Copper–zinc superoxide dismutase (Sod1) is a critical antioxidant enzyme that rids the cell of reactive oxygen through the redox cycling of a catalytic copper ion provided by its copper chaperone (Ccs). Ccs must first acquire this copper ion, directly or indirectly, from the influx copper transporter, Ctr1. The three proteins of this transport pathway ensure careful trafficking of copper ions from cell entry to target delivery, but the intricacies remain undefined. Biochemical examination of each step in the pathway determined that the activation of the target (Sod1) regulates the Ccs·Ctr1 interaction. Ccs stably interacts with the cytosolic C-terminal tail of Ctr1 (Ctr1c) in a copper-dependent manner. This interaction becomes tripartite upon the addition of an engineered immature form of Sod1 creating a stable Cu(I)-Ctr1c·Ccs·Sod1 heterotrimer in solution. This heterotrimer can also be made by the addition of a preformed Sod1·Ccs heterodimer to Cu(I)-Ctr1c, suggestive of multiple routes to the same destination. Only complete Sod1 activation (i.e. active site copper delivery and intra-subunit disulfide bond formation) breaks the Sod1·Ccs·Ctr1c complex. The results provide a new and extended view of the Sod1 activation pathway(s) originating at cellular copper import. Electronic supplementary material The online version of this article (10.1007/s10534-019-00206-3) contains supplementary material, which is available to authorized users.

Edited by Ruma BanerjeeCopper (Cu) is essential for the survival of aerobic organisms through its interaction with molecular oxygen (O 2 ). However, Cu's chemical properties also make it toxic, requiring specific cellular mechanisms for Cu uptake and handling, mediated by Cu chaperones. CCS1, the budding yeast (S. cerevisiae) Cu chaperone for Cu-zinc (Zn) superoxide dismutase (SOD1) activates by directly promoting both Cu delivery and disulfide formation in SOD1. The complete mechanistic details of this transaction along with recently proposed molecular chaperone-like functions for CCS1 remain undefined. Here, we present combined structural, spectroscopic, kinetic, and thermodynamic data that suggest a multifunctional chaperoning role(s) for CCS1 during SOD1 activation. We observed that CCS1 preferentially binds a completely immature form of SOD1 and that the SOD1⅐CCS1 interaction promotes high-affinity Zn(II) binding in SOD1. Conserved aromatic residues within the CCS1 C-terminal domain are integral in these processes. Previously, we have shown that CCS1 delivers Cu(I) to an entry site at the SOD1⅐CCS1 interface upon binding. We show here that Cu(I) is transferred from CCS1 to the entry site and then to the SOD1 active site by a thermodynamically driven affinity gradient. We also noted that efficient transfer from the entry site to the active site is entirely dependent upon the oxidation of the conserved intrasubunit disulfide bond in SOD1. Our results herein provide a solid foundation for proposing a complete molecular mechanism for CCS1 activity and reclassification as a first-of-its-kind "dual chaperone."Copper (Cu) 2 is required for the activation of dioxygen, a function essential for the survival of aerobic organisms (1). Figure 4. Kinetic analysis of CCS1 mediated copper delivery to sites on SOD1. Stopped-flow kinetic traces at 260 nm and corresponding curve fittings (red line) obtained upon rapid mixing of Cu(I)-Ccs (20 M) with equal volumes of apo H46R/H48Q (X,Zn-SOD1 SH ) or WT E,Zn-SOD1 SH (20 M) under aerobic or anaerobic conditions are shown. The decreased absorbance at 260 nm (top left) corresponds to the copper ion moving from the cysteine coordination of CCS1 and/or SOD1 and entering the histidine coordination of the SOD1 active site. Increased absorbance at 260 nm shows the copper ion transfer from CCS1 cysteines to the SOD1⅐CCS1 2Cys/1His entry site.

Summary Lineage-defining transcription factors (LTFs) play key roles in small-cell lung cancer (SCLC) pathophysiology. Delineating the LTF-regulated genes operative in SCLC could provide a road map to identify SCLC dependencies. We integrated chromatin landscape and transcriptome analyses of patient-derived SCLC preclinical models to identify super-enhancers (SEs) and their associated genes in the ASCL1-, NEUROD1-, and POU2F3-high SCLC subtypes. We find SE signatures predict LTF-based classification of SCLC, and the SE-associated genes are enriched with those defined as common essential genes in DepMap. In addition, in ASCL1-high SCLC, we show ASCL1 complexes with NKX2-1 and PROX1 to co-regulate genes functioning in NOTCH signaling, catecholamine biosynthesis, and cell-cycle processes. Depletion of ASCL1 demonstrates it is a key dependency factor in preclinical SCLC models and directly regulates multiple DepMap-defined essential genes. We provide LTF/SE-based subtype-specific gene sets for SCLC for further therapeutic investigation.

Zinc (II) ions (hereafter simplified as zinc) are important for the structural and functional activity of many proteins. For Cu, Zn superoxide dismutase (Sod1), zinc stabilizes the native structure of each Sod1 monomer, promotes homo-dimerization and plays an important role in activity by “softening” the active site so that copper cycling between Cu(I) and Cu(II) can rapidly occur. Previously, we have reported that binding of Sod1 by its copper chaperone (Ccs) stabilizes a conformation of Sod1 that promotes site-specific high-affinity zinc binding. While there are a multitude of Sod1 mutations linked to the familial form of amyotrophic lateral sclerosis (fALS), characterizations by multiple research groups have been unable to realize strong commonalities among mutants. Here, we examine a set of fALS-linked Sod1 mutations that have been well-characterized and are known to possess variation in their biophysical characteristics. The zinc affinities of these mutants are evaluated here for the first time and then compared with the previously established value for wild-type Sod1 zinc affinity. Ccs does not have the same ability to promote zinc binding to these mutants as it does for the wild-type version of Sod1. Our data provides a deeper look into how (non)productive Sod1 maturation by Ccs may link a diverse set of fALS-Sod1 mutations.

Copper ions (i.e., copper) are a critical part of several cellular processes, but tight regulation of copper levels and trafficking are required to keep the cell protected from this highly reactive transition metal. Cu, Zn superoxide dismutase (Sod1) protects the cell from the accumulation of radical oxygen species by way of the redox cycling activity of copper in its catalytic center. Multiple posttranslational modification events, including copper incorporation, are reliant on the copper chaperone for Sod1 (Ccs). The high-affinity copper uptake protein (Ctr1) is the main entry point of copper into eukaryotic cells and can directly supply copper to Ccs along with other known intracellular chaperones and trafficking molecules. This review explores the routes of copper delivery that are utilized to activate Sod1 and the usefulness and necessity of each.