Characterising somatic mutation profiles in human breast cancer (HBC) is essential for understanding tumour progression and guiding therapeutic strategies. We performed genomic and transcriptomic analyses to profile the mutational landscape of HBC. In addition to our primary analysis of HBC, we conducted comparative genomic analyses to evaluate the extent to which these mutational processes are recapitulated in canine mammary tumour (CMT), a widely proposed translational model. APOBEC3 (A3)-associated mutations were extensive in HBC but largely absent in CMT, likely due to structural differences in A3 proteins and lower basal expression. Transcriptomic stratification of HBC by A3 activity uncovered that tumours with A3 activity showed a strong association with the PAM50-HER2-Enriched (HER2E) subtype, and FGFR4 was significantly correlated with the expression and enzymatic function of APOBEC3A/B. These findings demonstrate that CMT is an unsuitable model for A3-mediated mutagenesis, emphasising the need to consider the limitations of cross-species mutational modelling in comparative oncology. Moreover, this study identifies a potential regulatory association between FGFR4 and A3, offering insights into the underlying mechanisms of A3-driven mutagenesis and suggesting that the FGFR4-A3 activity could be considered alongside other molecular biomarkers for the classification of the HER2E subtype.
Bacteriophage genomes are densely packed with coding sequences and frequently encode genes of unknown function. Unbiased phage functional genomics approaches are therefore needed, particularly for large lytic phages. Here, we harness the mariner transposase to develop phage transposon mutagenesis and sequencing (phage TnSeq), which enables pooled sequencing to identify both fitness-conferring and dispensable genes. Using the Pseudomonas aeruginosa-infecting nucleus-forming jumbo phage ΦKZ (280,334 bp; 371 predicted genes), we show that ∼110 genes are fitness-conferring via phage TnSeq, identifying many known and previously unknown essential genes. Moreover, this phage harbors ∼261 non-essential genes, including some capsid and tail proteins, many of which are important for fitness across different clinical isolates or conditions. Phage TnSeq was also extended to a base-modified phage. Together, phage TnSeq is a scalable technology that can identify essential phage genes, generate knockouts in all non-essential genes, and sensitively assign the quantitative fitness contributions of every gene in parallel.
Industrial-scale production of 6'-sialyllactose (6'-SL), a sialylated human milk oligosaccharide, has recently attracted increasing attention. As a crucial enzyme for both microbial synthesis approaches and enzymatic catalysis, several α2,6-sialyltransferases (α2,6-SiaTs) have been reported and characterized. However, the low expression levels and poor stability of α2,6-SiaTs remain major bottlenecks in the construction of efficient 6'-SL production systems. In this study, we applied a deep-learning-based protein generative model, ProteinMPNN, to redesign an α2,6-SiaT from Photobacterium sp. JT-ISH-224 (pst6-224), resulting in a marked improvement in its heterologous expression in Escherichia coli BL21(DE3). Furthermore, machine learning-guided combinatorial mutagenesis was employed to restore catalytic activity in the redesigned enzyme. The expression level of the final variant was increased by approximately tenfold compared with that of the wild-type pst6-224, and a higher 6'-SL titer was achieved in a 1 L cascade catalytic system starting from CMP. This work demonstrates the potential of machine learning-assisted protein engineering and provides an α2,6-SiaT variant with high expression levels for the biological synthesis of 6'-SL.
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Viral attachment mediated by host cell surface receptors is the first step in viral infection. As a key cell surface receptor, heparan sulfate (HS) mediates the attachment and entry of numerous non-enveloped viruses in livestock, thereby serving as a crucial molecular target for studying virus-host interactions. Based on the structural scaffold of a nanobody (Nb; PDB: 7TJC), we rationally designed and constructed a mutant Nb targeting HS, designated HS-Mut-Nb1, using molecular docking, site-directed mutagenesis, molecular dynamics (MD) simulations, and experimental characterization. Molecular docking indicated that the active site of wild-type Nb for HS binding was located within the cavity jointly formed by the complementarity-determining region 3 (CDR3) and the framework regions (FRs) of the wild-type Nb. A comprehensive analysis integrating virtual alanine scanning, site-directed mutagenesis, and MD simulations revealed that the combination of three point mutations (Phe47Arg, Asp99Tyr, and Tyr108Pro) significantly enhanced the binding affinity of Mut-Nb1 for HS, with a calculated binding free energy (ΔG) of -83.26 ± 3.06 kcal/mol. Enzyme-linked immunosorbent assay (ELISA) results further confirmed that Mut-Nb1 exhibited high affinity for HS (KD = 65.87 nM) and specificity (positive/negative ratio, P/N = 3.84; cross-reactivity, CR < 6.60%). This study not only provides novel candidate molecules for elucidating the mechanism of HS-virus interactions and developing related inhibitors but also offers a reference for the rapid construction of mutant Nbs.
Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) has been shown to bind cytosolic nucleic acid sensors including retinoic acid-inducible gene I (RIG-I) and cyclic GMP-AMP synthase (cGAS) in innate immunity. As a scaffolding protein enhancing protein-protein and protein-RNA interactions during cellular stress response, we examine whether G3BP1 could bind additional innate immune signaling molecules and investigate the relevance and mechanisms underlying these interactions. To define G3BP1 function in dsRNA-triggered anti-viral signaling and identify additional interacting molecules, we generated siRNA- and CRISPER-Cas9-mediated G3BP1-knockdown and knockout HEK293T cells, conducted biochemical experiments in human and murine cells including co-immunoprecipitation, domain mutagenesis and mapping, as well as LC-MS/MS analyses to study protein post-translational modifications. We also performed p(I:C)-stimulation assays to measure interferon production, pharmacological inhibition studies to determine enzyme-substrate specificity, as well as confocal immunofluorescence microscopy to examine protein subcellular localization. Our study reveals a role for G3BP1 in binding TBK1 and IRF3, two important molecules in innate immune signaling leading to the production of IFN-β. Deficiency in G3BP1 reduces TBK1-IRF3 complex formation and affects IRF3 phosphorylation, nuclear translocation, and the production of IFN-β during p(I:C) stimulation while the phosphorylation of TBK1 was largely preserved. We further show that G3BP1 constitutively associates with IRF3 and recruits TBK1 upon p(I:C) stimulation. These findings suggest that G3BP1 acts as a scaffold to recruit activated TBK1 and facilitates its activation of IRF3. Domain mapping experiments indicated that the C-terminal RGG region of G3BP1 is critical for binding IRF3 and TBK1. LC-MS/MS analysis revealed human G3BP1 to be arginine-methylated at R435 and R460 residues upon p(I:C)-stimulation, and mutagenesis experiments indicated that R460 is required for its efficient binding of TBK1. We further show that the arginine methyltransferase PRMT5 associates with and promotes the symmetric arginine-dimethylation of G3BP1 and pharmacological inhibition of PRMT5 impaired G3BP1-TBK1 interaction. Together, our data indicate that G3BP1 could bind PRMT5, TBK1 and IRF3 and reveal G3BP1 as a signaling hub that constitutively binds IRF3 and is arginine-methylated by PRMT5 to recruit TBK1 for IRF3 phosphorylation and activation and the induction of interferon production during host antiviral response.
To bypass the availability limitation and metabolic crosstalk associated with native reduced nicotinamide adenine dinucleotide (NADH) pools in L-alanine production, a non-natural cofactor system may offer a compelling strategy to secure independent reducing power. Here, we engineered an alanine dehydrogenase (AlaDH) from Geobacillus kaustophilus to shift its cofactor preference from NAD to the non-natural cofactor nicotinamide cytosine dinucleotide (NCD). Through three rounds of iterative mutagenesis and screening, an optimal triple mutant, A225P/V165A/S219E (designated as AlaDH*), was obtained. AlaDH* exhibited an 83-fold improvement in NCD preference, retaining 62% of the catalytic efficiency toward NCD relative to the wild-type enzyme toward NAD. Crystal structure analysis of the AlaDH*-NCD complex combined with site-directed mutagenesis revealed that cofactor binding cavity shrinkage and protein surface electrostatic map alterations contribute to NCD preference. Molecular dynamics simulations provided further insights into the mechanism of cofactor selectivity. Finally, we successfully constructed a formate-driven system by using NCD-preferring formate dehydrogenase (FDH*) and AlaDH*, demonstrating a dedicated reductive amination of pyruvate independent of NADH supply. Our results provide a new opportunity to engineer amino acid dehydrogenases for a more efficient production of amino acids, laying the foundation for future development of advanced cell factories by using NCD-linked enzymes.
Transaminases provide an effective approach for the asymmetric synthesis of chiral amines. However, native ω-transaminases often suffer from low catalytic efficiency and poor substrate concentration tolerance, limiting industrial applicability. Targeting the synthesis of (S)-1-Boc-3-aminopiperidine, this study engineered the (S)-selective ω-transaminase CC1012 from Caulobacter sp. D5 through active pocket remodeling and substrate tunnel engineering. Hydrophobic remodeling of the small binding pocket yielded the single mutant M1 (Q25F) with a 27-fold improvement in intrinsic catalytic efficiency (kcat/Km). Conservation analysis and virtual saturation mutagenesis further identified R424 and D320 as key residues in the loop region at the substrate tunnel entrance. Iterative saturation mutagenesis on these sites using M1 as a template produced the optimal mutant M3 (Q25F/D320M/R424L), exhibiting a 45-fold enhancement in kcat/Km over the wild type, achieving 98% conversion with ee > 99.9% at 1000 mM substrate concentration. This study provides a superior biocatalyst for the efficient synthesis of (S)-1-Boc-3-aminopiperidine.
Ulvan lyases play a key role in marine polysaccharide degradation, but how members of the PL40 family achieve their specific product profiles remains poorly understood. Here, we show that variations in the architecture of the substrate-binding cleft dictate whether these enzymes generate disaccharides or longer oligosaccharides as their dominant products. We focused on JpPL40A, a previously uncharacterized ulvan lyase from the marine bacterium Jejuia pallidilutea, and found that it works best at 40 °C and pH 8.0. Using a combination of homology modeling and site-directed mutagenesis, the catalytic mechanism was elucidated, identifying a functional Tyr/His pair (Y260/H429) with R459 responsible for neutralizing the substrate's carboxyl group. Notably, comparative structural analysis revealed that a unique loop region adjacent to substrate binding subsites +5 and +6 dictates its product profile, leading to the predominant generation of unsaturated hexasaccharides. This loop's crucial role was confirmed via truncation mutagenesis, which shifted the main product to disaccharides, and further validated by the discovery of a homologous lyase sharing this structural feature. These results reveal a new structural feature that controls product specificity, helping us better understand how PL40 enzymes work and offering a starting point for designing ulvan lyases that make specific products.
While our understanding of kinesin autoinhibition mechanisms is advancing, key insights into kinesin activation, especially autoinhibition relieve by cargo-motor adaptors, remain unclear. JIP3 and JIP4 are adaptors that activate kinesin-1 and dynein-dynactin motors, enabling bidirectional transport along microtubules. Here, we characterized the interaction between the kinesin-1 heavy chain, KIF5B, and both JIP3 and JIP4 (JIP3/4), using mutagenesis and binding experiments associated with modelling. We identified the minimal regions of JIP3/4 and KIF5B required for interaction. JIP3/4 binds to the CC4 coiled-coil domain of KIF5B, with no significant interaction detected with the distal tail. Conversely, KIF5B-CC4 binds to the RH1 domain of JIP3/4, with the preceding disordered N-terminus modulating the force of the binding. Notably, JIP3 and JIP4 exhibit ∼40-fold differences in affinity for KIF5B. We performed extensive site-directed mutagenesis on KIF5B-binding regions of JIP3/JIP4 targeting accessible charged residues and sequence differences between JIP3 and JIP4. This revealed multiple weak-affinity binding sites that collectively define an extensive KIF5B-binding surface on the RH1 domain of JIP3. Importantly, the KIF5B-binding surface on JIP3 differs from that of the DLIC (dynein light intermediate chain), confirming no competition between the two motors. Altogether, the fragment and mutant mapping of KIF5B binding to JIP3 and JIP4 presented here provides insights into how the auto-inhibited form of kinesin-1 might be relieved with the CC4 region playing a central role.
The ecological adaptations of insect pests, such as multi-niche colonization and physiological resistance to conventional chemicals, pose severe challenges to the sustainable production of sweet potato (Ipomoea batatas). The tobacco whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), and the sweet potato weevil, Cylas formicarius (Coleoptera: Brentidae), form a highly destructive, spatially separated pest complex. In this study, we evaluated the dual-niche pathogenicity of a Beauveria bassiana (Hypocreales: Cordycipitaceae) isolate, BbCF-2, generated via spaceflight mutagenesis, against C. formicarius and B. tabaci under controlled laboratory conditions. The mutated strain exhibited enhanced colony expansion and a high sporulation capacity (2.72 × 108 conidia/mL). Bioassays revealed that BbCF-2 possesses significantly increased virulence compared to the wild-type strain, capable of overcoming the distinct physiological and physical barriers of both targeted pests. Against the highly sclerotized subterranean C. formicarius adults, BbCF-2 achieved 92.68% mortality at 15 days post-inoculation at 1 × 108 conidia/mL, with an LC50 of 8.452 × 103 conidia/mL and an LT50 of 6.305 days. Concurrently, against the canopy-dwelling B. tabaci, the isolate demonstrated rapid lethal mycosis with an LT50 of 6.718 days, effectively reducing the adult vector population prior to their typical dispersal timeframe. These results demonstrate that the spaceflight-mutated BbCF-2 strain exhibits broad pathogenicity. By simultaneously targeting both foliar and soil-dwelling pests, this single-agent biological control strategy shows potential for integrated pest management, pending greenhouse and field evaluation.
The regulatory relationship between transcription factor MAX and splicing factor SF3A3 in hepatocellular carcinoma (HCC) is unknown. We investigated whether MAX directly regulates SF3A3 and their functional role in HCC progression. MAX and SF3A3 expression were analyzed in The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-HCC) dataset (UALCAN, GEPIA) and validated in 33 paired HCC and adjacent non-tumor tissues by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Expression and prognostic significance were assessed across cancer stages, grades, and nodal status. Functional roles were evaluated in Hep3B (high expression) and PLC/PRF/5 (low expression) cells using shRNA-mediated knockdown and overexpression, respectively. Cell proliferation (cell counting kit-8, colony formation), migration (wound healing, Transwell), invasion (Matrigel Transwell), and epithelial-mesenchymal transition (EMT) (Western blot for E-cadherin, N-cadherin, and vimentin) were assessed. Mechanistic studies included chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR), luciferase reporter assays with site-directed mutagenesis, and Myc inhibition. In vivo tumor growth was evaluated using a xenograft mouse model. MAX and SF3A3 were overexpressed in HCC tissues compared to normal liver, and high expression was correlated with reduced patient survival, advanced cancer stage, higher tumor grade, and nodal metastasis. A significant positive correlation between MAX and SF3A3 expression was observed. Functional assays demonstrated that MAX or SF3A3 overexpression promoted HCC cell proliferation, migration, invasion, and EMT, while knockdown suppressed these phenotypes. MAX directly bound the SF3A3 promoter (P3 E-box) and activated its transcription in a Myc-dependent manner. Overexpression of MAX or SF3A3 promoted malignant phenotypes, while SF3A3 knockdown reversed MAX-driven oncogenic effects in vitro and reduced tumor growth in vivo. This study establishes a novel MAX-SF3A3 regulatory axis in which MAX directly binds the SF3A3 promoter and activates its transcription in a Myc-dependent manner, driving HCC cell proliferation, migration, invasion, EMT, and tumor growth. Targeting the MAX-SF3A3 axis represents a potential therapeutic strategy for HCC.
Anterior-posterior patterning in Drosophila relies on positional information supplied by the graded distributions of three maternally supplied transcription factors originally identified in large-scale mutagenesis screens. Here, I examine the development of embryos in which these three maternal gradients have been flattened, but set at different levels. Following such manipulations, expression patterns at the blastoderm stage are uniform along the anterior-posterior axis, but their specific nature depends on the combination and concentration level of the three systems. Each of the three gradients are then examined in contexts in which the others are flattened but set at different levels. This reveals that each system generates a greater fraction of the wild-type pattern than that deleted when that system is removed by loss-of-function mutations, with the exact pattern depending on the levels to which the two remaining flattened systems are set. These simplified patterns are then used to analyze interactions between the maternal systems and to investigate how the pattern of gap and pair rule genes are modulated by network interactions downstream of the maternal inputs themselves.
Gallbladder cancer (GBC), the most common malignancy of the biliary tract, exhibits a high propensity for lymphatic system invasion. However, the mechanisms underlying lymphatic metastasis in GBC remain poorly understood. Here, we demonstrate that C-C motif chemokine ligand 21 (CCL21) is significantly upregulated in GBC and positively correlates with lymphatic vessel density and lymph node metastasis. Both in vivo and in vitro experiments confirm that CCL21/CCR7 axis promotes lymphangiogenesis and lymphatic metastasis in GBC. Mechanistically, CCL21/CCR7 modulates Vascular Endothelial Growth Factor-D (VEGF-D) mRNA N6-Methyladenosine (m6A) modification in an AlkB homolog 5 (ALKBH5)-dependent manner, thereby regulating VEGF-D protein expression. Site-directed mutagenesis experiments reveal that m6A modification sites in the coding sequence (CDS) region of VEGF-D mRNA are critical for VEGF-D protein regulation. Collectively, our findings highlight that CCL21/CCR7 regulates VEGF-D expression via m6A modification, driving lymphatic metastasis in GBC.
Plant-parasitic nematodes pose a severe threat to global agricultural production, creating an urgent demand for green and highly efficient nematicides. In this study, we investigated the nematicidal activity and underlying action mechanism of 4-(diethylamino)salicylaldehyde (DSA) against Ditylenchus destructor. DSA exhibited significant nematicidal activity with a 72 h LC₅₀ of 1.951 μg/mL. At 10 μg/mL, DSA completely inhibited nematode infection, comparable to fluopyram. DSA damaged the body wall, induced reactive oxygen species accumulation and lipid peroxidation, and inhibited antioxidant enzymes. Multi-omics analysis revealed that DSA disrupted glycolysis and the TCA cycle, suppressed pyruvate dehydrogenase activity, and reduced ATP levels. Molecular docking and site-directed mutagenesis confirmed that DSA directly targets the PDH E1α subunit via Arg93 and Tyr92. Notably, DSA showed low toxicity to mammals, birds, and silkworm, with only moderate toxicity to zebrafish. Collectively, this study reveals a PDH-targeted, multi-pathway synergistic nematicidal mechanism of DSA, providing a theoretical basis and a promising lead compound for green nematicide development.
Replicative DNA polymerases are responsible for the high-fidelity duplication of genomes in all organisms. The overall DNA replication process is highly accurate, with deoxyribonucleoside monophosphate (dNMP) errors occurring on the order of 1 in tens of millions of replicated bases. However, DNA polymerases are more prone to misincorporating ribonucleoside monophosphates (rNMPs) in place of their cognate dNMPs, with measurements on the order of 1 in a couple thousand base pairs synthesized. Owing in part to the high concentration of rNTPs relative to dNTPs in vivo, rNMPs may represent the most common nucleotide requiring removal from genomic DNA in organisms spanning all domains of life. Cells employ a set of pathways, including ribonucleotide excision repair (RER), to address abundant genomic rNMPs. Defects in ribonucleotide excision repair result in mutagenesis, neurological disorders, and embryonic lethality. In this review, we discuss the mechanisms for how ribonucleotides are misincorporated by DNA polymerases, how RNase H enzymes recognize and cleave RNA-DNA hybrids, and the process of ribonucleotide excision repair. Further, we review and discuss how ribonucleotide error correction occurs in eukarya, archaea, and bacteria, providing perspectives across a broad range of organisms and ribonucleotide resolution pathways.
Polyamines, well-known regulators of the mitochondrial calcium (Ca2+) uniporter channel, show unexpected effects when binding the channel from within the matrix. Using cryo-EM, molecular dynamics simulations, and mutagenesis experiments, we determine that polyamines achieve such regulation by binding within the pore to a ring of negative residues forming a matrix gate, inhibiting Ca2+ conduction. In whole-mitoplast electrophysiology assays, matrix polyamines cause a gradual increase in Ca2+ currents during prolonged conduction, due to relief of this inhibition. Notably, this electrostatic binding increases 3-fold as the inner membrane depolarizes, preventing Ca2+ efflux. Additionally, we also identify that phospholipids form part of the Ca2+ conduction pathway through MCU. Because we find significant variability in matrix polyamine content across mouse organs, this unexpected mechanism for sculpting the mitochondrial Ca2+ waveform suggests a tissue-specific regulation of metabolism.
Lung cancer in individuals who have never smoked (LCINS) represents a clinically and biologically distinct subset of non-small cell lung cancer, driven predominantly by oncogenic alterations rather than tobacco-related mutagenesis. This review aims to summarize current and emerging targeted and immune-based therapeutic strategies in LCINS individuals. These patients present a molecular profile that differs substantially from tobacco-associated disease and has direct consequences for treatment selection. Evidence published over the past five years has clarified how these molecular features shape treatment response and resistance in this setting. Particular attention is given to tumors with alterations in epidermal growth factor receptor, anaplastic lymphoma kinase, c-ros oncogene 1, rearranged during transfection, Mesenchymal-Epithelial Transition (MET) exon 14 skipping mutation, human epidermal growth factor receptor 2, valine-to-glutamic acid substitution at codon 600 of the BRAF gene (BRAF V600E), and neurotrophic tyrosine receptor kinase, which together comprise the dominant driver landscape in never-smoker lung cancer. Although third-generation tyrosine kinase inhibitors have markedly improved response rates in several of these subgroups, long-term disease control is frequently compromised by acquired resistance, and heterogeneous drug exposure, particularly in the central nervous system. By contrast, immune checkpoint inhibitors have yielded limited benefit, in keeping with the low mutational burden and generally low baseline immune activation observed in most LCINS tumors. As a result, alternative approaches such as antibody-drug conjugates, bispecific antibodies, and adoptive cellular therapies are being evaluated to address gaps left by existing treatments.
Carbapenem resistance in Acinetobacter baumannii is usually mediated by acquired carbapenemases. Here we investigated an alternative mechanism involving variants of the intrinsic Acinetobacter-derived cephalosporinase (ADC). Eighteen clonally related clinical isolates collected between 2019 and 2024 in a French long-term care facility were analysed together with four epidemiologically related isolates from additional hospitals. Antimicrobial susceptibility testing, whole-genome sequencing, cloning experiments, site-directed mutagenesis and enzyme kinetic analyses were performed to assess the contribution of ADC variants to carbapenem susceptibility. All isolates were resistant to broad-spectrum cephalosporins, and 72% and 83% were resistant to imipenem and meropenem, respectively. Whole-genome sequencing identified a novel ADC variant, designated ADC-200, differing from ADC-30 by a single amino-acid substitution (S318T) and located downstream of ISAba1. Cloning experiments demonstrated that ADC-200 production increased meropenem MICs compared with reference ADC enzymes. Introduction of the S318T substitution into ADC-50 reproduced this phenotype, indicating that this residue plays a key role in modulating meropenem susceptibility. Kinetic analyses showed increased catalytic efficiency for meropenem hydrolysis in ADC variants harbouring the S318T substitution. SNP-based phylogenetic analysis revealed clonal dissemination of ADC-200-producing isolates belonging to international clone 2 across several institutions. Our findings demonstrate that naturally occurring ADC variants can contribute to reduced susceptibility to meropenem in epidemic A. baumannii lineages. Although this mechanism alone confers only moderate increases in carbapenem MICs, its occurrence in widespread clones highlights the importance of genomic surveillance to detect emerging resistance determinants beyond classical carbapenemases.
β-cell dysfunction is a key factor in the progression of diabetes. It was reported that lipotoxicity impaired β-cell mitochondrial function. However, the molecular mechanisms regulating lipotoxicity-induced β-cell mitochondrial dysfunction remain unclear. Islet cells were isolated from diabetic db/db and control db/m mice. Palmitic acid (PA) was used to treat MIN6 cells to construct a lipotoxicity-induced β-cell model. Co-IP was employed to verify the interaction between Sirt1 and Trim67. Enrichment of Klf5, Klf2, Klf6, and Ahr on the Trim67 promoter was analyzed using DNA pull-downs. Binding of Klf6 to the Trim67 promoter region was detected using ChIP and dual-luciferase reporter assays. Cell proliferative capacity and mitochondrial function were measured by CCK-8, EdU, JC-1, Seahorse XF-96 and ATP detection kits. Protein expression was tested using RT-qPCR and western blotting. Sirt1 deacetylase activity was measured fluorometrically. Ubiquitination sites were mapped using site-directed mutagenesis. In vivo experiments were performed using AAV8-mediated Trim67 overexpression in the db/db mice. Trim67 was downregulated in pancreatic islet of diabetic mice and in PA-induced β-cells. AAV8-mediated Trim67 overexpression in db/db mice significantly improved glucose tolerance, enhanced insulin secretion, and preserved islet integrity. Overexpression of Trim67 alleviated PA-induced mitochondrial dysfunction in β-cells. Moreover, Trim67 was transcriptionally activated by Klf6 in PA-induced β-cells. Trim67 increased Sirt1 stability through K63-linked ubiquitination at lysines 491 and 591 to mediate the Sirt1/Pgc-1α pathway, which in turn alleviates PA-induced mitochondrial dysfunction in β-cells. Klf6 transcriptionally activated Trim67 to regulate the Sirt1/Pgc-1α axis, thereby alleviating PA-induced mitochondrial dysfunction in β-cells. This study offers new insights into the treatment of diabetes.