1=Rhushikesh A. |last2=Worth |first2=Andrew J. |last3=Zengeya |first3=Thomas T. |last4=Shrimp |first4=Jonathan H. |last5=Garlick |first5=Julie M. |last6=Roberts |first6=Allison M. |last7=Montgomery |first7=David C. |last8=Sourbier |first8=Carole |last9=Gibbs |first9=Benjamin K. |last10=Mesaros |first10=Clementina |last11=Tsai |first11=Yien Che |last12=Das |first12=Sudipto |last13=Chan |first13=King C. |last14=Zhou |first14=Ming |last15=Andresson |first15=Thorkell |date=February 2017 |title=Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling |journal=Cell Chemical Biology |language=en |volume=24 |issue=2 |pages=231–242 |doi=10.1016/j.chembiol.2017.01.002 |pmc=5864104 |pmid=28163016 |doi-access=free}}</ref><ref name=":1" /><ref name=":8" /> This complete reversal of charge is thought to disrupt ionic interactions both within the protein itself and with negatively charged components of nucleotides, proteins and small molecules.<ref name=":8" /> Such alterations can occur at multiple lysine residues within a single protein, although their overall frequency varies considerably across the proteome.<ref name=":7" /> In mouse liver, for example, about half of all malonylated proteins contain a single site, while the frequency decreases sharply beyond four sites and only a few are extensively modified, the most heavily modified enzyme being carbamoyl‑phosphate synthetase 1 (CPS1) of the urea cycle with 31 sites.<ref name=":7" />

In the context of other lysine acylations, malonylation can be positioned as follows:

While acetylation neutralizes lysine's positive charge, malonylation introduces a negative one, placing it among the acidic acylations alongside methylmalonylation, succinylation, glutarylation, 3‑hydroxy‑3‑methylglutarylation, 3‑methylglutaconylation, and 3‑methylglutarylation.<ref name=":8" /><ref name=":13">{{Cite journal |last1=Baldensperger |first1=Tim |last2=Glomb |first2=Marcus A. |date=2021-04-29 |title=Pathways of Non-enzymatic Lysine Acylation |journal=Frontiers in Cell and Developmental Biology |language=English |volume=9 |doi=10.3389/fcell.2021.664553 |issn=2296-634X |pmc=8116961 |pmid=33996820 |doi-access=free |article-number=664553}}</ref><ref name=":15" /> In size, malonylation (three carbons) is bulkier than acetylation (two) but smaller than succinylation (four) and glutarylation (five).<ref name=":8" /> As a result, such acidic acyl modifications, as discussed for malonylation and succinylation, are expected to exert a greater impact than acetylation at the same lysine site.<ref name=":7" />

Each modification arises from the corresponding acyl-CoA derivative.<ref name=":20">{{Cite journal |last1=Zhang |first1=Zhihong |last2=Tan |first2=Minjia |last3=Xie |first3=Zhongyu |last4=Dai |first4=Lunzhi |last5=Chen |first5=Yue |last6=Zhao |first6=Yingming |date=January 2011 |title=Identification of lysine succinylation as a new post-translational modification |journal=Nature Chemical Biology |language=en |volume=7 |issue=1 |pages=58–63 |doi=10.1038/nchembio.495 |issn=1552-4450 |pmc=3065206 |pmid=21151122 |bibcode=2011NatCB...7...58Z }}</ref><ref name=":11" /><ref name=":15" /><ref name=":16">{{Cite journal |last1=Tan |first1=Minjia |last2=Peng |first2=Chao |last3=Anderson |first3=Kristin A. |last4=Chhoy |first4=Peter |last5=Xie |first5=Zhongyu |last6=Dai |first6=Lunzhi |last7=Park |first7=Jeongsoon |last8=Chen |first8=Yue |last9=Huang |first9=He |last10=Zhang |first10=Yi |last11=Ro |first11=Jennifer |last12=Wagner |first12=Gregory R. |last13=Green |first13=Michelle F. |last14=Madsen |first14=Andreas S. |last15=Schmiesing |first15=Jessica |date=April 2014 |title=Lysine Glutarylation Is a Protein Posttranslational Modification Regulated by SIRT5 |journal=Cell Metabolism |language=en |volume=19 |issue=4 |pages=605–617 |doi=10.1016/j.cmet.2014.03.014 |pmc=4108075 |pmid=24703693 |doi-access=free}}</ref> Malonyl‑CoA is produced in cytosol and mitochondria by acetyl‑CoA carboxylase (ACC) and, in mitochondria, also by acyl-CoA synthetase family member 3 (ACSF3);<ref name=":6" /> succinyl‑CoA stems from the TCA cycle and amino acid catabolism;<ref name=":1" /><ref name=":11" /> glutaryl‑CoA from amino acid catabolism;<ref name=":1" /> and methylmalonyl‑CoA from amino acid and odd‑chain fatty acid metabolism, which accumulates in vitamin B12 deficiency and methylmalonic acidemias.<ref name=":15" /> Malonyl‑CoA is far less reactive toward proteins than succinyl‑CoA or glutaryl‑CoA because, like acetyl‑CoA, its shorter carbon chain cannot support the intramolecular catalysis needed to form a reactive cyclic anhydride intermediate, which in turn enables modification over a broader pH range.<ref name=":17" /> Malonyl, succinyl, and glutaryl groups are removed by Sirtuin 5 (SIRT5), which shows little activity toward acetylation.<ref name=":8" />

Malonylation occurs mainly in mitochondria but also in the cytosol and nucleus.<ref name=":4" /> In mouse liver, about 60% of malonylated proteins are mitochondrial, whereas in human fibroblasts the distribution is more even.<ref name=":4" /> Succinylation and glutarylation are likewise enriched in mitochondria but not exclusive to them.<ref name=":8" /> The relative abundance of these modifications reflects acyl-CoA availability: acetylation is most common, succinylation reaches 10–30 % of acetylation levels, malonylation is at least tenfold less frequent, and glutarylation occurs only in trace amounts.<ref name=":13" /> In addition to their differing frequencies, malonylation, succinylation, and acetylation can also target the same lysine site.<ref name=":7" /> In mouse liver mitochondria, about 85 % of succinylation sites overlap with at least one of these modifications, and ~6 % can contain all three, mainly in proteins involved in fatty acid oxidation, glutaryl-CoA degradation, and ketogenesis.<ref name=":7" /> In contrast, only 55 % of malonylation sites overlap with succinylation, while about 45 % are unique.<ref name=":7" /> These distinct patterns suggest a specific regulatory role for malonylation among lysine acyl modifications.<ref name=":7" /><div style="overflow:auto"> {| class="wikitable center" |+Selected lysine acyl modifications ! colspan="2" | !Acetylation !Malonylation !Methylmalonylation !Succinylation !Glutarylation |- | rowspan="2" |Functional group |Chemical formula |C₂H₃O |C₃H₃O₃ |C₄H₅O₃ |C₄H₄O₄ |C₅H₆O₄ |- |Condensed structural formula |–CO–CH3 |–CO–CH₂–COOH |–CO–CH(CH₃)–COOH |–CO–(CH₂)₂–COOH |–CO–(CH₂)₃–COOH |- | colspan="2" |Bulkiness |Two-carbon group<ref name=":8" /> |Three-carbon group<ref name=":8" /> |Four-carbon group |Four-carbon group<ref name=":8" /> |Five-carbon group<ref name=":16" /> |- | colspan="2" |Charge shift | +1 → 0<ref name=":8" /> | colspan="4" | +1 → -1<ref name=":8" /><ref name=":15" /> |- | colspan="2" |Donor |Acetyl-CoA<ref name=":8" /> |Malonyl-CoA<ref name=":11" /> |Methylmalonyl-CoA<ref name=":15" /> |Succinyl-CoA<ref name=":20" /> |Glutaryl-CoA<ref name=":8" /> |- | colspan="2" |Frequency | colspan="5" |Acetyl-CoA < Succinyl-CoA < Malonyl-CoA < Glutaryl-CoA<ref name=":13" /> |- | colspan="2" |Non-enzymatic acylation: Reactivity |No anhydride ring formation<ref name=":17" /> |No anhydride ring formation<ref name=":17">{{Cite journal |last1=Wagner |first1=Gregory R. |last2=Bhatt |first2=Dhaval P. |last3=O'Connell |first3=Thomas M. |last4=Thompson |first4=J. Will |last5=Dubois |first5=Laura G. |last6=Backos |first6=Donald S. |last7=Yang |first7=Hao |last8=Mitchell |first8=Grant A. |last9=Ilkayeva |first9=Olga R. |last10=Stevens |first10=Robert D. |last11=Grimsrud |first11=Paul A. |last12=Hirschey |first12=Matthew D. |date=April 2017 |title=A Class of Reactive Acyl-CoA Species Reveals the Non-enzymatic Origins of Protein Acylation |journal=Cell Metabolism |language=en |volume=25 |issue=4 |pages=823–837.e8 |doi=10.1016/j.cmet.2017.03.006 |pmc=5399522 |pmid=28380375 |doi-access=free}}</ref> |Unknown |Highly reactive five‑membered cyclic anhydride intermediate<ref name=":17" /> |Highly reactive six‑membered cyclic anhydride intermediate<ref name=":17" /> |- | colspan="2" |Enzymatic deacylation: Enzymes (Eraser) | * Nucleus: HDAC1, HDAC2, HDAC3, Sirtuin 1,<ref name=":18">{{Cite journal |last1=Curcio |first1=Antonio |last2=Rocca |first2=Roberta |last3=Alcaro |first3=Stefano |last4=Artese |first4=Anna |date=2024-05-10 |title=The Histone Deacetylase Family: Structural Features and Application of Combined Computational Methods |journal=Pharmaceuticals |language=en |volume=17 |issue=5 |page=620 |doi=10.3390/ph17050620 |issn=1424-8247 |pmc=11124352 |pmid=38794190 |doi-access=free}}</ref> Sirtuin 2<ref name=":18" /> * Cytoplasm: Sirtuin 1,<ref name=":18" /> Sirtuin 2<ref name=":18" /> * Mitochondria: Sirtuin 3<ref name=":18" /> | colspan="4" |Global: Sirtuin 5<ref name=":8" /><ref name=":15" /> |- | colspan="2" |Pathways | * Nearly all enzymes involved in core metabolic pathways linked to insulin secretion<ref>{{Cite journal |last1=Shang |first1=Shuang |last2=Liu |first2=Jing |last3=Hua |first3=Fang |date=2022-12-29 |title=Protein acylation: mechanisms, biological functions and therapeutic targets |journal=Signal Transduction and Targeted Therapy |language=en |volume=7 |issue=1 |article-number=396 |doi=10.1038/s41392-022-01245-y |issn=2059-3635 |pmc=9797573 |pmid=36577755 |doi-access=free}}</ref> * Gene regulation | * Glucose metabolism<ref name=":5" /> * Fatty acid metabolism<ref name=":5" /> * Glutaryl-CoA degregation<ref name=":7">{{Cite journal |last1=Nishida |first1=Yuya |last2=Rardin |first2=Matthew J. |last3=Carrico |first3=Chris |last4=He |first4=Wenjuan |last5=Sahu |first5=Alexandria K. |last6=Gut |first6=Philipp |last7=Najjar |first7=Rami |last8=Fitch |first8=Mark |last9=Hellerstein |first9=Marc |last10=Gibson |first10=Bradford W. |last11=Verdin |first11=Eric |date=July 2015 |title=SIRT5 Regulates both Cytosolic and Mitochondrial Protein Malonylation with Glycolysis as a Major Target |journal=Molecular Cell |language=en |volume=59 |issue=2 |pages=321–332 |doi=10.1016/j.molcel.2015.05.022 |pmc=4571487 |pmid=26073543 |doi-access=free}}</ref> * Ketogenesis<ref name=":7" /> | * Glutathione synthesis<ref name=":15" /> * Urea cycle<ref name=":15" /> * Arginine biosynthesis<ref name=":15" /> * Oxidoreductase activity<ref name=":15" /> | * Oxioreductase activity<ref name=":8" /> * Amino acid metabolism<ref name=":8" /> * Fatty acid metabolism<ref name=":8" /> * Glutaryl-CoA degregation<ref name=":7" /> * Ketogenesis<ref name=":7" /> | * Oxioreductase activity<ref name=":8" /> * Amino acid metabolism<ref name=":8" /> * Fatty acid metabolism<ref name=":8" /> * Glutaryl-CoA degregation<ref name=":7" /> * Ketogenesis<ref name=":7" /> |} </div>

== Malonyl-CoA as donor == {{Main|Malonyl-CoA}} Malonyl-CoA, the donor for lysine malonylation, cannot cross membranes and must be synthesized locally in each cellular compartment.<ref name=":3">{{Cite journal |last1=Bowman |first1=Caitlyn E. |last2=Rodriguez |first2=Susana |last3=Selen Alpergin |first3=Ebru S. |last4=Acoba |first4=Michelle G. |last5=Zhao |first5=Liang |last6=Hartung |first6=Thomas |last7=Claypool |first7=Steven M. |last8=Watkins |first8=Paul A. |last9=Wolfgang |first9=Michael J. |date=June 2017 |title=The Mammalian Malonyl-CoA Synthetase ACSF3 Is Required for Mitochondrial Protein Malonylation and Metabolic Efficiency |journal=Cell Chemical Biology |language=en |volume=24 |issue=6 |pages=673–684.e4 |doi=10.1016/j.chembiol.2017.04.009 |pmc=5482780 |pmid=28479296 |doi-access=free}}</ref>

* In the cytosol, acetyl-CoA carboxylase (ACC) generates malonyl-CoA from acetyl-CoA and CO₂ and is responsible for the majority of the cellular malonyl-CoA pool.<ref name=":2">{{Cite journal |last1=Bowman |first1=Caitlyn E. |last2=Wolfgang |first2=Michael J. |date=January 2019 |title=Role of the malonyl-CoA synthetase ACSF3 in mitochondrial metabolism |journal=Advances in Biological Regulation |language=en |volume=71 |pages=34–40 |doi=10.1016/j.jbior.2018.09.002 |pmc=6347522 |pmid=30201289}}</ref> The amount of malonyl-CoA in the cytosol is tightly regulated by the opposing activities of ACC and malonyl-CoA decarboxylase (MCD), which catalyzes the reverse reaction to produce acetyl-CoA and CO₂.<ref name=":4">{{Cite journal |last1=Colak |first1=Gozde |last2=Pougovkina |first2=Olga |last3=Dai |first3=Lunzhi |last4=Tan |first4=Minjia |last5=te Brinke |first5=Heleen |last6=Huang |first6=He |last7=Cheng |first7=Zhongyi |last8=Park |first8=Jeongsoon |last9=Wan |first9=Xuelian |last10=Liu |first10=Xiaojing |last11=Yue |first11=Wyatt W. |last12=Wanders |first12=Ronald J.A. |last13=Locasale |first13=Jason W. |last14=Lombard |first14=David B. |last15=de Boer |first15=Vincent C.J. |date=November 2015 |title=Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation |journal=Molecular & Cellular Proteomics |language=en |volume=14 |issue=11 |pages=3056–3071 |doi=10.1074/mcp.M115.048850 |pmc=4638046 |pmid=26320211 |doi-access=free}}</ref> Cytosolic malonyl-CoA plays a key role in regulating fatty acid metabolism.<ref name=":2" /> Although malonyl-CoA itself cannot enter mitochondria, malonate produced through non-enzymatic hydrolysis of cytosolic malonyl-CoA may cross membranes and contribute to the mitochondrial malonyl-CoA pool.<ref name=":2" /> * In mitochondria, the malonyl-CoA pool is generated by acyl-CoA synthetase family member 3 (ACSF3), which catalyzes the thioesterification of malonate and CoA, and by a mitochondrial isoform of acetyl-CoA carboxylase 1 (mtACC1), which produces malonyl-CoA through the carboxylation of acetyl-CoA and CO₂.<ref name=":6">{{Cite journal |last1=Monteuuis |first1=Geoffray |last2=Suomi |first2=Fumi |last3=Kerätär |first3=Juha M. |last4=Masud |first4=Ali J. |last5=Kastaniotis |first5=Alexander J. |date=2017-11-15 |title=A conserved mammalian mitochondrial isoform of acetyl-CoA carboxylase ACC1 provides the malonyl-CoA essential for mitochondrial biogenesis in tandem with ACSF3 |url=https://portlandpress.com/biochemj/article/474/22/3783/49536/A-conserved-mammalian-mitochondrial-isoform-of |journal=Biochemical Journal |language=en |volume=474 |issue=22 |pages=3783–3797 |doi=10.1042/BCJ20170416 |issn=0264-6021 |pmid=28986507 |url-access=subscription}}</ref> Complementing these synthetic activities, MCD likeweise operates in mitochondria, where it converts malonyl-CoA back to acetyl-CoA and CO₂.<ref name=":3" /> Mitochondrial malonyl‑CoA is essential for local protein malonylation as well as for mitochondrial fatty acid synthesis (mtFAS).<ref name=":3" /><ref name=":6" /> * In the nucleus, malonyl-CoA is synthesized by ACC1, which is mainly cytoplasmic, suggesting a local and possibly unconventional function.<ref name=":1" /> The extent of malonylation increases with malonyl‑CoA availability particularly under conditions such as metabolic stress or enzyme deficiencies, for example malonyl‑CoA decarboxylase deficiency.<ref name=":4" /><ref>{{Cite journal |last1=Cheng |first1=Mei-Ling |last2=Yang |first2=Cheng-Hung |last3=Wu |first3=Pei-Ting |last4=Li |first4=Yi-Chin |last5=Sun |first5=Hao-Wei |last6=Lin |first6=Gigin |last7=Ho |first7=Hung-Yao |date=2023-02-23 |title=Malonyl-CoA Accumulation as a Compensatory Cytoprotective Mechanism in Cardiac Cells in Response to 7-Ketocholesterol-Induced Growth Retardation |journal=International Journal of Molecular Sciences |language=en |volume=24 |issue=5 |page=4418 |doi=10.3390/ijms24054418 |issn=1422-0067 |pmc=10002498 |pmid=36901848 |doi-access=free}}</ref>

== Mechanism == Non-enzymatic malonylation occurs spontaneously through direct transfer of a malonyl group from malonyl‑CoA to the ε-amino group (–NH₂) of a deprotonated lysine residue, without enzyme involvement.<ref name=":14" /> Only the deprotonated lysine residue can react in this way because its ε-amino group carries a free electron pair that can attack the carbonyl carbon of the highly reactive malonyl-CoA thioester, whose electron-withdrawing carboxyl group further increases its reactivity.<ref name=":14" /> Since the lysine residue has a pK_a of about 10.5, however, it exists almost entirely in its protonated form at physiological pH (~7.4), with less than 0.1% deprotonated as calculated from the Henderson–Hasselbalch equation. Local protein microenvironments, such as near negatively charged residues or within hydrophobic pockets, can additionally enable lysine deprotonation, while broader conditions such as the more alkaline pH (~8.0) of the mitochondrial matrix increase the fraction of deprotonated lysine residues to about 0.3%,{{refn|name=help1|group=note|as calculated from the Henderson–Hasselbalch equation}} thereby favoring non-enzymatic malonylation.<ref name=":14" /><ref name=":1" /> In compartments with near-neutral pH (~7.2), such as the cytosol or nucleus, lysine residues are therefore almost fully protonated and rely more on enzymatic malonylation there, suggesting that both mechanisms contribute to the overall malonylation pattern in cells.<ref name=":1" />

In enzymatic malonylation, protonated lysine residues (–NH₃⁺), which is the form in which they almost all exist (≈ 99.9%){{refn|name=help1|group=note|as calculated from the Henderson–Hasselbalch equation}} at physiological pH (~7.4), can also be modified.<ref name=":1" /><ref name=":22" /> Structural similarities between acetyl-CoA and malonyl-CoA suggest that certain lysine acetyltransferases (KATs) may also catalyze malonylation.<ref name=":8" /> KAT2A (GCN5) has been experimentally linked to histone malonylation and is currently the strongest candidate, while p300 has also been proposed and is known to mediate other acyl modifications such as crotonylation.<ref name=":1" /><ref name=":0" /> Analogous to the GCN5 acetylation mechanism, the ε-amino group is thought to be transiently deprotonated by a catalytic base within the enzyme's active site, thereby enabling the same reaction with malonyl-CoA as in non-enzymatic malonylation.<ref name=":22">{{Cite journal |last1=Albaugh |first1=Brittany N. |last2=Denu |first2=John M. |date=February 2021 |title=Catalysis by protein acetyltransferase Gcn5 |journal= Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms|volume=1864 |issue=2 |doi=10.1016/j.bbagrm.2020.194627 |issn=1876-4320 |pmc=7854473 |pmid=32841743 |article-number=194627}}</ref><ref name=":1" /> However, specific enzymes known as malonyltransferases have not yet been definitively identified.<ref name=":1" />

The demalonylation is catalyzed by the enzyme Sirtuin 5 (SIRT5), a class III histone deacetylase that requires NAD⁺ for activity but is inhibited by nicotinamide.<ref name=":11" /> SIRT5 is globally expressed in mitochondrial, cytoplasmic, and nuclear compartments, and can also remove other negatively charged acyl modifications.<ref name=":8" /><ref name=":15">{{Cite journal |last1=Head |first1=PamelaSara E. |last2=Myung |first2=Sangho |last3=Chen |first3=Yong |last4=Schneller |first4=Jessica L. |last5=Wang |first5=Cindy |last6=Duncan |first6=Nicholas |last7=Hoffman |first7=Pauline |last8=Chang |first8=David |last9=Gebremariam |first9=Abigael |last10=Gucek |first10=Marjan |last11=Manoli |first11=Irini |last12=Venditti |first12=Charles P. |date=2022-05-25 |title=Aberrant methylmalonylation underlies methylmalonic acidemia and is attenuated by an engineered sirtuin |journal=Science Translational Medicine |language=en |volume=14 |issue=646 |article-number=eabn4772 |doi=10.1126/scitranslmed.abn4772 |issn=1946-6234 |pmc=10468269 |pmid=35613279}}</ref> It catalyzes the demalonylation in the following reaction:<ref name=":8" /> {{center|1=malonyl-lysine-protein + NAD⁺ → lysine-protein + ''O''-malonyl-ADP-ribose + nicotinamide}} Proteomic profiling of mouse liver revealed that SIRT5 regulates about 16% of all identified malonyl-lysine sites, the majority of which contain only a single malonylated lysine residue.<ref name=":7" /> The proteins regulated in this way are mainly involved in glycolysis, gluconeogenesis, fatty acid oxidation, and the urea cycle.<ref name=":7" /> The moderate reduction in malonylation observed upon SIRT5 knockdown in turn suggests the presence of additional, unidentified demalonylases.<ref name=":5" /> It has also been proposed that demalonylases and deacetylases function less as dedicated regulatory enzymes and more as part of a protein quality-control mechanism.<ref name=":2" />

== Malonylated proteins == Proteomic analysis revealed malonylated proteins were enriched in pathways related to glucose and fatty acid metabolism, as well as the urea cycle, involving both mitochondrial and cytosolic enzymes.<ref name=":5" /><ref name=":7" /> Malonylation was also detected on nuclear proteins such as histone H2B.<ref name=":1" />

Below is a list of selected proteins that have been experimentally verified to undergo malonylation:

* Acetyl-CoA carboxylase 1 (ACC1)<ref>{{Cite journal |last1=Cao |first1=Huanyi |last2=Cai |first2=Qingxian |last3=Guo |first3=Wanrong |last4=Su |first4=Qiao |last5=Qin |first5=Hancheng |last6=Wang |first6=Tian |last7=Xian |first7=Yingxin |last8=Zeng |first8=Longyi |last9=Cai |first9=Mengyin |last10=Guan |first10=Haixia |last11=Chen |first11=Sifan |last12=Liang |first12=Hua |last13=Xu |first13=Fen |date=April 2023 |title=Malonylation of Acetyl-CoA carboxylase 1 promotes hepatic steatosis and is attenuated by ketogenic diet in NAFLD |journal=Cell Reports |language=en |volume=42 |issue=4 |article-number=112319 |doi=10.1016/j.celrep.2023.112319 |pmid=37002924 |doi-access=free}}</ref> * Carbamoyl phosphate synthetase 1 (CPS1)<ref name=":7" /><ref name=":12">{{cite bioRxiv |last1=Le Questel |first1=Enora |title=Diurnal regulation of Acyl-CoA synthetase 3 (ACSF3) underlies daily mitochondrial lysine-malonylation and hepatic metabolism |date=2024-09-06 |language=en |biorxiv=10.1101/2024.09.03.607283 |last2=Besnard |first2=Charlène |last3=Atger |first3=Florian |last4=Foucher |first4=Yolène |last5=Tollec |first5=Alwéna |last6=Pakulska |first6=Victoria |last7=Rodrigues Oliveira |first7=Arsênio |last8=Clotteau |first8=Chloé |last9=Gourdel |first9=Mathilde }}</ref> * Carnitine palmitoyltransferase 1 (CPT1)<ref name=":4" /> * Enolase 1 (ENO1)<ref name=":11">{{Cite journal |last1=Peng |first1=Chao |last2=Lu |first2=Zhike |last3=Xie |first3=Zhongyu |last4=Cheng |first4=Zhongyi |last5=Chen |first5=Yue |last6=Tan |first6=Minjia |last7=Luo |first7=Hao |last8=Zhang |first8=Yi |last9=He |first9=Wendy |last10=Yang |first10=Ke |last11=Zwaans |first11=Bernadette M.M. |last12=Tishkoff |first12=Daniel |last13=Ho |first13=Linh |last14=Lombard |first14=David |last15=He |first15=Tong-Chuan |date=December 2011 |title=The First Identification of Lysine Malonylation Substrates and Its Regulatory Enzyme |journal=Molecular & Cellular Proteomics |language=en |volume=10 |issue=12 |article-number=M111.012658 |doi=10.1074/mcp.M111.012658 |pmc=3237090 |pmid=21908771 |doi-access=free}}</ref> * Formyltetrahydrofolate dehydrogenase, 10- (ALDH1L1)<ref name=":12" /> * Fructose bisphosphate aldolase B (ALDOB)<ref name=":5" /> * Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)<ref name=":0" /> * Histone H2B<ref name=":1" /> * Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD)<ref name=":4" /> * Very long-chain acyl-CoA dehydrogenase (VLCAD)<ref name=":4" />

== Clinical relevance == In the metabolic disorder combined malonic and methylmalonic aciduria (CMAMMA), the mitochondrial enzyme ACSF3 is defective, which contributes to the mitochondrial malonyl-CoA pool by converting malonate.<ref name=":6" /> The reduced availability of the donor malonyl-CoA leads to a decrease in mitochondrial lysine malonylation.<ref name=":3" /> In mouse models, this hypomalonylation has been shown to disrupt key metabolic pathways such as glycolysis, gluconeogenesis, fatty acid oxidation and NADPH metabolism, ultimately impairing energy balance.<ref name=":12" />

In the metabolic disorder malonic aciduria, the enzyme malonyl-CoA decarboxylase (MCD) is defective, required for the conversion of malonyl-CoA to acetyl-CoA.<ref>{{Cite journal |last1=Zhang |first1=J. M. |last2=Hao |first2=L. L. |last3=Qiu |first3=W. J. |last4=Zhang |first4=H. W. |last5=Chen |first5=T. |last6=Ji |first6=W. J. |last7=Zhang |first7=Y. |last8=Liu |first8=F. |last9=Gu |first9=X. F. |last10=Yang |first10=S. H. |last11=Han |first11=L. S. |date=2024-10-01 |title=Clinical, biochemical and genetic characteristics and long-term follow-up of five patients with malonyl-CoA decarboxylase deficiency |journal=Brain and Development |language=English |volume=46 |issue=9 |pages=286–293 |doi=10.1016/j.braindev.2024.07.001 |issn=0387-7604 |pmid=39069445 |doi-access=free}}</ref> This leads to accumulation of malonyl-CoA and a marked increase in lysine malonylation.<ref name=":4" /> Proteomic and functional analyses have shown that this hypermalonylation impairs mitochondrial respiration and reduces fatty acid oxidation capacity, suggesting a direct role for protein malonylation in the disease's metabolic dysfunction.<ref name=":4" /> Clinical similarities between MCD and ACSF3 defects suggest their involvement in a shared pathway.<ref name=":3" />

Malonylation also occurs on nuclear proteins, including histones, where it regulates chromatin-associated processes.<ref name=":1">{{Cite journal |last1=Zhang |first1=Ran |last2=Bons |first2=Joanna |last3=Scheidemantle |first3=Grace |last4=Liu |first4=Xiaojing |last5=Bielska |first5=Olga |last6=Carrico |first6=Chris |last7=Rose |first7=Jacob |last8=Heckenbach |first8=Indra |last9=Scheibye-Knudsen |first9=Morten |last10=Schilling |first10=Birgit |last11=Verdin |first11=Eric |date=March 2023 |title=Histone malonylation is regulated by SIRT5 and KAT2A |journal=iScience |language=en |volume=26 |issue=3 |article-number=106193 |doi=10.1016/j.isci.2023.106193 |pmc=9985052 |pmid=36879797 |bibcode=2023iSci...26j6193Z |doi-access=free}}</ref> Histone malonylation has been shown to increase ribosomal RNA (rRNA) expression and nucleolar size, both of which are features associated with cellular aging.<ref name=":1" /> Aged mouse tissues exhibit globally increased malonylation, potentially due to elevated expression of acetyl-CoA carboxylase and reduced activity of the deacylase SIRT5, which depends on declining NAD⁺ levels.<ref name=":1" /> These findings suggest a role for histone malonylation in the epigenetic regulation of aging processes.<ref name=":1" />

In type 2 diabetes, lysine malonylation is significantly elevated in liver tissue, as shown in obese mouse models such as db/db and ob/ob mice.<ref name=":5" /> Many of the affected proteins are involved in glucose and lipid metabolism, and malonylation of glycolytic enzymes has been shown to suppress their activity, leading to reduced glycolytic flux.<ref name=":5" /><ref name=":7" /> Six of the ten glycolytic enzymes are malonylated at sites regulated by the demalonylase SIRT5, which counteracts this inhibition and may serve as a therapeutic target, along with other yet unidentified enzymes that regulate malonylation.<ref name=":7" /><ref name=":5" />

In osteoarthritis, SIRT5 expression decreases in cartilage during aging while lysine malonylation increases.<ref name=":21">{{Cite journal |last1=Liu |first1=Huanhuan |last2=Binoy |first2=Anupama |last3=Ren |first3=Siqi |last4=Martino |first4=Thomas C. |last5=Miller |first5=Anna E. |last6=Willis |first6=Craig R. G. |last7=Veerabhadraiah |first7=Shivakumar R. |last8=Bons |first8=Joanna |last9=Rose |first9=Jacob P. |last10=Schilling |first10=Birgit |last11=Jurynec |first11=Michael J. |last12=Zhu |first12=Shouan |date=September 2025 |title=Regulation of Chondrocyte Metabolism and Osteoarthritis Development by Sirt5 Through Protein Lysine Malonylation |journal=Arthritis & Rheumatology |language=en |volume=77 |issue=9 |pages=1216–1227 |doi=10.1002/art.43164 |issn=2326-5191 |pmc=12354001 |pmid=40176311 |doi-access=free}}</ref> In mice, the combination of Sirt5 deficiency and high-fat diet–induced obesity exacerbates joint degeneration, accompanied by widespread hypermalonylation of glycolytic enzymes and impaired chondrocyte metabolism.<ref name=":21" /> A rare missense mutation in human SIRT5 (F101L) found in familial osteoarthritis further confirms a direct link between hypermalonylation and osteoarthritis.<ref name=":21" />

In resting macrophages, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds to inflammatory mRNAs such as TNFα and suppresses their translation.<ref name=":0" /> In activated macrophages, lysine malonylation functions as a regulatory signal during inflammatory responses.<ref name=":0" /> Inflammatory stimulation with lipopolysaccharide (LPS) increases cytosolic malonyl-CoA levels and leads to malonylation of GAPDH at lysine 213.<ref name=":0" /> Malonylation disrupts this binding, thereby promoting translation of pro-inflammatory cytokines like TNFα.<ref name=":0" /> These findings establish lysine malonylation as a link between cellular metabolism and immune activation.<ref name=":0" />

Inhibition of fatty acid synthase (FASN) increases malonyl-CoA levels in endothelial cells, leading to lysine malonylation of mTOR at lysine 1218.<ref name=":9">{{Cite journal |last1=Bruning |first1=Ulrike |last2=Morales-Rodriguez |first2=Francisco |last3=Kalucka |first3=Joanna |last4=Goveia |first4=Jermaine |last5=Taverna |first5=Federico |last6=Queiroz |first6=Karla C.S. |last7=Dubois |first7=Charlotte |last8=Cantelmo |first8=Anna Rita |last9=Chen |first9=Rongyuan |last10=Loroch |first10=Stefan |last11=Timmerman |first11=Evy |last12=Caixeta |first12=Vanessa |last13=Bloch |first13=Katarzyna |last14=Conradi |first14=Lena-Christin |last15=Treps |first15=Lucas |date=December 2018 |title=Impairment of Angiogenesis by Fatty Acid Synthase Inhibition Involves mTOR Malonylation |journal=Cell Metabolism |language=en |volume=28 |issue=6 |pages=866–880.e15 |doi=10.1016/j.cmet.2018.07.019 |pmc=8057116 |pmid=30146486 |doi-access=free}}</ref> This impairs the kinase activity of mTOR complex 1, which reduces endothelial proliferation and ultimately leads to impaired angiogenesis.<ref name=":9" /> The effect was seen in both normal vessel development and disease-related angiogenesis, such as retinal neovascularization in a mouse model of retinopathy of prematurity (ROP).<ref name=":9" /> These findings link malonylation to angiogenic regulation via mTOR signaling.<ref name=":9" />

== Research == Malonyllysine is chemically unstable and can decarboxylate to acetyllysine upon heating, which complicates its analysis and may lead to misidentification.<ref name=":10">{{Cite journal |last1=Jing |first1=Yihang |last2=Bergholtz |first2=Sarah E. |last3=Omole |first3=Anthony |last4=Kulkarni |first4=Rhushi A. |last5=Zengeya |first5=Thomas T. |last6=Yoo |first6=Euna |last7=Meier |first7=Jordan L. |date=2022-01-05 |title=Synthesis and Evaluation of a Stable Isostere of Malonyllysine** |journal=ChemBioChem |language=en |volume=23 |issue=1 |article-number=e202100491 |doi=10.1002/cbic.202100491 |issn=1439-4227 |pmc=10262540 |pmid=34652056 |doi-access=free}}</ref> In tandem mass spectrometry, this reaction is associated with a characteristic 44 Da loss corresponding to CO₂ release.<ref name=":11" /> To overcome this, a stable tetrazole-based malonyllysine isostere was developed that resists such decomposition.<ref name=":10" /> It is compatible with peptide synthesis and shows reduced but detectable recognition by malonyl-specific antibodies and SIRT5, allowing studies of malonylation without decarboxylation artifacts.<ref name=":10" />

== See also == * Sirtuine

== Notes == {{Reflist|group=note}}

== References == {{reflist}}{{Protein posttranslational modification}} {{Posttranslational modification}}

Category:Post-translational modification Category:Protein structure Category:Organic reactions Category:Cell biology