Tobacco carcinogens metabolism-related genes (TCMGs) could generate reactive metabolites of tobacco carcinogens, which subsequently contributed to multiple diseases. However, the association between genetic variants in TCMGs and bladder cancer susceptibility remains unclear. In this study, we derived TCMGs from metabolic pathways of polycyclic aromatic hydrocarbons and tobacco-specific nitrosamines, and then explored genetic associations between TCMGs and bladder cancer risk in two populations: a Chinese population of 580 cases and 1101 controls, and a European population of 5930 cases and 5468 controls, along with interaction and joint analyses. Expression patterns of TCMGs were sourced from Nanjing Bladder Cancer (NJBC) study and publicly available datasets. Among 43 TCMGs, we observed that rs7087341 T > A in AKR1C2 was associated with a reduced risk of bladder cancer in the Chinese population [odds ratio (OR) = 0.84, 95% confidence interval (CI) = 0.72-0.97, P = 1.86 × 10-2]. Notably, AKR1C2 rs7087341 showed an interaction effect with cigarette smoking on bladder cancer risk (Pinteraction = 5.04 × 10-3), with smokers carrying the T allele increasing the risk up to an OR of 3.96 (Ptrend < 0.001). Genetically, rs7087341 showed an allele-specific transcriptional regulation as located at DNA-sensitive regions of AKR1C2 highlighted by histone markers. Mechanistically, rs7087341 A allele decreased AKR1C2 expression, which was highly expressed in bladder tumors that enhanced metabolism of tobacco carcinogens, and thereby increased DNA adducts and reactive oxygen species formation during bladder tumorigenesis. These findings provided new insights into the genetic mechanisms underlying bladder cancer.

The mechanisms underlying the complex correlation between immunoglobulin A nephropathy (IgAN) and inflammatory bowel disease (IBD) remain unclear. This study aimed to identify the optimal cross-talk genes, potential pathways, and mutual immune-infiltrating microenvironments between IBD and IgAN to elucidate the linkage between patients with IBD and IgAN. The IgAN and IBD datasets were obtained from the Gene Expression Omnibus (GEO). Three algorithms, CIBERSORTx, ssGSEA, and xCell, were used to evaluate the similarities in the infiltrating microenvironment between the two diseases. Weighted gene co-expression network analysis (WGCNA) was implemented in the IBD dataset to identify the major immune infiltration modules, and the Boruta algorithm, RFE algorithm, and LASSO regression were applied to filter the cross-talk genes. Next, multiple machine learning models were applied to confirm the optimal cross-talk genes. Finally, the relevant findings were validated using histology and immunohistochemistry analysis of IBD mice. Immune infiltration analysis showed no significant differences between IBD and IgAN samples in most immune cells. The three algorithms identified 10 diagnostic genes, MAPK3, NFKB1, FDX1, EPHX2, SYNPO, KDF1, METTL7A, RIDA, HSDL2, and RIPK2; FDX1 and NFKB1 were enhanced in the kidney of IBD mice. Kyoto Encyclopedia of Genes and Genomes analysis showed 15 mutual pathways between the two diseases, with lipid metabolism playing a vital role in the cross-talk. Our findings offer insights into the shared immune mechanisms of IgAN and IBD. These common pathways, diagnostic cross-talk genes, and cell-mediated abnormal immunity may inform further experimental studies.

Pentachlorophenol (PCP) is a widely used pesticide. However, whether PCP and its metabolite chloranil have endocrine-disrupting effects by inhibiting placental 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1) remains unclear. The study used in vitro assays with human and rat placental microsomes to measure 3β-HSD activity as well as human JAr cells to evaluate progesterone production. The results showed that PCP exhibited moderate inhibition of human 3β-HSD1, with an IC50 value of 29.83 μM and displayed mixed inhibition in terms of mode of action. Conversely, chloranil proved to be a potent inhibitor, demonstrating an IC50 value of 147 nM, and displaying a mixed mode of action. PCP significantly decreased progesterone production by JAr cells at 50 μM, while chloranil markedly reduced progesterone production at ≥1 μM. Interestingly, PCP and chloranil moderately inhibited rat placental homolog 3β-HSD4, with IC50 values of 27.94 and 23.42 μM, respectively. Dithiothreitol (DTT) alone significantly increased human 3β-HSD1 activity. Chloranil not PCP mediated inhibition of human 3β-HSD1 activity was completely reversed by DTT and that of rat 3β-HSD4 was partially reversed by DTT. Docking analysis revealed that both PCP and chloranil can bind to the catalytic domain of 3β-HSDs. The difference in the amino acid residue Cys83 in human 3β-HSD1 may explain why chloranil is a potent inhibitor through its interaction with the cysteine residue of human 3β-HSD1. In conclusion, PCP is metabolically activated to chloranil as a potent inhibitor of human 3β-HSD1.

Catalytic activity is undoubtedly a key focus in enzyme engineering. The complicated reaction conditions hinder some enzymes from industrialization even though they have relatively promising activity. This has occurred to some dehydrogenases. Hydroxysteroid dehydrogenases (HSDHs) specifically catalyze the conversion between hydroxyl and keto groups, and hold immense potential in the synthesis of steroid medicines. We underscored the importance of 7α-HSDH activity, and analyzed the overall robustness and underlying mechanisms. Employing a high-throughput screening approach, we comprehensively assessed a mutation library, and obtained a mutant with enhanced enzymatic activity and overall stability/tolerance. The superior mutant (I201M) was identified to harbor improved thermal stability, substrate susceptibility, cofactor affinity, as well as the yield. This mutant displayed a 1.88-fold increase in enzymatic activity, a 1.37-fold improvement in substrate tolerance, and a 1.45-fold increase in thermal stability when compared with the wild type (WT) enzyme. The I201M mutant showed a 2.25-fold increase in the kcat/KM ratio (indicative of a stronger binding affinity for the cofactor). This mutant did not exhibit the highest enzyme activity compared with all the tested mutants, but these improved characteristics contributed synergistically to the highest yield. When a substrate at 100 mM was present, the 24 h yield by I201M reached 89.7%, significantly higher than the 61.2% yield elicited by the WT enzyme. This is the first report revealing enhancement of the catalytic efficiency, cofactor affinity, substrate tolerance, and thermal stability of NAD(H)-dependent 7α-HSDH through a single-point mutation. The mutated enzyme reached the highest enzymatic activity of 7α-HSDH ever reported. High enzymatic activity is undoubtedly crucial for enabling the industrialization of an enzyme. Our findings demonstrated that, when compared with other mutants boasting even higher enzymatic activity, mutants with excellent overall robustness were superior for industrial applications. This principle was exemplified by highly active enzymes such as 7α-HSDH.

Digitoxin, an important secondary metabolite of Digitalis purpurea, is a commonly used cardiotonic in clinical practice. 3β-Hydroxysteroid dehydrogenase(3βHSD) is a key enzyme involved in the biosynthesis of digitoxin. It belongs to the short-chain dehydrogenase/reductase(SDR) family, playing a role in the biosynthesis of cardiac glycosides by oxidizing and isomerizing the precursor sterol. In this study, two 3βHSD genes were cloned from D. purpurea. The results showed that the open reading frame(ORF) of Dp3βHSD1 was 780 bp, encoding 259 amino acid residues. The ORF of Dp3βHSD2 was 774 bp and encoded 257 residues. Dp3βHSD1/2 had the cofactor binding site TGxxxA/GxG and the catalytic site YxxxK. In vitro experiments confirmed that Dp3βHSD1/2 catalyzed the generation of progesterone from pregnenolone, and Dp3βHSD1 had stronger catalytic capacity than Dp3βHSD2. The expression level of Dp3βHSD1 was much higher than that of Dp3βHSD2 in leaves, and digitoxin was only accumulated in leaves. The results implied that Dp3βHSD1 played a role in the dehydrogenation of pregnenolone to produce progesterone in the biosynthesis of digitoxin. This study provides a reference for further exploring the biosynthetic pathway of cardiac glycosides in D. purpurea.

BACKGROUND: 7α-Hydroxysteroid dehydrogenase (7α-HSDH) plays a pivotal role in vivo in the biotransformation of secondary bile acids and has great potential in industrial biosynthesis due to its broad substrate specificity. In this study, we expressed and characterized a novel thermostable 7α-HSDH (named Sa 7α-HSDH).
METHODS: The DNA sequence was derived from the black bear gut microbiome metagenomic sequencing data, and the coding sequence of Sa 7α-HSDH was chemically synthesized. The heterologous expression of the enzyme was carried out using the pGEX-6p-1 vector. Subsequently, the activity of the purified enzyme was studied by measuring the absorbance change at 340 nm. Finally, the three-dimensional structure was predicted with AlphaFold2.
RESULTS: Coenzyme screening results confirmed it to be NAD(H) dependent. Substrate specificity test revealed that Sa 7α-HSDH could catalyze taurochenodeoxycholic acid (TCDCA) with catalytic efficiency (kcat/Km) 3.81 S-1 mM-1. The optimum temperature of Sa 7α-HSDH was measured to be 75°C, confirming that it belongs to thermophilic enzymes. Additionally, its thermostability was assessed using an accelerated stability test over 32 hours. The catalytic activity of Sa 7α-HSDH remained largely unchanged for the first 24 hours and retained over 90% of its functionality after 32 hours at 50°C. Sa 7α-HSDH exhibited maximal activity at pH 10. The effect of metal ions-K+, Na+, Mg2+ and Cu2+-on the enzymatic activity of Sa 7α-HSDH was investigated. Only Mg2+ was observed to enhance the enzyme\'s activity by 27% at a concentration of 300 mM. Neither K+ nor Na+ had a significant influence on activity. Only Cu2+ was found to reduce enzyme activity.
CONCLUSIONS: We characterized the thermostable 7α-HSDH, which provides a promising biocatalyst for bioconversion of steroids at high reaction temperatures.

The use of additives, especially colorants, in food and pharmaceutical industry is increasing dramatically. Currently, additives are classified as contaminants of emerging concern (CECs). Concerns have been raised about the potential hazards of food additives to reproductive organs and fertility. The present study investigates the reproductive toxicity of tartrazine (TRZ), a synthetic colorant, in male rats and aims to explore the curative effect of Ginkgo biloba extract (EGb) against TRZ-induced testicular toxicity. Twenty-four rats were divided into four groups: the control (0.5 ml distilled water), the EGb group (100 mg/kg EGb alone), the TRZ group (7.5 mg/kg TRZ alone), and the TRZ-EGb group (7.5 mg/kg TRZ plus 100 mg/kg EGb). The doses were administered orally in distilled water once daily for 28 days. Toxicity studies of TRZ investigated testicular redox state, serum gonadotropins, and testosterone levels, testicular 17 ß-hydroxysteroid dehydrogenase activity, sperm count and quality, levels of inflammatory cytokines, and caspase-3 expression as an apoptotic marker. Also, histopathological alterations of the testes were examined. TRZ significantly affected the testicular redox status as indicated by the increase in malondialdehyde and the decrease in reduced glutathione, superoxide dismutase, and catalase. It also disrupted serum gonadotropins (follicle stimulating hormone and luteinizing hormone) and testosterone levels and the activity of testicular 17ß-hydroxysteroid dehydrogenase. Additionally, TRZ adversely affected sperm count, motility, viability, and abnormality. Levels of tumor necrosis factor-α, interleukin-1β, interleukin-6, and expression of caspase-3 were increased in the testes. Histopathological examination of the testes supported the alterations mentioned above. Administration of EGb significantly ameliorated TRZ-induced testicular toxicity in rats. In conclusion, EGb protected against TRZ-induced testicular toxicity through antioxidant, anti-inflammatory, and anti-apoptotic mechanisms.

A highly selective hydrogenation of 3-keto in steroids to 3-hydroxyl steroids catalyzed by hydroxysteroid dehydrogenases (HSDHs) was demonstrated. The Ct3α-HSDH-catalyzed hydrogenation generated 3α-hydroxyl steroids as the main enantiopure isomers in high yields, while the Ss3β-HSDH catalytic system afforded 3β-hydroxyl steroids in excellent yields. In both catalytic systems, the hydrogenation proceeded regioselectively at 3-keto with 7-, 11-, 17-, and 20-keto almost unreacted, and chemoselectively with the C═C bond and ester group unattacked. Our HSDH-promoted hydrogenation showed advantages like high regio-, chemo-, and enantioselectivity, good yields, mild conditions, a wide substrate scope, and being suitable for gram-scale synthesis. Notably, bioactive molecules like dehydroepiandrosterone, brienolone, and alfaxalone were obtained facilely in high yields via our hydrogenation approach.

Hydroxysteroid dehydrogenases (HSDHs) are crucial for bile acid metabolism and influence the size of the bile acid pool and gut microbiota composition. HSDHs with high activity, thermostability, and substrate selectivity are the basis for constructing engineered bacteria for disease treatment. In this study, we designed mutations at the cofactor binding site involving Thr15 and Arg16 residues of HSDH St-2-2. The T15A, R16A, and R16Q mutants exhibited 7.85-, 2.50-, and 4.35-fold higher catalytic activity than the wild type, respectively, while also displaying an altered substrate preference (from taurocholic acid (TCA) to taurochenodeoxycholic acid (TCDCA)). These mutants showed lower Km and higher kcat values, indicating stronger binding to the substrate and resulting in 3190-, 3123-, and 3093-fold higher kcat/Km values for TCDCA oxidation. Furthermore, the Tm values of the T15A, R16A, and R16Q mutants were found to increase by 4.3 °C, 6.0 °C, and 7.0 °C, respectively. Molecular structure analysis indicated that reshaped internal hydrogens and surface mutations could improve catalytic activity and thermostability, and altered interactions among the catalytic triad, cofactor binding sites, and substrates could change substrate preference. This work provides valuable insights into modifying substrate preference as well as enhancing the catalytic activity and thermostability of HSDHs by targeting the cofactor binding site.

Lipedema is a debilitating chronic condition predominantly affecting women, characterized by the abnormal accumulation of fat in a symmetrical, bilateral pattern in the extremities, often coinciding with hormonal imbalances.
Despite the conjectured role of sex hormones in its etiology, a definitive link has remained elusive. This study explores the case of a patient possessing a mutation deletion within the C-terminal region of Aldo-keto reductases Member C2 (AKR1C2), Ser320PheTer2, that could lead to heightened enzyme activity. A cohort of 19 additional lipedema patients and 2 additional affected family members14 were enrolled in this study. The two additional affected family members are relatives of the patient with the AKR1C1 L213Q variant, which is included in the 19 cohorts and described in literature.
Our investigation revealed that AKR1C2 was overexpressed, as quantified by qPCR, in 5 out of 21 (24%) lipedema patients who did not possess mutations in the AKR1C2 gene. Collectively, these findings implicate AKR1C2 in the pathogenesis of lipedema, substantiating its causative role.
This study demonstrates that the activating mutation in the enzyme or its overexpression is a causative factor in the development of lipedema. Further exploration and replication in diverse populations will bolster our understanding of this significant connection.