Methylation Step Research Articles

The Role of Exogenous microRNAs on Human Health: The Plant-Human Trans-Kingdom Hypothesis.

MicroRNAs (miRNAs) are small, endogenous, single-stranded RNAs that act on gene silencing at the post-transcriptional level by binding to a target messenger RNA (mRNA), leading to its degradation or inhibiting translation into functional proteins. The key role of miRNAs in development, proliferation, differentiation andapoptosis has been deeply investigated, revealing that deregulation in their expression is critical in various diseases, such as metabolic disorders and cancer. Since these small molecules initially evolved as a mechanism of protection against viruses and transposable elements, the fascinating hypothesis that they can move between organisms both of the same or different species has been postulated. Trans-kingdom is the term used to define the migration that occurs between species. This mechanism has been well analyzed between plants and their pests, in order to boost defense and increase pathogenicity, respectively. Intriguingly, in the last decades, the plant-human trans-kingdom migration via food intake hypothesis arose. In particular, various studies highlighted the ability of exogenous miRNAs, abundant in the mainly consumed plant-derived food, to enter the human body affecting gene expression. Notably, plant miRNAs can resist the strict conditions of the gastrointestinal tract through a methylation step that occurs during miRNA maturation, conferring high stability to these small molecules. Recent studies observed the anti-tumoral, immune modulator and anti-inflammatory abilities of trans-kingdom interaction between plant and human. Here, we depict the existing knowledge and discuss the fascinating plant-human trans-kingdom interaction, highlighting first the eventual role of plant miRNAs from foods on our somatic gene identity card and then the potential impact of using plant miRNAs as novel therapeutic avenues.

Structural insights into phosphatidylethanolamine N-methyltransferase PmtA mediating bacterial phosphatidylcholine synthesis.

Phosphatidylethanolamine N-methyltransferase (PmtA) catalyzes the biosynthesis of phosphatidylcholine (PC) from phosphatidylethanolamine (PE). Although PC is one of the major phospholipids constituting bilayer membranes in eukaryotes, certain bacterial species encode PmtA, a membrane-associated methyltransferase, to produce PC, which is correlated with cellular stress responses, adaptability to environmental changes, and symbiosis or virulence with eukaryotic hosts. Depending on the organism, multiple PmtAs may be required for producing monomethyl- and dimethyl-PE derivatives along with PC, whereas in organisms such as Rubellimicrobium thermophilum, a single enzyme is sufficient to direct all three methylation steps. In this study, we present the x-ray crystal structures of PmtA from R. thermophilum in complex with dimethyl-PE and S-adenosyl-l-homocysteine, as well as in its lipid-free form. Moreover, we demonstrate that the enzyme associates with the cellular membrane via electrostatic interactions facilitated by a group of critical basic residues and can successively methylate PE and its methylated derivatives, culminating in the production of PC.

An Alternative Method for Synthesizing N,2,3-Trimethyl-2H-indazol-6-amine as a Key Component in the Preparation of Pazopanib

Due to its application as an anti-cancer drug, pazopanib (1) has attracted the interest of many researchers, and several studies on pazopanib synthesis have been reported over the years. This paper provides a novel route for synthesizing N,2,3-trimethyl-2H-indazol-6-amine (5), which is a crucial building block in the synthesis of pazopanib from 3-methyl-6-nitro-1H-indazole (6). By alternating between the reduction and two methylation steps, compound 5 was obtained in a yield comparable (55%) to what has been reported (54%). It is noteworthy that the last step of N2-methylation also yielded N,N,2,3-tetramethyl-2H-indazol-6-amine (5′) as a novel compound. Furthermore, the data presented in this paper can serve as a valuable resource for future research aimed at further refining the process of synthesizing pazopanib and its derivatives.

Lysine methylation steps into another step of the central dogma.

Lysine methylation steps into another step of the central dogma.

Continuous Flow Synthesis of the Nerve Agent Antidote Pralidoxime (2‐PAM) Iodide

AbstractPralidoxime salts are important medical countermeasures towards different organophosphorus compounds, either nerve agents or pesticides, as they act as acetylcholinesterase reactivators, avoiding health issues and even death that can result from intoxication with such chemicals. In this work, we present an expeditious study focuses on the optimization of a two‐step continuous‐flow synthesis of 2‐PAM. Initial attempts to replace the solvent composition in the oxime formation reaction with acetonitrile resulted in lower yields, highlighting the significance of a polar protic solvent. Subsequent optimization under continuous‐flow conditions revealed the successful execution of oxime formation at room temperature with a residence time of 20 minutes, achieving an impressive 96 % conversion, surpassing literature‐reported conditions. The methylation step required a high residence time of 60 minutes at 120 °C for a substantial conversion of 78 %. The two‐step continuous‐flow process demonstrated robustness, yielding 75 %, with space‐time yields of 0.32 g/h and 0.18 g/h. Further concentration adjustments increased space‐time yields to 3.2 g/h and 2.2 g/h. Despite solvent incompatibility, a strategic alteration of the reaction sequence allowed for a successful cascade process, integrating the methylation step in acetonitrile and the oxime formation in water. The continuous‐flow system, incorporating a heat exchanger, exhibited stability, maintaining full conversion and a space‐time yield of 0.3 g/h over extended periods. This systematic optimization not only enhances the efficiency of 2‐PAM synthesis but also provides insights into a scalable and practical continuous‐flow cascade process.

The seeds of its regulation: Natural antisense transcripts as single-gene control switches in neurodegenerative disorders

Several proteins play critical roles in vulnerability or resistance to neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and frontotemporal dementia (FTD). Regulation of these proteins is critical to maintaining healthy neurohomeostasis. In addition to transcription factors regulating gene transcription and microRNAs regulating mRNA translation, natural antisense transcripts (NATs) regulate mRNA levels, splicing, and translation. NATs’ roles are significant in regulating key protein-coding genes associated with neurodegenerative disorders. Elucidating the functions of these NATs could prove useful in treating or preventing diseases. NAT activity is not restricted to mRNA translation; it can also regulate DNA (de)methylation and other gene expression steps. NATs are noncoding RNAs (ncRNAs) encoded by DNA sequences overlapping the pertinent protein genes. These NATs have complex structures, including introns and exons, and therefore bind their target genes, precursor mRNAs (pre-mRNAs), and mature RNAs. They can occur at the 5’- or 3’-ends of a mRNA-coding sequence or internally to a parent gene. NATs can downregulate translation, e.g., microtubule-associated protein tau (MAPT) antisense-1 gene (MAPT-AS1), or upregulate translation, e.g., β-Amyloid site Cleaving Enzyme 1 (BACE1) antisense gene (BACE1-AS). Regulation of NATs can parallel pathogenesis, wherein a “pathogenic” NAT (e.g., BACE1-AS) is upregulated under pathogenic conditions, while a “protective” NAT (e.g., MAPT-AS1) is downregulated under pathogenic conditions. As a relatively underexplored endogenous control mechanism of protein expression, NATs may present novel mechanistic targets to prevent or ameliorate aging-related disorders.

Determination of fatty acid composition of Silurus triostegus Heckel, 1843 and Arabibarbus grypus Heckel, 1843 species living in Ilısu Dam Lake (Batman/TÜRKİYE)

In this study, the fatty acid compounds in the muscle tissues of Mesopotamian catfish (Silurus triostegus, Heckel, 1843) and Shabut (Arabibarbus grypus, Heckel, 1843), which live in Ilısu Dam Lake on the Tigris River and have economic importance. For fatty acid analysis, muscle tissues were taken from the samples obtained from local fishermen, placed in tubes and lipids were obtained by applying chloroform/methanol method. The methylation steps of fatty acids in the samples were performed in accordance with TS EN ISO 12966:2 (TS EN ISO 12966-2., 2017) method. The total fatty acid content of saturated fatty acids (SFA) value was detected as 27,97 ± 0,26 in A.grypus, 32.46 ± 0.37 in S. triostegus, The value of Monounsaturated Fatty Acids (MUFA) varied between 42.63 ± 0.6% in A. grypus and 41.5 ± 0.51% in S. triostegus. Total polyunsaturated polyunsaturated acids (PUFA) ranged between 21.83 ± 1.49 for A. grypus and 25.82 ± 1.94 for S. triostegus. MUFA values in all samples were higher than SFA and PUFA values for both fish species. Docosa Hexaenoic Acid (DHA) and Eicosa Pentaenoic Acid (EPA) are commonly consumed omega-3 types. A. grypus and S. triostegus are within the recommended limits for EPA (C20:5n3) and DHA (C22:6n3). For A. grypus, ω3/ω6 values were (1,06); for S. triostegus ω3/ω6 values were (1,38). When the ω3/ω6 values of both fish species were analysed for human health, it was determined that the ω3/ω6 values of omega fatty acids A. grypus and S. triostegus were above 1. As a result information on the composition of 20 types of fatty acids and ω3/ω6 amounts were obtained in muscle tissue of A. grypus and S. triostegus species living in Ilısu Dam Lake and most preferred by local people. The levels of fat and its components in these species, which may cause different problems when accumulated in the body as a result of changing consumption habits, were indicated in the study.

Electrophilic Functionalization of a Hexaphosphabenzene Ligand in [(Cp*Mo)2(μ,η6 : 6-P6)

The electrophilic functionalization of the triple-decker sandwich complex [{Cp*Mo}2(μ,η6:6-P6)] (A) and its mono-oxidized counterpart [{Cp*Mo}2(μ,η6:6-P6)][SbF6] (B) with reactive main-group electrophiles as well as radical scavengers is shown to be a reliable method for the selective functionalization of the hexaphosphabenzene ligand. Depending on the electrophile used, the regioselectivity of the functionalization can be adjusted. Using group 16 electrophiles, the trisubstituted compounds [{Cp*Mo}2{(μ,η3 : 3-P3)(μ,η1 : 1 : 1 : 1-1,3-(SePh)2-2-Br-P3)}][TEF] (1), [{Cp*Mo}2(μ,η3 : 3-P3)(μ,η1 : 1 : 1 : 1-1,2,3-(EPh)3-P3)][SbF6] (E=S (2), Se (3)) as well as the side product [{Cp*Mo}2(μ,η4:4-P4)(μ,η1 : 1-P(SPh)2)][SbF6] (4) are obtained. By switching to phosphenium ions as group 15 electrophiles, the ring-inserted products [{Cp*Mo}2(μ,η3 : 3 : 2 : 2-P7R2)][TEF] (R=Cy (5), iPr (6)) are isolated, showing an unprecedented P7R2 structural motif. Furthermore, the reaction with MeOTf yields the dimeric [{Cp*Mo}4(1,4-Me2-μ4,η1 : 1 : 1 : 1 : 1 : 1-P6)(μ,η3 : 3-P3)2][TEF]2 (7) as the first example of a complex featuring two interconnected cyclo-P6 middle deck ligands. Finally, by combination of the methylation step with Ph2Se2, the mixed group 14/16 complex [{Cp*Mo}2{(μ,η3 : 3-P3)(μ,η1 : 1 : 1 : 11,2-(SePh)2-3-Me-P3)}][OTf] (8) is obtained.

Metagenomic insights into phenanthrene biodegradation in electrical field-governed biofilms for groundwater bioremediation

Electrical fields (EFs)-assisted in-situ bioremediation of petroleum-contaminated groundwater, such as polycyclic aromatic hydrocarbons, has drawn increasing attention. However, the long-term stability, the EFs influence, and metabolic pathways are still poorly understood, hindering the further development of robust technology design. Herein, a series of EFs was applied to the phenanthrene-contaminated groundwater, and the corresponding system performance was investigated. The highest removal capacity of phenanthrene (phe) (7.63 g/(m3·d)) was achieved with EF_0.8 V biofilm at a hydrolytic retention time of 0.5 d. All the biofilms with four EFs exhibited a high removal efficiency of phe over 80% during a 100-d continuous-flow operation. Intermediates analysis revealed the main pathways of phe degradation: phthalate and salicylate via hydroxylation, methylation, carboxylation, and ring cleavage steps. Synergistic effects between phe-degraders (Dechloromonas), fermentative bacteria (Delftia), and electroactive microorganisms (Geobacter) were the main contributors to the complete phe mineralization. Genes encoding various proteins of methyl-accepting (mcp), response regulator (cheABDRY), and type IV pilus (pilABCMQV) were dominant, revealing the importance of cell motility and extracellular electron transfer. Metagenomics analysis unveiled phe-degrading genes, including ring reduction enzymes (bamBCDE), carboxylase of aromatics (ubiD), and methyltransferase protein (ubiE, pcm). These findings offered a molecular understanding of refractory organics’ decompositions in EFs-governed biotechnology.

An α, β-unsaturated ketone alkylation and efficient reduction protocol for the synthesis of 3α-hydroxy-3β-methyl-4,4-dimethyl-5α-21-bromo-19-nor-pregnan-20-one

Alkyl-substituted testosterone derivatives are promising platforms for new drug discovery in medicinal chemistry. This approach provides a simple and efficient method for introducing an alkyl substituent to steroids at the C-4 position. In this study, the novel compound 3α-hydroxy-3β-methyl-4,4-dimethyl-5α-21-bromo-19-nor-pregnan-20-one is synthesized from 19-nor-testosterone. The protocol involves methylation of the dienolate, reduction of the alkene, the Grignard reaction of the carbonyl group, a Wittig reaction, hydroboration with BH3, the oxidation and bromination with a 49% overall yield. For the methylation and reduction steps, the effects of the base, solvent, and reactant ratio on the conversion and yield are investigated. The structures of the synthesized compounds are determined by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) (electrospray ionization (ESI)).

Rational Design of Highly Potent SARS-CoV-2 nsp14 Methyltransferase Inhibitors.

The search for new drugs against COVID-19 and its causative agent, SARS-CoV-2, is one of the major trends in the current medicinal chemistry. Targeting capping machinery could be one of the therapeutic concepts based on a unique mechanism of action. Viral RNA cap synthesis involves two methylation steps, the first of which is mediated by the nsp14 protein. Here, we rationally designed and synthesized a series of compounds capable of binding to both the S-adenosyl-l-methionine and the RNA-binding site of SARS-CoV-2 nsp14 N7-methyltransferase. These hybrid molecules showed excellent potency, high selectivity toward various human methyltransferases, nontoxicity, and high cell permeability. Despite the outstanding activity against the enzyme, our compounds showed poor antiviral performance in vitro. This suggests that the activity of this viral methyltransferase has no significant effect on virus transcription and replication at the cellular level. Therefore, our compounds represent unique tools to further explore the role of the SARS-CoV-2 nsp14 methyltransferase in the viral life cycle and the pathogenesis of COVID-19.

Engineering a Carboxyl Methyltransferase for the Formation of a Furan-Based Bioplastic Precursor.

FtpM from Aspergillus fumigatus was the first carboxyl methyltransferase reported to catalyse the dimethylation of dicarboxylic acids. Here the creation of mutant R166M that can catalyse the quantitative conversion of bio-derived 2,5-furandicarboxylic acid (FDCA) to its dimethyl ester (FDME), a bioplastics precursor, was reported. Wild type FtpM gave low conversion due to its reduced catalytic efficiency for the second methylation step. An AlphaFold 2 model revealed a highly electropositive active site, due to the presence of 4 arginine residues, postulated to favour the binding of the dicarboxylic acid over the intermediate monoester. The R166M mutation improved both binding and turnover of the monoester to permit near quantitative conversion to the target dimethyl ester product. The mutant also had improved activity for other diacids and a range of monoacids. R166M was incorporated into 2 multienzyme cascades for the synthesis of the bioplastics precursor FDME from bioderived 5-hydroxymethylfurfural (HMF) as well as from poly(ethylene furanoate) (PEF) plastic, demonstrating the potential to recycle waste plastic.

Integrating machine learning and bioinformatics analysis to m6A regulator-mediated methylation modification models for predicting glioblastoma patients' prognosis and immunotherapy response.

Epigenetic regulations of immune responses are essential for cancer development and growth. As a critical step, comprehensive and rigorous explorations of m6A methylation are important to determine its prognostic significance, tumor microenvironment (TME) infiltration characteristics and underlying relationship with glioblastoma (GBM). To evaluate m6A modification patterns in GBM, we conducted unsupervised clustering to determine the expression levels of GBM-related m6A regulatory factors and performed differential analysis to obtain m6A-related genes. Consistent clustering was used to generate m6A regulators cluster A and B. Machine learning algorithms were implemented for identifying TME features and predicting the response of GBM patients receiving immunotherapy. It is found that the m6A regulatory factor significantly regulates the mutation of GBM and TME. Based on Europe, America, and China data, we established m6Ascore through the m6A model. The model accurately predicted the results of 1206 GBM patients from the discovery cohort. Additionally, a high m6A score was associated with poor prognoses. Significant TME features were found among the different m6A score groups, which demonstrated positive correlations with biological functions (i.e., EMT2) and immune checkpoints. m6A modification was important to characterize the tumorigenesis and TME infiltration in GBM. The m6Ascore provided GBM patients with valuable and accurate prognosis and prediction of clinical response to various treatment modalities, which could be useful to guide patient treatments.

Elucidating the Kinetic Mechanism of Human METTL16.

Methyltransferase-like protein 16 (METTL16) is one of four catalytically active, S-adenosylmethionine (SAM)-dependent m6A RNA methyltransferases in humans. Well-known methylation targets of METTL16 are U6 small nuclear RNA (U6 snRNA) and the MAT2A mRNA hairpins; however, METTL16 binds to other RNAs, including the 3' triple helix of the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Herein, we investigated the kinetic mechanism and biochemical properties of METTL16. METTL16 is a monomer in complex with either the MALAT1 triple helix or U6 snRNA and binds to these RNAs with respective dissociation constants of 31 nM and 18 nM, whereas binding to the methylated U6 snRNA product is 1.1 μM. The MALAT1 triple helix, on the other hand, is not methylated by METTL16 under in vitro conditions. Using the U6 snRNA to study methylation steps, preincubation and isotope partitioning assays indicated an ordered-sequential mechanism, whereby METTL16 binds U6 snRNA before SAM. The apparent dissociation constant for the METTL16·U6 snRNA·SAM ternary complex is 126 μM. Steady-state kinetic assays established a kcat of 0.07 min-1, and single-turnover assays established a kchem of 0.56 min-1. Furthermore, the methyltransferase domain of METTL16 methylated U6 snRNA with an apparent dissociation constant of 736 μM and a kchem of 0.42 min-1, suggesting that the missing vertebrate conserved regions weaken the ternary complex but do not induce any rate-limiting conformational rearrangements of the U6 snRNA. This study helps us to better understand the catalytic activity of METTL16 in the context of its biological functions.

Functional Diversification and Structural Origins of Plant Natural Product Methyltransferases.

In plants, methylation is a common step in specialized metabolic pathways, leading to a vast diversity of natural products. The methylation of these small molecules is catalyzed by S-adenosyl-l-methionine (SAM)-dependent methyltransferases, which are categorized based on the methyl-accepting atom (O, N, C, S, or Se). These methyltransferases are responsible for the transformation of metabolites involved in plant defense response, pigments, and cell signaling. Plant natural product methyltransferases are part of the Class I methyltransferase-superfamily containing the canonical Rossmann fold. Recent advances in genomics have accelerated the functional characterization of plant natural product methyltransferases, allowing for the determination of substrate specificities and regioselectivity and further realizing the potential for enzyme engineering. This review compiles known biochemically characterized plant natural product methyltransferases that have contributed to our knowledge in the diversification of small molecules mediated by methylation steps.

Rapid Synthesis of Kaolinite Nanoscrolls through Microwave Processing.

Kaolinite nanoscrolls (NScs) are halloysite-like nanotubular structures of great interest due to their ability to superimpose halloysite’s properties and applicability. Especially attractive is the ability of these NScs to serve as reaction vessels for the uptake and conversion of different chemical species. The synthesis of kaolinite NScs, however, is demanding due to the various processing steps that lead to extended reaction times. Generally, three intercalation stages are involved in the synthesis, where the second step of methylation dominates others in terms of duration. The present research shows that introducing microwave processing throughout the various steps can simplify the procedure overall and reduce the synthesis period to less than a day (14 h). The kaolinite nanoscrolls were obtained using two final intercalating agents, aminopropyl trimethoxy silane (APTMS) and cetyltrimethylammonium chloride (CTAC). Both produce abundant NScs, as corroborated by microscopy measurements as well as the surface area of the final products; APTMS intercalated NScs were 63.34 m2/g, and CTAC intercalated NScs were 73.14 m2/g. The nanoscrolls averaged about 1 μm in length with outer diameters of APTMS and CTAC intercalated samples of 37.3 ± 8.8 nm and 24.9 ± 6.1 nm, respectively. The availability of methods for the rapid production of kaolinite nanoscrolls will lead to greater utility of these materials in technologically significant applications.

Coculture engineering for efficient production of vanillyl alcohol in Escherichia coli

Vanillyl alcohol is a precursor of vanillin, which is one of the most widely used flavor compounds. Currently, vanillyl alcohol biosynthesis still encounters the problem of low efficiency. In this study, coculture engineering was adopted to improve production efficiency of vanillyl alcohol in E. coli. First, two pathways were compared for biosynthesis of the immediate precursor 3, 4-dihydroxybenzyl alcohol in monocultures, and the 3-dehydroshikimate-derived pathway showed higher efficiency than the 4-hydroxybenzoate-derived pathway. To enhance the efficiency of the last methylation step, two strategies were used, and strengthening S-adenosylmethionine (SAM) regeneration showed positive effect while strengthening SAM biosynthesis showed negative effect. Then, the optimized pathway was assembled in a single cell. However, the biosynthetic efficiency was still low, and was not significantly improved by modular optimization of pathway genes. Thus, coculturing engineering strategy was adopted. At the optimal inoculation ratio, the titer reached 328.9 mg/L. Further, gene aroE was knocked out to reduce cell growth and improve 3,4-DHBA biosynthesis of the upstream strain. As a result, the titer was improved to 559.4 mg/L in shake flasks and to 3.89 g/L in fed-batch fermentation. These are the highest reported titers of vanillyl alcohol so far. This work provides an effective strategy for sustainable production of vanillyl alcohol.

Copper Hydride-Catalyzed Enantioselective Olefin Hydromethylation.

The enantioselective installation of a methyl group onto a small molecule can result in the significant modification of its biological properties. While hydroalkylation of olefins represents an attractive approach to introduce alkyl substituents, asymmetric hydromethylation protocols are often hampered by the incompatibility of highly reactive methylating reagents and a lack of general applicability. Herein, we report an asymmetric olefin hydromethylation protocol enabled by CuH catalysis. This approach leverages methyl tosylate as a methyl source compatible with the reducing base-containing reaction environment, while a catalytic amount of iodide ion transforms the methyl tosylate in situ into the active reactant, methyl iodide, to promote the hydromethylation. This method tolerates a wide range of functional groups, heterocycles, and pharmaceutically relevant frameworks. Density functional theory studies suggest that after the stereoselective hydrocupration, the methylation step is stereoretentive, taking place through an SN2-type oxidative addition mechanism with methyl iodide followed by a reductive elimination.

Two O-Methyltransferases Mediate Multiple Methylation Steps in the Biosynthesis of Coumarins in Cnidium monnieri.

Coumarins with methoxy groups such as osthole (1), xanthotoxin (2), bergapten (3), and isopimpinellin (4) are typical bioactive ingredients of many medicinal plants. The methylation steps remain widely unknown. Herein, we report the discovery of two methyltransferases in the biosynthesis of O-methyl coumarins in Cnidium monnieri by transcriptome mining, heterologous expression, and in vitro enzymatic assays. The results reveal that (i) CmOMT1 catalyzes the methylation of osthenol (8) as the final step in the biosynthesis of 1, (ii) CmOMT2 shows the highest efficiency and preference for methylating xanthotoxol (11) to form 2, and (iii) CmOMT1 and CmOMT2 also efficiently transform bergaptol (10) and 8-hydroxybergapten (13) into 3 or 4, suggesting the CmOMTs mediate multistep methylations in the biosynthesis of linear furanocoumarins in C. monnieri.

Metabolic Labeling-Based Chemoproteomics Establishes Choline Metabolites as Protein Function Modulators.

Choline is an essential nutrient for mammalian cells. Our understanding of the cellular functions of choline and its metabolites, independent of their roles as choline lipid metabolism intermediates, remains limited. In addition to fundamental cellular physiology, this knowledge has implications for cancer biology because elevated choline metabolite levels are a hallmark of cancer. Here, we establish a mammalian choline metabolite-interacting proteome by utilizing a photocrosslinkable choline probe. To design this probe, we performed metabolic labeling experiments with structurally diverse choline analogues that resulted in the serendipitous discovery of a choline lipid headgroup remodeling mechanism involving sequential dealkylation and methylation steps. We demonstrate that phosphocholine inhibits the binding of one of the proteins identified, the attractive anticancer target p32, to its endogenous ligands and to the promising p32-targeting anticancer agent, Lyp-1. Our results reveal that choline metabolites play vital roles in cellular physiology by serving as modulators of protein function.