Efficiency, selectivity and robustness of the nuclear pore complex transport

All materials enter or exit the cell nucleus through nuclear pore complexes (NPCs), efficient transport devices that combine high selectivity and throughput. A central feature of this transport is the binding of cargo-carrying soluble transport factors to flexible, unstructured proteinaceous filaments called FG-nups that line the NPC. We have modeled the dynamics of transport factors and their interaction with the flexible FG-nups as diffusion in an effective potential, using both analytical theory and computer simulations. We show that specific binding of transport factors to the FG-nups facilitates transport and provides the mechanism of selectivity. We show that the high selectivity of transport can be accounted for by competition for both binding sites and space inside the NPC, which selects for transport factors over other macromolecules that interact only non-specifically with the NPC. We also show that transport is relatively insensitive to changes in the number and distribution of FG-nups in the NPC, due mainly to their flexibility; this accounts for recent experiments where up to half of the total mass of the NPC has been deleted, without abolishing the transport. Notably, we demonstrate that previously established physical and structural properties of the NPC can account for observed features of nucleocytoplasmic transport. Finally, our results suggest strategies for creation of artificial nano-molecular sorting devices.Comment: 38 pages, six figure

Discovery and characterisation of a novel toxin from Dendroaspis angusticeps, named Tx7335, that activates the potassium channel KcsA

Due to their central role in essential physiological processes, potassium channels are common targets for animal toxins. These toxins in turn are of great value as tools for studying channel function and as lead compounds for drug development. Here, we used a direct toxin pull-down assay with immobilised KcsA potassium channel to isolate a novel KcsA-binding toxin (called Tx7335) from eastern green mamba snake (Dendroaspis angusticeps) venom. Sequencing of the toxin by Edman degradation and mass spectrometry revealed a 63 amino acid residue peptide with 4 disulphide bonds that belongs to the three-finger toxin family, but with a unique modification of its disulphide-bridge scaffold. The toxin induces a dose-dependent increase in both open probabilities and mean open times on KcsA in artificial bilayers. Thus, it unexpectedly behaves as a channel activator rather than an inhibitor. A charybdotoxinsensitive mutant of KcsA exhibits similar susceptibility to Tx7335 as wild-type, indicating that the binding site for Tx7335 is distinct from that of canonical pore-blocker toxins. Based on the extracellular location of the toxin binding site (far away from the intracellular pH gate), we propose that Tx7335 increases potassium flow through KcsA by allosterically reducing inactivation of the channel

Preferential Phosphorylation of R-domain Serine 768 Dampens Activation of CFTR Channels by PKA

CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (Po) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted Po-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals

The replication initiator of the cholera pathogen's second chromosome shows structural similarity to plasmid initiators

Abstract The conserved DnaA-oriC system is used to initiate replication of primary chromosomes throughout the bacterial kingdom; however, bacteria with multipartite genomes evolved distinct systems to initiate replication of secondary chromosomes. In the cholera pathogen, Vibrio cholerae, and in related species, secondary chromosome replication requires the RctB initiator protein. Here, we show that RctB consists of four domains. The structure of its central two domains resembles that of several plasmid replication initiators. RctB contains at least three DNA binding winged-helix-turn-helix motifs, and mutations within any of these severely compromise biological activity. In the structure, RctB adopts a head-to-head dimeric configuration that likely reflects the arrangement in solution. Therefore, major structural reorganization likely accompanies complex formation on the head-to-tail array of binding sites in oriCII. Our findings support the hypothesis that the second Vibrionaceae chromosome arose from an ancestral plasmid, and that RctB may have evolved additional regulatory features

Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region

Acknowledgements We thank all members of the Di Virgilio lab for their feedback and discussion; V Delgado-Benito (Di Virgilio lab, MDC, Berlin) for her contribution to the project development; L Keller (Di Virgilio lab, MDC, Berlin) for support with cloning, mutagenesis, and mice genotyping; C Brischetto (Scheidereit Lab, MDC, Berlin) for assistance with confocal microscopy; Aberdeen Proteomics facility (University of Aberdeen) for the mass spec analysis of Aph-induced hRIF1 phosphorylation; and the MDC FACS Core Facility and Dr. HP Rahn for support with cell sorting. Aliquots of ATRi and ATMi were gener- ously provided by AG Henssen (MDC and ECRC, Berlin). Figures 1B and D, 2A, and 4C contain items created with BioRender.com. This work was supported by ERC grant 638897 (to MDV), the Helmholtz- Gemeinschaft Zukunftsthema 'Immunology and Inflammation' ZT-0027 (to MDV), P41 GM109824 and P41 GM103314 (to BTC), and Cancer Research UK awards C1445/A19059 and DRCPGM\100,013 (to ADD and SH).Peer reviewedPublisher PD

Excess of health care use in general practice and of comorbid chronic conditions in cancer patients compared to controls

<p>Abstract</p> <p>Background</p> <p>The number of cancer patients and the number of patients surviving initial treatments is expected to rise. Traditionally, follow-up monitoring takes place in secondary care. The contribution of general practice is less visible and not clearly defined.</p> <p>This study aimed to compare healthcare use in general practice of patients with cancer during the follow-up phase compared with patients without cancer. We also examined the influence of comorbid conditions on healthcare utilisation by these patients in general practice.</p> <p>Methods</p> <p>We compared health care use of N=8,703 cancer patients with an age and gender-matched control group of patients without cancer from the same practice. Data originate from the Netherlands Information Network of General Practice (LINH), a representative network consisting of 92 general practices with 350,000 enlisted patients. Health care utilisation was assessed using data on contacts with general practice, prescription and referral rates recorded between 1/1/2001 and 31/12/2007. The existence of additional comorbid chronic conditions (ICPC coded) was taken into account.</p> <p>Results</p> <p>Compared to matched controls, cancer patients had more contacts with their GP-practice (19.5 vs. 11.9, p<.01), more consultations with the GP (3.5 vs. 2.7, p<.01), more home visits (1.6 vs. 0.4, p<.01) and they got more medicines prescribed (18.7 vs. 11.6, p<.01) during the follow-up phase. Cancer patients more often had a chronic condition than their matched controls (52% vs. 44%, p<.01). Having a chronic condition increased health care use for both patients with and without cancer. Cancer patients with a comorbid condition had the highest health care use.</p> <p>Conclusion</p> <p>We found that cancer patients in the follow-up phase consulted general practice more often and suffered more often from comorbid chronic conditions, compared to patients without cancer. It is expected that the number of cancer patients will rise in the years to come and that primary health care professionals will be more involved in follow-up care. Care for comorbid chronic conditions, communication between specialists and GPs, and coordination of tasks then need special attention.</p

First dose behavioral tolerance to phencyclidine on food-rewarded bar pressing behavior in the rat

The behavioral effects of single doses of phencyclidine (PCP) were examined in drug-naive adult male Holtzman rats trained to press a bar on a fixed ratio (4) schedule (FR 4 ), i.e., a reward of sugarsweetened milk was earned on every fourth bar press. Groups of rats (four to eight rats per group) received specific doses of PCP which were held constant for each group throughout the study. Dose-response curves for PCP given in doses of 1.0, 1.8, 2.4, and 3.2 mg/kg IP were first determined and then redetermined at weekly intervals. A drug-free interval of 7–8 days was maintained between injections given weekly over a period of 4 weeks. The final dose of PCP was administered after a 4-week drug-free period. Evidence was obtained for first dose behavioral tolerance as shown by the significantly shortened duration of suppression of bar pressing on subsequent injections. Although subsequent weekly effects of equal doses of PCP showed no significant differences, they all differed significantly from the first injection. The reduced response to PCP was shown to be due to learned behavioral tolerance as demonstrated when PCP (3.2 mg/kg IP) was given to drug-naive animals in their home cages and 1 week later given the second dose in the operant behavioral situation. Under these circumstances, the second dose of PCP showed a similarly protracted depression of FR 4 responding as other animals given the drug for the first time in the operant situation. Subsequent weekly injections in the operant situation produced similar behavioral tolerance.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46412/1/213_2004_Article_BF00426513.pd

Enhancement of Transport Selectivity through Nano-Channels by Non-Specific Competition

The functioning of living cells requires efficient and selective transport of materials into and out of the cell, and between different cellular compartments. Much of this transport occurs through nano-scale channels that do not require large scale molecular re-arrangements (such as transition from a ‘closed’ to an ‘open’ state) and do not require a direct input of metabolic energy during transport. Nevertheless, these ‘always open’ channels are highly selective and pass only their cognate molecules, while efficiently excluding all others; indeed, these channels can efficiently transport specific molecules even in the presence of a vast excess of non-specific molecules. Such biological transporters have inspired the creation of artificial nano-channels. These channels can be used as nano-molecular sorters, and can also serve as testbeds for examining modes of biological transport. In this paper, we propose a simple kinetic mechanism that explains how the selectivity of such ‘always open’ channels can be based on the exclusion of non-specific molecules by specific ones, due to the competition for limited space inside the channel. The predictions of the theory account for the behavior of the nuclear pore complex and of artificial nanopores that mimic its function. This theory provides the basis for future work aimed at understanding the selectivity of various biological transport phenomena