Genetic code expansion reveals site-specific lactylation in living cells reshapes protein function
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Hui Ye
PROVIDER: PXD048127 | iProX | Tue Dec 26 00:00:00 GMT 2023
REPOSITORIES: iProX
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Project description:Still in its infancy, the functions of lactylation remain elusive. To address this, we established a comprehensive workflow for lactylation studies that integrates the discovery of lactylation sites with proteomics, the expression of site-specifically lactylated proteins in living cells via genetic code expansion (GCE), and the evaluation of the resulting biological consequences. Specifically, we developed a wet-and-dry-lab combined proteomics strategy, and identified highly conserved lactylation at ALDOA-K147. Driven by its potential biological significance, we site-specifically expressed this lactylated ALDOA in mammalian cells and interrogated the biological changes. We discovered that it not only inhibited enzyme activity but also elicited gain-of-function effects——it dramatically reshaped the functionality of ALDOA by improving stability, enhancing nuclear translocation and affecting gene expression. Further, we demonstrated broad applicability of this workflow to study distinct histone lactylation sites. Together, we anticipate its wide uses in elucidating causative links between site-specific lactylation and target-centric or cell-wide changes.
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Project description:Toxoplasma gondii, a single-celled protozoan parasite (phylum Apicomplexa) and remains the causative pathogen of one of the most wide-spread infectious diseases, toxoplasmosis. To continuously proliferate in warm-blooded animals, this parasite undergoes repeated cycles of invasion, division and induction of host cell rupture. Lactate, which is derived from cellular metabolic pathways, is widely considered not only an energy source in organisms but also a regulatory molecule that participates in gene activation. Lysine lactylation is a new type of protein posttranslational modification (PTM) that was recently associated with chromatin remodeling, but the lysine lactylation of histone and nonhistone proteins has not yet been studied in parasites. To examine the prevalence and function of lactylation in T. gondii parasites, we mapped the lactylation proteome of proliferating tachyzoite cells and found 1,964 lactylation sites on 955 proteins in the T. gondii RH strain. The lactylated proteins were distributed in multiple subcellular compartments and were closely related to a wide variety of biological processes, including mRNA splicing, the citrate cycle (TCA cycle), aminoacyl-tRNA biosynthesis, glyoxylate and dicarboxylate metabolism, glycolysis/gluconeogenesis, oxidative phosphorylation, RNA transport and the HIF-1 signaling pathway. Lysine lactation might regulate numerous cellular processes to aid the establishment of a hospitable environment that allows the survival, replication and dissemination of the parasite. These study offers the first data of the global lactylation proteome and provides a basis for further functional dissection of important proteins associated with T. gondii development and pathogenicity.
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Project description:An orthogonal aminoacyl-tRNA synthetase/tRNA pair is a key prerequisite for site-specific incorporation of unnatural amino acids. Due to its high codon suppression efficiency and full orthogonality, the pyrrolysyl-tRNA synthetase/pyrrolysyl-tRNA pair is currently the ideal system for genetic code expansion in both eukaryotes and prokaryotes. There is a pressing need to discover or engineer other fully orthogonal translation systems that allow unnatural amino acids with distinct scaffolds and functionalities to be incorporated into a wide range of living organisms efficiently. Here, through rational chimera design by transplanting the key orthogonal components from the pyrrolysine system, we create multiple chimeric tRNA synthetase/chimeric tRNA pairs, including chimera histidine, phenylalanine, and alanine systems. We further show that these engineered chimeric systems are orthogonal and highly efficient with comparable flexibility to the pyrrolysine system. In addition, the chimera phenylalanine system can incorporate a group of phenylalanine, tyrosine and tryptophan analogues efficiently in both E. coli and mammalian cells. These aromatic amino acids analogous exhibit unique properties and characteristics, including fluorescence, post-translation modification. To our knowledge, most of these molecules have never been shown to be incorporated with fully orthogonal pairs. Therefore, these chimera pairs offer the potential for incorporation of de novo unnatural amino acids into target proteins for a variety of applications.
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Project description:Dysregulation of Th17 differentiation was implicated in multiple inflammatory and autoimmune diseases including autoimmune uveitis. In the current study, we have provided evidence indicating that lactate-derived lactylation plays important roles in regulating Th17 differentiation. The lactylation level of CD4+ T cells was upregulated in EAU mice and inhibiting lactylation resulted in impaired EAU progression. We characterized the global lactylome of CD4+ T cells of normal and EAU mice. We found that the differentially lactylated proteins were enriched in pathways related to immune responses including leukocyte differentiation. Importantly, our results show that the lactylation level of Ikzf1 (K164) functions in regulating Th17 differentiation by differentially modulating gene expression patterns which are related to CD4+ T cell differentiation by CUT& Tag analysis. In view of the above mentioned well-documented evidence, Ikzf1 lactylation might represent an important regulator for Th17 differentiation in autoimmune uveitis.
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Project description:Protein post-translational modification regulates near every aspect of cellular processes in eukaryotes. However, the identification of new protein post-translational modification is very challenging. Here, using genetically encoded unnatural amino acids as chemical probes, we report the identification and validation of a previously unreported form of protein post-translational modification, aminoacylated lysine ubiquitination, in which the modification occurs on the α-amine group of aminoacylated lysine. We identify more than two thousands of unique aminoacylated lysine ubiquitination sites on all 20 aminoacylated lysines in two human cell lines. The aminoacylated lysine ubiquitination modifications can mediate rapid protein degradation, complementing the canonical lysine ubiquitination-mediated proteome degradation. Furthermore, we demonstrate that the ubiquitin conjugating enzyme UBE2W acts as a writer of aminoacylated lysine ubiquitination and facilitates the ubiquitination of internal α-amine groups on proteins using an unexpected enzymatic activity. More broadly, the discovery and validation of aminoacylated lysine ubiquitination paves the way for the identification and verification of new protein post-translation modifications with the genetic code expansion strategy.
2022-07-20 | PXD035166 |
Project description:We developed a method using site-specific genetic code expansion strategy in conjunction with APEX2-mediated proximity labeling to decipher interactors of hPTMs in living cells via quantitative proteomics. The method allows site-specific incorporation of a PTM-modified amino acid into histones that fused to APEX2, facilitating the labeling of the PTM-modified histones to binding proteins in vivo. Thus, this system will be a promising alternative strategy for interpreting the interactomes of hPTMs in cellular contexts.
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Project description:Aggregation of phosphorylated tau is associated with cognitive impairment in Alzheimers disease patients. In AD a metabolic shift due to the Warburg effect results in increased lactate production. Lactate can induce a post-translational modification on proteins that conjugates lactyl groups to lysine residues known as lactylation. Our findings suggest that tau lactylation links metabolic dysregulation with tauopathies and could serve as a novel diagnostic and therapeutic target.
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Project description:Longstanding evidence implicates glioma stem cells (GSCs) as the major driver for glioma propagation and recurrence. GSCs have a distinctive metabolic landscape characterized by elevated glycolysis. Lactate accumulation resulting from enhanced glycolytic activity can drive lysine lactylation to regulate protein functions, suggesting that elucidating the lactylation landscape in GSCs could provide insights into glioma biology. Herein, we demonstrated that global lactylation was significantly elevated in GSCs compared to differentiated glioma cells (DGCs). PTBP1, a central regulator of RNA processing, was hyperlactylated in GSCs, and SIRT1 induced PTBP1 delactylation. PTBP1-K436 lactylation supported glioma progression and GSC maintenance. Mechanistically, K436 lactylation inhibited PTBP1 proteasomal degradation by attenuating the interaction with TRIM21. Moreover, PTBP1 lactylation enhanced its RNA-binding capacity and facilitated PFKFB4 mRNA stabilization, which further increased glycolysis. Together, these findings uncovered a lactylation-mediated mechanism in GSCs driven by metabolic reprogramming that induces aberrant epigenetic modifications to further stimulate glycolysis, resulting in a vicious cycle to exacerbate tumorigenesis.
2024-11-05 | GSE262995 | GEO
Project description:A generalizable strategy with programmable site-specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a Single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications, and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and β-hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation, and established linkages for chromatin acylation mark-defined proteome, genome and functions. This led to the identification of GLYR1 as a distinct interacting protein in modulating H3K56cr’s gene body localization as well as the discovery of an elevated super-enhancer repertoire underlying bhb-mediated chromatin modulations. SiTomics offers a platform technology for elucidating the “metabolites-modification-regulation” axis, which is widely applicable for multi-omics profiling and functional dissection of modifications beyond acylations and proteins beyond histones.
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Project description:A generalizable strategy with programmable site-specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a Single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications, and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and β-hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation, and established linkages for chromatin acylation mark-defined proteome, genome and functions. This led to the identification of GLYR1 as a distinct interacting protein in modulating H3K56cr’s gene body localization as well as the discovery of an elevated super-enhancer repertoire underlying bhb-mediated chromatin modulations. SiTomics offers a platform technology for elucidating the “metabolites-modification-regulation” axis, which is widely applicable for multi-omics profiling and functional dissection of modifications beyond acylations and proteins beyond histones.
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