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.

The Escherichia coli multidrug transporter MdfA contains a membrane-embedded charged residue (Glu-26) that was shown to play an important role in substrate recognition. To identify additional determinants of multidrug recognition we isolated 58 intragenic second-site mutations that restored the function of inactive MdfA E26X mutants. In addition, two single-site mutations that enhanced the activity of wild-type MdfA were identified. Most of the mutations were found in two regions, the cytoplasmic half of transmembrane segments (TMs) 4, 5, and 6 (cluster 1) and the periplasmic half of TM 1 and 2 (cluster 2). The identified residues were mutated to cysteines in the background of a functional cysteineless MdfA, and substrate protection against alkylation was analyzed. The results support the suggestion that the two clusters are involved in substrate recognition. Using inverted membrane vesicles we observed that a proton electrochemical gradient (⌬˜H؉, inside positive and acidic) enhanced the substrate-protective effect in the cytoplasmic region, whereas it largely reduced this effect in the periplasmic side of MdfA. Therefore, we propose that substrates interact with two sites in MdfA, one in the cytoplasmic leaflet of the membrane and the other in the periplasmic leaflet. Theoretically, these domains could constitute a large part of the multidrug pathway through MdfA.

The resistance of cells to many drugs simultaneously (multidrug resistance) often involves the expression of membrane transporters (Mdrs); each recognizes and expels a broad spectrum of chemically unrelated drugs from the cell. The Escherichia coli Mdr transporter MdfA is able to transport differentially charged substrates in exchange for protons. This includes neutral compounds, namely chloramphenicol and thiamphenicol, and lipophilic cations such as tetraphenylphosphonium and ethidium. Here we show that the chloramphenicol and thiamphenicol transport reactions are electrogenic, whereas the transport of several monovalent cationic substrates is electroneutral. Therefore, unlike with positively charged substrates, the transmembrane electrical potential (negative inside) constitutes a major part of the driving force for the transport of electroneutral substrates by MdfA. These results demonstrate an unprecedented ability of a single secondary transporter to catalyze discrete transport reactions that differ in their electrogenicity and are governed by different components of the proton motive force.

Mirror touch and sense of self 2 Highlights• Sense of bodily self is strongly linked with mirroring mechanisms• Mirroring for self-and other-related information differs at early and later stages • Depersonalisation specifically alters mirroring for self-related events• Mirroring thus depends on how connected one feels to one's bodily self Mirror touch and sense of self 3 AbstractOur sense of self is thought to develop through sensory-motor contingencies provided, not only by observing one's own body, but also by mirroring interactions with others. This suggests that there is a strong link between mirroring mechanisms and the bodily self. The present study tested whether this link is expressed at early, implicit stages of the mirroring process or at later, more cognitive stages. We also provide, to the best of our knowledge, the first demonstration of how inter-individual differences in our sense of bodily self may affect mirroring mechanisms. We used somatosensory event-related potentials (SEPs) to investigate the temporal dynamics of mirroring highly self-related information (viewed touch on one's own face) compared to other-related information (viewed touch on a stranger's face), in individuals with low and high levels of depersonalisation, a mental condition characterised by feeling detached or estranged from one's self and body. For the low-depersonalisation group, mirroring for self-related events (P45) preceded mirroring for other-related events (N80). At later stages (P200), mirroring was stronger for other-related than self-related events. This shows that early, implicit and later, more cognitive processes play different relative roles in mirroring self-and other-related bodily events. Critically, mirroring differed in the high-depersonalisation group, specifically for self-related events. An absence of early, implicit mirroring for self-related events over P45 suggests that the associated processes may be the neural correlates of the disembodiment experienced in depersonalisation. A lack of differential mirroring for self-and other-related events over P200 may reflect compensatory mechanisms that redress deficiencies in mirroring at earlier Mirror touch and sense of self 4 stages, which may break down to give rise to symptoms of depersonalisation.Alternatively, or in addition, they may represent an attenuation of processes related to self-other distinction. Our study thus shows that mirroring, especially for events on one's own face, can be strongly affected by how connected the observer feels to their own bodily self.

According to the current topology model of the Escherichia coli multidrug transporter MdfA, it contains a membrane-embedded negatively charged residue, Glu26, which was shown to play an important role in substrate recognition. To further elucidate the role of this substrate recognition determinant, various Glu26 replacements were characterized. Surprisingly, studies with neutral MdfA substrates showed that, unlike many enzymatic systems where the size and chemical properties of binding site residues are relatively defined, MdfA tolerates a variety of changes at position 26, including size, hydrophobicity, and charge. Moreover, although efficient transport of positively charged substrates requires a negative charge at position 26 (Glu or Asp), neutralization of this charge does not always abrogate the interaction of MdfA with cationic drugs, thus demonstrating that the negative charge does not play an essential role in the multidrug transport mechanism. Collectively, these results suggest a link between the broad substrate specificity profile of multidrug transporters and the structural and chemical promiscuity at their substrate recognition pockets.

The Hippo pathway is an evolutionarily conserved pathway that controls cell proliferation, organ size, tissue regeneration and stem cell self-renewal. Here we show that it also regulates the DNA damage response. At high cell density, when the Hippo pathway is active, DNA damage-induced apoptosis and the activation of the tyrosine kinase c-Abl were suppressed. At low cell density, overexpression of the Hippo pathway kinase large tumor suppressor 2 (Lats2) inhibited c-Abl activity. This led to reduced phosphorylation of downstream c-Abl substrates, the transcription coactivator Yes-associated protein (Yap) and the tumor suppressor p73. Inhibition of c-Abl by Lats2 was mediated through Lats2 interaction with and phosphorylation of c-Abl. Lats2 knockdown, or expression of c-Abl mutants that escape inhibition by Lats2, enabled DNA damage-induced apoptosis of densely plated cells, while Lats2 overexpression inhibited apoptosis in sparse cells. These findings explain a long-standing enigma of why densely plated cells are radioresistant. Furthermore, they demonstrate that the Hippo pathway regulates cell fate decisions in response to DNA damage.

Proteasomes are large intracellular complexes responsible for the degradation of cellular proteins. The altered protein homeostasis of cancer cells results in increased dependency on proteasome function. The cellular proteasome composition comprises the 20S catalytic complex that is frequently capped with the 19S regulatory particle in forming the 26S proteasome. Proteasome inhibitors target the catalytic barrel (20S) and thus this inhibition does not allow the deconvolution of the distinct roles of 20S versus 26S proteasomes in cancer progression. We examined the degree of dependency of cancer cells specifically to the level of the 26S proteasome complex. Oncogenic transformation of human and mouse immortalized cells with mutant Ras induced a strong posttranscriptional increase of the 26S proteasome subunits, giving rise to high 26S complex levels. Depletion of a single subunit of the 19S RP was sufficient to reduce the 26S proteasome level and lower the cellular 26S/20S ratio. Under this condition the viability of the Ras-transformed MCF10A cells was severely compromised. This observation led us to hypothesize that cancer cell survival is dependent on maximal utilization of its 26S proteasomes. We validated this possibility in a large number of cancer cell lines and found that partial reduction of the 26S proteasome level impairs viability in all cancer cells examined and was not correlated with cell doubling time or reduction efficiency. Interstingly, normal human fibroblasts are refractory to the same type of 26S proteasome reduction. The suppression of 26S proteasomes in cancer cells activated the UPR and caspase-3 and cells stained positive with Annexin V. In addition, suppression of the 26S proteasome resulted in cellular proteasome redistribution, cytoplasm shrinkage, and nuclear deformation, the hallmarks of apoptosis. The observed tumor cell-specific addiction to the 26S proteasome levels sets the stage for future strategies in exploiting this dependency in cancer therapy.

The Escherichia coli multidrug transporter MdfA contains a single membrane-embedded charged residue (Glu-26) that plays a critical role in the recognition of cationic substrates (Edgar, R., and Bibi, E. (1999) EMBO J. 18, 822-832). Using an inactive mutant (MdfA-E26T), we isolated a spontaneous second-site mutation (MdfA-E26T/V335E) that re-established the recognition of cationic drugs by the transporter. Only a negative charge at position 335 was able to restore the functioning of the inactive mutant MdfA-E26T. Intriguingly, the two genetically interacting residues are located at remote and distinct regions along the secondary structure of MdfA. Glu-26 is located in the periplasmic half of transmembrane helix 1, and as shown here, the complementing charge at position 335 resides within the cytoplasmic loop connecting transmembrane helices 10 and 11. The spatial relation between the two residues was investigated by cross-linking. A functional split version of MdfA devoid of cysteines was constructed and introduced with a cysteine pair at positions 26 and 335. Strikingly, the results indicate that residues 26 and 335 are spatially adjacent, suggesting that they both constitute parts of the multidrug recognition pocket of MdfA. The fact that electrostatic interactions are preserved with cationic substrates even if the critical acidic residue is placed on another face of the pocket reveals an additional dimension of promiscuity in multidrug recognition and transport.