Mitochondrial Reactive Oxygen Species Formation Determines ACSL4/LPCAT2-Mediated Ferroptosis.

Ferroptosis is a form of oxidative cell death that is characterized by enhanced lipid peroxidation and mitochondrial impairment. The enzymes acyl-CoA synthetase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase (LPCAT) play an essential role in the biosynthesis of polyunsaturated fatty acid (PUFA)-containing phospholipids, thereby providing the substrates for lipid peroxidation and promoting ferroptosis. To examine the impact of mitochondria in ACSL4/LPCAT2-driven ferroptosis, HEK293T cells overexpressing ACSL4 and LPCAT2 (OE) or empty vector controls (LV) were exposed to 1S, 3R-RSL3 (RSL3) for induction of ferroptosis. The ACSL4/LPCAT2 overexpression resulted in higher sensitivity against RSL3-induced cell death compared to LV-transfected controls. Moreover, mitochondrial parameters such as mitochondrial reactive oxygen species (ROS) formation, mitochondrial membrane potential, and mitochondrial respiration deteriorated in the OE cells, supporting the conclusion that mitochondria play a significant role in ACSL4/LPCAT2-driven ferroptosis. This was further confirmed through the protection of OE cells against RSL3-mediated cell death by the mitochondrial ROS scavenger mitoquinone (MitoQ), which exerted protection via antioxidative properties rather than through previously reported metabolic effects. Our findings implicate that mitochondrial ROS production and the accompanying organelle disintegration are essential for mediating oxidative cell death initiated through lipid peroxidation in ferroptosis.

Three-dimensional growth reveals fine-tuning of 5-lipoxygenase by proliferative pathways in cancer.

The leukotriene (LT) pathway is positively correlated with the progression of solid malignancies, but the factors that control the expression of 5-lipoxygenase (5-LO), the central enzyme in LT biosynthesis, in tumors are poorly understood. Here, we report that 5-LO along with other members of the LT pathway is up-regulated in multicellular colon tumor spheroids. This up-regulation was inversely correlated with cell proliferation and activation of PI3K/mTORC-2- and MEK-1/ERK-dependent pathways. Furthermore, we found that E2F1 and its target gene MYBL2 were involved in the repression of 5-LO during cell proliferation. Importantly, we found that this PI3K/mTORC-2- and MEK-1/ERK-dependent suppression of 5-LO is also existent in tumor cells from other origins, suggesting that this mechanism is widely applicable to other tumor entities. Our data show that tumor cells fine-tune 5-LO and LT biosynthesis in response to environmental changes repressing the enzyme during proliferation while making use of the enzyme under cell stress conditions, implying that tumor-derived 5-LO plays a role in the manipulation of the tumor stroma to quickly restore cell proliferation.

Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression.

The access to information on the dynamic behaviour of large proteins is usually hindered as spectroscopic methods require the site-specific attachment of biophysical probes. A powerful emerging tool to tackle this issue is amber codon suppression. Till date, its application on large and complex multidomain proteins of MDa size has not been reported. Herein, we systematically investigate the feasibility to introduce different non-canonical amino acids into a 540 kDa homodimeric fatty acid synthase type I by genetic code expansion with subsequent fluorescent labelling. Our approach relies on a microplate-based reporter assay of low complexity using a GFP fusion protein to quickly screen for sufficient suppression conditions. Once identified, these findings were successfully utilized to upscale both the expression scale and the protein size to full-length constructs. These fluorescently labelled samples of fatty acid synthase were subjected to initial biophysical experiments, including HPLC analysis, activity assays and fluorescence spectroscopy. Successful introduction of such probes into a molecular machine such as fatty acid synthases may pave the way to understand the conformational variability, which is a primary intrinsic property required for efficient interplay of all catalytic functionalities, and to engineer them.