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.

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.

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 thatE2F1and its target geneMYBL2were 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.

17Amber codon suppression is a powerful tool to site-specifically modify proteins to 18 generate novel biophysical probes. Yet, its application on large and complex 19 multidomain proteins is challenging, leading to difficulties during structural and 20 conformational characterization using spectroscopic methods. The animal fatty acid 21 synthase type I is a 540 kDa homodimer displaying large conformational variability. 22As the key enzyme of de novo fatty acid synthesis, it attracts interest in the fields of 23 obesity, diabetes and cancer treatment. Substrates and intermediates remain 24 covalently bound to the enzyme during biosynthesis and are shuttled to all catalytic 25 domains by the acyl carrier protein domain. Thus, conformational variability of animal 26 FAS is an essential aspect for fatty acid biosynthesis. We investigate this 27 multidomain protein as a model system for probing amber codon suppression by 28 genetic encoding of non-canonical amino acids. The systematic approach relies on a 29 microplate-based reporter assay of low complexity, that was used for quick screening 30 of suppression conditions. Furthermore, the applicability of the reporter assay is 31 demonstrated by successful upscaling to both full-length constructs and increased 32 expression scale. The obtained fluorescent probes of murine FAS type I could be 33 subjected readily to a conformational analysis using single-molecule fluorescence 34 resonance energy transfer. 35All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.