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Starch serves as a vital energy reserve in plants. During its biosynthesis, malto-oligosaccharides (MOS) are essential primers. One of the key pathways for MOS production involves plastidial α-glucan phosphorylase (PHS1/Pho1) and disproportionating enzyme (DPE1). However, the functional relationship between these enzymes is unclear. Here, we demonstrate that rice PHS1 and DPE1 assemble into a multimeric complex. Cryo-EM structures of the PHS1-DPE1 complex reveal an assembly mechanism and suggest a potential substrate tunnel. Biochemical assays show the complex dramatically enhances catalytic efficiency over individual enzymes. Single-molecule fluorescence resonance energy transfer (smFRET) visualizes conformational dynamics, enabling rapid substrate transfer between the enzymes. We further identify the unique L80 loop in PHS1 as a potential regulator. Its deletion reduces catalytic efficiency and prolongs conformational state lifetimes during substrate transfer, thereby reducing the production of longer MOSs. Our findings establish that the PHS1-DPE1 complex facilitates efficient MOS primer synthesis through efficient substrate transfer or diffusion between the two enzymes, providing mechanistic insight into a critical step of starch biosynthesis with agronomic implications.

The search for rational synthetic pathways for steroid drugs from accessible plant sources represents an important and relevant objective. Yucca gloriosa presents significant interest as a source of tigogenin-a valuable sapogenin for steroid hormonal drug production. Along with traditionally utilized leaves, the flowers of this plant contain 1.25-1.48% tigogenin and possess several technological advantages: simpler extraction of the target product due to lower lipophilic substance content and softer raw material texture. Endogenous β-glucosidase plays a key role in the tigogenin production process, catalyzing the conversion of oligofurostanoside to oligospirostanoside. This work investigated the properties of β-glucosidase from flowers of Yucca gloriosa cultivated in Georgia. Two enzyme forms differing in substrate specificity were identified. The first form (30% ammonium sulfate saturation fraction) hydrolyzes both natural oligofurostanoside and synthetic substrate 4-nitrophenyl-β-D-glucopyranoside, whereas the second form (80% saturation fraction) cleaves only the synthetic substrate. For the first β-glucosidase form, which has practical significance for tigogenin production, optimal operating conditions were determined: pH6.3 and temperature 47°C. Notably, the temperature optimum for the flower enzyme (47°C) proved higher than for the leaf enzyme (37°C). Heating at 57°C for 10 minutes reduces enzyme activity by 29%, while at 67°C it reduces activity by 79%.

The kappa class of glutathione S-transferases 1 (GSTK1) is a vital regulatory factor in metabolic diseases. This study was conducted to investigate the regulatory effects of GSTK1 on renal ectopic fat deposition (EFD) and lipotoxic injury in diabetic nephropathy (DN) . HK-2 cells under high glucose(HG) / high fatty acid (HFA) stimulation, diabetic mice and human renal biopsy tissues were used. GSTK1 plasmid, GSTK1 siRNA and OSBPL8 siRNA were applied in vitro. Lipid accumulation was analyzed in the renal tissue of type 2 DN patients, diabetic mice and HK-2 cells under HG/HFA stimulation. The expression of GSTK1, DGAT1, ACAT1, CPT-1, BECLIN1, LC3II, ATG5 and RAB7 in renal tubular cells of diabetic mice and HK-2 cells under HG/HFA condition decreased significantly. Metformin treatment restored the expression of GSTK1 in diabetic mice. Additionally, the GSTK1 pharmacological modulator metformin relieved lipophagy dysfunction and promoted fatty acid (FA) β-oxidation enzyme CPT-1. In vitro, GSTK1 plasmid reduced lipid accumulation, fibrosis and inflammation and up-regulated the expression of CPT1 in HK-2 cells, but GSTK1 plasmid had no effect on lipid metabolizing enzymes (ACAT1, DGAT1) . In addition, GSTK1 plasmid could obviously restore lipophagy. However, pretreatment of HK-2 cells with the AMPK inhibitor Compound C, GSTK1 siRNA or OSBPL8 siRNA negated the activating effects of GSTK1 on lipophagy. This study indicated that GSTK1 could contribute to alleviate EFD in DN tubular cell through increasing the expression of FA β-oxidation enzyme CPT-1 and restoring lipophagy via AMPK-OSBPL8 pathway.

This study investigated the α-glucosidase (α-G) and α-amylase (α-A) inhibitory properties of black highland barley anthocyanins (BHA) and its main monomer, cyanidin-3-O-glucoside (C3G), using an integrated approach combining in vitro simulated digestion, enzyme kinetics, multi-spectroscopic analyses, and molecular docking. Despite substantial degradation of anthocyanins during digestion, the intestinal digesta retained potent α-G inhibitory activity. The crude BHA extract exhibited stronger inhibition than C3G, with IC50 values of 8.31 μg/mL for α-G and 14.91 μg/mL for α-A. Kinetic studies revealed reversible, mixed-type inhibition for BHA, whereas C3G acted via a non-competitive mechanism. Multiple techniques demonstrated that the inhibitors bind to the enzymes, alter their conformation, and promote aggregation, with molecular docking attributing these effects to hydrogen bonding and hydrophobic interactions at the active site. These findings elucidate the mechanisms by which BHA and C3G modulate carbohydrate-hydrolyzing enzymes, highlighting BHA as a potent functional food ingredient for postprandial glycemic control.

High-altitude pulmonary hypertension (HAPH), classified as Group 3 pulmonary hypertension, is a significant threat to the health of high-altitude populations. The scarcity of studies in diverse populations has become a research bottleneck, limiting diagnostic and therapeutic advances. In this first proteomic study focusing on the eastern Pamir Plateau (Kizilsu Kyrgyz Autonomous Prefecture, Xinjiang), plasma samples were analyzed using data-independent acquisition (DIA) mass spectrometry. Differential expression analysis in parallel with weighted gene co-expression network analysis was performed to identify core pathways and hub proteins, and gene set enrichment analysis was used for quality assessment. Integrative analysis of the two methods was used to select candidates for validation by enzyme-linked immunosorbent assay (ELISA) in an independent cohort. Among >&#x2009;1400 detected proteins, 123 were differentially expressed and 45 were identified as hub proteins significantly associated with HAPH. Extracellular matrix (ECM) remodeling- and angiogenesis-related proteins were upregulated, whereas proteins related to enzyme activity, iron metabolism, and inflammatory responses were downregulated. Integrative analysis identified 23 core proteins, with ECM-receptor interaction and TGF-&#x3b2;/Smad signaling identified as key pathways. ELISA confirmed that plasma levels of THBS2, LOXL1, and POSTN were significantly elevated in patients with HAPH (P&#x2009;<&#x2009;0.05). Among these, THBS2 and LOXL1 levels were positively correlated with mPAP (THBS2: r&#x2009;=&#x2009;0.389, 95% CI: 0.034-0.657, P&#x2009;=&#x2009;0.033; LOXL1: r&#x2009;=&#x2009;0.457, 95% CI: 0.115-0.701, P&#x2009;=&#x2009;0.011). ECM remodeling is closely associated with HAPH in this indigenous high-altitude population. THBS2, LOXL1, and POSTN show potential as biomarkers and therapeutic targets.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder, affecting nearly 0.3% of the global population. Its pathology is primarily linked to dopaminergic neuronal loss in the substantia nigra, leading to hallmark motor impairments such as tremor, rigidity, and bradykinesia. A defining molecular feature of PD is the aberrant aggregation of &#x3b1;-synuclein, alongside dysregulation of proteins such as MAO-B, COMT, and LRRK2, which collectively contribute to disease progression. Within the current research, these proteins were designated as docking targets to explore the enzyme-modulating activity and the therapeutic promise of steroidal alkaloid candidates from the genus Fritillaria, a taxon long recognized in traditional medicine for its neuroprotective properties. Docking analyses revealed that among 70 compounds analysed, compound 65 exhibited strong MAO-B inhibitory activity (binding energy&#x2009;-&#x2009;11&#xa0;kcal/mol), compound 5 demonstrated pronounced COMT inhibition (-&#x2009;9&#xa0;kcal/mol), and compound 42 emerged as a promising dual-acting agent capable of targeting both enzymes. Favorable physicochemical attributes, including optimal lipophilicity, low polar surface area, and blood-brain barrier permeability, further support their suitability. These findings identify preliminary computational leads that warrant further experimental validation for potential future development.

The synergistic cardioprotective effects of monoester alkaloids and ginsenosides constitute the pharmacological basis of Shenfu decoction (SFD) for chronic heart failure. However, the underlying molecular interactions remain unclear. This study aimed to identify the key bioactive components of SFD, elucidate their molecular targets, and define the mechanistic pathways underlying their synergistic cardioprotective effects. We employed an ACE-dependent Ang I-stimulated cardiomyocyte hypertrophy model in vitro and a transverse aortic constriction (TAC) mouse model in vivo to evaluate pharmacological efficacy. Integrated transcriptomic-proteomic profiling identified downstream effectors, validated by genetic knockdown. The molecular targets were investigated using limited proteolysis-mass spectrometry (Lip-MS), molecular docking, surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), and enzyme activity assays. Benzoylaconitine (BAC) and ginsenoside Rb1 (Rb1) were identified as the dominant cardioprotective constituents of SFD. Pharmacological assessments demonstrated that BAC and Rb1 synergistically attenuated Ang &#x2160;-induced cardiomyocyte hypertrophy and fibrosis, and mitigated pathological cardiac remodeling in TAC mice. Integrated transcriptomic and proteomic analyses revealed that their combined treatment reduced mitochondrial reactive oxygen species (ROS) generation, suppressed mitochondrial fission, and restored mitochondrial homeostasis. Mechanistically, BAC promoted the transcription of Discs Large MAGUK Scaffold Protein 1 (DLG1), whereas Rb1 slowed its protein degradation, thereby synergistically upregulating DLG1 expression and improving mitochondrial function. Lip-MS further demonstrated that BAC specifically targeted angiotensin-converting enzyme (ACE) and inhibited its activity, whereas Rb1 targeted and activated ACE2, thereby rebalancing the renin-angiotensin-aldosterone system (RAAS) between the AT1R and MAS axes. This dual-target modulation ultimately contributed to the upregulation of DLG1, preserved mitochondrial integrity, and ameliorated maladaptive ventricular remodeling. BAC and Rb1 exert synergistic cardioprotection by targeting ACE and ACE2, restoring RAAS homeostasis, enhancing DLG1 expression, and sustaining mitochondrial stability, thereby attenuating pathological cardiac remodeling. These findings highlight the potential of BAC and Rb1 as a dual-target synergistic therapy for heart failure.

Considering the devastating effect of salt stress on crops the present study investigates the regulatory role of exogenous silymarin in enhancing antioxidant defense system and morpho-physiology of Brassica napus under salt stress. Twenty-three-day-old rapeseed (Brassica napus cv. BARI Sarisha-18) plants were supplemented with a foliar spray of 250&#xa0;ppm silymarin followed by two doses of NaCl, viz. 75 and 150&#xa0;mM. This growing condition was maintained for the following 30&#xa0;days. Salinity resulted in reduced biomass production, growth attributes, and relative water content of rapeseed plants with increased levels of Na+ ions, hydrogen peroxide (H2O2), lipid peroxidation, electrolyte leakage (EL), and proline (Pro) content. This led to oxidative damage by suppressing the activities of antioxidant enzymes. Silymarin reduced lipid peroxidation, H2O2, EL, and Pro content by 23, 15, 9, and 17%, respectively in 150&#xa0;mM NaCl-stressed plants compared to their corresponding controls. The activities of glyoxalase, as well as antioxidant enzymes such as ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, catalase, glutathione peroxidase, glutathione S-transferase, peroxidase, lipoxygenase, and superoxide dismutase were also upregulated due to silymarin application. Findings from the current study suggest that salt-induced rapeseed plants exhibited reduced growth attributes alongside elevated oxidative stress markers, including both enzymatic and non-enzymatic antioxidants. Furthermore, it highlights the potential of silymarin as a potential growth regulator and antioxidant that can enhance salt tolerance in rapeseed plants by reducing the harmful effects of reactive oxygen species.

Up to 80% of diffuse midline gliomas (DMGs) are characterized by a lysine to methionine driver mutation (K27M) in the tail of histone variant H3.3, pointing to likely roles for epigenetic mechanisms in K27M-driven tumorigenesis. Understanding the effects of mutant histone H3.3 on the complex patterns of histone modifications and interactions with chromatin structure and modifying enzymes is essential to developing effective combination treatment therapies for K27M DMG such as targeting multiple epigenetic enzymes at once. Here, using a genomics approach, we identified combinatorial patterns of epigenetic modifications that are affected by mutant H3.3 in DMG. We also characterized a strong association between H3.3 and the structural chromatin regulator CTCF, finding that mutant H3.3 leads to ectopic binding of CTCF at many additional sites across the genome in DMG. Notably, a number of these ectopic CTCF binding events occur within the HOX gene loci and are associated with an increase in H3K27me3 levels at bivalent domains and a decrease in HOX gene expression. We also find an association of H3.3 and CTCF at genomic sites adjacent to regions with active or repressive modifications, suggesting a potential role for these two factors in segmenting the chromatin and regulating, perhaps insulating, different types of domains. Together our data suggest that H3.3 K27M both affects epigenetic marks and chromatin organization in part through interaction with CTCF and point to a potentially novel contributory role for CTCF in promoting oncogenesis in DMG. These findings could have potential implications for designing therapy regimens to more effectively target the chromatin changes and genomic instability observed in H3.3K27M glioma cells.

Ovarian cancer (OC) remains the most lethal malignancy within the spectrum of gynecological cancers globally. While protein S-palmitoylation has been extensively implicated in tumor progression, its specific functional contributions and molecular mechanisms in the context of OC pathogenesis remain to be fully elucidated. This article aims to explore the prognostic effect associated with palmitoylation in OC. In this study, palmitoylation-related genes (PRGs) were defined as genes encoding enzymes directly involved in the palmitoylation/depalmitoylation process, as well as genes whose functions, subcellular localization, or signaling are regulated by this modification. Based on this definition, PRGs comprising enzymes and regulated substrates, were identified from public transcriptomic databases. By intersecting ovarian cancer (OC)-associated and palmitoylation-linked differentially expressed genes (DEGs), candidate targets were pinpointed. A prognostic risk model was then constructed using LASSO and Cox regression analyses on the TCGA-OV cohort (N&#x2009;=&#x2009;378) and validated in the GSE51088 cohort (N&#x2009;=&#x2009;152). This model was integrated into a predictive nomogram and further characterized through pathway enrichment, immune infiltration, checkpoint analysis, drug screening, and mutation profiling. Finally, identified markers were validated via RT-qPCR in clinical samples. Through intersecting DEGs1 and DEGs2, we obtained 24 candidate biomarkers. Four PRGs (HSPG2, BRD4, RARRES1, and SCGB1D2) were identified to construct a prognostic risk model. The risk score, alongside ethnicity and tumor stage, served as an independent prognostic indicator, integrated into a robust nomogram. Mechanistically, high-risk cohorts were characterized by dysregulated ribosome and translation initiation pathways, altered infiltration of seven immune cell types, and significant variations in seven checkpoints (e.g., CTLA4, CD274). Additionally, the model predicted sensitivities for 131 drugs and captured a high TP53 mutation rate. RT-qPCR validation confirmed the upregulation of HSPG2, SCGB1D2, and BRD4, and the downregulation of RARRES1 in OC tissues, showing high consistency with bioinformatic predictions (P&#x2009;<&#x2009;0.05). This study identified HSPG2, BRD4, RARRES1, and SCGB1D2, which served as prognostic markers reflecting the palmitoylation-related biological landscape in OC that could lay the foundation for innovative therapeutic strategies.

The functional properties and application performance of poly-&#x3b3;-glutamic acid (&#x3b3;-PGA) are strongly dependent on its molecular weight (MW). However, the precise control of MW remains challenging, significantly hindering its high-value applications in the food, agricultural, cosmetic, and pharmaceutical sectors. Heterologous enzyme expression has emerged as a powerful and specific strategy for regulating the MW of &#x3b3;-PGA. In this study, &#x3b3;-DL-glutamyl hydrolase (BvPgdS45) from Bacillus velezensis CAU263 was heterologously expressed in Komagataella phaffii, achieving an activity of 102.7 IU/mL following high-cell-density fermentation. BvPgdS45 exhibited optimal activity at a pH of 7.0 and temperature of 50 &#x2103;. Subsequently, its effects on &#x3b3;-PGA degradation property were evaluated. The weight-average molecular weight (Mw) of &#x3b3;-PGA decreased from 3.3&#xd7;106Da to 1.1&#xd7;105Da after 3h of enzymatic hydrolysis at enzyme dosages ranging from 0 IU/mL to 10 IU/mL. Furthermore, &#x3b3;-PGA with different MW was produced using B. velezensis CAU263 via in situ enzymatic hydrolysis. Products with Mw ranging from 5.1&#xd7;105 Da to 2.1&#xd7;106 Da were generated, while the &#x3b3;-PGA yield increased from 62.6g/L to 73.7g/L in a 5L fermenter. Overall, this study demonstrates the effectiveness of BvPgdS45 expressed in K. phaffii for degrading &#x3b3;-PGA and provides an efficient in situ hydrolysis strategy for the precise regulation of its MW.

5-Fluorouracil (5-FU) is a widely utilized antimetabolite in colorectal cancer chemotherapy, primarily exerting its cytotoxic effects by irreversibly inhibiting thymidylate synthase (TS). This inhibition leads to a reduction in deoxythymidine monophosphate (dTMP), which disrupts DNA synthesis and repair. A major challenge in 5-FU treatment is the dose-limiting toxicity of chemotherapeutic-induced intestinal mucositis. Da-Bu-Pi Decoction (DBPD), a well-established formula in traditional Chinese medicine for treating spleen-stomach deficiency, is often used to enhance spleen qi and balance the middle jiao. While growing clinical evidence points to the therapeutic benefits of DBPD in alleviating 5-FU-induced mucositis, the underlying molecular mechanisms remain largely undefined. To elucidate the molecular mechanisms underlying chemotherapy-induced intestinal mucositis (CIM) induced by 5-FU, with a focus on the disruption of bile acid metabolism and the downregulation of the key detoxifying enzyme DPYD expression. DBPD was administered orally to C57BL/6 mice with 5-FU-induced intestinal mucositis over a seven-day period. The evaluation of intestinal damage included assessments of diarrhea, morphology, intestinal barrier function and inflammatory factors, alongside techniques such as immunofluorescence, immunohistochemistry, transmission electron microscopy and Western blot. Transcriptome analysis of mouse ileal tissue was applied to reveal differentially expressed genes (DEGs) in different treated mice, and bile acid metabolism-related genes (UDP-glucuronosyltransferase 1A1 (UGT1A1), UGT1A9, Farnesoid x receptor (FXR), TGR5) and DPYD, a key detoxification enzyme for 5-FU, were confirmed by qRT-PCR. Additionally, changes of DCA and LCA were measured using ELISA. Bioinformatics helped delineate the association of these genes with pan-cancer versus normal tissues. Meanwhile, 5-FU-induced intestinal epithelial cells (HIEC) were treated with serum containing DBPD. It was further explored how DBPD modulates the UGT1A1/TGR5/FXR signaling pathway and enhances DPYD activity to reduce apoptosis and intestinal barrier damage in 5-FU induced HIEC. AlphaFold3 and further bioinformatics analysis predicted the binding interactions and expression correlations among UGT1A1, UGT1A9 and FXR. DBPD is protective by reducing inflammation and intestinal barrier dysfunction in 5-FU-induced intestinal mucositis. Transcriptome analysis and in vivo validation highlighted the crucial function of bile acid metabolism-related pathways and DPYD in 5-FU-induced CIM. 5-FU increased the levels of deoxycholic acid (DCA) and lithocholic acid (LCA) in the mouse ileum. DBPD activated the UGT1A1/TGR5/FXR pathway and up-regulated DPYD to suppress the pathological accumulation of these specific cytotoxic bile acids within the local intestinal microenvironment and ameliorate 5-FU-induced CIM. It was indicated by the analysis of bioinformatics that low levels of UGT1A1, UGT1A9, and FXR exhibited a connection with poor prognosis within colorectal cancer. In vitro studies confirmed that DBPD significantly improved the UGT1A1/TGR5/FXR pathway and increased the expression of DPYD in 5-FU treated HIEC, thereby improving intestinal barrier integrity and alleviating apoptosis. Further validation using the FXR inhibitor (Gly-&#x3b2;-MCA) showed that DBPD ameliorated Gly-&#x3b2;-MCA induced HIEC apoptosis and barrier attenuation. Finally, AlphaFold3 and bioinformatics predictions suggested potential binding interactions between UGT1A1, UGT1A9 and FXR with positively correlated expression.

Drought threatens apple (Malus domestica) growth and productivity, often leading to irreversible economic losses. C1-papain family cysteine proteases are key enzymes in plant responses to abiotic stress, yet their specific roles under drought conditions remain largely uncharacterized in apple. Sequence analysis identified MdCP37 as a member of the C1-papain family. Here, we demonstrate that overexpression of MdCP37 (OE) in transgenic apple increases drought sensitivity, while RNAi-mediated silencing of MdCP37 enhances drought tolerance. Under drought stress, MdCP37-OE lines displayed reduced antioxidant enzyme activity and suppressed expression of drought-responsive genes, whereas RNAi lines exhibited opposite trends. Moreover, MdCP37 accelerated chlorophyll and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) degradation and promoted leaf senescence under drought conditions. Collectively, our findings establish MdCP37 as a negative regulator of drought tolerance in apple and offer theoretical support for improving drought resilience in apple breeding programs, particularly in arid regions.

Nanozyme-based aptasensors still face challenges, including low catalytic activity and inverse signal correlation, which hinder their advancement in the field of food safety. To address these issues, this study developed a novel nanozyme-based colorimetric aptasensor for the high-performance detection of AFB1. Initially, a gold-silver alloy nanoframework (Au-Ag NFs) was synthesized using liposomes as a template, followed by the deposition of platinum nanoclusters on its surface, resulting in the successful preparation of the Au-Ag NFs@Pt NCs nanozyme. This nanozyme exhibited outstanding oxidase-like activity, efficiently catalyzing the oxidation of TMB, leading to a significant increase in absorbance at 652&#x202f;nm and a visually observable blue color. The AFB1-specific aptamer was covalently immobilized onto the nanozyme surface through Au-S bonds, forming the Au-Ag NFs@Pt NCs@apt complex and enabling the construction of an integrated "recognition-catalysis" sensing system. Upon target recognition by the aptamer, conformational changes in the Au-Ag NFs@Pt NCs @apt-target complex reduced the masking effect of the aptamer on the surface of the nanoenzyme, thereby enhancing oxidase-like activity. Ultimately, this colorimetric aptasensor achieved an enzyme-like "turn-on" detection of AFB1. The aptasensor achieved a detection limit as low as 22.3&#x202f;pg/mL, and demonstrated excellent selectivity, stability, and satisfactory recovery in spiked peanut samples. This work not only provides a promising new approach for on-site rapid screening of AFB1, but also offers novel composite material insights for designing high-performance nanozyme sensing platforms targeting other analytes.