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The fermentative production of valuable chemicals from lignocellulosic feedstocks has attracted considerable attention. Although Saccharomyces cerevisiae is a promising microbial host, it lacks the ability to efficiently metabolize xylose, a major component of lignocellulosic feedstocks. The xylose oxidative pathway offers advantages such as simplified metabolic regulation and fewer enzymatic steps. Specifically, the pathway involves the conversion of xylose into 2-keto-3-deoxy-xylonate, which can be channeled into two distinct pathways, the Dahms pathway and the Weimberg pathway. However, the growth of yeast on xylose as the sole carbon source through the xylose oxidative pathway has not been achieved, limiting its utilization. We successfully engineered S. cerevisiae to metabolize xylose as its sole carbon source via the xylose oxidative pathways, achieved by enhancing enzyme activities through iron metabolism engineering and rational enzyme selection. We found that increasing the supply of the iron-sulfur cluster to activate the bottleneck enzyme XylD by BOL2 disruption and tTYW1 overexpression facilitated the growth of xylose and the production of ethylene glycol at 1.5 g/L via the Dahms pathway. Furthermore, phylogenetic analysis of xylonate dehydratases led to the identification of a highly active homologous enzyme. A strain possessing the Dahms pathway with this highly active enzyme exhibited reduced xylonate accumulation. Furthermore, the introduction of enzymes based on phylogenetic tree analysis allowed for the utilization of xylose as the sole carbon source through the Weimberg pathway. This study highlights the potential of iron metabolism engineering and phylogenetic enzyme selection for the development of non-native metabolic pathways in yeast. Key points: • A 1.5 g/L ethylene glycol was produced via the Dahms pathway in S. cerevisiae. • Enzyme activation enabled growth on xylose via both the Dahms and Weimberg pathways. • Tested enzymes in this study may expand the application of xylose oxidative pathway. [ABSTRACT FROM AUTHOR]

Acute pancreatitis (AP) is a disease characterized by an acute inflammatory response in the pancreas. This is caused by the abnormal activation of pancreatic enzymes by a variety of etiologic factors, which results in a localized inflammatory response. The symptoms of this disease include abdominal pain, nausea and vomiting and fever. These symptoms are induced by a hyperinflammatory response and oxidative stress. In recent years, research has focused on developing anti-inflammatory and antioxidative therapies for the treatment of acute pancreatitis (AP). However, there are still limitations to this approach, including poor drug stability, low bioavailability and a short half-life. The advent of nanotechnology has opened up a novel avenue for the management of acute pancreatitis (AP). Nanomaterials can serve as an efficacious vehicle for conventional pharmaceuticals, enhancing their targeting ability, improving bioavailability and prolonging their half-life. Moreover, they can also exert a direct therapeutic effect. This review begins by introducing the general situation of acute pancreatitis (AP). It then discusses the pathogenesis of acute pancreatitis (AP) and the current status of treatment. Finally, it considers the literature related to the treatment of acute pancreatitis (AP) by nanomaterials. The objective of this study is to provide a comprehensive review of the existing literature on the use of nanomaterials in the treatment of acute pancreatitis (AP). In particular, the changes in inflammatory markers and therapeutic outcomes following the administration of nanomaterials are examined. This is done with the intention of offering insights that can inform subsequent research and facilitate the clinical application of nanomaterials in the management of acute pancreatitis (AP). [ABSTRACT FROM AUTHOR]

AbstractMononuclear non-heme iron enzymes catalyze a wide array of important oxidative transformations. They are correspondingly diverse in both structure and mechanism. Despite significant evolutionary distance, it is becoming increasingly apparent that these enzymes nonetheless illustrate a compelling case of mechanistic convergence via the formation of peroxo species bridging metal and substrate. Aromatic amino acid hydroxylases and 2-oxoglutarate (2OG)-dependent enzymes, for example, form bridged acyl- or alkylperoxo intermediates en route to highly oxidizing ferryl species, while catechol dioxygenases utilize such ‘bridged’ peroxos directly. Analogous acylperoxoiron intermediates have also been demonstrated to precede a perferryl oxidant in biomimetic systems. Herein, we synthesize the results of structural, spectroscopic and computational studies on these systems to gain insight into the shared chemical logic that drives iron-peracid formation and reactivity. In all cases, reactions are tuned via the electron-donating properties of coordinating ligands. Second-sphere residues have also been demonstrated to modulate the orientation of the bridge, thereby influencing reaction outcomes. The effect of carboxylic acid addition to relevant biomimetic catalyst reactions further underscores these fundamental chemical principles. Altogether, we provide a comprehensive analysis of the cross-cutting mechanisms that guide peroxo formation and subsequent oxidative chemistry performed by non-heme mononuclear iron catalysts. [ABSTRACT FROM AUTHOR]

AbstractSalinity is a major abiotic stress that negatively affects agricultural land, significantly reducing crop yields. It alters the fundamental structure of the soil, causing a decrease in porosity, reduced aeration, and impaired water movement. Piriformospora indica, multifaceted fungi can enhance plant tolerance under abiotic stress conditions. The present study examined the effects of Piriformospora indica on the growth of Solanum melongena L. under saline conditions in a greenhouse, assessing parameters such as proline accumulation, lipid peroxidation, chlorophyll content, stomatal behavior, antioxidant activity, and phenotypic traits under salt stress Results of the present study showed significant improvement in phenotypic traits of Piriformospora indica colonized plants under saline conditions. Solanum melongena L. plants treated with 200 mM NaCl had swollen, deformed guard cells and closed stomata, while colonized plants maintained normal stomatal structure and their stomata remained open. Additionally, untreated plants exhibited higher malondialdehyde levels, indicating greater lipid peroxidation, while Piriformospora indica-colonized plants showed reduced oxidative damage, increased chlorophyll content, and enhanced peroxidase activity under saline conditions. The salt tolerance mediated by Piriformospora indica likely involves lipid desaturation, activation of antioxidant enzymes to counter reactive oxygen species, enhanced metabolism, improved nutrient uptake, proline accumulation, and increased phytohormone production. [ABSTRACT FROM AUTHOR]

Enzyme replacement therapy (ERT) is the only approved disease-modifying treatment modality for Pompe disease, a rare, inherited metabolic disorder caused by a deficiency in the acid α -glucosidase (GAA) enzyme that catabolizes lysosomal glycogen. First-generation recombinant human GAA (rhGAA) ERT (alglucosidase alfa) can slow the progressive muscle degeneration characteristic of the disease. Still, most patients experience diminished efficacy over time, possibly because of poor uptake into target tissues. Next-generation ERTs aim to address this problem by increasing bis-phosphorylated high mannose (bis-M6P) N -glycans on rhGAA as these moieties have sufficiently high receptor binding affinity at the resultant low interstitial enzyme concentrations after dosing to drive uptake by the cation-independent mannose 6-phosphate receptor on target cells. However, some approaches introduce bis-M6P onto rhGAA via non-natural linkages that cannot be hydrolyzed by natural human enzymes and thus inhibit the endolysosomal glycan trimming necessary for complete enzyme activation after cell uptake. Furthermore, all rhGAA ERTs face potential inactivation during intravenous delivery (and subsequent non-productive clearance) as GAA is an acid hydrolase that is rapidly denatured in the near-neutral pH of the blood. One new therapy, cipaglucosidase alfa plus miglustat, is hypothesized to address these challenges by combining an enzyme enriched with naturally occurring bis-M6P N -glycans with a small-molecule stabilizer. Here, we investigate this hypothesis by analyzing published and new data related to the mechanism of action of the enzyme and stabilizer molecule. Based on an extensive collection of in vitro , preclinical, and clinical data, we conclude that cipaglucosidase alfa plus miglustat successfully addresses each of these challenges to offer meaningful advantages in terms of pharmacokinetic exposure, target-cell uptake, endolysosomal processing, and clinical benefit. [ABSTRACT FROM AUTHOR]

Background: Gentamicin (GM) administration is associated with decreased metabolism, increased oxidative stress, and induction of nephrotoxicity. Sambucus nigra L., containing flavonoids, anthocyanins, and phytosterols, possesses antioxidant and anti-inflammatory potential. Objectives: The present study aimed to investigate the nephroprotective and anti-inflammatory potential of lyophilized Sambucus nigra fruit extract (S. nigra extract) to reduce acute oxidative stress and residual toxicity of GM in a 7-day experimental model in Balb/c rodents. Methods: The S. nigra extract was lyophilized (300 rpm; 10 min; −45 °C) to improve pharmacological properties. Balb/c mice were divided into four (n = 6) groups: controls; S. nigra extract per os (120 mg kg−1 day−1 bw); GM (200 mg kg−1 day−1 bw) (4); and GM + S. nigra therapy. The activities of antioxidant and renal enzymes, cytokines, and levels of oxidative stress biomarkers—Hydroxiproline, CysC, GST, KIM-1, PGC-1α, MDA, GSPx—were analyzed by ELISA tests. The ROS and RNS levels, as well as 5-MSL-protein oxidation, were measured by EPR spectroscopy. Results: The antioxidant-protective effect of S. nigra extract (120 mg kg−1) was demonstrated by reduced MDA, ROS, and RNS and increased activation of endogenous enzymes. Furthermore, S. nigra extract significantly reduced the expression of IL-1β, IL-6, IL-10, TNF-α, IFN-γ, and KIM-1 and regulated collagen/protein (PGC-1α and albumin) deposition in renal tissues. Conclusions: Histological evaluation confirmed that S. nigra (120 mg kg−1) attenuated renal dysfunction and structural damage by modulating oxidative stress and acute inflammation and could be used as an anti-fibrotic alternative in GM nephrotoxicity. [ABSTRACT FROM AUTHOR]

Heat stress hinders the growth and productivity of sweetpotato plants, predominantly through oxidative damage to cellular membranes. Therefore, the development of efficient approaches for mitigating heat-related impairments is essential for the long-term production of sweetpotatoes. Melatonin has been recognised for its capacity to assist plants in dealing with abiotic stress conditions. This research aimed to investigate how different doses of exogenous melatonin influence heat damage in sweetpotato plants. Heat stress drastically affected shoot and root fresh weight by 31.8 and 44.5%, respectively. This reduction resulted in oxidative stress characterised by increased formation of hydrogen peroxide (H2O2) by 804.4%, superoxide ion (O2 •−) by 211.5% and malondialdehyde (MDA) by 234.2%. Heat stress also reduced chlorophyll concentration, photosystem II efficiency (Fv/Fm) by 15.3% and gaseous exchange. However, pre-treatment with 100 mmol L-1 melatonin increased growth and reduced oxidative damage to sweetpotato plants under heat stress. In particular, melatonin decreased H2O2, O2 •− and MDA by 64.8%, 42.7% and 38.2%, respectively. Melatonin also mitigated the decline in chlorophyll levels and improved stomatal traits, gaseous exchange and Fv/Fm (13%). Results suggested that the favorable outcomes of melatonin treatment can be associated with elevated antioxidant enzyme activity and an increase in non-enzymatic antioxidants and osmo-protectants. Overall, these findings indicate that exogenous melatonin can improve heat stress tolerance in sweetpotatoes. This study will assist researchers in further investigating how melatonin makes sweetpotatoes more resistant to heat stress. [ABSTRACT FROM AUTHOR]

There is a variety of electrophiles in the environment. In addition, there are precursor chemicals that undergo metabolic activation by enzymes and conversion to electrophiles in the body. Although electrophiles covalently bind to protein nucleophiles, they also form adducts associated with adaptive or toxic responses. Low molecular weight compounds containing sulfur are capable of blocking such adduct formation by capturing the electrophiles. In this review, we present our findings on the capture and inactivation of electrophiles by: (i) intracellular glutathione, (ii) reactive sulfur species, and (iii) extracellular cysteine (formed during the production of sulfur adducts). These actions not only substantially suppress electrophilic activity but also regulate protein adduct formation. [ABSTRACT FROM AUTHOR]

Normal tissues typically maintain partial oxygen pressure within a range of 3–10% oxygen, ensuring homeostasis through a well-regulated oxygen supply and responsive vascular network. However, in solid tumors, rapid growth often outpaces angiogenesis, creating a hypoxic microenvironment that fosters tumor progression, altered metabolism and resistance to therapy. Hypoxic tumor regions experience uneven oxygen distribution with severe hypoxia in the core due to poor vascularization and high metabolic oxygen consumption. Cancer cells adapt to these conditions through metabolic shifts, predominantly relying on glycolysis, and by upregulating antioxidant defenses to mitigate reactive oxygen species (ROS)-induced oxidative damage. Hypoxia-induced ROS, resulting from mitochondrial dysfunction and enzyme activation, exacerbates genomic instability, tumor aggressiveness, and therapy resistance. Overcoming hypoxia-induced ROS cancer resistance requires a multifaceted approach that targets various aspects of tumor biology. Emerging therapeutic strategies target hypoxia-induced resistance, focusing on hypoxia-inducible factors, ROS levels, and tumor microenvironment subpopulations. Combining innovative therapies with existing treatments holds promise for improving cancer outcomes and overcoming resistance mechanisms. [ABSTRACT FROM AUTHOR]

This work constructed an immobilized laccase - peroxymonosulfate (PMS) activation synergistic catalytic system for enhancing the efficient removal of reactive blue 19 (RB19). Superparamagnetic CuFe 2 O 4 /kaolin (CF/K) composites were prepared by one-step solvothermal and calcination methods and then modified with chitosan as immobilized carriers for laccase. The enzyme activity of CuFe 2 O 4 /kaolin-CTS-Laccase (CF/K-CTS-Lac) could reach 316.3 U/g under optimal immobilization conditions of 3 h and 2.6 mg/mL. CF/K-CTS-Lac exhibited improved pH stability and thermal stability in comparison to free laccase. The CF/K-CTS-Lac/PMS synergistic catalytic system achieved 98.4 % decolourization of RB19 within 90 min, which was 1.7 and 1.9 times higher than that of the CF/K-CTS-Lac system and CF/K-CTS/PMS system, respectively. Enhanced RB19 degradation efficiency attributed to synergistic catalysis by immobilized laccase and PMS activation. Free radical quenching experiments showed that hydroxyl radicals (•OH), sulfate radicals (SO 4 •-), superoxide radicals (•O 2 -) and singlet oxygen (1O 2) were involved in the synergistic catalytic system, with •O 2 - and 1O 2 playing a major role. Our investigations are expected to provide new insights into the construction of synergistic catalytic systems for pollutants. [Display omitted] • Immobilized laccase - PMS activation synergistic catalytic system was constructed. • Laccase and CuFe 2 O 4 nanoparticles were uniformly and firmly dispersed on kaolin. • CuFe 2 O 4 /kaolin-CTS-Laccase demonstrates superior sustainability and catalytic activity. • •OH, SO 4 •-, •O 2 - and 1O 2 are involved in synergistic catalytic degradation of RB19. [ABSTRACT FROM AUTHOR]

Drought stress (DS) is a major devastating factor affecting plant growth and development worldwide. Potassium (K) is considered a vigorous moiety and stress alleviator, which crop cultivars need for better yield. It is also helpful in alleviating the DS-induced negative consequences by regulating various morphological, physiological, biochemical, and molecular mechanisms in plants. Particularly, the K application improves plant tolerance against DS by improving plant growth parameters, photosynthetic pigments, cell turgor pressure, osmotic pressure, nutritional balance, compatible solutes, and the plant's antioxidant defense system. Apart from its role as a constituent of the plant structure, biochemical processes such as protein synthesis, carbohydrate metabolism, and enzyme activation are also regulated by K. However, the exact K-mediated molecular mechanisms of DS tolerance are still unclear and require more investigation. The present review aims to provide insight into the role of K in regulating various morphological and physico-chemical aspects under DS. It also emphasizes the crosstalk of K with other nutrients and phytohormones, as well as molecular mechanisms for K homeostasis under DS. We have also shed light on genomics analysis to discover K transporter's novel genes in different plant species. [Display omitted] • Drought stress is a prominent constraint that consequently limits crop yield. • Potassium has been used on crop fields to increase crop productivity under stressed conditions. • Potassium modulates various developmental aspects, such as plant growth, yield, and quality. • Potassium provides cellular signaling alone/combined with phytohormones and with other nutrients. • We shed light on genomics of potassium transporter's genes and their role in molecular breeding. [ABSTRACT FROM AUTHOR]

The occurrence and development of tumors are accompanied by the abnormal expression of specific microRNAs (miRNAs). Therefore, miRNAs are considered as an important biomarker. The establishment of efficient, simple and sensitive miRNA imaging methods in living cells will contribute to the early diagnosis, treatment and drug development of diseases. In this study, we developed an endogenous enzyme-initiated AND logic circuit using gold nanocubes (AuNCs) as carriers for simultaneous detection of miRNA-21 and miRNA-210 in cells. Apurinic/apyrimidinic endonuclease 1 (APE1) and telomerase (TE), which are overexpressed in cancer cells, act as control switches in a logic circuit that enables sensitive in situ analysis of intracellular miRNAs without additional external intervention. At the same time, due to the lack of necessary enzymes as activation switches, the DNA circuit in normal cells remains in an inactive state. This strategy effectively reduces the risk of false positive signal generation. Our research results show that the logic circuit can not only distinguish between cancer cells and normal cells, and able to distinguish between different types of cancer cells. This finding provides a promising approach to accurately identify cell types. The specific overexpression of APE1 and telomerase in tumors acts as the on-off switch of the circuit, effectively reducing non-specific activation. [Display omitted] • The AND-gate nanomachine can detect low-abundance miRNAs. • The target can be recycled to achieve fluorescence signal amplification. • Fluorescence method can realize in situ imaging analysis of two miRNAs in cells. [ABSTRACT FROM AUTHOR]

Salvia miltiorrhiza is a model medicinal plant that is typically used to treat cardiovascular and cerebrovascular diseases. The primary active medicinal ingredients of S. miltiorrhiza are salvianolic acids and tanshinones. Jasmonate (JA) is a vital phytohormone that regulates secondary metabolism. The exogenous application of methyl jasmonate (MeJA) can promote the accumulation of active ingredients in S. miltiorrhiza. Here, we identified a MeJA-responsive SmbHLH59 gene that encodes for a bHLH IIIe family transcription factor. The overexpression of SmbHLH59 in S. miltiorrhiza increased the contents of salvianolic acids and tanshinones, while the opposite effect was observed when SmbHLH59 was knocked out via CRISPR. Meanwhile, Sm bHLH59 was observed to activate the expressions of SmPAL1 , SmC4H1 , SmHPPR1 , SmCPS1 , and SmKSL1 genes by binding to the E/G-box elements of their promoters. Further investigations demonstrated that Sm JAZ1 and Sm JAZ8 interacted with Sm bHLH59 to inhibit its activation of these five genes. In summary, a JA signaling pathway component (Sm bHLH59) was identified that strongly enhanced the accumulation of salvianolic acids and tanshinones through the direct activation of multiple enzyme genes in their biosynthetic pathways. Consequently, this study enriches our knowledge toward further elucidating the molecular mechanisms behind the regulation of JA in the secondary metabolism of S. miltiorrhiza. [ABSTRACT FROM AUTHOR]

[Display omitted] • A synthetic methylsuccinate metabolism pathway was demonstrated in Escherichia coli. • An adaptive laboratory evolution led to E. coli growing on methylsuccinate as the sole carbon source. • A novel flavoprotein-mediated electron transfer pathway was identified and validated in vivo. • The engineered strain can grow efficiently on methylsuccinate with a specific growth rate of 0.11 h-1. • This strain can be used to screen methane activation enzymes and synthetic methanotrophy. Methylsuccinate is a branched-chain, 5-carbon (C5) dicarboxylate that can be generated from the O 2 -independent activation of methane via fumarate addition. However, no established metabolic pathway enables growth and product synthesis from methylsuccinate. Here, we report a synthetic pathway that converts methylsuccinate into two precursor metabolites: pyruvate and acetyl-CoA. The pathway was constructed through rational design and validated both in vitro and in vivo using E. coli as the host. Subsequently, growth on methylsuccinate as the sole carbon source was achieved using two parallel strategies: adaptive laboratory evolution and enzyme mining. Through the latter approach, we identified a heterologous electron transfer pathway mediated by previously uncharacterized enzymes and integrated into E. coli enabling the conversion of methylsuccinyl-CoA to mesaconyl-C4-CoA. The engineered strain demonstrated efficient growth on various C5 dicarboxylates including methylsuccinate, mesaconate, and itaconate, with a specific growth rate of 0.11 h−1 on methylsuccinate. This study represents an important step toward achieving synthetic methanotrophy, as the engineered strain can serve as a platform for screening potential methane activation enzymes and ultimately as a production chassis for the bioconversion of methane into various value-added products. [ABSTRACT FROM AUTHOR]

Improving lignocellulose degradation and organic matter conversion in agricultural and livestock wastes remains a great challenge. Here, the contribution of humic acid (HA) to lignocellulose degradation was investigated, focusing on the abundance of key microbial species and carbohydrate-active enzymes during aerobic composting. The results demonstrated that the addition of HA not only increased the complexity of the microbial network, but also enhanced the positive interaction between microorganism. The abundance of phylum Actinobacteria related to lignin degradation was significantly increased, especially genus Nocardiopsis (50.97 %), and Nocardiopsis was significantly positively correlated with HA and humus (HS) (p < 0.05). Additionally, the abundance of GH (43.45%) and AA (5.88%) enzymes and the activation of metabolic pathways of AA, carbohydrates and energy were significantly increased (p < 0.05). Remarkably, the quantity of lignocellulose-degrading genes and carbohydrate-active enzymes experienced a marked boost (p < 0.05), with the peak abundance observed in Nocardiopsis. The structural equation model revealed that the addition of HA boosted the abundance of Nocardiopsis , which in turn amplified lignocellulose degradation by up-regulating lignocellulose degradation genes and enhancing carbohydrase activity, and facilitating the conversion of HA and FA. The lignocellulose degradation experiment verified that Nocardiopsis alba exhibited good ability in the degradation of cellulose and hemicellulose. These findings provided a novel perspective on the mechanisms underlying lignocellulose degradation, and broaden the understanding of the ecophysiological role of Nocardiopsis in composting system. [Display omitted] • The addition of humic acid (HA) raises the abundance of Nocardiopsis by 50.97%. • HA addition boosts lignocellulose gene abundance and CAZyme activity. • GH, AA enzymes are up with HA addition by 43.4 and 5.88%, respectively. • HA addition enhances microbial network complexity, emphasizing Nocardiopsis. [ABSTRACT FROM AUTHOR]

• Aedes aegypti from fernando de noronha remains susceptible to pyrethroids. • Non-irradiated aedes aegypti show glutathione S-transferases (GSTs) activity changes in both male and female mosquitoes. • Ionizing radiation boosts GST activity in aedes aegypti , showing a stress response. • Gamma-induced GST activity may improve sterile male fitness when SIT and insecticides are combined. This study characterizes the Aedes aegypti population from Fernando de Noronha Island, Pernambuco, Brazil, prior to implementing the Sterile Insect Technique (SIT). The main objective was to assess changes in glutathione S-transferase (GST) enzyme activity, previously linked to cypermethrin resistance in this population, in 2010. GST activity was measured in both male and female mosquitoes, masse produced in lab, after exposure to ionizing radiation. The populational evaluation after six years showed a complete susceptibility to cypermethrin, deltamethrin and lambda-cyhalothrin, although GST activity remained altered, increasing furthermore following irradiation (50 % higher in irradiated males and 31 % higher in irradiated females compared to non-irradiated controls). This stress response to gamma radiation suggesting implications for the effectiveness and viability of sterile males, particularly when SIT is combined with chemical insecticides. These findings enhance our understanding of radiation's impact on metabolic responses of the sterile males and provide valuable insights for refining integrated control strategies in vector management programs. [Display omitted] [ABSTRACT FROM AUTHOR]

Interleukin-2 (IL-2) is a T cell growth factor that is essential for the proliferation of T cells and the generation of effector and memory cells. The antitumor activity of high-dose IL-2 therapy requires maintaining the affinity between IL-2 and IL2-Rα, which can also bring serious toxic side effects. To address this issue, we designed ZGP-Cysteamine-IL-2-K64C and (ZGP-Cysteamine) 2 -IL-2-(K43C, K64C) based on the strategy of FAPα enzyme-activated prodrugs, and investigated their anti-tumour activity and side effects. In vitro FAPα enzyme cleavage results indicated that the side-chain modified ZGP-Cysteamine moiety could be precisely recognized and cleaved by FAPα, thereby restoring the activity of native IL-2 capable of binding to IL-2Rα in the tumour microenvironment, where it promotes the expansion of CD8+ T cells. Meanwhile, surface plasmon resonance analysis revealed that, compared to wt-IL-2, both ZGP-Cysteamine-IL-2-K64C and (ZGP-Cysteamine) 2 -IL-2-(K43C, K64C) exhibited significantly reduced affinity for IL-2Rα, while their affinity for IL-2Rβγ remained unchanged. Remarkably, ZGP-Cysteamine-IL-2-K64C and (ZGP-Cysteamine) 2 -IL-2-(K43C, K64C) almost completely eliminated the pulmonary edema and vascular permeability. Furthermore, the combination of ZGP-Cysteamine-IL-2-K64C and PD-1 blockade showed robust anti-tumour activity in mice tumour models. Our study provides new insights into the structural design of IL-2 prodrug with low side effect and robust anti-tumour efficacy. • A novel IL-2 prodrug can effectively release parent IL-2 through specific cleavage by FAPα enzyme in tumour microenviroment. • ZGP-Cysteamine molecule eliminated toxicity by markedly inhibiting the formation of IL-2-IL-2Rα complexes. • IL-2 prodrug undergoes activation by the FAPα enzyme within the TME, releasing the Z -Gly-Pro-OH group and restoring the natural IL-2 activity. • ZGP-Cysteamine-IL-2-K64C+PD-1 combination therapy significantly shows robust anti-tumour activity in mice tumuor models. [ABSTRACT FROM AUTHOR]

Mutations that increase LRRK2 kinase activity have been linked to Parkinson's disease and Crohn's disease. LRRK2 is also activated by lysosome damage. However, the endogenous cellular mechanisms that control LRRK2 kinase activity are not well understood. In this study, we identify signaling through stimulator of interferon genes (STING) as an activator of LRRK2 via the conjugation of ATG8 to single membranes (CASM) pathway. We furthermore establish that multiple chemical stimuli that perturb lysosomal homeostasis also converge on CASM to activate LRRK2. Although CASM results in the lipidation of multiple ATG8 protein family members, we establish that LRRK2 lysosome recruitment and kinase activation are highly dependent on interactions with the GABARAP member of this family. Collectively, these results define a pathway that integrates multiple stimuli at lysosomes to control the kinase activity of LRRK2. Aberrant activation of LRRK2 via this pathway may be of relevance in both Parkinson's and Crohn's diseases., (© 2025 Bentley-DeSousa et al.)

Ca 2+ /calmodulin-dependent protein kinase II α (CaMKIIα) "autonomous" activation induced by Thr286 phosphorylation has a crucial role in synaptic plasticity. Previous studies showed that in Alzheimer's disease brain, CaMKIIα autophosphorylation at Thr286 is reduced while the level of cysteine-oxidized CAMKIIα is elevated. We performed tryptic mapping of the oxidized CaMKIIα and discovered the formation of a disulfide between the N-terminal Cys6 and the regulatory domain Cys280. The apparent pK a values of Cys6 and Cys280 are 7.1 and 7.7, respectively, lower than the 8.5 for free Cys. The low apparent pK a of Cys6 facilitates the oxidation of its thiol to the sulfenic acid at physiological pH. The thiolate of Cys280 can then attack the sulfenic acid to form a disulfide. Using an antibody against phosphorylated Thr286, we showed that disulfide formation prevents Thr286 phosphorylation. CaMKIIα autonomous activation induced by disulfide formation is much lower than the autonomous activation induced by phosphorylation. The decreased autonomous activation may contribute to the synaptic impairment of Alzheimer's disease. We also generated a CaMKIIα mutant in which Cys6 was mutated to Ser6. This mutation prevented disulfide formation and restored autonomous activation induced by phosphorylation. Our findings provide insight into the mechanistic details of CaMKIIα autonomous activation induced by disulfide formation that may contribute to the impairment of long-term potentiation in Alzheimer's disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Obesity increases the risk and mortality of breast cancer through dysregulated secretion of proinflammatory cytokines and tumor adipokines that induce an inflammatory breast microenvironment. Resistin is an adipokine secreted by adipocytes, immune cells, and predominantly macrophages, which contributes to cancer progression, but its molecular mechanism in cancer is not completely described. In this study, we analyzed the relationship of resistin on breast cancer prognosis and tumor progression and the effect in vitro of resistin on p38 and ERK1/2 activation in breast cancer cell lines. By bioinformatic analysis, we found that resistin is overexpressed in the basal subtype triple-negative breast cancer and is related to poor prognosis. In addition, we demonstrated a positive correlation between RETN and MAPK3 expression in basal triple-negative breast cancer. Importantly, we found amplifications of the RETN gene in at least 20 % of metastatic samples from patients with breast cancer. Most samples with RETN amplifications metastasized to bone and showed high expression of IL-8 (CXCL8) and IL-6 (IL6). Finally, resistin could be considered a prognostic marker for basal triple-negative breast cancer, and we also proposed the possibility that resistin-induced cell migration involves the activation of MAPK in breast cancer cells., Competing Interests: Declaration of competing interest Please check the following as appropriate: X All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version. X This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue. X The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript The following authors have affiliations with organizations with direct or indirect financial interest in the subject matter discussed in the manuscript., (Copyright © 2024 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Cezanne-2 (Cez2) is a deubiquitinylating (DUB) enzyme involved in the regulation of ubiquitin-driven cellular signaling and selectively targets Lys11-linked polyubiquitin chains. As a representative member of the ovarian tumor (OTU) subfamily DUBs, it performs cysteine proteolytic isopeptide bond cleavage; however, its exact catalytic mechanism is not yet resolved. In this work, we used different computational approaches to get molecular insights into the Cezanne-2 catalytic mechanism. Extensive molecular dynamics (MD) simulations were performed for 12 μs to model free Cez2 and the diubiquitin (diUb) substrate-bound protein-protein complex in two different charge states of Cez2, each corresponding to a distinct reactive state in its catalytic cycle. The simulations were analyzed in terms of the relevant structural parameters for productive enzymatic catalysis. Reactive diUb-Cez2 complex configurations were identified, which lead to isopeptide bond cleavage and stabilization of the tetrahedral oxyanion intermediate. The reliability of these complexes was further assessed by quantum mechanics/molecular mechanics (QM/MM) optimizations. The results show that Cez2 follows a modified cysteine protease mechanism involving a catalytic Cys210/His367 dyad, with the oxyanion hole to be a part of the "C-loop," and polarization of His367 by the formation of a strictly conserved water bridge with Glu173. The third residue has a dual role in catalysis as it mediates substrate binding and polarization of the catalytic dyad. A similar mechanism was identified for Cezanne-1, the paralogue of Cez2. In general, our simulations provide valuable molecular information that may help in the rational design of selective inhibitors of Cez2 and closely related enzymes.

mTOR plays a crucial role in PI3K/AKT/mTOR signaling. We hypothesized that mTOR activation mechanisms driving oncogenesis can advise effective therapeutic designs. To test this, we combined cancer genomic analysis with extensive molecular dynamics simulations of mTOR oncogenic variants. We observed that conformational changes within mTOR kinase domain are associated with multiple mutational activation events. The mutations disturb the α-packing formed by the kαAL, kα3, kα9, kα9b, and kα10 helices in the kinase domain, creating cryptic pocket. Its opening correlates with opening of the catalytic cleft, including active site residues realignment, favoring catalysis. The cryptic pocket created by disrupted α-packing coincides with the allosteric pocket in PI3Kα can be harmoniously fitted by the PI3Kα allosteric inhibitor RLY-2608, suggesting that analogous drugs designed based on RLY-2608 can restore the packed α-structure, resulting in mTOR inactive conformation. Our results exemplify that knowledge of detailed kinase activation mechanisms can inform innovative allosteric inhibitor development.

Reductase expression is a potential indicator of cellular pathology. Single-detection systems for reductases have been developed, however, the development of dual-detection systems remain largely unexplored. We rationally designed a dual-lock fluorescent probe that exhibited a high signal-to-noise ratio with a fluorescence Off-On response exclusively for the simultaneous activity of two reductases, NTR and hNQO1, which are overexpressed in cancer hypoxia. The system comprised a naphthalimide fluorophore with dual-lock control mediated by PET and ICT, a trimethyl-locked quinone group sensitive to hNQO1, and a nitrobenzyl carbamate group sensitive to NTR. This study employed a hypoxia model in HeLa cells to demonstrate that our developed dual-lock system detected hypoxia more effectively than single-detection systems. Moreover, it enabled noninvasive real-time monitoring of hypoxia in zebrafish embryos. Consequently, the dual-lock fluorescent probe, which strategically provides a fluorescence response only when both NTR and NQO1 are active, offers a novel diagnostic platform for both in vitro and in vivo applications, effectively detecting hypoxia and monitoring various pathological states.

Aims: Hepsin (HPN), a Type II transmembrane serine protease (TTSP), is involved in hepatocyte metabolism and various diseases. It undergoes autoactivation on the surface of human hepatoma cells, a mechanism not observed in other cell types. This study aims to explore HPN activation and surface expression in endometrial epithelial cells., Materials and Methods: We studied HPN zymogen activation and cell surface expression in human embryonic kidney 293 and endometrial epithelial AN3CA and Ishikawa cells using site-directed mutagenesis, Western blotting, flow cytometry, and immunostaining. Treatments with brefeldin A (BFA) and monensin, along with co-transfection assays, were employed to assess HPN activation and expression before reaching the cell surface. We also analyzed the activation and expression of TMPRSS2 and TMPRSS13 and examined the effect of the serine protease inhibitor HAI-1 on these proteases., Key Findings: HPN zymogen autoactivates in the endoplasmic reticulum (ER) and Golgi apparatus. Its active form reduces cell surface expression through trans-autodegradation, a mechanism also applicable to in TMPRSS2 and TMPRSS13. Additionally, HAI-1 interacts with these TTSPs in different ways: it inhibits HPN activation and stabilizes its cell-surface expression; it inhibits TMPRSS2 activation without affecting its cell-surface expression; and it facilitates TMPRSS13 activation, protecting it from degradation and stabilizing its cell surface expression., Significance: These results revealed an intracellular autoactivation and expression mechanism of HPN, TMPRSS2, and TMPRSS13, differing from the extracellular activated TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation, potentially aiding in treating TTSP-related endometrial diseases., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The nonsense-mediated mRNA decay (NMD) pathway triggers the degradation of defective mRNAs and governs the expression of mRNAs with specific characteristics. Current understanding indicates that NMD is often significantly suppressed during viral infections to protect the viral genome. In numerous viruses, this inhibition is achieved through direct or indirect interference with the RNA helicase UPF1, thereby promoting viral replication and enhancing pathogenesis. In this study, we employed biochemical, biophysical assays and cellular investigations to explore the interplay between UPF1 and the nucleocapsid (Np) protein of SARS-CoV-2. We evaluated their direct interaction and its impact on inhibiting cellular NMD. Furthermore, we characterized how this interaction affects UPF1's enzymatic function. Our findings demonstrate that Np inhibits the unwinding activity of UPF1 by physically obstructing its access to structured nucleic acid substrates. Additionally, we showed that Np binds directly to UPF2, disrupting the formation of the UPF1/UPF2 complex essential for NMD progression. Intriguingly, our research also uncovered a surprising pro-viral role of UPF1 and an antiviral function of UPF2. These results unveil a novel, multi-faceted mechanism by which SARS-CoV-2 evades the host's defenses and manipulates cellular components. This underscores the potential therapeutic strategy of targeting Np-UPF1/UPF2 interactions to treat COVID-19., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Tissue factor (TF) and protease-activated receptor 2 (PAR2) have been associated with the progression of cancer, while integrins are essential for the adhesion and migration of cancer cells. This study aimed to explore the cross-talk between the TF:FVIIa complex, PAR2 signaling, and the expression of integrin α1 in cervical cancer cells. Utilizing data from The Cancer Genome Atlas (TCGA), the research examined the relationship between the TF and PAR2 genes and the integrin α1 gene (ITGA1) in reproductive cancers, revealing a positive correlation between integrin α1 expression and both TF and PAR2 genes. Analyses through Western blotting and RT-PCR demonstrated that TF:FVIIa complex transactivates PAR2, which significantly increases the phosphorylation of MEK1/2 and subsequently elevates integrin α1 expression. Inhibition of either PAR2 or MEK1/2 resulted in a decrease in the FVIIa-induced increase in integrin α1 expression. Additionally, cell migration studies indicated that elevated expression of integrin α1, mediated by the TF:FVIIa/PAR2 pathway, was linked to enhanced cell migration, which could be inhibited by blocking integrin α1. This investigation uncovers a novel signaling pathway in HeLa cells, highlighting the significance of the TF:FVIIa:PAR2 axis in modulating integrins that are vital for cancer progression, thereby offering insights for potential targeted therapeutic approaches in cancer treatment., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Paranitharan Nagarajan reports financial support was provided by Council of Scientific & Industrial Research (CSIR). Velayuthaprabhu Shanmugam reports financial support was provided by Rashtriya Uchchatar Shiksha Abhiyan (RUSA 2.0 BEICH). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Endothelin (ET)-1 contributes to melanoma progression via cell proliferation, invasion, and migration. We previously reported that annexin A2 (AnxA2) binds to ET receptors. In this study, we aimed to further investigate role of AnxA2 in melanoma cell proliferation after ET-1 stimulation. AnxA2 knockdown inhibited ET-1-stimulated cell proliferation and AKT activation in SK-MEL28 melanoma cells. ET-1 stimulation phosphorylated serine on AnxA2, and AnxA2 Ser25 phosphorylation-deficient mutant (AnxA2 S25A) cells showed lower ET-1-stimulated cell proliferation and AKT activation than the rescue AnxA2 knockdown (AnxA2 res) and AnxA2 Ser11 phosphorylation-deficient mutant (AnxA2 S11A) cells. Although AnxA2 S25A was localized to the plasma membrane, it exhibited lower colocalization with ET receptors than AnxA2 res and AnxA2 S11A on the plasma membrane. These results suggest that phosphorylation of AnxA2 Ser25 affects the colocalization of AnxA2 and ETRs and plays an important role in cell proliferation and AKT activation in ET-1 stimulated melanoma cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Treatments which inhibit or inactivate Cdk1/cyclin B in metaphase-arrested mammalian cells and budding yeast are described. These treatments induce the cells to exit mitosis and return to interphase, though without chromosome segregation or cytokinesis, and they provide the basis for a method to identify enzymes or other proteins which act "downstream" from Cdk1 inactivation and to elucidate the roles of those proteins in mitotic exit. In this method, inactivation of Cdk1 is combined with inhibition or inactivation of a protein of interest and the effects are observed. This approach should be particularly useful for determining which protein phosphatases are involved in the transition from mitosis to G1-phase and for identifying their substrates., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Vascular smooth muscle cells are important in intimal hyperplasia. Thrombospondin-1 is a matricellular protein involved in the vascular injury response. Statins are cholesterol lowering drugs that have beneficial cardiovascular effects. Statis have been shown to inhibit smooth muscle migration through the mevalonate pathway. This effect is thought to be mediated by small G protein Ras and Rho turnover which requires many hours. While many patients undergoing treatment for vascular disease are on statins, many are not. Thus immediate pretreatment with statins before surgery may be beneficial. We hypothesized that statins have effects independent of the mevalonate pathway and thus have an immediate effect., Methods: Human vascular smooth muscle cells were pretreated for 20 h (long-term) or 20 min (short-term) with fluvastatin, or mevalonolactone plus fluvastatin. Thrombospondin-1-induced migration, activation of p42/p44 extracellular signal-regulated kinase, c-Src, focal adhesion kinase and PI3 kinase was determined. The effect of fluvastatin on thrombospondin-1-induced expression of THBS1 , FOS , HAS2 and TGFB2 was examined., Results: Both treatments inhibited thrombospondin-1-induced chemotaxis back to the control group. Mevalonolactone reversed the long-term statin effect by increasing migration but had no effect on the short-term statin response. p42/p44 extracellular signal-regulated kinase was activated by thrombospondin-1 and both treatments augmented activation. Neither treatment affected c-Src activity, but both inhibited focal adhesion kinase and PI3 kinase activity. Only long-term statin treatment inhibited THBS1 expression while both treatments inhibited FOS and TGFB2 expression. Neither treatment affected HAS2 . FOS knockdown inhibited thrombospondin-1-induced HAS2 but not TGFβ2 gene expression., Conclusion: Long-term fluvastatin inhibited thrombospondin-1-induced chemotaxis through the mevalonate pathway while short-term fluvastatin inhibited chemotaxis through an alternate mechanism. Short-term stains have immediate effects independent of the mevalonate pathway. Acute local treatment with statins followed by longer term therapy may limit the vascular response to injury., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

AMP-activated protein kinase (AMPK) is an energy-sensing serine/threonine kinase involved in metabolic regulation. It is phosphorylated by the upstream liver kinase B1 (LKB1) or calcium/calmodulin-dependent kinase kinase 2 (CaMKKβ). In cultured cells, AMPK activation correlates with LKB1 activity. The phosphorylation activates AMPK, shifting metabolism toward catabolism and promoting mitogenesis. In muscles, inactivity reduces AMPK activation, shifting the phenotype of oxidative muscles toward a more glycolytic profile. Here, we compared the basal level of AMPK activation in glycolytic and oxidative muscles and analyzed whether this relates to LKB1 or CaMKKβ. Using Western blotting, we assessed AMPK expression and phosphorylation in soleus, gastrocnemius (GAST), extensor digitorum longus (EDL), and heart from C57BL6J mice. We also assessed LKB1 and CaMKKβ expression, and CaMKKβ activity in tissue homogenates. AMPK activation was higher in oxidative (soleus and heart) than in glycolytic muscles (gastrocnemius and EDL). This correlated with AMPK α1-isoform expression, but not LKB1 and CaMKKβ. LKB1 expression was sex dependent and lower in male than female muscles. CaMKKβ expression was very low in skeletal muscles and did not phosphorylate AMPK in muscle lysates. The higher AMPK activation in oxidative muscles is in line with the fact that activated AMPK maintains an oxidative phenotype. However, this could not be explained by LKB1 and CaMKKβ. These results suggest that the regulation of AMPK activation is more complex in muscle than in cultured cells. As AMPK has been proposed as a therapeutic target for several diseases, future research should consider AMPK isoform expression and localization, and energetic compartmentalization. NEW & NOTEWORTHY It is important to understand how AMP-activated kinase, AMPK, is regulated, as it is a potential therapeutic target for several diseases. AMPK is activated by liver kinase B1, LKB1, and calcium/calmodulin-dependent kinase kinase 2, CaMKKβ. In cultured cells, AMPK activation correlates with LKB1 expression. In contrast, we show that AMPK-activation was higher in oxidative than glycolytic muscle, without correlating with LKB1 or CaMKKβ expression. Thus, AMPK regulation is more complex in highly compartmentalized muscle cells.