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Formation of disulphide bonds within the mammalian endoplasmic reticulum (ER) requires the combined activities of Ero1α and protein disulphide isomerase (PDI). As Ero1α produces hydrogen peroxide during oxidation, regulation of its activity is critical in preventing ER-generated oxidative stress. Here, we have expressed and purified recombinant human Ero1α and shown that it has activity towards thioredoxin and PDI. The activity towards PDI required the inclusion of glutathione to ensure sustained oxidation. By carrying out site-directed mutagenesis of cysteine residues, we show that Ero1α is regulated by non-catalytic disulphides. The midpoint reduction potential (E°′) of the regulatory disulphides was calculated to be approximately −275 mV making them stable in the redox conditions prevalent in the ER. The stable regulatory disulphides were only partially reduced by PDI (E°′∼−180 mV), suggesting either that this is a mechanism for preventing excessive Ero1α activity and oxidation of PDI or that additional factors are required for Ero1α activation within the mammalian ER.

The TIM10 chaperone facilitates the insertion of hydrophobic proteins at the mitochondrial inner membrane. Here we report the novel molecular mechanism of TIM10 assembly. This process crucially depends on oxidative folding in mitochondria and involves: (i) import of the subunits in a Cys-reduced and unfolded state; (ii) folding to an assembly-competent structure maintained by intramolecular disulfide bonding of their four conserved cysteines; and (iii) assembly of the oxidized zinc-devoid subunits to the functional complex. We show that intramolecular disulfide bonding occurs in vivo, whereas intermolecular disulfides observed in vitro are abortive intermediates in the assembly pathway. This novel mechanism of compartment-specific redox-regulated assembly is crucial for the formation of a functional TIM10 chaperone.Cysteine has unique biological functions by using its sulfhydryl (ϪSH) group in the active site of an enzyme, in chelating metals, or as the active site of disulfide reshuffling. For example, in the case of the molecular chaperone, Hsp33 activity is regulated by a redox switch with its inactive form reduced and zinc-coordinated and its active form turned on by oxidation and disulfide formation (1). The transcription factor OxyR is similarly activated through the formation of a disulfide bond and inactivated by enzymatic reduction with glutaredoxin (2). Disulfide bond formation in general is an essential step in the folding of many proteins, and it is catalyzed in vivo by the dsb system in the bacterial periplasm (3) and the functionally related PDI/Ero1 (4) system in the ER of eukaryotic cells. Although a mitochondrial intermembrane space sulfhydryl oxidase, Erv1p, has been identified (5), there has been no report suggesting disulfide bond formation in mitochondria. Here we demonstrate that the mitochondrial intermembrane space can allow oxidative folding events, challenging the commonly accepted notion that this compartment is in complete redox equilibrium with the reducing cytosol. We show that substrates for this oxidation event are Tim9 and Tim10, the subunits of the TIM10 chaperone that mediates hydrophobic protein insertion at the inner mitochondrial membrane (6 -9). This complex binds to the hydrophobic segments of the precursor (10) at an early import stage as the precursor emerges from the outer membrane protein import channel (translocase of the outer membrane, TOM 1 complex). Subsequently, the precursor is carried across the intermembrane space and passed onto the TIM22 membrane-embedded complex that facilitates insertion (11-13) through a twin pore involving two voltage-dependent steps (14).As all of the TIM subunits are imported themselves from the cytosol, correct assembly to their respective complex is essential for their function. Tim9 and Tim10 partner each other specifically to form the TIM10 complex, but the structural basis and the mechanism of this assembly process remain unclear. Although the "twin CX3C" motif common to all of the small Tim proteins is thought to be important for...

IntroductionThe forkhead transcription factor FOXM1 coordinates expression of cell cycle-related genes and plays a pivotal role in tumorigenesis and cancer progression. We previously showed that FOXM1 acts downstream of 14-3-3ζ signaling, the elevation of which correlates with a more aggressive tumor phenotype. However, the role that FOXM1 might play in engendering resistance to endocrine treatments in estrogen receptor-positive (ER+) patients when tumor FOXM1 is high has not been clearly defined yet.MethodsWe analyzed FOXM1 protein expression by immunohistochemistry in 501 ER-positive breast cancers. We also mapped genome-wide FOXM1, extracellular signal-regulated kinase 2 and ERα binding events by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) in hormone-sensitive and resistant breast cancer cells after tamoxifen treatment. These binding profiles were integrated with gene expression data derived from cells before and after FOXM1 knockdown to highlight specific FOXM1 transcriptional networks. We also modulated the levels of FOXM1 and newly discovered FOXM1-regulated genes and examined their impact on the cancer stem-like cell population and on cell invasiveness and resistance to endocrine treatments.ResultsFOXM1 protein expression was high in 20% of the tumors, which correlated with significantly reduced survival in these patients (P = 0.003 by logrank Mantel-Cox test). ChIP-seq analyses revealed that FOXM1 binding sites were enriched at the transcription start site of genes involved in cell-cycle progression, maintenance of stem cell properties, and invasion and metastasis, all of which are associated with a poor prognosis in ERα-positive patients treated with tamoxifen. Integration of binding profiles with gene expression highlighted FOXM1 transcriptional networks controlling cell proliferation, stem cell properties, invasion and metastasis. Increased expression of FOXM1 was associated with an expansion of the cancer stem-like cell population and with increased cell invasiveness and resistance to endocrine treatments. Use of a selective FOXM1 inhibitor proved very effective in restoring endocrine therapy sensitivity and decreasing breast cancer aggressiveness.ConclusionsCollectively, our findings uncover novel roles for FOXM1 and FOXM1-regulated genes in promoting cancer stem-like cell properties and therapy resistance. They highlight the relevance of FOXM1 as a therapeutic target to be considered for reducing invasiveness and enhancing breast cancer response to endocrine treatments.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0436-4) contains supplementary material, which is available to authorized users.