Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids 1,2. To date, ferroptosis has been believed to be restrained only by the phospholipid hydroperoxide (PLOOH)-reducing enzyme glutathione peroxidase 4 (GPX4) 3,4 and radicaltrapping antioxidants (RTAs) 5,6. The factors which underlie a given cell type's sensitivity to ferroptosis 7 is, however, critical to understand the pathophysiological role of ferroptosis and how it may be exploited for cancer treatment. Although metabolic constraints 8 and phospholipid composition 9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been yet been identified that account for ferroptosis resistance. We undertook an expression cloning approach to identify genes able to complement GPX4 loss. These efforts uncovered the flavoprotein "apoptosis inducing factor mitochondria-associated 2 (AIFM2)" as a previously unrecognized anti-ferroptotic gene. AIFM2, hereafter renamed "ferroptosis-suppressor-protein 1" (FSP1), initially described as a pro-apoptotic gene 11 , confers an unprecedented protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that ferroptosis suppression by FSP1 is mediated via ubiquinone (CoQ10): its reduced form ubiquinol traps lipid peroxyl radicals that mediate lipid peroxidation, while FSP1 catalyses its regeneration by using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. Conclusively, FSP1/CoQ10/NAD(P)H exists as a standalone parallel system, which cooperates with GPX4 and glutathione (GSH) to suppress phospholipid peroxidation (pLPO) and ferroptosis. program NEUROPROTEKT (03VP04260), as well as the m4 Award provided by the Bavarian Ministry of Economic Affairs, Regional Development and Energy (StMWi) to M.C., the Cancer Research UK

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Mdm2 is a major negative regulator of the tumor suppressor p53 protein, a protein that plays a crucial role in maintaining genome integrity. Inactivation of p53 is the most prevalent defect in human cancers. Inhibitors of the Mdm2-p53 interaction that restore the functional p53 constitute potential nongenotoxic anticancer agents with a novel mode of action. We present here a 2.0 Å resolution structure of the Mdm2 protein with a bound stapled p53 peptide. Such peptides, which are conformationally and proteolytically stabilized with all-hydrocarbon staples, are an emerging class of biologics that are capable of disrupting protein-protein interactions and thus have broad therapeutic potential. The structure represents the first crystal structure of an i, i + 7 stapled peptide bound to its target and reveals that rather than acting solely as a passive conformational brace, a staple can intimately interact with the surface of a protein and augment the binding interface.

Intensive anticancer drug discovery efforts have been made to develop small molecule inhibitors of the p53-MDM2 and p53-MDMX interactions. We present here the structures of the most potent inhibitors bound to MDM2 and MDMX that are based on the new imidazo-indole scaffold. In addition, the structure of the recently reported spiro-oxindole inhibitor bound to MDM2 is described. The structures indicate how the substituents of a small molecule that bind to the three subpockets of the MDM2/X-p53 interaction should be optimized for effective binding to MDM2 and/or MDMX. While the spiro-oxindole inhibitor triggers significant ligand-induced changes in MDM2, the imidazo-indoles share similar binding modes for MDMX and MDM2, but cause only minimal induced-fit changes in the structures of both proteins. Our study includes the first structure of the complex between MDMX and a small molecule and should aid in developing efficient scaffolds for binding to MDMX and/or MDM2.

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The oncoprotein Mdm2, and the recently intensely studied, homologues protein Mdmx, are principal negative regulators of the p53 tumor suppressor. The mechanisms by which they regulate the stability and activity of p53 are not fully established. We have determined the crystal structure of the N-terminal domain of Mdmx bound to a 15-residue p53 peptide. The structure reveals that although the principle features of the Mdm2-p53 interaction are preserved in the Mdmx-p53 complex, the Mdmx hydrophobic cleft on which the p53 peptide binds is significantly altered: a part of the cleft is blocked by sidechains of Met and Tyr of the p53-binding pocket of Mdmx. Thus specific inhibitors of Mdm2-p53 would not be optimal for binding to Mdmx. Our binding assays show indeed that nutlins, the newly discovered, potent antagonists of the Mdm2-p53 interaction, are not capable to efficiently disrupt the Mdmx-p53 interaction. To achieve full activation of p53 in tumor cells, compounds that are specific for Mdmx are necessary to complement the Mdm2 specific binders.

structure ͉ cell growth T he insulin-like growth factor-binding protein (IGFBP) family comprises six soluble proteins (IGFBP1-6) of Ϸ250 residues that bind to IGFs with nanomolar affinities (1-4). Because of their sequence homology, IGFBPs are assumed to share a common overall fold and are expected to have closely related IGF-binding determinants. Each IGFBP can be divided into three distinct domains of approximately equal lengths: highly conserved cysteinerich N and C domains and a central linker domain unique to each IGFBP species. Both the N and C domains participate in the binding to IGFs, although the specific roles of each of these domains in IGF binding have not been decisively determined (1-13). The C-terminal domain may be responsible for preferences of IGFBPs for one species of IGF over the other (2, 3-7, 9-13); the C-terminal domain is also involved in regulation of the IGF-binding affinity through interaction with extracellular matrix components (1,2,14) and is most probably engaged in mediating IGF1-independent actions (1, 4, 14). The central linker domain is the least conserved region and has never been cited as part of the IGF-binding site for any IGFBP. This domain is the site of posttranslational modifications, specific proteolysis (4), and the acid-labile subunit (1) and extracellular matrix associations (1, 2, 14) known for IGFBPs. Proteolytic cleavage in this domain is believed to produce loweraffinity N-and C-terminal fragments that cannot compete with IGF receptors for IGFs, and, thus, the proteolysis is assumed to be the predominant mechanism for IGF release from IGFBPs (4, 9).However, recent studies indicate that the resulting N-and Cterminal fragments still can inhibit IGF activity and have functional properties that differ from those of the intact proteins (1, 3, 5, 9).The structure of the N-terminal domain of IGFBP-5, free (15) and complexed to IGF1 (16), was solved some time ago. More recently, low-resolution structures of the C-terminal domain of IGFBP6 (12) and its binding surface on IGF2 (3, 12) have been determined with NMR spectroscopy. There is, however, no x-ray structure of a ternary complex of the C-terminal domain of any IGFBPs bound to both the N-terminal domain and IGF, although the C-terminal fragment of IGFBP4 was crystallized recently (9), and also the x-ray structure of the isolated C-terminal fragment of IGFBP1 has been solved (17). We recently reported the x-ray structure of the ternary complex of the N-and C-terminal domains of IGFBP4 bound to IGF1 (10) and described ordered structures for the N-terminal domain of IGFBP4 and IGF1. The C domain was represented by disconnected patches of electron density and could not be interpreted. We describe here the long-sought, highresolution x-ray structure of a complex of the N-and C-terminal domains of IGFBP4 bound to IGF1. We also present the structure of the C-terminal domain of IGFBP1 bound to the N-terminal domain of IGFBP4 and IGF1 and the structure of the binary complex of the N-terminal domain of IGFBP4 (residues 1-92)...

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1–HIND interaction, cannot use certain non-canonical 5′ splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier.