Copper is an essential cofactor for all organisms, and yet it becomes toxic if concentrations exceed a threshold maintained by evolutionarily conserved homeostatic mechanisms. How excess copper induces cell death, however, is unknown. Here, we show in human cells that copper-dependent, regulated cell death is distinct from known death mechanisms and is dependent on mitochondrial respiration. We show that copper-dependent death occurs by means of direct binding of copper to lipoylated components of the tricarboxylic acid (TCA) cycle. This results in lipoylated protein aggregation and subsequent iron-sulfur cluster protein loss, which leads to proteotoxic stress and ultimately cell death. These findings may explain the need for ancient copper homeostatic mechanisms.

Human cancer cell lines are the workhorse of cancer research. Although cell lines are known to evolve in culture, the extent of the resultant genetic and transcriptional heterogeneity and its functional consequences remain understudied. Here we use genomic analyses of 106 human cell lines grown in two laboratories to show extensive clonal diversity. Further comprehensive genomic characterization of 27 strains of the common breast cancer cell line MCF7 uncovered rapid genetic diversification. Similar results were obtained with multiple strains of 13 additional cell lines. Notably, genetic changes were associated with differential activation of gene expression programs and marked differences in cell morphology and proliferation. Barcoding experiments showed that cell line evolution occurs as a result of positive clonal selection that is highly sensitive to culture conditions. Analyses of single-cell-derived clones demonstrated that continuous instability quickly translates into heterogeneity of the cell line. When the 27 MCF7 strains were tested against 321 anti-cancer compounds, we uncovered considerably different drug responses: at least 75% of compounds that strongly inhibited some strains were completely inactive in others. This study documents the extent, origins and consequences of genetic variation within cell lines, and provides a framework for researchers to measure such variation in efforts to support maximally reproducible cancer research.

Protein degradation is an essential and highly regulated process. The proteasomal degradation of the tumor suppressors p53 and p73 is regulated by both polyubiquitination and by an ubiquitin-independent process. Here, we show that this ubiquitin-independent process is mediated by the 20S proteasomes and is regulated by NQO1. NQO1 physically interacts with p53 and p73 in an NADH-dependent manner and protects them from 20S proteasomal degradation. Remarkably, the vast majority of NQO1 in cells is found in physical association with the 20S proteasomes, suggesting that NQO1 functions as a gatekeeper of the 20S proteasomes. We further show that this pathway plays a role in p53 accumulation in response to ionizing radiation. Our findings provide the first evidence for in vivo degradation of p53 and p73 by the 20S proteasomes and its regulation by NQO1 and NADH level. Protein degradation determines the outcome of many cellular physiological processes (Coux et al. 1996). Degradation of proteins by the proteasomes occurs via various pathways (Verma and Deshaies 2000;Pickart and Cohen 2004). The most intensely studied one is the ubiquitin-26S proteasome pathway (Hershko 1996;Hershko and Ciechanover 1998;Goldberg 2003). The tumor suppressor p53 is a very labile protein that undergoes Mdm2 and ubiquitin-dependent 26S proteasomal degradation (Haupt et al. 1997;Kubbutat et al. 1997). Recently, we reported that degradation of p53 also occurs in an Mdm2 and ubiquitin-independent manner (Asher et al. 2002b). This pathway of p53 degradation is regulated by NAD(P)H quinone oxidoreductase 1 (NQO1) (Asher et al. 2001(Asher et al. , 2002a(Asher et al. ,b, 2003(Asher et al. , 2004), yet the underlying molecular mechanisms that control p53 degradation remained elusive. Results and DiscussionTo investigate the role of NQO1 in proteasomal degradation, we followed NQO1 distribution in fractionated mouse liver extracts. Ammonium sulfate precipitation and gel-filtration chromatography of liver extracts revealed that the majority of NQO1 cofractionated with the 20S proteasomes (Fig. 1A). These fractions are devoid of the 26S proteasomes that were excluded by the differential ammonium sulfate precipitation (Fig. 1A, IB: 26S, anti TBP1 a subunit of the 19S). These results suggest that the vast majority of cellular NQO1 is found in a large protein complex that possibly includes the 20S proteasomes.To further study this possibility, the 20S-containing fractions were pooled and fractionated by anion exchange chromatography according to a standard 20S purification protocol (Friguet et al. 2002). Remarkably, NQO1 was detected in the 0.3 M NaCl fraction containing the 20S proteasomes (Fig. 1B). Electrophoresis of the 0.3 M NaCl fraction on a nondenaturing PAGE, followed by peptidase activity assay, showed that the purified 20S is functional (Fig. 1C, Activity panel). Immunoblot analysis with anti NQO1 antibody revealed that NQO1 comigrated with the 20S proteasomes, but not with the 26S proteasomes (Fig. 1C). Finally, a coimmunopercipitation experim...

The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome inhibitor sensitivity. When cells were forced to use oxidative phosphorylation rather than glycolysis, they became proteasome inhibitor-resistant. This mitochondrial state, however, creates a unique vulnerability: sensitivity to the small-molecule compound elesclomol. Genome-wide CRISPR/Cas9 screening showed that a single gene, encoding the mitochondrial reductase FDX1, could rescue elesclomol-induced cell death. Enzymatic function and NMR-based analyses further showed that FDX1 is the direct target of elesclomol, which promotes a unique form of copper-dependent cell death. These studies elucidate a fundamental mechanism by which cells adapt to proteotoxic stress and suggests strategies to mitigate proteasome inhibitor-resistance.

NAD(P)H quinone oxidoreductase 1 (NQO1) is a ubiquitous flavoenzyme that catalyzes two-electron reduction of quinones to hydroquinones utilizing NAD(P)H as an electron donor. NQO1 binds and stabilizes several short-lived proteins including the tumor suppressors p53 and p73 and the enzyme ornithine decarboxylase (ODC). Dicoumarol is a widely used potent competitive inhibitor of NQO1 enzymatic activity, which competes with NAD(P)H for binding to NQO1. Dicoumarol also disrupts the binding of NQO1 to p53, p73, and ODC and induces their ubiquitin-independent proteasomal degradation. We report here the crystal structure of human NQO1 in complex with dicoumarol at 2.75 A resolution. We have identified the interactions of dicoumarol with the different residues of NQO1 and the conformational changes imposed upon dicoumarol binding. The most prominent conformational changes that occur in the presence of dicoumarol involve Tyr 128 and Phe 232 that are present on the surface of the NQO1 catalytic pocket. On the basis of the comparison of the NQO1 structure in complex with different NQO1 inhibitors and our previous analysis of NQO1 mutants, we propose that the specific conformation of Tyr 128 and Phe 232 is important for NQO1 interaction with p53 and other client proteins.