site-specific-lysine-lactylation-via-genetic-code-expansion-e-coli

Site-Specific Lysine Lactylation via Genetic Code Expansion in <em>E. coli </em>and Mammalian Cells

Lactylation is a recently discovered post-translational modification (PTM) with diverse biological functions. Since its initial identification in 2019 as ...

Previous studies have suggested associations between testosterone, genetic factors, and a series of complex diseases, but the associations with the lifespan phenotype, such as health span, remain unclear.

Sex-Specific Associations of Testosterone and Genetic Factors With Health Span.

Peptide drug development has made great progress in the last decade thanks to new production, modification, and analytic technologies. Peptides have been produced and modified using both chemical and biological methods, together with novel design and delivery strategies, which have helped to overcome the inherent drawbacks of peptides and have allowed the continued advancement of this field. A wide variety of natural and modified peptides have been obtained and studied, covering multiple therapeutic areas. This review summarizes the efforts and achievements in peptide drug discovery, production, and modification, and their current applications. We also discuss the value and challenges associated with future developments in therapeutic peptides.

Therapeutic peptides: current applications and future directions.

Lactylation was initially discovered on human histones. Given its nascence, its occurrence on nonhistone proteins and downstream functional consequences remain elusive. Here we report a cyclic immonium ion of lactyllysine formed during tandem mass spectrometry that enables confident protein lactylation assignment. We validated the sensitivity and specificity of this ion for lactylation through affinity-enriched lactylproteome analysis and large-scale informatic assessment of nonlactylated spectral libraries. With this diagnostic ion-based strategy, we confidently determined new lactylation, unveiling a wide landscape beyond histones from not only the enriched lactylproteome but also existing unenriched human proteome resources. Specifically, by mining the public human Meltome Atlas, we found that lactylation is common on glycolytic enzymes and conserved on ALDOA. We also discovered prevalent lactylation on DHRS7 in the draft of the human tissue proteome. We partially demonstrated the functional importance of lactylation: site-specific engineering of lactylation into ALDOA caused enzyme inhibition, suggesting a lactylation-dependent feedback loop in glycolysis.

Cyclic immonium ion of lactyllysine reveals widespread lactylation in the human proteome.

A proximity-enabled protein cross-linking strategy with additional spatiotemporal control is highly desirable. Here, we report an oxidation-induced protein cross-linking strategy involving the incorporation of a vinyl thioether group into proteins in both  and mammalian cells via genetic code expansion. We demonstrated that vinyl thioether can be selectively induced by exogenously added oxidant or by reactive oxygen species from the cellular environment, as well as by photocatalysts, and converted into a Michael acceptor, enabling fluorescence labeling and protein cross-linking.

Oxidation-Induced Protein Cross-Linking in Mammalian Cells.

Genetically encoding proximal-reactive unnatural amino acids (PrUaas), such as fluorosulfate-l-tyrosine (FSY), into natural proteins of interest (POI) confer the POI with the ability to covalently bind to its interacting proteins (IPs). The PrUaa-incorporated POIs hold promise for blocking undesirable POI-IP interactions. Selecting appropriate PrUaa anchor sites is crucial, but it remains challenging with the current methodology, which heavily relies on crystallography to identify the proximal residues between the POIs and the IPs for the PrUaa anchorage. To address the challenge, here, we propose a footprinting-directed genetically encoded covalent binder (footprinting-GECB) approach. This approach employs carbene footprinting, a structural mass spectrometry (MS) technique that quantifies the extent of labeling of the POI following the addition of its IP, and thus identifies the responsive residues. By genetically encoding PrUaa into these responsive sites, POI variants with covalent bonding ability to its IP can be produced without the need for crystallography. Using the POI-IP model, KRAS/RAF1, we showed that engineering FSY at the footprint-assigned KRAS residue resulted in a KRAS variant that can bind irreversibly to RAF1. Additionally, we inserted FSY at the responsive residue in RAF1 upon footprinting the oncogenic KRAS/RAF1, which lacks crystal structure, and generated a covalent binder to KRAS. Together, we demonstrated that by adopting carbene footprinting to direct PrUaa anchorage, we can greatly expand the opportunities for designing covalent protein binders for PPIs without relying on crystallography. This holds promise for creating effective PPI inhibitors and supports both fundamental research and biotherapeutics development.

Carbene Footprinting Directs Design of Genetically Encoded Proximity-Reactive Protein Binders.

Lysine lactylation has recently been discovered and demonstrated to be an essential player in immunity, cancer and neurodegenerative diseases. Genetic code expansion (GCE) technique is powerful in uncovering lactylation functions, since it allows site-specific incorporation of lactyllysine (Klac) into proteins of interest (POIs) in living cells. However, the inefficient uptake of Klac into cells, due to its high hydrophilicity, results in limited expression of lactylated POIs. To address this challenge, here we designed esterified Klac derivatives, exemplified by ethylated Klac (KlacOEt), to enhance Klac's lipophilicity and improve its cellular uptake. The expression level of site-specifically lactylated POIs was doubled using KlacOEt in both  and HEK293T cells. Immunoprecipitation mass spectrometry analysis verified the significantly increased yield of the precisely lactylated fructose-bisphosphate aldolase A using KlacOEt. Furthermore, in conjunction with the Target Responsive Accessibility Profiling approach, we found that lactylation at ALDOA-K147 altered the protein's conformation, which may explain the lactylation-induced reduction in enzyme activity. Together, we demonstrate that, through enhancing the yield of lactylated proteins with Klac esters via GCE, we are able to site-specifically reveal the effects of lactylation on POIs' interactions, conformations and activities using a suite of functional proteomics and biochemical tools.

Lactyllysine Esterification Enables Efficient Lactylprotein Expression via Genetic Code Expansion and Supports Functional Proteomics Studies.

Abnormal Notch1 expression has an important role in tumorigenesis. However, upstream control mechanisms for Notch1 are still insufficiently understood. Acute myeloid leukemia (AML) is one of the most common and lethal blood malignancies with limited possibilities for treatment. Thus, new therapeutic targets are urgently needed to improve current ineffective therapies. Herein, high Annexin A1 (ANXA1) expression is found correlated with hyperproliferation of AML cells, and then ANXA1 is identified as a novel negative regulator of Notch1 function in AML. Mechanistically, ANXA1 directly bound to the intracellular domain of Notch1 (NICD) to target this tumor suppressor for degradation. Furthermore, NICD executed its tumor suppressive function through activation of the p15 promoter. Thus, ablation of the Notch1-p15-mediated tumor suppression by ANXA1 provided a novel mechanism of AML proliferation. In human AML patients, a mutual exclusive relation is discovered between ANXA1 and Notch1/p15, corroborating mechanistic discovery. On the basis of these results, it is reasonably speculated that targeting ANXA1 would provide an effective approach for treatment of AML. In support of this new therapeutic paradigm, provided proof-of-concept data by antagonizing ANXA1 using NICD inhibitory peptides.

Identification of ANXA1 as a Novel Upstream Negative Regulator of Notch1 Function in AML.

Herein, we report a Rh(III)-catalyzed triple C-H activation-annulation of Phe-based peptides with alkynes for the preparation of fluorescent peptides. The robustness of this protocol is reflected by a broad substrate scope, high atom- and step-economy, and excellent chemo- and site-selectivity. An in situ generated polycyclic aromatic hydrocarbon carbocation as a fluorophore exhibits good fluorescence properties (maximum emission wavelength up to 628 nm) and low cell cytotoxicity. The synthetic utility of this method is further demonstrated by versatile product applications in bioconjugation with the protein BSA and specifically targeting lysosomes and mitochondria of live mammalian cells.

Triple C-H Activation/Annulation: In Situ Construction of Fluorescent Peptides.

Protein lactylation is an emerging field. To advance the exploration of its biological functions, here we develop a comprehensive workflow that integrates proteomics to identify lactylated sites, genetic code expansion (GCE) for the expression of site-specifically lactylated proteins in living cells, and an integrated functional analysis (IFA) platform to evaluate their biological effects. Using a combined wet-and-dry-lab proteomics strategy, we identify a conserved lactylation at ALDOA-K147, which we hypothesize plays a significant biological role. Expression of this site-specifically lactylated ALDOA in mammalian cells reveals that this modification not only inhibits enzymatic activity but also induces gain-of-function effects. These effects reshaped ALDOA functionality by enhancing protein stability, promoting nuclear translocation, regulating adhesion-related gene expression, altering cell morphology and modulating ALDOA-interacting proteins. Our findings highlight the utility of the GCE-based workflow in establishing causal relationships between specific lactylation events and both target-specific and cell-wide changes, advancing our understanding of protein lactylation's functional impact.

Genetic code expansion reveals site-specific lactylation in living cells reshapes protein functions.

Dissecting the histological heterogeneity of ovarian carcinosarcoma and high-grade serous ovarian cancer in primary and metastatic tumors by single-cell transcriptomic analysis.

Fluorine-containing organic compounds are vital in bioactive molecules and materials, with allylic -difluorides as key motifs in drugs. Herein, we report an efficient photocatalytic strategy for the synthesis of allylic -difluorides using bench-stable oxime esters as dual alkyl or aryl/iminyl radical precursors. The system exhibits a broad substrate scope with 39-87% yields and gram-scale scalability. Meanwhile, this methodology exhibits high regioselectivity and  selectivity. Additionally, the products can be further transformed into other functionalized products.

Article	Year
Sex-Specific Associations of Testosterone and Genetic Factors With Health Span. Frontiers in endocrinology\| PubMed ID: 34899607	2021
Therapeutic peptides: current applications and future directions. Signal transduction and targeted therapy\| PubMed ID: 35165272	2022
Cyclic immonium ion of lactyllysine reveals widespread lactylation in the human proteome. Nature methods\| PubMed ID: 35761067	2022
Oxidation-Induced Protein Cross-Linking in Mammalian Cells. ACS synthetic biology\| PubMed ID: 37000479	2023
Carbene Footprinting Directs Design of Genetically Encoded Proximity-Reactive Protein Binders. Analytical chemistry\| PubMed ID: 38684118	2024
Lactyllysine Esterification Enables Efficient Lactylprotein Expression via Genetic Code Expansion and Supports Functional Proteomics Studies. Journal of proteome research\| PubMed ID: 39316072	2024
Identification of ANXA1 as a Novel Upstream Negative Regulator of Notch1 Function in AML. Advanced science (Weinheim, Baden-Wurttemberg, Germany)\| PubMed ID: 39447086	2024
Triple C-H Activation/Annulation: In Situ Construction of Fluorescent Peptides. Organic letters\| PubMed ID: 39632563	2024
Genetic code expansion reveals site-specific lactylation in living cells reshapes protein functions. Nature communications\| PubMed ID: 39779673	2025
Dissecting the histological heterogeneity of ovarian carcinosarcoma and high-grade serous ovarian cancer in primary and metastatic tumors by single-cell transcriptomic analysis. Chinese medical journal\| PubMed ID: 40709520	2025
Photocatalytic Radical Relay Strategy for the Synthesis of Allylic -Difluorides via 1,4-Difunctionalization of -Difluorobuta-1,3-dienes. Organic letters\| PubMed ID: 41588581	2026

Nanxi Wang

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