Structural basis for the interaction of protein S1 with the Escherichia coli ribosome

In Gram-negative bacteria, the multi-domain protein S1 is essential for translation initiation, as it recruits the mRNA and facilitates its localization in the decoding centre. In sharp contrast to its functional importance, S1 is still lacking from the high-resolution structures available for Escherichia coli and Thermus thermophilus ribosomes and thus the molecular mechanism governing the S1–ribosome interaction has still remained elusive. Here, we present the structure of the N-terminal S1 domain D1 when bound to the ribosome at atomic resolution by using a combination of NMR, X-ray crystallography and cryo-electron microscopy. Together with biochemical assays, the structure reveals that S1 is anchored to the ribosome primarily via a stabilizing π-stacking interaction within the short but conserved N-terminal segment that is flexibly connected to domain D1. This interaction is further stabilized by salt bridges involving the zinc binding pocket of protein S2. Overall, this work provides one hitherto enigmatic piece in the ′ribosome puzzle′, namely the detailed molecular insight into the topology of the S1–ribosome interface. Moreover, our data suggest novel mechanisms that have the potential to modulate protein synthesis in response to environmental cues by changing the affinity of S1 for the ribosome.     

Semi-automated radioassay for determination of dihydropyrimidine dehydrogenase (DPD) activity screening cancer patients for DPD deficiency, a condition associated with 5-fluorouracil toxicity

Dihydropyrimidine dehydrogenase (DPD) catalyzes the reduction of the naturally occurring pyrimidines, uracil and thymine, and the fluoropyrimidine anticancer drug, 5-fluorouracil (FUra) to 5,6-dihydropyrimidines. Previous studies have demonstrated that cancer patients who are DPD deficient exhibit severe toxicity (including death) following treatment with FUra. To date, the direct measurement of DPD enzyme activity has been the only reliable method to identify DPD deficient cancer patients. We now report a semi-automated radioassay for measuring DPD activity in human peripheral lymphocytes. Following incubation of lymphocyte cytosol (at a fixed protein concentration of 200 μg) with [6-¹⁴C]FUra at timepoints ranging from 0 to 30 min, samples are ethanol precipitated, filtered and analyzed by HPLC. Determination of radioactivity is accomplished using an in-line flow scintillation analyzer with automatic quantitation of peaks. This method provides the first specific assay for DPD enzyme activity which is rapid, reproducible and sensitive enough to be used in the routine screening of cancer patients for DPD deficiency prior to treatment with FUra.     

Differential effects of postconditioning on myocardial stunning and infarction: a study in conscious dogs and anesthetized rabbits

Postconditioning, i.e., brief intermittent episodes of myocardial ischemia-reperfusion performed at the onset of reperfusion, reduces infarct size after prolonged ischemia. Our goal was to determine whether postconditioning is protective against myocardial stunning. Accordingly, conscious chronically instrumented dogs (sonomicrometry, coronary balloon occluder) were subjected to a control sequence (10 min coronary artery occlusion, CAO, followed by coronary artery reperfusion, CAR) and a week apart to postconditioning with four cycles of brief CAR and CAO performed at completion of the 10 min CAO. Three postconditioning protocols were investigated, i.e., 15 s CAR/15 s CAO (n=5), 30 s CAR/30 s CAO (n=7), and 1 min CAR/1 min CAO (n=6). Left ventricular wall thickening was abolished during CAO and similarly reduced during subsequent stunning in control and postconditioning sequences (e.g., at 1 h CAR, 33+/-4 vs. 34+/-4%, 30+/-4 vs. 30+/-4%, and 33+/-4 vs. 32+/-4% for 15 s postconditioning, 30 s postconditioning, and 1 min postconditioning vs. corresponding control, respectively). We confirmed this result in anesthetized rabbits by demonstrating that shortening of left ventricular segment length was similarly depressed after 10 min CAO in control and postconditioning sequences (4 cycles of 30 s CAR/30 s CAO). In additional rabbits, the same postconditioning protocol significantly reduced infarct size after 30 min CAO and 3 h CAR (39+/-7%, n=6 vs. 56+/-4%, n=7 of the area at risk in postconditioning vs. control, respectively). Thus, contrasting to its beneficial effects on myocardial infarction, postconditioning does not protect against myocardial stunning in dogs and rabbits. Conversely, additional episodes of ischemia-reperfusion with postconditioning do not worsen myocardial stunning.     

Sympathetic control after cardiac resynchronization therapy: responders versus nonresponders

Cardiac resynchronization therapy (CRT) decreases muscle sympathetic nerve activity (MSNA) in patients with severe congestive heart failure (CHF) and cardiac asynchrony. Whether this affects equally patients who clinically respond or not to CRT is unknown. We tested the hypothesis that the favorable effects of CRT on MSNA disappear on CRT interruption only in those who respond to CRT. Twenty-three consecutive CHF patients participated in the study, among whom 16 presented a symptomatic improvement by one or more New York Heart Association (NYHA) functional classes 15 +/- 5 mo after CRT (responders), and seven had not improved after 12 +/- 4 mo of CRT (nonresponders). MSNA and echocardiographic recordings were obtained in random order during atrio-right ventricular pacing (ARV), without stimulation in patients who were not pacemaker dependent (OFF, n = 17), and during atrio-biventricular pacing (BIV). Responders had a longer 6-min walking distance, a lower NYHA class and brain natriuretic peptide levels, and a better quality of life than did nonresponders (all P < 0.05). MSNA increased by 25 +/- 7% in the responders, whereas it remained unchanged in the nonresponders, when shifting from the BIV mode to a nonsynchronous condition (ARV and OFF modes) (P < 0.01). Cardiac output decreased by 0.7 +/- 0.2 l/min in the responders but did not change when shifting from the BIV mode to the nonsynchronous pacing mode in the nonresponders (P < 0.01). In conclusion, reversible sympathoinhibition is a marker of the clinical response to CRT.     

Heterogeneous nucleotide occupancy stimulates functionality of phage shock protein F, an AAA+ transcriptional activator

The catalytic AAA+ domain (PspF1-275) of an enhancer-binding protein is necessary and sufficient to contact sigma54-RNA polymerase holoenzyme (Esigma54), remodel it, and in so doing catalyze open promoter complex formation. Whether ATP binding and hydrolysis is coordinated between subunits of PspF and the precise nature of the nucleotide(s) bound to the oligomeric forms responsible for substrate remodeling are unknown. We demonstrate that ADP stimulates the intrinsic ATPase activity of PspF1-275 and propose that this heterogeneous nucleotide occupancy in a PspF1-275 hexamer is functionally important for specific activity. Binding of ADP and ATP triggers the formation of functional PspF1-275 hexamers as shown by a gain of specific activity. Furthermore, ATP concentrations congruent with stoichiometric ATP binding to PspF1-275 inhibit ATP hydrolysis and Esigma54-promoter open complex formation. Demonstration of a heterogeneous nucleotide-bound state of a functional PspF1-275.Esigma54 complex provides clear biochemical evidence for heterogeneous nucleotide occupancy in this AAA+ protein. Based on our data, we propose a stochastic nucleotide binding and a coordinated hydrolysis mechanism in PspF1-275 hexamers.     

Phenotypic and genetic consequences of size selection at the larval stage in the Pacific oyster (Crassostrea gigas)

The life histories of oysters in the genus Crassostrea, like those of most marine bivalves, are typified by high fecundity and low survival in nature. Rearing conditions in hatcheries however ensure optimized density, diet, and temperature. Hatcheries are becoming increasingly important for the production of juveniles in aquaculture, and their culture practices often include culling of slow growing larvae to reduce and synchronize the time taken to reach settlement. Because previous studies have found substantial genetic variation for early life developmental traits in Crassostrea gigas, these culling practices are likely to cause highly different selective pressures in hatcheries from those in the natural environment. We studied the phenotypic and genetic impact of such culling practices in a factorial cross between 10 males and 3 females subjected to progressive culling of the smallest 50% of larvae, compared with a non-culled control. Measurements were made on larval growth, survival, time taken to attain pediveliger stage and settlement success. Culling had a larger effect on the variance of these larval traits than on their means. The larvae in culled cultures were approximately 10% larger than those in controls, whereas the coefficient of variation was reduced by 30-40%. Culling also reduced the mean time to settlement by 12% and its variance by 55%. Using a multiplexed set of microsatellite markers to trace parentage, we also estimated the variance in reproductive success in a controlled experiment to quantify the consequences of intensive hatchery rearing practices. We also focused on changes in effective population size and genetic structure over time (and developmental stages). Our results show a loss of genetic diversity following removal of the smallest larvae by culling, as well as temporally varying genetic structure of the larval population. This supports the existence of genetic variability in early life developmental traits in C. gigas. Culling in hatcheries, like size-related selective pressures in the wild, are likely to have a significant genetic impact, through their effects on the timing of settlement.     

Complex minisatellite rearrangements generated in the total or partial absence of Rad27/hFEN1 activity occur in a single generation and are Rad51 and Rad52 dependent

Genomes contain tandem repeat blocks that are at risk of expansion or contraction. The mechanisms of destabilization of the human minisatellite CEB1 (arrays of 36- to 43-bp repeats) were investigated in a previously developed model system, in which CEB1-0.6 (14 repeats) and CEB1-1.8 (42 repeats) alleles were inserted into the genome of Saccharomyces cerevisiae. As in human cells, CEB1 is stable in mitotically growing yeast cells but is frequently rearranged in the absence of the Rad27/hFEN1 protein involved in Okazaki fragments maturation. To gain insight into this mode of destabilization, the CEB1-1.8 and CEB1-0.6 human alleles and 47 rearrangements derived from a CEB1-1.8 progenitor in rad27Delta cells were sequenced. A high degree of polymorphism of CEB1 internal repeats was observed, attesting to a large variety of homology-driven rearrangements. Simple deletion, double deletion, and highly complex events were observed. Pedigree analysis showed that all rearrangements, even the most complex, occurred in a single generation and were inherited equally by mother and daughter cells. Finally, the rearrangement frequency was found to increase with array size, and partial complementation of the rad27Delta mutation by hFEN1 demonstrated that the production of novel CEB1 alleles is Rad52 and Rad51 dependent. Instability can be explained by an accumulation of unresolved flap structures during replication, leading to the formation of recombinogenic lesions and faulty repair, best understood by homology-dependent synthesis-strand displacement and annealing.     

Tracking the opioid receptors on the way of desensitization

Opioid receptors belong to the super family of G-protein coupled receptors (GPCRs) and are the targets of numerous opioid analgesic drugs. Prolonged use of these drugs results in a reduction of their effectiveness in pain relief also called tolerance, a phenomenon well known by physicians. Opioid receptor desensitization is thought to play a major role in tolerance and a lot of work has been dedicated to elucidate the molecular basis of desensitization. As described for most of GPCRs, opioid receptor desensitization involves their phosphorylation by kinases and their uncoupling from G-proteins realized by arrestins. More recently, opioid receptor trafficking was shown to contribute to desensitization. In this review, our knowledge on the molecular mechanisms of desensitization and recent progress on the role of opioid receptor internalization, recycling or degradation in desensitization will be reported. A better understanding of these regulatory mechanisms would be helpful to develop new analgesic drugs or new strategies for pain treatment by limiting opioid receptor desensitization and tolerance.     

Importance of the adaptor (membrane fusion) protein hairpin domain for the functionality of multidrug efflux pumps

Drug efflux pumps of Gram-negative bacteria are tripartite export machineries located in the bacterial envelopes contributing to multidrug resistance. Protein structures of all three components have been determined, but the exact interaction sites are still unknown. We could confirm that the hybrid system composed of Pseudomonas aeruginosa channel tunnel OprM and the Escherichia coli inner membrane complex, formed by adaptor protein (membrane fusion protein) AcrA and transporter AcrB of the resistance nodulation cell division (RND) family, is not functional. However, cross-linking experiments show that the hybrid exporter assembles. Exchange of the hairpin domain of AcrA with the corresponding hairpin from adaptor protein MexA of P. aeruginosa restored the functionality. This shows the importance of the MexA hairpin domain for the functional interaction with the OprM channel tunnel. On the basis of these results, we have modeled the interaction of the hairpin domain and the channel tunnel on a molecular level for AcrA and TolC as well as MexA and OprM, respectively. The model of two hairpin docking sites per TolC protomer corresponding with hexameric adaptor proteins was confirmed by disulfide cross-linking experiments. The role of this interaction for functional efflux pumps is discussed.     

What Triggers Reproductive Life? Effects of Adolescent Cohabitation,Social Novelty and Aggression in a Monogamous Mouse

Cohabitation during childhood has been described as a powerful inhibitor of later sexual interest in animals including humans (the 'Westermarck effect'), serving as a brother–sister incest avoidance mechanism. Mound-building mice Mus spicilegus display a strong social inhibition of reproduction, responsible for the absence of reproduction in over-wintering tumuli. To better understand the mechanisms responsible for triggering reproduction in this monogamous species, we formed 100 experimental couples of juveniles (35 d) and surveyed reproduction for 45 d. As expected, very few couples reproduced, which confirms the role of social familiarity in the inhibition of reproduction. Temporary separation (1 h or 24 h) of the two partners had little effect on reproductive success. However, pairing with a new partner, with or without prior isolation, significantly triggered reproduction. Observations of the first encounter between new partners revealed more agonistic and less affiliative behaviour than in controls (reunion of familiar partners). Interestingly, when the new partner was a sibling of the previous one, the behavioural analysis revealed an intermediate level of aggression, indicating that kinship with the previous partner was perceived and had consequences on social behaviour. Mice could therefore choose a new partner based on its relatedness to the previous mate. Mutual tolerance between new partners during the dyadic encounter was negatively correlated with subsequent reproduction. These results demonstrate the paramount role of social novelty in triggering reproduction in this monogamous mouse, and suggest a link between agonistic behaviour and sexual motivation. In the field, mound-building mice may need to engage in agonistic interactions so as to overcome the long-lasting social inhibition of reproduction in overwintering mounds.