Biosynthesis of Enfumafungin-type Antibiotic Reveals an Unusual Enzymatic Fusion Pattern and Unprecedented C-C Bond Cleavage.

Enfumafungin-type antibiotics, represented by enfumafungin and fuscoatroside, belong to a distinct group of triterpenoids derived from fungi. These compounds exhibit significant antifungal properties with ibrexafungerp, a semisynthetic derivative of enfumafungin, recently gaining FDA's approval as the first oral antifungal drug for treating invasive vulvar candidiasis. Enfumafungin-type antibiotics possess a cleaved E-ring with an oxidized carboxyl group and a reduced methyl group at the break site, suggesting unprecedented C-C bond cleavage chemistry involved in their biosynthesis. Here, we show that a 4-gene (fsoA, fsoD, fsoE, fsoF) biosynthetic gene cluster is sufficient to yield fuscoatroside by heterologous expression in Aspergillus oryzae. Notably, FsoA is an unheard-of terpene cyclase-glycosyltransferase fusion enzyme, affording a triterpene glycoside product that relies on enzymatic fusion. FsoE is a P450 enzyme that catalyzes successive oxidation reactions at C19 to facilitate a C-C bond cleavage, producing an oxidized carboxyl group and a reduced methyl group that have never been observed in known P450 enzymes. Our study thus sets the important foundation for the manufacture of enfumafungin-type antibiotics using biosynthetic approaches.

VHL enhances 9-cis-retinoic acid treatment by down-regulating retinoid X receptor α in renal cell carcinomas.

Renal cell carcinoma (RCC) is the most common malignant kidney tumors in adults. Von Hippel-Lindau (VHL) gene is deficient in >50% of RCC cases, but the role of VHL as a potential therapeutic target in RCC has not been well established. In the present study, 9-cis-Retinoic acid, which is a potent natural agonist of retinoid X receptors (RXRs), was found to decrease the viability of VHL-proficient RCC cells, but had little effect on VHL-deficient RCC cells. In addition, it was demonstrated that VHL transcriptionally regulated RXRα in a hypoxia-inducible factor-α independent manner. Moreover, a negative correlation was observed between the expressions of VHL and RXRα in RCC tissues. Collectively, these data indicate that VHL-proficient RCC patients may be more sensitive to treatment with 9-cis-retinoic acid, which acts by regulating RXRα expression, compared with VHL-deficient RCC patients. The findings of the present study demonstrate a novel function of VHL and highlight the potential of VHL expression as a therapeutic modality for the optimized treatment of RCC patients.

Biosynthesis of Biscognienyne†B Involving a Cytochrome P450-Dependent Alkynylation.

The alkyne is a biologically significant moiety found in many natural products and a versatile functional group widely used in modern chemistry. Recent studies have revealed the biosynthesis of acetylenic bonds in fatty acids and amino acids. However, the molecular basis for the alkynyl moiety in acetylenic prenyl chains occurring in a number of meroterpenoids remains obscure. Here, we identify the biosynthetic gene cluster and characterize the biosynthetic pathway of an acetylenic meroterpenoid biscognienyne B based on heterologous expression, feeding experiments, and in vitro assay. This work shows that the alkyne moiety is constructed by an unprecedented cytochrome P450 enzyme BisI, which shows promiscuous activity towards C5 and C15 prenyl chains. This finding provides an opportunity for discovery of new compounds, featuring acetylenic prenyl chains, through genome mining, and it also expands the enzyme inventory for de novo biosynthesis of alkynes.

Identification of nestin as a urinary biomarker for acute kidney injury.

OBJECTIVES: Acute kidney injury (AKI) is a common complication in hospitalized patients and the incidence of AKI is rapidly increasing. Despite the advances in treatment of AKI, many patients still progress to end-stage renal disease and depend on dialysis. Therefore, early diagnosis and adequate treatment of AKI could improve prognosis. METHODS: We established rat models of AKI induced by cisplatin nephrotoxicity and renal ischemia-reperfusion (I/R). Urine samples were collected, labeled with isobaric tags for relative and absolute quantification agents, and then subjected to nano-LC-MS/MS-based proteomic analysis. Results of the proteomic study were confirmed by Western blot. We also performed RNAi to silence nestin and investigate its role in renal I/R injury. We then validated its clinical application by studying urine nestin levels in AKI patients with cardiovascular surgeries. RESULTS: Our proteomic analysis showed that fetuin-A, nestin, hamartin and T-kininogen were differentially expressed in the urine samples of rats after cisplatin or I/R treatment. Western blot confirmed the differential expression of these proteins in animal models and ELISA confirmed the differential expression of nestin in human urine samples. To explore the expression of nestin in the development of AKI, our results showed that nestin was primarily detected in the glomeruli and barely detected in tubular cells but increased in tubular cells during I/R- and cisplatin-induced AKI. The urine nestin-to-creatinine ratio increased earlier than serum creatinine in AKI patients with postcardiovascular surgeries. The role of nestin in AKI might be related to the p53 signaling pathway. CONCLUSIONS: Thus, our results demonstrated that urinary nestin could be a urinary biomarker for patients with AKI and its role in AKI might be related to the p53 signaling pathway.

Hypoxia-inducible factor 1α mediates the down-regulation of superoxide dismutase 2 in von Hippel-Lindau deficient renal clear cell carcinoma.

Hypoxia-inducible factor 1α (HIF-1α) is an oxygen-sensitive subunit of HIF-1, the master transcription factor for cellular response to hypoxia. Down-regulation of the mitochondrial enzyme superoxide dismutase 2 (SOD2) contributes to the stabilization of HIF-1α under hypoxia due to the decreased dismutation of superoxide radical. Here we report that HIF-1α could also regulate the expression of SOD2. We found that both stabilization of HIF-1α expression under nomoxia caused by pVHL deficiency and hypoxia treatment significantly reduced SOD2 expression, and shRNAs specifically against HIF-1α restored SOD2 expression in both circumstances. Further analyses with luciferase reporter assay and chromatin immunoprecipitation assay revealed that HIF-1α inhibited and directly bound to the hypoxia-responsive element in SOD2 promoter. These findings indicated the existence of a positive feedback between HIF-1α and SOD2 and provided new clues for understanding the molecular mechanisms of hypoxia adaptation.

Recent advances in dissecting the demethylation reactions in natural product biosynthesis.

Demethylation is a chemical process widely distributed in nature to remove a methyl group from an organic molecule, which is a key aspect of diverse biological processes including biosynthesis of natural products, degradation of plant biomass and epigenetic regulation. This process is facilitated by diverse demethylases via distinct mechanisms. Recent studies have disclosed some novel demethylation reactions as well as their underlying demethylases in the biosynthesis of bacterial sterols, fungal terpenoids, and plant alkaloids. This article focuses on current advances in dissecting the demethylation reactions in biosynthesis of natural products and aims to point out the enzymatic mechanisms, which will further enhance our knowledge and understanding of demethylation process in nature.

VHL deficiency augments anthracycline sensitivity of clear cell renal cell carcinomas by down-regulating ALDH2.

The von Hippel-Lindau (VHL) is deficient in ∼70% of clear-cell renal cell carcinomas (ccRCC), which contributes to the carcinogenesis and drug resistance of ccRCC. Here we show that VHL-deficient ccRCC cells present enhanced cytotoxicity of anthracyclines in a hypoxia-inducible factor-independent manner. By subtractive proteomic analysis coupling with RNAi or overexpression verification, aldehyde dehydrogenase 2 (ALDH2) is found to be transcriptionally regulated by VHL and contributes to enhanced anthracyclines cytotoxicity in ccRCC cells. Furthermore, VHL regulates ALDH2 expression by directly binding the promoter of -130 bp to -160 bp to activate the transcription of hepatocyte nuclear factor 4 alpha (HNF-4α). In addition, a positive correlation is found among the protein expressions of VHL, HNF-4α and ALDH2 in ccRCC samples. These findings will deepen our understanding of VHL function and shed light on precise treatment for ccRCC patients.

Inhibitor of DNA Binding 1 Is Induced during Kidney Ischemia-Reperfusion and Is Critical for the Induction of Hypoxia-Inducible Factor-1α.

In this study, rat models of acute kidney injury (AKI) induced by renal ischemia-reperfusion (I/R) and HK-2 cell models of hypoxia-reoxygenation (H/R) were established to investigate the expression of inhibitor of DNA binding 1 (ID1) in AKI, and the regulation relationship between ID1 and hypoxia-inducible factor 1 alpha (HIF-1α). Through western blot, quantitative real-time PCR, immunohistochemistry, and other experiment methods, the induction of ID1 after renal I/R in vivo was observed, which was expressed mainly in renal tubular epithelial cells (TECs). ID1 expression was upregulated in in vitro H/R models at both the protein and mRNA levels. Via RNAi, it was found that ID1 induction was inhibited with silencing of HIF-1α. Moreover, the suppression of ID1 mRNA expression could lead to decreased expression and transcription of HIF-1α during hypoxia and reoxygenation. In addition, it was demonstrated that both ID1 and HIF-1α can regulate the transcription of twist. This study demonstrated that ID1 is induced in renal TECs during I/R and can regulate the transcription and expression of HIF-1α.

Hypoxia inducible factor-1α suppresses Peroxiredoxin 3 expression to promote proliferation of CCRCC cells.

Peroxiredoxin 3 (Prx3) is a mitochondrial member of the antioxidant family of thioredoxin peroxidases that uses mitochondrial thioredoxin 2 as a source of reducing equivalents to scavenge hydrogen peroxide (H2O2). Here, we report that the protein levels of Prx3 are significantly reduced in VHL-deficient clear cell renal cell carcinoma (CCRCC). Furthermore, stabilization of HIF-1α protein, caused either by VHL deficiency under normoxia, or by hypoxia, significantly reduced Prx3 expression. Luciferase-reporter and chromatin-immunoprecipitation assays indicated that HIF-1α binds to the hypoxia-responsive elements of PRDX3 promoter and represses its transcription. Finally, shRNA-based assays suggested that Prx3 downregulation is required for the HIF-1α-dependent proliferation of CCRCC cells. Taken together, our results shed new light onto the mechanism of HIF-1α-dependent proliferation in CCRCC cells.