No abstract

Nosiheptide is a bicyclic thiopeptide natural product that exhibits potent antibiotic activity against a number of clinically important Gram‐positive pathogens. It is biosynthesized from a ribosomally produced core peptide that is then modified extensively to contain a number of thiazole rings and dehydrated amino acids, as well as other modifications. It also contains a unique side‐ring structure composed of a 3,4‐dimethylindolic acid bridge connected to the side chains of Glu6 and Cys8 of the core peptide via ester and thioester linkages, respectively. In addition to the core peptide, encoded by the nosM gene, the biosynthesis of the side‐ring structure requires the actions of NosI, J, K, L, and N. NosN is annotated as a class C radical S‐adenosylmethionine (SAM) methylase, but little is known about how the enzyme functions or what its true substrate is. By designing, synthesizing and testing small peptides as substrate mimics, we show that the true function of NosN is to transfer a C1 unit from SAM to C4 of 3‐methyl‐2‐indolic acid (MIA) with concomitant formation of a bond between the carboxylate of Glu6 of the core peptide and the nascent C1 unit. In addition, using full‐length peptides, we show that NosN completes installation of the side‐ring structure immediately after NosK appends MIA to the core peptide. Using a substrate mimic containing a rigid structure, we also identify and characterize two reaction‐based adducts containing SAM fused to C4 of MIA The two SAM‐adducts are derived from a consensus radical‐containing species proposed to be the key intermediate in our proposed catalytic mechanism of NosN. Support or Funding Information This work was supported by NIH (GM‐122595 and AI‐133318 to S.J.B.). S.J.B. is an investigator of the Howard Hughes Medical Institute. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The tumor microenvironment in glioblastoma provides cancer cells with favorable conditions to proliferate and invade surrounding tissues. Macrophages comprise a large portion of the glioblastoma tumor microenvironment (TME) both in terms of volume and function. These cells have been reported to influence tumor progression by modulating immune responses, remodeling extracellular matrix, and providing nutrients to cancer cells among numerous other functions. Radiation therapy forms one of the pillars of glioblastoma management along with surgical resection and chemotherapy. Here we investigated the effects of radiation on macrophage iron metabolism. Using mouse bone-marrow-derived macrophages (BMDMs) we performed in-vitro 59Fe radiotracer assays to study how radiation exposure modified iron trafficking in these cells. We found that low dose radiation at 0.25, 0.5, or 2 Gy from a 60Co source stimulated iron release from the BMDMs with maximal release occurring at 0.5 Gy. Moreover, we observed that iron release was dependent on the amount of serum present in culture media with cells cultured in 20% fetal bovine serum (FBS) showing reduced iron release profiles compared to those cultured in 10% or 1% FBS. Since glioblastoma patients exhibit sexually dimorphic survival outcomes, we investigated whether these radiation-induced responses occurred in a sexually dimorphic pattern. At radiation doses of 0.25 Gy we observed that male macrophages tended to release more iron than female macrophages despite no differences in iron uptake between the sexes – raising the question as to whether differential iron trafficking in response to treatment contributes to the poorer survival outcomes observed in males. Our data suggest that delineating how supporting cells such as macrophages respond to glioblastoma treatment regimens may provide insights into addressing mechanisms of treatment resistance and further our understanding of the sexual dimorphism observed in patient outcomes.