Modification Sites Research Articles

RNA methylase RBM15 facilitates malignant progression of colorectal cancer through regulating E2F2 in an m6A modification-dependent manner.

Recently, RBM15 has emerged as an oncogenic factor in a majority of tumors. However, the mechanism is unclear that accounts for how RBM15-induces colorectal cancer (CRC) progression and it is in need of further study. We determined RBM15 expression through the UALCAN database and RT-qPCR. The role of RBM15 in inducing the malignant and aggressive cancerous phenotype was characterized based on the results of the western blot, RT-qPCR, CCK-8 and transwell assays. The target genes of RBM15 were screened by LinkedOmics. m6A methylation kit was applied to analyze the methylation levels of mRNA. SRAMP website was employed to predict m6A sites of targeted mRNA. RIP, dual luciferase reporter gene and actinomycin D assay were conducted to verify the interactions between RBM15 and its targeted gene, and the presence of m6A modification site of its targeted mRNA, respectively. We confirmed the augmentation of RBM15 expression in CRC, which also has a high clinical diagnostic value for CRC. Functionally, RBM15 silencing clearly restrained malignant cellular processes in CRC cells. Mechanistically, RBM15 bound to E2F2 which increased its m6A binding and stabilized the corresponding E2F2 mRNA formation. Excessive E2F2 largely restored the repression malignant phenotype of tumor cells caused by RBM15 silencing. RBM15 regulated E2F2 in an m6A modification-dependent manner thereby boosting malignant cellular processes in CRC. The RBM15/E2F2 axis may be a novel target for CRC therapy.

2D-CEX-FcRn-MS to Study Structure/Function Relation of mAb Charge Variants.

The automated elucidation of the interplay between monoclonal antibody (mAb) structure and function using two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS) is reported. Charge variants, induced through forced degradation, are resolved by first-dimension (1D) cation-exchange chromatography (CEX) and subsequently collected in loops installed on a multiple heart-cutting valve prior to transfer to second-dimension (2D) neonatal crystallizable fragment receptor (FcRn) affinity chromatography coupled with MS. As such, binding affinity of the latter mAb variants can elegantly be assessed and a first glimpse of identity provided. To maximize MS sensitivity, charge variants are unfolded upon eluting from the 2D affinity column by postcolumn addition of a denaturing solution. Further structural details, i.e., modification sites and chain distribution, are unraveled by a multidimensional LC-MS (mD-LC-MS) setup incorporating 1D CEX and parallel online middle-up and bottom-up LC-MS analysis in the subsequent dimensions. Identified charge variants could be ranked according to their affinity for FcRn. Binding is predominantly impacted by heavy chain (HC) M253 oxidation and to a lesser extend, M429 oxidation. Oxidation of both HCs more drastically affects FcRn interaction compared to single-chain oxidation, and the more oxidation, the less binding. Other modifications, such as HC glycosylation, HC N385/390, and N326 deamidation or HC C-terminal processing, are not shown to affect binding. The streamlined platform is challenged against the established workflow involving offline collection of charge variants and structural and functional assessment by, respectively, LC-MS and enzyme-linked immunosorbent assay (ELISA). A decent correlation is demonstrated between the binding affinity measured with ELISA and 2D FcRn affinity chromatography. In addition, throughput is improved (7-fold), material requirements are substantially reduced (2 orders of magnitude), and sample preparation artifacts and loss are minimized. With the simultaneous determination of mAb structure and function, the current study takes the concept of multiattribute analysis to the next level, thereby contributing to the future development of safer and more effective antibody therapeutics.

ModXNA: A Modular Approach to Parametrization of Modified Nucleic Acids for Use with Amber Force Fields.

Modified nucleic acids have surged as a popular therapeutic route, emphasizing the importance of nucleic acid research in drug discovery and development. Beyond well-known RNA vaccines, antisense oligonucleotides and aptamers can incorporate various modified nucleic acids to target specific biomolecules for various therapeutic activities. Molecular dynamics simulations can accelerate the design and development of these systems with noncanonical nucleic acids by observing intricate dynamic properties and relative stability on the all-atom level. However, modeling these modified systems is challenging due to the time and resources required to parametrize components outside default force field parameters. Here, we present modXNA, a tool to derive and build modified nucleotides for use with Amber force fields. Several nucleic acid systems varying in size and number of modification sites were used to evaluate the accuracy of modXNA parameters, and results indicate the dynamics and structure are preserved throughout the simulations. We detail the protocol for quantum mechanics charge derivation and describe a workflow for implementing modXNA in Amber molecular dynamics simulations, which includes updates and added features to CPPTRAJ.

Phosphorylation of the HMGN1 Nucleosome Binding Domain Decreases Helicity and Interactions with the Acidic Patch.

Intrinsically disordered proteins are abundant in the nucleus and are prime sites for posttranslational modifications that modulate transcriptional regulation. Lacking a defined three-dimensional structure, intrinsically disordered proteins populate an ensemble of several conformational states, which are dynamic and often altered by posttranslational modifications, or by binding to interaction partners. Although there is growing appreciation for the role that intrinsically disordered regions have in regulating protein-protein interactions, we still have a poor understanding of how to determine conformational population shifts, their causes under various conditions, and how to represent and model conformational ensembles. Here, we study the effects of serine phosphorylation in the nucleosome-binding domain of an intrinsically disordered protein - HMGN1 - using NMR spectroscopy, circular dichroism and modelling of protein complexes. We show that phosphorylation induces local conformational changes in the peptide backbone and decreases the helical propensity of the nucleosome binding domain. Modelling studies using AlphaFold3 suggest that phosphorylation disrupts the interface between HMGN1 and the nucleosome acidic patch, but that the models over-predict helicity in comparison to experimental data. These studies help us to build a picture of how posttranslational modifications might shift the conformational populations of disordered regions, alter access to histones, and regulate chromatin compaction.

PreMLS: The undersampling technique based on ClusterCentroids to predict multiple lysine sites.

The translated protein undergoes a specific modification process, which involves the formation of covalent bonds on lysine residues and the attachment of small chemical moieties. The protein's fundamental physicochemical properties undergo a significant alteration. The change significantly alters the proteins' 3D structure and activity, enabling them to modulate key physiological processes. The modulation encompasses inhibiting cancer cell growth, delaying ovarian aging, regulating metabolic diseases, and ameliorating depression. Consequently, the identification and comprehension of post-translational lysine modifications hold substantial value in the realms of biological research and drug development. Post-translational modifications (PTMs) at lysine (K) sites are among the most common protein modifications. However, research on K-PTMs has been largely centered on identifying individual modification types, with a relative scarcity of balanced data analysis techniques. In this study, a classification system is developed for the prediction of concurrent multiple modifications at a single lysine residue. Initially, a well-established multi-label position-specific triad amino acid propensity algorithm is utilized for feature encoding. Subsequently, PreMLS: a novel ClusterCentroids undersampling algorithm based on MiniBatchKmeans was introduced to eliminate redundant or similar major class samples, thereby mitigating the issue of class imbalance. A convolutional neural network architecture was specifically constructed for the analysis of biological sequences to predict multiple lysine modification sites. The model, evaluated through five-fold cross-validation and independent testing, was found to significantly outperform existing models such as iMul-kSite and predML-Site. The results presented here aid in prioritizing potential lysine modification sites, facilitating subsequent biological assays and advancing pharmaceutical research. To enhance accessibility, an open-access predictive script has been crafted for the multi-label predictive model developed in this study.

Asymmetric Nanopore Sensor for Logic Detection of Dam and M.SssI Methyltransferases in Combination of DNA Walker and Autocatalytic Hybridization Reaction.

The detection of DNA methyltransferase (MTase) was crucial for understanding gene expression regulation, cancer mechanisms, and various biological processes, contributing significantly to disease diagnosis and drug development. Herein, a nanopore sensor based on cascaded signal amplification of DNA walker and autocatalytic hybridization reaction (AHR) was developed for the ultrasensitive determination of various MTases. In the presence of Dam MTase, the hairpin structure HD underwent methylation and cleavage by DpnI endonuclease, forming T-DNA fragments. These T-DNA fragments were used to activate the DNA walker, which moved across the surface of magnetic beads step by step, generating a large quantity of initiator I by cleaving the substrate. The initiator I subsequently activated the AHR. The AHR included a hybridization chain reaction (HCR) amplifier and a catalytic hairpin assembly (CHA) convertor. The HCR amplifier generated multiple novel CHA triggers, which activated the CHA convertor. This, in turn, stimulated the HCR amplifier, creating an AHR circuit that resulted in the formation of numerous DNA nanowires. These DNA nanowires were adsorbed onto the G4-PAMAM-modified nanopore surface under the influence of an electric field, thereby altering the surface charge of the nanopore and changing the ionic rectification curve. The detection limit of the Dam MTase nanopore sensor reached 0.0002 U/mL. By modification of the recognition sites of the probes, this nanopore system could also be used for the detection of M.SssI MTase. Moreover, a four-input parallel concatenated logic circuit (AND//INHIBIT-OR) had been constructed and applied for the multivariate detection of Dam MTase and M.SssI MTase, presenting a novel conceptual model for advancing the construction of nanopore logic gate systems and their applications in biosensing.

A Comparison of DNA-Methylation during Protoplast Culture of Ponkan Mandarin (Citrus reticulata Blanco) and Tobacco (Nicotiana tabacum L.)

The epigenetic variation in protoplast regeneration is a topic that has attracted interest recently. To elucidate the role of DNA methylation in the regeneration of protoplasts from the ponkan (Citrus reticulata), this study employs the methylation-sensitive amplification polymorphism (MSAP) molecular marker technique to analyze changes in DNA methylation levels and patterns during the isolation and culture of protoplasts from ponkan and tobacco. Additionally, differential DNA methylation fragments are cloned, sequenced, and subjected to bioinformatics analysis. The results reveal that, for non-regenerable ponkan mesophyll protoplasts, DNA methylation levels increase by 3.98% after isolation and then show a trend of initial decrease followed by an increase during culture. In contrast, for regenerable ponkan callus protoplasts and tobacco mesophyll protoplasts, DNA methylation levels decrease by 1.75% and 2.33%, respectively, after isolation. During culture, the DNA methylation levels of ponkan callus protoplasts first increase and then decrease, while those of tobacco mesophyll protoplasts show an opposite trend of initial decrease followed by an increase. Regarding DNA methylation patterns, ponkan mesophyll protoplasts exhibit primarily hypermethylation changes accompanied by a small amount of gene demethylation, whereas ponkan callus protoplasts are dominated by demethylation changes with some genes undergoing hypermethylation. The methylation exhibits dynamic changes in protoplast isolation regeneration. By recovering, cloning, sequencing, and performing BLASTn alignment analysis on specific methylation modification sites in the ponkan, 18 DNA sequences with high homology are identified which are found to be involved in various biological functions, thereby establishing a foundational basis for genetic editing in protoplasts.

Enzymatically Cyclic Activated Biosensor Based on a Tetrahedral DNA Framework for Precise Tumor in Situ Molecular Imaging.

The development of stimulus-responsive and amplification-based strategies is crucial for achieving improved spatial specificity and enhanced sensitivity in tumor molecular imaging, addressing challenges such as off-tumor signal leakage and limited biomarker content. Therefore, a cyclically activated enzymatic biosensor based on the modification of an AP site within a tetrahedral framework DNA (AP-tFNA) was rationally developed for tumor cell-specific molecular imaging using the endogenous enzyme apurinic/apyrimidinic endonuclease 1 (APE1) as a target, exhibiting superior spatial specificity and high sensitivity. APE1, which predominantly localizes within the nucleus in normal cells but exhibits cytosolic and nucleus expression in cancer cells, can specifically recognize and cleave the AP site in AP-tFNA, resulting in the separation of the fluorophore and quenching group, thereby inducing a fluorescence signal. Additionally, upon completion of the excision of one AP site in AP-tFNA, APE1 is released, thereby initiating a subsequent cycle of hydrolytic cleavage reactions. The experimental results demonstrated that AP-tFNA enables precise differentiation of tumor cells both in vitro and in vivo. In particular, the AP-tFNA can monitor drug resistance in neuroblastoma cells and classify the risk for neuroblastoma patients at the clinical plasma level.

Inhibition of bacteria adhesion and biofilm formation using a precisely structured nitric oxide-releasing coating with repeatedly renewing antimicrobial and antifouling ability

Bacterial adhesion and colonization usually causes the formation of biofilm that cannot be easily eradicated by common antibiotics and disinfectants. Surface covalent immobilization of nitric oxide (NO)-releasing polymer is an effective surface modification strategy to combat the threat from bacteria. Although surface covalent strategy avoided the leaching of toxic NO donors, the existence of hydrophobic NO donors on topsurface could inevitably accelerate the microorganism adhesion and accumulation, and the NO-releasing period of time was still very limited. In this work, we prepared an antimicrobial and antibiofilm surface via tethering a precision-structured NO-releasing copolymer brush to an organic-inorganic hybrid hard coating (> 5H pencil hardness) on one end. The precision-structured diblock copolymer brush was composed of a surface antifouling block and a subsurface antimicrobial block of NO-releasing units. A typical effect of “kill two birds with one stone” was observed, the NO releasing subsurface segment within a polymeric matrix prolongs NO release while also preventing surface fouling caused by hydrophobic NO donors on top surface. The impacts of polymer architecture on bioactivity was detailed investigated, and the block copolymer brush exhibited the optimal inhibitory effects against bacteria colonization and biofilm formation. The developed bioactive diblock copolymer brush was grafted from a hard hybrid persistent coating that embeds considerable initiation sites for surface modification. Thanks to the presence of initiator throughout the hybrid network, the initiation layer could initiate polymerization repeatedly after the polymer brushes are worn off at high pressure. Thus, the coating exhibited repeatedly renewing antimicrobial and antifouling ability after multiple abrasion cycles. This work not only developed a novel strategy for regulating NO releasing via modulating the polymer architecture, but provided a feasible route for obtaining a robust initiator layer for synthesizing polymer brush for antimicrobial and antifouling applications.

Modulating effects of fodder grasses extracts on antibiotic sensitivity and biofilm production in avian pathogenic Escherichia coli strains

Extracts of certain fodder grasses may be viewed as powerful agents against infections induced by avian pathogenic Escherichia coli strains. Here we demonstrated ability of Galega orientalis and Rhaponticum carthamoides extracts, alone or in combination with antibiotics, to inhibit growth, viability and biofilm formation in avian pathogenic Escherichia coli strains with different sensitivity to antibiotics and non-pathogenic laboratory strain E. coli BW25113 as well as its mutant derivatives. Modulation of motility and production of extracellular structures in the presence of the extracts correlated with their anti-biofilm effects. Interestingly, an increase in antibacterial action of kanamycin, streptomycin, ciprofloxacin, and cefotaxime on both biofilms and planktonic cultures of the studied strains was observed in the presence of the extracts, including antibiotic resistant APEC strain #45. The extracts alone showed weak prooxidant activity which could contribute to modification of redox-sensitive sites of various regulatory circuits, resulting to synergetic effects in combination with antibiotics.

Dynamic landscape of m6A modifications and related post-transcriptional events in muscle-invasive bladder cancer

BackgroundMuscle-invasive bladder carcinoma (MIBC) is a serious and more advanced stage of bladder carcinoma. N6-Methyladenosine (m6A) is a dynamic and reversible modifications that primarily affects RNA stability and alternative splicing. The dysregulation of m6A in MIBC can be potential target for clinical interventions, but there have been limited studies on m6A modifications in MIBC and their associations with post-transcriptional regulatory processes.MethodsPaired tumor and adjacent-normal tissues were obtained from three patients with MIBC following radical cystectomy. The additional paired tissues for validation were obtained from patients underwent transurethral resection. Utilizing Nanopore direct-RNA sequencing, we characterized the m6A RNA methylation landscape in MIBC, with a focus on identifying post-transcriptional events potentially affected by changes in m6A sites. This included an examination of differential transcript usage, polyadenylation signal sites, and variations in poly(A) tail length, providing insights into the broader impact of m6A alterations on RNA processing in MIBC.ResultsThe prognostic-related m6A genes and m6A-risk model constructed by machine learning enables the stratification of high and low-risk patients with precision. A novel m6A modification site in the 3’ untranslated region (3’UTR) of IGLL5 gene were identified, characterized by a lower m6A methylation ratio, elongated poly(A) tails, and a notable bias in transcript usage. Furthermore, we discovered two particular transcripts, VWA1-203 and CEBPB-201. VWA1-203 displayed diminished m6A methylation levels, a truncated 3’UTR, and an elongated poly(A) tail, whereas CEBPB-201 showed opposite trends, highlighting the complex interplay between m6A modifications and RNA processing. Source code was provided on GitHub (https://github.com/lelelililele/Nanopore-m6A-analysis).ConclusionsThe state-of-the-art Nanopore direct-RNA sequencing and machine learning techniques enables comprehensive identification of m6A modification and provided insights into the potential post-transcriptional regulation mechanisms on the development and progression in MIBC.

Cell membrane-inspired COF/AAO hybrid nanofluidic membrane with ion current rectification properties for ultrasensitive detection of E. coli

Nanofluidic membranes hold great prospects in sensitive and rapid detection of bioanalysts, while remaining lacking robust and general approaches for specificity. In this work, we introduce a cell membrane-inspired nanofluidic membrane with excellent ion current rectification properties as a simple and robust platform for label-free and ultrasensitive biosensing. The asymmetric nanofluidic membrane is prepared by coupling a two-dimensional covalent organic framework (COF) membrane to an anodic aluminum oxide (AAO) nanochannel via hydrogen bonding. The prepared COF/AAO membrane presents selective ion transport due to the asymmetric structure and anion selectivity of COF. With the large surface area and abundant functional modification sites of COF, further grafting of the recognition unit can be easily achieved to mimic the recognition process of receptors and ligands on cell membranes. As a proof of concept, the application in bacterial detection is demonstrated by covalently bonded Concanavalin A as recognition units. The highly amplified ionic current based on ion current rectification properties of the COF/AAO membrane allows sensitive and selective bioanalysis. Specifically, the proposed asymmetric nanofluidic membrane presents a linear detection range from 10 to 106 CFU/mL with an ultralow detection limit of 7 CFU/mL for E. coli. This work reveals the great potential of COF/AAO hybrid nanofluidic membrane as a sensor for rapid and precise bioanalysis.

PH-Controlled Chemoselective Rapid Azo-Coupling Reaction (CRACR) Enables Global Profiling of Serotonylation Proteome in Cancer Cells.

Serotonylation has been identified as a novel protein posttranslational modification for decades, where an isopeptide bond is formed between the glutamine residue and serotonin through transamination. Transglutaminase 2 (also known as TGM2 or TGase2) was proven to act as the main "writer" enzyme for this PTM, and a number of key regulatory proteins (including small GTPases, fibronectin, fibrinogen, serotonin transporter, and histone H3) have been characterized as the substrates of serotonylation. However, due to the lack of pan-specific antibodies for serotonylated glutamine, the precise enrichment and proteomic profiling of serotonylation still remain challenging. In our previous research, we developed an aryldiazonium probe to specifically label protein serotonylation in a bioorthogonal manner, which depended on a pH-controlled chemoselective rapid azo-coupling reaction. Here, we report the application of a photoactive aryldiazonium-biotin probe for the global profiling of serotonylation proteome in cancer cells. Thus, over 1,000 serotonylated proteins were identified from HCT 116 cells, many of which are highly related to carcinogenesis. Moreover, a number of modification sites of these serotonylated proteins were determined, attributed to the successful application of our chemical proteomic approach. Overall, these findings provided new insights into the significant association between cellular protein serotonylation and cancer development, further suggesting that to target TGM2-mediated monoaminylation may serve as a promising strategy for cancer therapeutics.

Light-Activated Artificial CO2-Reductase: Structure and Activity.

Light-dependent reduction of carbon dioxide (CO2) into value-added products can be catalyzed by a variety of molecular complexes. Here we report a rare example of a structurally characterized artificial enzyme, resulting from the combination of a heme binding protein, heme oxygenase, with cobalt-protoporphyrin IX, with good activity for the photoreduction of CO2 to carbon monoxide (CO). Using a copper-based photosensitizer, thus making the photosystem free of noble metals, a large turnover frequency value of ∼616 h-1, a turnover value of ∼589, after 3 h reaction, and a CO vs H2 selectivity of 72% were obtained, establishing a record among previously reported artificial CO2 reductases. Thorough photophysical studies allowed tracking of reaction intermediates and provided insights into the reaction mechanism. Thanks to a high-resolution crystal structure of the artificial enzyme, both in the absence and in the presence of the protein-bound CO2 substrate, a rational site-directed mutagenesis approach was used to study the effect of some modifications of the active site on the activity.

Platinum modification of metallic cobalt defect sites for efficient electrocatalytic oxidation of 5-hydroxymethylfurfural

Co3O4 possesses both direct and indirect oxidation effects and is considered as a promising catalyst for the oxidation of 5-hydroxymethylfurfural (HMF). However, the enrichment and activation effects of Co3O4 on OH− and HMF are weak, which limits its further application. Metal defect engineering can regulate the electronic structure, optimize the adsorption of intermediates, and improve the catalytic activity by breaking the symmetry of the material, which is rarely involved in the upgrading of biomass. In this work, we prepare Co3O4 with metal defects and load the precious metal platinum at the defect sites (Pt-Vco). The results of in-situ characterizations, electrochemical measurements, and theoretical calculations indicate that the reduction of Co–Co coordination number and the formation of Pt–Co bond induce the decrease of electron filling in the antibonding orbitals of Co element. The resulting upward shift of the d-band center of Co combined with the characteristic adsorption of Pt species synergically enhances the enrichment and activation of organic molecules and OH− species, thus exhibiting excellent HMF oxidation activity (including a lower onset potential (1.14 V) and 19 times higher current density than pure Co3O4 at 1.35 V). In summary, this work explores the adsorption enhancement mechanism of metal defect sites modified by precious metal in detail, provides a new option for improving the HMF oxidation activity of cobalt-based materials, broadens the application field of metal defect based materials, and gives an innovative guidance for the functional utilization of metal defect sites in biomass conversion.

RNA infrastructure profiling illuminates transcriptome structure in crowded spaces.

RNAs fold into compact structures and undergo protein interactions in cells. These occluded environments can block reagents that probe the underlying RNAs. Probes that can analyze structure in crowded settings can shed light on RNA biology. Here, we employ 2'-OH-reactive probes that are small enough to access folded RNA structure underlying close molecular contacts within cells, providing considerably broader coverage for intracellular RNA structural analysis. The data are analyzed first with well-characterized human ribosomal RNAs and then applied transcriptome-wide to polyadenylated transcripts. The smallest probe acetylimidazole (AcIm) yields 80% greater structural coverage than larger conventional reagent NAIN3, providing enhanced structural information in hundreds of transcripts. The acetyl probe also provides superior signals for identifying m6A modification sites in transcripts, particularly in sites that are inaccessible to a standard probe. Our strategy enables profiling RNA infrastructure, enhancing analysis of transcriptome structure, modification, and intracellular interactions, especially in spatially crowded settings.

Research advances in protein lysine 2-hydroxyisobutyrylation: From mechanistic regulation to disease relevance.

Histone lysine 2-hydroxyisobutyrylation (Khib) was identified as a novel posttranslational modification in 2014. Significant progress has been made in understanding its roles in reproduction, development, and disease. Although 2-hydroxyisobutyrylation shares some overlapping modification sites and regulatory factors with other lysine residue modifications, its unique structure suggests distinct functions. This review summarizes the latest advancements in Khib, including its regulatory mechanisms, roles in mammalian physiological processes, and its relationship with diseases. This provides direction for further research on Khib and offers new perspectives for developing treatment strategies for related diseases.

Establishing the Comprehensive Structure−Activity Relationship of the Natural Antibiotic Kibdelomycin/Amycolamicin

Abstract: Kibdelomycin (KBD) and amycolamicin (AMM) are potent natural antibiotics effective against antibiotic‐resistant Gram‐positive pathogens, including vancomycin‐intermediate Staphylococcus aureus (S. aureus, VISA), methicillin‐resistant S. aureus (MRSA), and quinolone‐resistant S. aureus (QRSA). Their antibacterial activity stems from an unprecedented dual mechanism: the lower binding sites occupy the adenosine triphosphate (ATP) binding pocket of bacterial type II topoisomerases, while the upper binding sites disrupt the enzyme dimer interface. This dual action, combined with their unique chemical structures, positions KBD and AMM as promising scaffolds for developing new antibiotics. However, the structure‐activity relationship (SAR) of KBD/AMM remains underexplored due to their highly complex chemical structures. In this study, we utilized total synthesis to produce KBD/AMM analogs with various site modifications and evaluated their antimicrobial activities. Our findings establish the first comprehensive SAR for KBD/AMM, paving the way for the development of novel KBD/AMM‐based antibiotics.

Establishing the Comprehensive Structure-Activity Relationship of the Natural Antibiotic Kibdelomycin/Amycolamicin.

Kibdelomycin (KBD) and amycolamicin (AMM) are potent natural antibiotics effective against antibiotic-resistant Gram-positive pathogens, including vancomycin-intermediate Staphylococcus aureus (S. aureus, VISA), methicillin-resistant S. aureus (MRSA), and quinolone-resistant S. aureus (QRSA). Their antibacterial activity stems from an unprecedented dual mechanism: the lower binding sites occupy the adenosine triphosphate (ATP) binding pocket of bacterial type II topoisomerases, while the upper binding sites disrupt the enzyme dimer interface. This dual action, combined with their unique chemical structures, positions KBD and AMM as promising scaffolds for developing new antibiotics. However, the structure-activity relationship (SAR) of KBD/AMM remains underexplored due to their highly complex chemical structures. In this study, we utilized total synthesis to produce KBD/AMM analogs with various site modifications and evaluated their antimicrobial activities. Our findings establish the first comprehensive SAR for KBD/AMM, paving the way for the development of novel KBD/AMM-based antibiotics.

UBASH3B-mediated MRPL12 Y60 dephosphorylation inhibits LUAD development by driving mitochondrial metabolism reprogramming

BackgroundMetabolic reprogramming plays a pivotal role in tumorigenesis and development of lung adenocarcinoma (LUAD). However, the precise mechanisms and potential targets for metabolic reprogramming in LUAD remain elusive. Our prior investigations revealed that the mitochondrial ribosomal protein MRPL12, identified as a novel mitochondrial transcriptional regulatory gene, exerts a critical influence on mitochondrial metabolism. Despite this, the role and regulatory mechanisms underlying MRPL12’s transcriptional activity in cancers remain unexplored.MethodsHuman LUAD tissues, Tp53fl/fl;KrasG12D-driven LUAD mouse models, LUAD patient-derived organoids (PDO), and LUAD cell lines were used to explored the expression and function of MRPL12. The posttranslational modification of MRPL12 was analyzed by mass spectrometry, and the oncogenic role of key phosphorylation sites of MRPL12 in LUAD development was verified in vivo and in vitro.ResultsMRPL12 was upregulated in human LUAD tissues, Tp53fl/fl;KrasG12D-driven LUAD tissues in mice, LUAD PDO, and LUAD cell lines, correlating with poor patient survival. Overexpression of MRPL12 significantly promoted LUAD tumorigenesis, metastasis, and PDO formation, while MRPL12 knockdown elicited the opposite phenotype. Additionally, MRPL12 deletion in a Tp53fl/fl;KrasG12D-driven mouse LUAD model conferred a notable survival advantage, delaying tumor onset and reducing malignant progression. Mechanistically, we discovered that MRPL12 promotes tumor progression by upregulating mitochondrial oxidative phosphorylation. Furthermore, we identified UBASH3B as a specific binder of MRPL12, dephosphorylating tyrosine 60 in MRPL12 (MRPL12 Y60) and inhibiting its oncogenic functions. The decrease in MRPL12 Y60 phosphorylation impeded the binding of MRPL12 to POLRMT, downregulating mitochondrial metabolism in LUAD cells. In-depth in vivo, in vitro, and organoid models validated the inhibitory effect of MRPL12 Y60 mutation on LUAD.ConclusionThis study establishes MRPL12 as a novel oncogene in LUAD, contributing to LUAD pathogenesis by orchestrating mitochondrial metabolism reprogramming towards oxidative phosphorylation (OXPHOS). Furthermore, it confirms Y60 as a specific phosphorylation modification site regulating MRPL12’s oncogenic functions, offering insights for the development of LUAD-specific targeted drugs and clinical interventions.Graphical