Phylogenetic and transcriptomic study of aldo-keto reductases in Haemonchus contortus and their inducibility by flubendazole.

Aldo-keto reductases (AKRs), a superfamily of NADP(H)-dependent oxidoreductases, catalyze the oxidoreduction of a wide variety of eobiotic and xenobiotic aldehydes and ketones. In mammals, AKRs play essential roles in hormone and xenobiotic metabolism, oxidative stress, and drug resistance, but little is known about these enzymes in the parasitic nematode Haemonchus contortus. In the present study, 22 AKR genes existing in the H. contortus genome were investigated and a phylogenetic analysis with comparison to AKRs in Caenorhabditis elegans, sheep and humans was conducted. The constitutive transcription levels of all AKRs were measured in eggs, larvae, and adults of H. contortus, and their expression was compared in a drug-sensitive strain (ISE) and a benzimidazole-resistant strain (IRE) previously derived from the sensitive strain by imposing benzimidazole selection pressure. In addition, the inducibility of AKRs by exposure of H. contortus adults to benzimidazole anthelmintic flubendazole in vitro was tested. Phylogenetic analysis demonstrated that the majority of AKR genes in H. contortus lack orthologues in the sheep genome, which is a favorable finding for considering AKRs as potential drug targets. Large differences in the expression levels of individual AKRs were observed, with AKR1, AKR3, AKR8, and AKR10 being the most highly expressed at most developmental stages. Significant changes in the expression of AKRs during the life cycle and pronounced sex differences were found. Comparing the IRE and ISE strains, three AKRs were upregulated, and seven AKRs were downregulated in adults. In addition, the expression of three AKRs was induced by flubendazole exposure in adults of the ISE strain. Based on these results, AKR1, AKR2, AKR3, AKR5, AKR10 and AKR19 in particular merit further investigation and functional characterization with respect to their potential involvement in drug biotransformation and anthelmintic resistance in H. contortus.

Aldo-keto reductases: Role in cancer development and theranostics.

Aldo-keto reductases (AKRs) are a superfamily of enzymes that play crucial roles in various cellular processes, including the metabolism of xenobiotics, steroids, and carbohydrates. A growing body of evidence has unveiled the involvement of AKRs in the development and progression of various cancers. AKRs are aberrantly expressed in a wide range of malignant tumors. Dysregulated expression of AKRs enables the acquisition of hallmark traits of cancer by activating oncogenic signaling pathways and contributing to chemoresistance. AKRs have emerged as promising oncotherapeutic targets given their pivotal role in cancer development and progression. Inhibition of aldose reductase (AR), either alone or in combination with chemotherapeutic drugs, has evolved as a pragmatic therapeutic option for cancer. Several classes of synthetic aldo-keto reductase (AKR) inhibitors have been developed as potential anticancer agents, some of which have shown promise in clinical trials. Many AKR inhibitors from natural sources also exhibit anticancer effects. Small molecule inhibitors targeting specific AKR isoforms have shown promise in preclinical studies. These inhibitors disrupt the activation of oncogenic signaling by modulating transcription factors and kinases and sensitizing cancer cells to chemotherapy. In this review, we discuss the physiological functions of human AKRs, the aberrant expression of AKRs in malignancies, the involvement of AKRs in the acquisition of cancer hallmarks, and the role of AKRs in oncogenic signaling, and drug resistance. Finally, the potential of aldose reductase inhibitors (ARIs) as anticancer drugs is summarized.

Cancer to Cataracts: The Mechanistic Impact of Aldo-Keto Reductases in Chronic Diseases.

Aldo-keto reductases (AKRs) are a superfamily of promiscuous enzymes that have been chiseled by evolution to act as catalysts for numerous regulatory pathways in humans. However, they have not lost their promiscuity in the process, essentially making them a double-edged sword. The superfamily is involved in multiple metabolic pathways and are linked to chronic diseases such as cataracts, diabetes, and various cancers. Unlike other detoxifying enzymes such as cytochrome P450s (CYP450s), short-chain dehydrogenases (SDRs), and medium-chain dehydrogenases (MDRs), that participate in essential pathways, AKRs are more widely distributed and have members with interchangeable functions. Moreover, their promiscuity is ubiquitous across all species and participates in the resistance of pathogenic microbes. Moreover, the introduction of synthetic substrates, such as synthetic molecules and processed foods, results in unwanted "toxification" due to enzyme promiscuity, leading to chronic diseases.

Advances in aldo-keto reductases immobilization for biocatalytic synthesis of chiral alcohols.

Chiral alcohols are essential building blocks of numerous pharmaceuticals and fine chemicals. Aldo-keto reductases (AKRs) constitute a superfamily of oxidoreductases that catalyze the reduction of aldehydes and ketones to their corresponding alcohols using NAD(P)H as a coenzyme. Knowledge about the crucial roles of AKRs immobilization in the biocatalytic synthesis of chiral alcohols is expanding. Herein, we reviewed the characteristics of various AKRs immobilization approaches, the applications of different immobilization materials, and the prospects of continuous flow bioreactor construction by employing these immobilized biocatalysts for synthesizing chiral alcohols. Finally, the opportunities and ongoing challenges for AKR immobilization are discussed and the outlook for this emerging area is analyzed.

Aldo-keto reductase (AKR) superfamily website and database: An update.

The aldo-keto reductase (AKR) superfamily is a large family of proteins found across the kingdoms of life. Shared features of the family include 1) structural similarities such as an (α/ß)8-barrel structure, disordered loop structure, cofactor binding site, and a catalytic tetrad, and 2) the ability to catalyze the nicotinamide adenine dinucleotide (phosphate) reduced (NAD(P)H)-dependent reduction of a carbonyl group. A criteria of family membership is that the protein must have a measured function, and thus, genomic sequences suggesting the transcription of potential AKR proteins are considered pseudo-members until evidence of a functionally expressed protein is available. Currently, over 200 confirmed AKR superfamily members are reported to exist. A systematic nomenclature for the AKR superfamily exists to facilitate family and subfamily designations of the member to be communicated easily. Specifically, protein names include the root "AKR", followed by the family represented by an Arabic number, the subfamily-if one exists-represented by a letter, and finally, the individual member represented by an Arabic number. The AKR superfamily database has been dedicated to tracking and reporting the current knowledge of the AKRs since 1997, and the website was last updated in 2003. Here, we present an updated version of the website and database that were released in 2023. The database contains genetic, functional, and structural data drawn from various sources, while the website provides alignment information and family tree structure derived from bioinformatics analyses.

Nicotinate-curcumin improves NASH by inhibiting the AKR1B10/ACCα-mediated triglyceride synthesis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a prevalent chronic liver condition. However, the potential therapeutic benefits and underlying mechanism of nicotinate-curcumin (NC) in the treatment of NASH remain uncertain. METHODS: A rat model of NASH induced by a high-fat and high-fructose diet was treated with nicotinate-curcumin (NC, 20, 40 mg·kg- 1), curcumin (Cur, 40 mg·kg- 1) and metformin (Met, 50 mg·kg- 1) for a duration of 4 weeks. The interaction between NASH, Cur and Aldo-Keto reductase family 1 member B10 (AKR1B10) was filter and analyzed using network pharmacology. The interaction of Cur, NC and AKR1B10 was analyzed using molecular docking techniques, and the binding energy of Cur and NC with AKR1B10 was compared. HepG2 cells were induced by Ox-LDL (25 µg·ml- 1, 24 h) in high glucose medium. NC (20µM, 40µM), Cur (40µM) Met (150µM) and epalrestat (Epa, 75µM) were administered individually. The activities of ALT, AST, ALP and the levels of LDL, HDL, TG, TC and FFA in serum were quantified using a chemiluminescence assay. Based on the changes in the above indicators, score according to NAS standards. The activities of Acetyl-CoA and Malonyl-CoA were measured using an ELISA assay. And the expression and cellular localization of AKR1B10 and Acetyl-CoA carboxylase (ACCα) in HepG2 cells were detected by Western blotting and immunofluorescence. RESULTS: The results of the animal experiments demonstrated that NASH rat model induced by a high-fat and high-fructose diet exhibited pronounced dysfunction in liver function and lipid metabolism. Additionally, there was a significant increase in serum levels of FFA and TG, as well as elevated expression of AKR1B10 and ACCα, and heightened activity of Acetyl-CoA and Malonyl-CoA in liver tissue. The administration of NC showed to enhance liver function in rats with NASH, leading to reductions in ALT, AST and ALP levels, and decrease in blood lipid and significant inhibition of FFA and TG synthesis in the liver. Network pharmacological analysis identified AKR1B10 and ACCα as potential targets for NASH treatment. Molecular docking studies revealed that both Cur and NC are capable of binding to AKR1B10, with NC exhibiting a stronger binding energy to AKR1B10. Western blot analysis demonstrated an upregulation in the expression of AKR1B10 and ACCα in the liver tissue of NASH rats, accompanied by elevated Acetyl-CoA and Malonyl-CoA activity, and increased levels of FFA and TG. The results of the HepG2 cell experiments induced by Ox-LDL suggest that NC significantly inhibited the expression and co-localization of AKR1B10 and ACCα, while also reduced levels of TC and LDL-C and increased level of HDL-C. These effects are accompanied by a decrease in the activities of ACCα and Malonyl-CoA, and levels of FFA and TG. Furthermore, the impact of NC appears to be more pronounced compared to Cur. CONCLUSION: NC could effectively treat NASH and improve liver function and lipid metabolism disorder. The mechanism of NC is related to the inhibition of AKR1B10/ACCα pathway and FFA/TG synthesis of liver.

Role of AKR1B10 in inflammatory diseases.

Inflammation is an important pathophysiological process in many diseases; it has beneficial and harmful effects. When exposed to various stimuli, the body triggers an inflammatory response to eliminate invaded pathogens and damaged tissues to maintain homeostasis. However, uncontrollable persistent or excessive inflammatory responses may damage tissues and induce various diseases, such as metabolic diseases (e.g. diabetes), autoimmune diseases, nervous system-related diseases, digestive system-related diseases, and even tumours. Aldo-keto reductase 1B10 (AKR1B10) is an important player in the development and progression of multiple diseases, such as tumours and inflammatory diseases. AKR1B10 is upregulated in solid tumours, such as hepatocellular carcinoma (HCC), non-small cell lung carcinoma, and breast cancer, and is a reliable serum marker. However, information on the role of AKR1B10 in inflammation is limited. In this study, we summarized the role of AKR1B10 in inflammatory diseases, including its expression, functional contribution to inflammatory responses, and regulation of signalling pathways related to inflammation. We also discussed the role of AKR1B10 in glucose and lipid metabolism and oxidative stress. This study provides novel information and increases the understanding of clinical inflammatory diseases.

Machine learning-based algorithm identifies key mitochondria-related genes in non-alcoholic steatohepatitis.

BACKGROUND: Evidence suggests that hepatocyte mitochondrial dysfunction leads to abnormal lipid metabolism, redox imbalance, and programmed cell death, driving the onset and progression of non-alcoholic steatohepatitis (NASH). Identifying hub mitochondrial genes linked to NASH may unveil potential therapeutic targets. METHODS: Mitochondrial hub genes implicated in NASH were identified via analysis using 134 algorithms. RESULTS: The Random Forest algorithm (RF), the most effective among the 134 algorithms, identified three genes: Aldo-keto reductase family 1 member B10 (AKR1B10), thymidylate synthase (TYMS), and triggering receptor expressed in myeloid cell 2 (TREM2). They were upregulated and positively associated with genes promoting inflammation, genes involved in lipid synthesis, fibrosis, and nonalcoholic steatohepatitis activity scores in patients with NASH. Moreover, using these three genes, patients with NASH were accurately categorized into cluster 1, exhibiting heightened disease severity, and cluster 2, distinguished by milder disease activity. CONCLUSION: These three genes are pivotal mitochondrial genes implicated in NASH progression.

AKR1B10 expression characteristics in hepatocellular carcinoma and its correlation with clinicopathological features and immune microenvironment.

Hepatocellular carcinoma (HCC) represents a major global health threat with diverse and complex pathogenesis. Aldo-keto reductase family 1 member B10 (AKR1B10), a tumor-associated enzyme, exhibits abnormal expression in various cancers. However, a comprehensive understanding of AKR1B10's role in HCC is lacking. This study aims to explore the expression characteristics of AKR1B10 in HCC and its correlation with clinicopathological features, survival prognosis, and tumor immune microenvironment, further investigating its role and potential regulatory mechanisms in HCC. This study conducted comprehensive analyses using various bioinformatics tools and databases. Initially, differentially expressed genes related to HCC were identified from the GEO database, and the expression of AKR1B10 in HCC and other cancers was compared using TIMER and GEPIA databases, with validation of its specificity in HCC tissue samples using the HPA database. Furthermore, the relationship of AKR1B10 expression with clinicopathological features (age, gender, tumor size, staging, etc.) of HCC patients was analyzed using the TCGA database's LIHC dataset. The impact of AKR1B10 expression levels on patient prognosis was evaluated using Kaplan-Meier survival analysis and the Cox proportional hazards model. Additionally, the correlation of AKR1B10 expression with tumor biology-related signaling pathways and tumor immune microenvironment was studied using databases like GSEA, Targetscan, and others, identifying microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) that regulate AKR1B10 expression to explore potential regulatory mechanisms. Elevated AKR1B10 expression was significantly associated with gender, primary tumor size, and fibrosis stage in HCC tissues. High AKR1B10 expression indicated poor prognosis and served as an independent predictor for patient outcomes. Detailed mechanism analysis revealed a positive correlation between high AKR1B10 expression, immune cell infiltration, and pro-inflammatory cytokines, suggesting a potential DANCR-miR-216a-5p-AKR1B10 axis regulating the tumor microenvironment and impacting HCC development and prognosis. The heightened expression of AKR1B10 in HCC is not only related to significant clinical-pathological traits but may also influence HCC progression and prognosis by activating key signaling pathways and altering the tumor immune microenvironment. These findings provide new insights into the role of AKR1B10 in HCC pathogenesis and highlight its potential as a biomarker and therapeutic target.

CHES1 modulated tumorigenesis and senescence of pancreas cancer cells through repressing AKR1B10.

Pancreatic ductal adenocarcinoma (PDAC), is characteristic by a heterogeneous tumor microenvironment and gene mutations, conveys a dismal prognosis and low response to chemotherapy and immunotherapy. Here, we found that checkpoint suppressor 1 (CHES1) served as a tumor repressor in PDAC and was associated with patient prognosis. Functional experiments indicated that CHES1 suppressed the proliferation and invasion of PDAC by modulating cellular senescence. To further identify the downstream factor of CHES1 in PDAC, label-free quantitative proteomics analysis was conducted, which showed that the oncogenic Aldo-keto reductase 1B10 (AKR1B10) was transcriptionally repressed by CHES1 in PDAC. And AKR1B10 facilitated the malignant activity and repressed senescent phenotype of PDAC cells. Moreover, pharmaceutical inhibition of AKR1B10 with Oleanolic acid (OA) significantly induced tumor regression and sensitized PDAC cells to gemcitabine, and this combined therapy did not cause obvious side effects. Rescued experiments revealed that CHES1 regulated the tumorigenesis and gemcitabine sensitivity through AKR1B10-mediated senescence in PDAC. In summary, this study revealed that the CHES1/AKR1B10 axis modulated the progression and cellular senescence in PDAC, which might provide revenues for drug-targeting and senescence-inducing therapies for PDAC.

Berberine directly targets AKR1B10 protein to modulate lipid and glucose metabolism disorders in NAFLD.

ETHNOPHARMACOLOGICAL RELEVANCE: Berberine (BBR) is the main active component from Coptidis rhizome, a well-known Chinese herbal medicine used for metabolic diseases, especially diabetes for thousands of years. BBR has been reported to cure various metabolic disorders, such as nonalcoholic fatty liver disease (NAFLD). However, the direct proteomic targets and underlying molecular mechanism of BBR against NAFLD remain less understood. AIM OF THE STUDY: To investigate the direct target and corresponding molecular mechanism of BBR on NAFLD is the aim of the current study. MATERIALS AND METHODS: High-fat diet (HFD)-fed mice and oleic acid (OA) stimulated HepG2 cells were utilized to verify the beneficial impacts of BBR on glycolipid metabolism profiles. The click chemistry in proteomics, DARTS, CETSA, SPR and fluorescence co-localization analysis were conducted to identify the targets of BBR for NAFLD. RNA-seq and shRNA/siRNA were used to investigate the downstream pathways of the target. RESULTS: BBR improved hepatic steatosis, ameliorated insulin resistance, and reduced TG levels in the NAFLD models. Importantly, Aldo-keto reductase 1B10 (AKR1B10) was first proved as the target of BBR for NAFLD. The gene expression of AKR1B10 increased significantly in the NAFLD patients' liver tissue. We further demonstrated that HFD and OA increased AKR1B10 expression in the C57BL/6 mice's liver and HepG2 cells, respectively, whereas BBR decreased the expression and activities of AKR1B10. Moreover, the knockdown of AKR1B10 by applying shRNA/siRNA profoundly impacted the beneficial effects on the pathogenesis of NAFLD by BBR. Meanwhile, the changes in various proteins (ACC1, CPT-1, GLUT2, etc.) are responsible for hepatic lipogenesis, fatty acid oxidation, glucose uptake, etc. by BBR were reversed by the knockdown of AKR1B10. Additionally, RNA-seq was used to identify the downstream pathway of AKR1B10 by examining the gene expression of liver tissues from HFD-fed mice. Our findings revealed that BBR markedly increased the protein levels of PPARα while downregulating the expression of PPARγ. However, various proteins of PPAR signaling pathways remained unaffected post the knockdown of AKR1B10. CONCLUSIONS: BBR alleviated NAFLD via mediating PPAR signaling pathways through targeting AKR1B10. This study proved that AKR1B10 is a novel target of BBR for NAFLD treatment and helps to find new targets for the treatment of NAFLD by using active natural compounds isolated from traditional herbal medicines as the probe.

Precision Engineering of the Co-immobilization of Enzymes for Cascade Biocatalysis.

The design and orderly layered co-immobilization of multiple enzymes on resin particles remain challenging. In this study, the SpyTag/SpyCatcher binding pair was fused to the N-terminus of an alcohol dehydrogenase (ADH) and an aldo-keto reductase (AKR), respectively. A non-canonical amino acid (ncAA), p-azido-L-phenylalanine (p-AzF), as the anchor for covalent bonding enzymes, was genetically inserted into preselected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azide-alkyne cycloaddition for the immobilization of AKR and ADH enabled sequential dual-enzyme coating on porous microspheres. The ordered dual-enzyme reactor was subsequently used to synthesize (S)-1-(2-chlorophenyl)ethanol asymmetrically from the corresponding prochiral ketone, enabling the in situ regeneration of NADPH. The reactor exhibited a high catalytic conversion of 74 % and good reproducibility, retaining 80 % of its initial activity after six cycles. The product had 99.9 % ee, which that was maintained in each cycle. Additionally, the double-layer immobilization method significantly increased the enzyme loading capacity, which was approximately 1.7 times greater than that of traditional single-layer immobilization. More importantly, it simultaneously enabled both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.

A Novel lncRNA lncRNA-4045 Promotes the Progression of Hepatocellular Carcinoma by Affecting the Expression of AKR1B10.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been shown to be related to the occurrence and development of a variety of cancers including hepatocellular carcinoma (HCC). However, a large number of potential HCC-related lncRNAs remain undiscovered and are yet to be fully understood. METHODS: Differentially expressed lncRNAs were first obtained from the tumor tissues and adjacent normal tissues of five HCC patients using high-throughput microarray chips. Then the expression levels of 10 differentially expressed lncRNAs were verified in 50 pairs of tissue samples from patients with HCC by quantitative real-time PCR (qRT-PCR). The oncogenic effects of lncRNA-4045 (ENST00000524045.6) in HCC cell lines were verified through a series of in vitro experiments including CCK-8 assay, plate clone formation assay, transwell assay, scratch assay, and flow cytometry. Subsequently, the potential target genes of lncRNA-4045 were predicted by bioinformatics analysis, fluorescence in situ hybridization assay, and RNA sequencing. The mechanism of lncRNA-4045 in HCC was explored by WB assay as well as rescue and enhancement experiments. RESULTS: The results from microarray chips showed 1,708 lncRNAs to have been significantly upregulated and 2725 lncRNAs to have been significantly downregulated in HCC tissues. Via validation in 50 HCC patients, a novel lncRNA lncRNA-4045 was found significantly upregulated in HCC tissues. Additionally, a series of in vitro experiments showed that lncRNA-4045 promoted the proliferation, invasion, and migration of HCC cell lines, and inhibited the apoptosis of HCC cell lines. The results of qRT-PCR in HCC tissues showed that the expression levels of AKR1B10 were significantly positively correlated with lncRNA-4045. LncRNA-4045 knockdown significantly down-regulated AKR1B10 protein expression, and overexpression of lncRNA-4045 led to significant up-regulation of AKR1B10 protein in HCC cell lines. Lastly, down-regulation of AKR1B10 could partially eliminate the enhancement of cell proliferation induced by lncRNA-4045 overexpression, while up-regulation of AKR1B10 was shown to enhance those effects. CONCLUSION: LncRNA-4045 may promote HCC via enhancement of the expression of AKR1B10 protein.

A novel aldo-keto reductase gene, OsAKR1, from rice confers higher tolerance to cadmium stress in rice by an in vivo reactive aldehyde detoxification.

Elevated levels of cadmium (Cd) have the ability to impede plant development. Aldo-keto reductases (AKRs) have been demonstrated in a number of plant species to improve tolerance to a variety of abiotic stresses by scavenging cytotoxic aldehydes; however, only a few AKRs have been identified to improve Cd tolerance. The OsAKR1 gene was extracted and identified from rice here. After being exposed to Cd, the expression of OsAKR1 dramatically rose in both roots and shoots, although more pronounced in roots. According to a subcellular localization experiment, the nucleus and cytoplasm are where OsAKR1 is primarily found. Mutants lacking OsAKR1 exhibited Cd sensitive phenotype than that of the wild-type (WT) Nipponbare (Nip), and osakr1 mutants exhibited reduced capacity to scavenge methylglyoxal (MG). Furthermore, osakr1 mutants exhibited considerably greater hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels, and increased catalase (CAT) activity in comparison to Nip. The expression of three isomeric forms of CAT was found to be considerably elevated in osakr1 mutants during Cd stress, as demonstrated by quantitative real-time PCR analysis, when compared to Nip. These results imply that OsAKR1 controlled rice's ability to withstand Cd by scavenging harmful aldehydes and turning on the reactive oxygen species (ROS) scavenging mechanism.

Multi-strategy orthogonal enhancement and analysis of aldo-keto reductase thermal stability.

Given their outstanding efficiency and selectivity, enzymes are integral in various domains such as drug synthesis, the food industry, and environmental management. However, the inherent instability of natural enzymes limits their widespread industrial application. In this study, we underscore the efficacy of enhancing protein thermal stability through comprehensive protein design strategies, encompassing elements such as the free energy of protein folding, internal forces within proteins, and the overall structural design. We also demonstrate the efficiency and precision of combinatorial screening in the thermal stability design of aldo-keto reductase (AKR7-2-1). In our research, three single-point mutations and five combinatorial mutations were strategically introduced into AKR7-2-1, using multiple computational techniques. Notably, the E12I/S235I mutant showed a significant increase of 25.4 °C in its melting temperature (Tm). Furthermore, the optimal mutant, E12V/S235I, maintained 80 % of its activity while realizing a 16.8 °C elevation in Tm. Remarkably, its half-life at 50 °C was increased to twenty times that of the wild type. Structural analysis indicates that this enhanced thermal stability primarily arises from reduced oscillation in the loop region and increased internal hydrogen bonding. The promising results achieved with AKR7-2-1 demonstrate that our strategy could serve as a valuable reference for enhancing the thermal stability of other industrial enzymes.

Aldo-keto reductase-7A2 protects against atorvastatin-induced hepatotoxicity via Nrf2 activation.

Atorvastatin (ATO), as a cholesterol-lowering drug, was the world's best-selling drug in the early 2000s. However, ATO overdose-induced liver or muscle injury is a threat to many patients, which restricts its application. Previous studies suggest that ATO overdose is accompanied with ROS accumulation and increased lipid peroxidation, which are the leading causes of ATO-induced liver damage. This study is, therefore, carried out to investigate the roles of anti-oxidant pathways and enzymes in protection against ATO-induced hepatotoxicity. Here we show that in ATO-challenged HepG2 cells, the expression levels of transcription factor NFE2L2/Nrf2 (nuclear factor erythroid 2 p45-related factor 2) are significantly upregulated. When Nrf2 is pharmacologically inhibited or genetically inactivated, ATO-induced cytotoxicity is significantly aggravated. Aldo-keto reductase-7A (AKR7A) enzymes, transcriptionally regulated by Nrf2, are important for bioactivation and biodetoxification. Here, we reveal that in response to ATO exposure, mRNA levels of human AKR7A2 are significantly upregulated in HepG2 cells. Furthermore, knockdown of AKR7A2 exacerbates ATO-induced hepatotoxicity, suggesting that AKR7A2 is essential for cellular adaptive response to ATO-induced cell damage. In addition, overexpression of AKR7A2 in HepG2 cells can significantly mitigate ATO-induced cytotoxicity and this process is Nrf2-dependent. Taken together, these findings indicate that Nrf2-mediated AKR7A2 is responsive to high concentrations of ATO and contributes to protection against ATO-induced hepatotoxicity, making it a good candidate for mitigating ATO-induced side effects.

Near-infrared light-driven asymmetric photolytic reduction of ketone using inorganic-enzyme hybrid biocatalyst.

Effective photolytic regeneration of the NAD(P)H cofactor in enzymatic reductions is an important and elusive goal in biocatalysis. It can, in principle, be achieved using a near-infrared light (NIR) driven artificial photosynthesis system employing H2O as the sacrificial reductant. To this end we utilized TiO2/reduced graphene quantum dots (r-GQDs), combined with a novel rhodium electron mediator, to continuously supply NADPH in situ for aldo-keto reductase (AKR) mediated asymmetric reductions under NIR irradiation. This upconversion system, in which the Ti-O-C bonds formed between r-GQDs and TiO2 enabled efficient interfacial charge transfer, was able to regenerate NADPH efficiently in 64 % yield in 105 min. Based on this, the pharmaceutical intermediate (R)-1-(3,5-bis(trifluoromethyl)phenyl)ethan-1-ol was obtained, in 84 % yield and 99.98 % ee, by reduction of the corresponding ketone. The photo-enzymatic system is recyclable with a polymeric electron mediator, which maintained 66 % of its original catalytic efficiency and excellent enantioselectivity (99.9 % ee) after 6 cycles.

An NADH/NAD+-favored aldo-keto reductase facilitates avilamycin A biosynthesis by primarily catalyzing oxidation of avilamycin C.

Avilamycins, which possess potent inhibitory activity against Gram-positive bacteria, are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes. Among these structurally related oligosaccharide antibiotics, avilamycin A serves as the main bioactive component in veterinary drugs and animal feed additives, which differs from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. However, the mechanisms underlying assembly and modification of the oligosaccharide chain to diversify individual avilamycins remain poorly understood. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C. Remarkably, the ratio of these two components produced by AviZ1 depends on the utilization of specific redox cofactors, namely NADH/NAD+ or NADPH/NADP+. These findings are inspired by gene disruption and complementation experiments and are further supported by in vitro enzymatic activity assays, kinetic analyses, and cofactor affinity studies on AviZ1-catalyzed redox reactions. Additionally, the results from sequence analysis, structure prediction, and site-directed mutagenesis of AviZ1 validate it as an NADH/NAD+-favored aldo-keto reductase that primarily oxidizes avilamycin C to form avilamycin A by utilizing abundant NAD+ in vivo. Building upon the biological function and catalytic activity of AviZ1, overexpressing AviZ1 in S. viridochromogenes is thus effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study represents, to our knowledge, the first characterization of biochemical reactions involved in avilamycin biosynthesis and contributes to the construction of high-performance strains with industrial value.IMPORTANCEAvilamycins are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes, which can be used as veterinary drugs and animal feed additives. Avilamycin A is the most bioactive component, differing from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. Currently, the biosynthetic pathway of avilamycins is not clear. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C. More importantly, AviZ1 exhibits a unique NADH/NAD+ preference, allowing it to efficiently catalyze the oxidation of avilamycin C to form avilamycin A using abundant NAD+ in cells. Thus, overexpressing AviZ1 in S. viridochromogenes is effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study serves as an enzymological guide for rational strain design, and the resulting high-performance strains have significant industrial value.

Decoding selectivity: computational insights into AKR1B1 and AKR1B10 inhibition.

Understanding selectivity mechanisms of inhibitors towards highly homologous proteins is of paramount importance in the design of selective candidates. Human aldo-keto reductases (AKRs) pertain to a superfamily of monomeric oxidoreductases, which serve as NADPH-dependent cytosolic enzymes to catalyze the reduction of carbonyl groups to primary and secondary alcohols using electrons from NADPH. Among AKRs, AKR1B1 is emerging as a promising target for cancer treatment and diabetes, despite its high structural similarity with AKR1B10, which leads to severe adverse events. Therefore, it is crucial to understand the selectivity mechanisms of AKR1B1 and AKR1B10 to discover safe anticancer candidates with optimal therapeutic efficacy. In this study, multiple computational strategies, including sequence alignment, structural comparison, Protein Contacts Atlas analysis, molecular docking, molecular dynamics simulation, MM-GBSA calculation, alanine scanning mutagenesis and pharmacophore modeling analysis were employed to comprehensively understand the selectivity mechanisms of AKR1B1/10 inhibition based on selective inhibitor lidorestat and HAHE. This study would provide substantial evidence in the design of potent and highly selective AKR1B1/10 inhibitors in future.

A three-level regulatory mechanism of the aldo-keto reductase subfamily AKR12D.

Modulation of protein function through allosteric regulation is central in biology, but biomacromolecular systems involving multiple subunits and ligands may exhibit complex regulatory mechanisms at different levels, which remain poorly understood. Here, we discover an aldo-keto reductase termed AKRtyl and present its three-level regulatory mechanism. Specifically, by combining steady-state and transient kinetics, X-ray crystallography and molecular dynamics simulation, we demonstrate that AKRtyl exhibits a positive synergy mediated by an unusual Monod-Wyman-Changeux (MWC) paradigm of allosteric regulation at low concentrations of the cofactor NADPH, but an inhibitory effect at high concentrations is observed. While the substrate tylosin binds at a remote allosteric site with positive cooperativity. We further reveal that these regulatory mechanisms are conserved in AKR12D subfamily, and that substrate cooperativity is common in AKRs across three kingdoms of life. This work provides an intriguing example for understanding complex allosteric regulatory networks.