The dynamic topological states of chromosomal DNA regulate many cellular fundamental processes universally in all three domains of life, that is, bacteria, archaea, and eukaryotes. DNA-binding proteins maintain the regional and global supercoiling of the chromosome and thereby regulate the chromatin architecture that ultimately influences the gene expression network and other DNA-centric molecular events in various microenvironments and growth phases. DNA-binding small molecules are pivotal weapons for treating a wide range of cancers. Recent advances in single-molecule biophysical tools have uncovered the fact that many DNA-binding ligands not only alter the regional DNA supercoiling but also modulate the overall morphology of DNA. Here we provide insight into recent advances in atomic force microscopy (AFM) acquired DNA structural change induced by therapeutically important mono- and bis-intercalating anticancer agents as well as DNA-adduct-forming anticancer drugs. We also emphasize the growing evidence of the mechanistic relevance of changes in DNA topology in the anticancer cellular responses of DNA-targeting chemotherapeutic agents.
Hypoxia-inducible factor prolyl hydroxylase domain 2 (PHD2) is an important oxygen sensor in animals. By using the CO-releasing molecule-2 (CORM-2) as an in situ CO donor, we demonstrate that CO is an inhibitor of PHD2. This report provides further evidence about the emerging role of CO in oxygen sensing and homeostasis.
Cancer is the second leading cause of death-1 in 6 deaths globally is due to cancer. Cancer metabolism is a complex and one of the most actively researched area in cancer biology. Metabolic reprogramming in cancer cells entails activities that involve several enzymes and metabolites to convert nutrient into building blocks that alter energy metabolism to fuel rapid cell division. Metabolic dependencies in cancer generate signature metabolites that have key regulatory roles in tumorigenesis. In this minireview, we highlight recent advances in the popular methods ingrained in biochemistry research such as stable and flux isotope analysis, as well as radioisotope labeling, which are valuable in elucidating cancer metabolites. These methods together with analytical tools such as chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry have helped to bring about exploratory work in understanding the role of important as well as obscure metabolites in cancer cells. Information obtained from these analyses significantly contribute in the diagnosis and prognosis of tumors leading to potential therapeutic targets for cancer therapy.
Certain enzymes of the glycoside hydrolase family 20 (GH20) exert transglycosylation activity and catalyze the transfer of β-N-acetylglucosamine (GlcNAc) from a chitobiose donor to lactose to produce lacto-N-triose II (LNT2), a key human milk oligosaccharide backbone moiety. The present work is aimed at increasing the transglycosylation activity of two selected hexosaminidases, HEX1 and HEX2, to synthesize LNT2 from lactose and chitobiose. Peptide pattern recognition analysis was used to categorize all GH20 proteins in subgroups. On this basis, we identified a series of proteins related to HEX1 and HEX2. By sequence alignment, four additional loop sequences were identified that were not present in HEX1 and HEX2. Insertion of these loop sequences into the wild-type sequences induced increased transglycosylation activity for three out of eight mutants. The best mutant, HEX1GTEPG , had a transglycosylation yield of LNT2 on the donor that was nine times higher than that of the wild-type enzyme. Homology modeling of the enzymes revealed that the loop insertion produced a more shielded substrate-binding pocket. This shielding is suggested to explain the reduced hydrolytic activity, which in turn resulted in the increased transglycosylation activity of HEX1GTEPG .
The hypoxia-inducible factors (HIFs) are key transcription factors in determining cellular responses involving alterations in protein levels in response to limited oxygen availability in animal cells. 2-Oxoglutarate-dependent oxygenases play key roles in regulating levels of HIF and its transcriptional activity. We describe MS-based proteomics studies in which we compared the results of subjecting human breast cancer MCF-7 cells to hypoxia or treating them with a cell-penetrating derivative (dimethyl N-oxalylglycine; DMOG) of the stable 2OG analogue N-oxalylglycine. The proteomic results are consistent with reported transcriptomic analyses and support the proposed key roles of 2OG-dependent HIF prolyl- and asparaginyl-hydroxylases in the hypoxic response. Differences between the data sets for hypoxia and DMOG might reflect context-dependent effects or HIF-independent effects of DMOG.
Understanding enzymatic Diels-Alder (DA) reactions that can form complex natural product scaffolds is of considerable interest. Sch 210972 1, a potential anti-HIV fungal natural product, contains a decalin core that is proposed to form through a DA reaction. We identified the gene cluster responsible for the biosynthesis of 1 and heterologously reconstituted the biosynthetic pathway in Aspergillus nidulans to characterize the enzymes involved. Most notably, deletion of cghA resulted in a loss of stereoselective decalin core formation, yielding both an endo (1) and a diastereomeric exo adduct of the proposed DA reaction. Complementation with cghA restored the sole formation of 1. Density functional theory computation of the proposed DA reaction provided a plausible explanation of the observed pattern of product formation. Based on our study, we propose that lipocalin-like CghA is responsible for the stereoselective intramolecular [4+2] cycloaddition that forms the decalin core of 1.