Lysine lactylation (Kla) links metabolism and gene regulation and plays a key role in multiple biological processes. However, the regulatory mechanism and functional consequence of Kla remain to be explored. Here, we report that HBO1 functions as a lysine lactyltransferase to regulate transcription. We show that HBO1 catalyzes the addition of Kla in vitro and intracellularly, and E508 is a key site for the lactyltransferase activity of HBO1. Quantitative proteomic analysis further reveals 95 endogenous Kla sites targeted by HBO1, with the majority located on histones. Using site-specific antibodies, we find that HBO1 may preferentially catalyze histone H3K9la and scaffold proteins including JADE1 and BRPF2 can promote the enzymatic activity for histone Kla. Notably, CUT&Tag assays demonstrate that HBO1 is required for histone H3K9la on transcription start sites (TSSs). Besides, the regulated Kla can promote key signaling pathways and tumorigenesis, which is further supported by evaluating the malignant behaviors of HBO1- knockout (KO) tumor cells, as well as the level of histone H3K9la in clinical tissues. Our study reveals HBO1 serves as a lactyltransferase to mediate a histone Kla-dependent gene transcription.

Lysine lactylation (Kla) links lactate metabolism to epigenetic regulation, playing a key role in modulation of gene expression in tumor and immune microenvironment. Our recent study shows that HBO1-mediated histone H3K9la activates the transcription of genes encoding tumorigenesis, suggesting the potential significance of intervening in this Kla site for tumor therapy. Evidence so far indicates that traditional deacetylases can catalyse the removal of Kla, however, the precise demodifying enzyme to histone H3K9la in vivo and functional consequence remain elusive. Herein, we combined an antibody-based proximity labeling approach with mass spectrometry analysis to identify SIRT3 as a major binder to histone H3K9la and showed the specific catalysis of SIRT3 for the removal of lactylation. Molecular docking further revealed the molecular mechanism of the binding of Histone H3K9la to SIRT3. More importantly, SIRT3 can specifically modulate gene transcription by regulating H3K9la, inhibiting the progression of esophageal squamous cancer cells (ESCC). Together, our work identifies the specific delactylase of H3K9la and reveals an H3K9la-mediated molecular mechanism catalysed by SIRT3 for gene transcription regulation in ESCC, and our findings provide an opportunity to investigate the physiological significance of Kla controlled by SIRT3 in cancer.

Tick-borne encephalitis virus (TBEV) is a major dangerous human pathogen, as TBEV infection can cause serious illness that can lead to irreversible neurological sequelae and even death. Langat virus (LGTV), a member of the tick-borne encephalitis virus (TBEV) serogroup, belongs to the family Flaviviridae, genus Flavivirus. Its nonstructural protein 5 (NS5) protein contains a methyltransferase (MTase) domain that can methylate RNA cap structures, which is critical for viral replication. We determined the structure of LGTV NS5 methyltransferase bound to S-adenosyl-L-homocysteine (SAH) at a 1.70 Å resolution. Sequence analysis and structural comparison of homologous MTases suggests that folds and structures are closely conserved throughout Flavivirus species and play important roles. This study provides the key structural information on LGTV MTase and the foundation for research on antiviral drugs targeting LGTV MTase.

Introduction: Flaviviruses, such as dengue, Zika, and Langat virus (LGTV), pose significant global health threats, highlighting the urgent need for broad-spectrum antiviral therapies. This study focuses on the NS3 helicase of LGTV, a key enzyme in viral replication, aiming to elucidate its structure and identify high-potency inhibitors to facilitate rational drug design.Methods: The study employed an integrated approach: 1) Structural Biology: Determining the crystal structure of apo LGTV helicase. 2) Comparative Analysis: Aligning the structure with Zika (ZIKV) and dengue (DENV) virus helicases to assess conformational flexibility. 3) Virtual Screening: Screening 11,027 compounds to identify high-affinity inhibitors. 4) Molecular Modeling: Validating binding modes and stability via molecular docking and dynamics simulations. 5) Experimental Validation: Assessing pan-flavivirus affinity of lead compounds using Isothermal Titration Calorimetry (ITC).Results and discussion: The cloverleaf-shaped architecture of LGTV helicase was resolved, revealing conserved ATP- and RNA-binding sites. LGTV exhibited an intermediate conformational flexibility upon ATP binding compared to ZIKV and DENV helicases. Virtual screening identified six high-affinity hits, notably the repurposed drug Zafirlukast. Zafirlukast demonstrated a dual-targeting mechanism, engaging both the ATPase pocket and RNA-binding cleft. Molecular dynamics confirmed stable binding, and ITC validated its broad-spectrum affinity across flaviviruses. The study establishes the LGTV helicase as a robust model for antiviral development. Zafirlukast emerges as a promising prototype for dual-target inhibitors, capable of simultaneously obstructing ATP hydrolysis and RNA unwinding. These findings provide a strong foundation for designing novel broad-spectrum therapeutics against neurotropic flaviviruses.

Background: Trichinosis is one of the most widespread parasitic infections worldwide. Trichinella spiralis not only infects humans but can also utilize wild anddomestic animals as hosts. The serine protease inhibitors secreted by Trichinella spiralis play a critical role in its invasion and immune evasion. Serpins can effectively inhibit host proteases, although the host can mount a strongimmune response against to these inhibitors. Results: In this study we analyzed the crystal structures of the serine protease inhibitors from Trichinella spiralis and Trichinella pseudospiralis, revealing that both serpins exhibit.structural characteristics typical of serine protease inhibitors. The similarity of both “breach” region and “shutter” region of the two serpins are very high, but the “hinge” region are different, the “hinge” of Tp-serpin is closed, while of Ts-serpin was partially inserted into sheet-A, suggesting that Tp-serpin had higher inhibition activity. Using alpha chymotrypsin as Ts-serpin and Tp-serpin protease targets, the two serpins enzyme inhibition activity were measured separately, by measuring the secondary inhibition rate constant, half inhibitory concentration IC50, inhibition of stoichiometric number parameters and confirmed both the serine protease inhibitory activity, and Tp-serpin slightly higher than that of Ts-serpin, but no inhibition activity of P1-P1’ mutant. Conclusion: In this study, the mechanism of enzyme inhibition activity of serpin was studied by means of structural biology and biochemistry comprehensively. These discoveries provide a theoretical foundation for a deeper understanding of the inhibition mechanisms of serpins and for the development of new drugs and vaccines against Trichinella spiralis infection.