Magdalena B. Murray

SignificanceFerroptosis is an oxidative form of cell death whose biochemical regulation remains incompletely understood. Cap'n'collar (CNC) transcription factors including nuclear factor erythroid-2-related factor 1 (NFE2L1/NRF1) and NFE2L2/NRF2 can both regulate oxidative stress pathways but are each regulated in a distinct manner, and whether these two transcription factors can regulate ferroptosis independent of one another is unclear. We find that NFE2L1 can promote ferroptosis resistance, independent of NFE2L2, by maintaining the expression of glutathione peroxidase 4 (GPX4), a key protein that prevents lethal lipid peroxidation. NFE2L2 can also promote ferroptosis resistance but does so through a distinct mechanism that appears independent of GPX4 protein expression. These results suggest that NFE2L1 and NFE2L2 independently regulate ferroptosis.

Ferroptosis is an iron-dependent cell death mechanism that may be important to prevent tumor formation and useful as a target for new cancer therapies. Transcriptional networks play a crucial role in shaping ferroptosis sensitivity by regulating the expression of transporters, metabolic enzymes, and other proteins. The Cap'n'collar (CNC) protein NFE2 like bZIP transcription factor 2 (NFE2L2, also known as NRF2) is a key regulator of ferroptosis in many cells and contexts. Emerging evidence indicates that the related CNC family members, BTB domain and CNC homolog 1 (BACH1) and NFE2 like bZIP transcription factor 1 (NFE2L1), also have roles in ferroptosis regulation. Here, we comprehensively review the role of CNC transcription factors in governing cellular sensitivity to ferroptosis. We describe how CNC family members regulate ferroptosis sensitivity through modulation of iron, lipid, and redox metabolism. We also use examples of ferroptosis regulation by CNC proteins to illustrate the flexible and highly context-dependent nature of the ferroptosis mechanism in different cells and conditions.

Mammalian cells can die by apoptosis or by one of several non-apoptotic mechanisms, such as ferroptosis. Here, we present a protocol to distinguish ferroptosis from other cell death mechanisms in cultured cells. We describe steps for seeding cells, administering mechanism-specific cell death inducers and inhibitors, and measuring cell death and viability. We then detail the use of molecular markers to verify mechanisms of cell death. This protocol can be used to identify and distinguish ferroptosis in 2D and 3D cultures. For complete details on the use and execution of this protocol, please refer to Ko, et al. (2019),1 Magtanong, et al. (2022),2 and Armenta, et al. (2022).3

Ferroptosis is a regulated nonapoptotic cell death process characterized by iron-dependent lipid peroxidation. Peroxidation of polyunsaturated fatty acid-containing phospholipids (PUFA-PL) is necessary for the execution of ferroptosis. Glutathione peroxidase 4 (GPX4) suppresses ferroptosis by reducing lipid hydroperoxides to lipid alcohols. GPX4 may be a useful target for drug development, highlighting the need to identify factors that govern GPX4 inhibitor sensitivity. In this study, we found that reduced GPX4 expression was sufficient to induce ferroptosis in multiple adherent (2D) cancer cell cultures. However, lower GPX4 protein levels did not consistently affect tumor xenograft growth in mice. Culturing cells as spheroids (3D) was sufficient to reduce sensitivity to pharmacologic GPX4 inhibition. Mechanistically, growth in 3D versus 2D conditions upregulated expression of the monounsaturated fatty acid (MUFA) biosynthetic gene stearoyl-CoA desaturase, altering the ratio of MUFA-PLs to PUFA-PLs in a direction favoring ferroptosis resistance. Similar shifts in MUFA-PL:PUFA-PL ratios were observed in xenograft tumors. Thus, lipidome remodeling in 3D growth conditions and in vivo may limit GPX4 inhibitor efficacy. SIGNIFICANCE: Changes in lipid composition can affect induction of ferroptosis, explaining why sensitivity of cancer cells in tissue culture does not reliably translate to more complex models and suggesting potential ferroptosis sensitization strategies.

HIV-1 virion production is driven by Gag and Gag-Pol (GP) proteins, with Gag forming the bulk of the capsid and driving budding, while GP binds Gag to deliver the essential virion enzymes protease, reverse transcriptase, and integrase. Virion GP levels are traditionally thought to reflect the relative abundances of GP and Gag in cells (;1:20), dictated by the frequency of a 21 programmed ribosomal frameshifting (PRF) event occurring in gag-pol mRNAs. Here, we exploited a panel of PRF mutant viruses to show that mechanisms in addition to PRF regulate GP incorporation into virions. First, we show that GP is enriched ;3-fold in virions relative to cells, with viral infectivity being better maintained at subphysiological levels of GP than when GP levels are too high. Second, we report that GP is more efficiently incorporated into virions when Gag and GP are synthesized in cis (i.e., from the same gag-pol mRNA) than in trans, suggesting that Gag/GP translation and assembly are spatially coupled processes. Third, we show that, surprisingly, virions exhibit a strong upper limit to trans-delivered GP incorporation; an adaptation that appears to allow the virus to temper defects to GP/Gag cleavage that may negatively impact reverse transcription. Taking these results together, we propose a "weighted Goldilocks"scenario for HIV-1 GP incorporation, wherein combined mechanisms of GP enrichment and exclusion buffer virion infectivity over a broad range of local GP concentrations. These results provide new insights into the HIV-1 virion assembly pathway relevant to the anticipated efficacy of PRF-targeted antiviral strategies.