Secret life of importin-β; solenoid flexibility as the key to transport through the nuclear pore

open2siThe current issue of Acta Crystallographica Section D features ‘Impact of the crystal- lization condition on importin- conformation’ by Tauchert et al. (2016), a significant advance in the area of nuclear transport that also has important implications for understanding the limitations of crystallization approaches. Active transport of macro- molecules into and out of the eukaryotic cell nucleus occurs through the nuclear envelope (NE)-embedded multiprotein subunit nuclear pore complexes (NPCs). The transport of most cargoes is dependent on solenoid proteins belonging to the Karyopherin family, of which importin (Imp) 1 is the prototype. Imp 1 is capable of recognizing specific nuclear import cargoes and transporting them across the NPC by interacting with the hydrophobic meshwork constituting the NPC core, formed by phenyl–alanine–glycine (FG) rich nucleoporins (nups). Cargo recognition can occur directly, or indirectly through adaptor proteins such as Imp or snurportin, and in either case complexes are dissociated upon binding of Ran-GTP to Imp 1 on the nucleoplasmic side of the NPC. Since the discovery of Imp 1 (Go ̈rlich et al., 1995), structural studies have helped elucidate many aspects of the molecular details of cargo and adaptor binding/release (Christie et al., 2016). Unanswered questions, however, include how Imp s achieve cargo transport across the NPC through interaction with nups (Liu & Stewart, 2005; Bayliss et al., 2000); Tauchert et al. provide an important, new slant on this question. Imp 1 binds to a plethora of different proteins, including cargoes, adaptors, RanGTP and nups. Previous studies have shown Imp 1 to be a solenoid formed by 19 HEAT repeats, each of which comprises two antiparallel helices connected by a turn (Cingolani et al., 1999); HEAT repeats are connected by short linkers and arranged in a superhelix, with very few long distance intraprotein interactions, enabling Imp 1 to undergo extensive changes in tertiary structure (overall protein shape), without alteration to secondary structure (HEAT repeats). Consistent with this idea, the comparison of Imp crystal structures to date reveals a wide range of conformations varying from very compact, heart-like structures (the ‘apo’ or nup-bound form) to more relaxed ones (e.g. bound to RanGTP). This observed structural variability has been postulated to be the direct effect of the binding of different partners to Imp 1 causing/inducing changes in Imp 1 folding, but X-ray scattering (SAXS) data shows that Imp 1 alone is more relaxed in solution than in crystal lattices (Fukuhara et al., 2004). Further, molecular dynamics (MD) simulations suggest that the apo form of Imp 1 undergoes remarkable conformational changes in solution, adopting a more extended S-shaped conformation that is quite distinct to that observed in its crystalline form (Zachariae & Grubmu ̈ller, 2008; Forwood et al., 2010). Significantly, two drastically different conformations of Imp 1 bound to the snurportin Imp 1 binding domain (IBB) were recently observed in the same crystallographic asymmetric unit (Bhardwaj & Cingolani, 2010); this both highlights Imp 1 flexibility, and importantly underlines the fact that structures obtained in crystals may not reflect the wide range of Imp 1 conformations in solution. Forwood et al. (2010) used crystal- lography/SAXS/MD to show that Imp 1 assumes various different conformations in solution, postulated to result from cumulative incremental structural changes along the entire length of the solenoid, and speculated to be integral to Imp 1’s ability to traverse the highly hydrophobic channel of the NPC. Indeed, MD simulations reveal that Imp 1 in water is extremely different from that in 50% 2,2,2-trifluoroethanol, where Imp 1 rapidly becomes more compact (Yoshimura et al., 2014); similar results have been electronic reprint http://dx.doi.org/10.1107/S2059798316008263 703 # 2016 International Union of Crystallography Acta Cryst. (2016). D72, 703–704 scientific commentaries Figure 1 either PEG or (NH4)2SO4 indicates that these properties do not apply, since Imp , although structurally related to Imp 1, is less flexible. The polar/apolar regions of PEG would appear to mimic nup FG repeats within the NPC, suggesting that Imp 1 traverses the NPC in an extended conformation, in contrast to what has been proposed previously (Halder et al., 2015; Yoshi- mura et al., 2014). The only crystal structures obtained so far between Imp 1 and NPC components used short FG-rich nup fragments, and thus do not shed light on the state of Imp 1–nups interaction within the core of the NPC (Liu & Stewart, 2005; Bayliss et al., 2000). Importantly, apart from giving an important new insight into this aspect of nuclear transport, Tauchert et al.’s study Effect of solvent on C. Thermophilum Imp 1 structure. The structures of Imp 1 obtained after PEG precipitation (red) or (NH4)2SO4 (blue) precipitation are superimposed. The structure shown clearly underlines the need for more on the right is rotated 180 C with respect to that on the left. obtained with both IBB-complexed and free Imp 1 in water compared to in methanol (Halder et al., 2015). This ability to undergo conformational changes appears to be the key to Imp 1-mediated transport across the NPC, since crosslinking to impair this flexibility impedes nuclear translocation (Yoshimura et al., 2014). Tauchert et al. extend these findings, proffering an inter- esting alternative point of view regarding the forces deter- mining different conformations of Imp 1 in crystalline form according to the hydrophobicity of the milieu. Tauchert et al. solve the structure of Imp 1 from the thermophilic fungus Chaetomium thermophilum in two physicochemically different conditions, taking advantage of the serendipitous S107P/ V134A mutant which crystallized in the presence of the hydrophilic inorganic salt (NH4)2SO4, adopting a much more compact structure than that of its wild-type counterpart crystallized in the presence of PEG (Fig. 1). These findings are confirmed in solution using SAXS, the important overall implication being that solvent hydrophobicity strongly affects Imp 1 conformation, and hence can be of key importance in the dominant conformation crystallized. Further, analyzing previous Imp 1 crystal structures, the authors find a strong correlation between the extent to which Imp 1 takes an extended conformation, and the amount of PEG in the buffer. Importantly, analysis of Imp crystal structure pairs solved in extensive Imp 1–nup complex struc- tures, with the proviso that the buffer systems used have to be considered critically (with a grain of salt perhaps?), and ideally should also be analysed in detail using complementary approaches such as SAXS.openAlvisi, Gualtiero; Jans, David AAlvisi, Gualtiero; Jans, David A

Synergy of importin α recognition and DNA binding by the yeast transcriptional activator GAL4

AbstractThe N-terminus of the yeast transcriptional activator GAL4 contains partially overlapping nuclear targeting and DNA binding functions. We have previously shown that GAL4 is recognised with high affinity by importin β and not by the conventional nuclear localisation sequence binding importin α subunit of the importin α/β heterodimer. The present study uses ELISA-based binding and electrophoretic mobility shift assays to show that recognition of GAL4 by importin α can occur, but only when GAL4 is bound to its specific DNA recognition sequence. Intriguingly, binding by importin α enhances DNA binding on the part of GAL4, implying a synergistic co-operation between these two functions. The results implicate a possible role for importin α in the nucleus additional to its established role in nuclear transport, as well as having implications for the use of GAL4 as a DNA carrier in gene therapy applications

Impaired nuclear import and viral incorporation of Vpr derived from a HIV long-term non-progressor

We previously reported an epidemiologically linked HIV-1 infected patient cohort in which a long-term non-progressor (LTNP) infected two recipients who then exhibited normal disease progression. Expression of patient-derived vpr sequences from each of the three cohort members in mammalian cells tagged with GFP revealed a significant reduction in Vpr nuclear import and virion incorporation uniquely from the LTNP, whereas Vpr from the two progressing recipients displayed normal localisation and virion incorporation, implying a link between efficient Vpr nuclear import and HIV disease progression. Importantly, an F72L point mutation in the LTNP was identified for the first time as being uniquely responsible for decreased Vpr nuclear import

Potential mechanisms underlying the decision to use a seat belt: a literature review

The purpose of this literature review was to serve as the background knowledgebase for a 5-year cooperative agreement between NHTSA and the University of Michigan. This discretionary cooperative agreement is intended to study promising lines of research that elucidate the mechanisms that underlie risk perception and can be applied to converting part-time belt users to full-time users. The overall goal of this cooperative agreement is to develop testable strategies, based on basic and applied research, for influencing risk perception to move motor vehicle occupants from part-time to full-time use of seat belts. Specific topics covered in this literature review are: individual belt user characteristics; social influences on belt use; applications from research on other risky behaviors; policy/enforcement/incentive; communication and education; and technology. Conclusions are drawn within each section and for the review overall.National Highway Traffic Safety Administrationhttp://deepblue.lib.umich.edu/bitstream/2027.42/110521/1/103147.pdfDescription of 103147.pdf : final repor

Dengue Non-structural Protein 5 Polymerase Complexes With Promyelocytic Leukemia Protein (PML) Isoforms III and IV to Disrupt PML-Nuclear Bodies in Infected Cells

Dengue virus (DENV) threatens almost 70% of the world's population, with no therapeutic currently available. The severe, potentially lethal forms of DENV disease (dengue hemorrhagic fever/dengue shock syndrome) are associated with the production of high level of cytokines, elicited as part of the host antiviral response, although the molecular mechanisms have not been fully elucidated. We previously showed that infection by DENV serotype 2 (DENV2) disrupts promyelocytic leukemia (PML) gene product nuclear bodies (PML-NBs) after viral protein translation in infected cells. Apart from playing a key role as the nucleating agent in forming PML-NBs, PML has antiviral activity against various viruses, including DENV. The present study builds on this work, showing for the first time that all four DENV serotypes elicit PML-NB breakdown. Importantly, we show for the first time that of the nuclear localizing proteins of DENV, DENV non-structural protein (NS) 5 polymerase alone is sufficient to elicit PML-NB disassembly, in part through complexing with PML isoforms III and IV, but not other PML isoforms or other PML-NB components. The results raise the possibility that PML-NB disruption by nuclear localized NS5 contributes to DENV's suppression of the host antiviral response.Fil: Giovannoni, Federico. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ladelfa, Maria Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Monte, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Jans, David A.. Monash University; AustraliaFil: Hemmerich, Peter. Leibniz Institute On Aging; AlemaniaFil: Garcia, Cybele. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Is a motivational interviewing based lifestyle intervention for obese pregnant women across Europe implemented as planned? Process evaluation of the DALI study

Background: Process evaluation is an essential part of designing and assessing complex interventions. The vitamin D and lifestyle intervention study (DALI) study is testing different strategies to prevent development of gestational diabetes mellitus among European obese pregnant women with a body mass index ≥29kg/m2. The intervention includes guidance on physical activity and/or healthy eating by a lifestyle coach trained in motivational interviewing (MI). The aim of this study was to assess the process elements: reach, dose delivered, fidelity and satisfaction and to investigate whether these process elements were associated with changes in gestational weight gain (GWG). Methods: Data on reach, dose delivered, fidelity, and satisfaction among 144 participants were collected. Weekly recruitment reports, notes from meetings, coach logs and evaluation questionnaires (n=110) were consulted. Fidelity of eight (out of twelve) lifestyle coach practitioners was assessed by analysing audio recorded counselling sessions using the MI treatment integrity scale. Furthermore, associations between process elements and GWG were assessed with linear regression analyses. Results: A total of 20% of the possible study population (reach) was included in this analysis. On average 4.0 (of the intended 5) face-to-face sessions were delivered. Mean MI fidelity almost reached \u27expert opinion\u27 threshold for the global scores, but was below \u27beginning proficiency\u27 for the behavioural counts. High variability in quality of MI between practitioners was identified. Participants were highly satisfied with the intervention, the lifestyle coach and the intervention materials. No significant associations were found between process elements and GWG. Conclusion: Overall, the intervention was well delivered and received by the study population, but did not comply with all the principles of MI. Ensuring audio recording of lifestyle sessions throughout the study would facilitate provision of individualized feedback to improve MI skills. A larger sample size is needed to confirm the lack of association between process elements and GWG. Trial registration: ISRCTN registry: ISRCTN70595832 ; Registered 12 December 2011

A Tri-National program for estimating the link between snow resources and hydrological droughts

To evaluate how summer low flows and droughts are affected by the winter snowpack, a Tri-National effort will analyse data from three catchments: Alpbach (Prealps, central Switzerland), Gudjaretis-Tskali (Little Caucasus, central Georgia), and Kamenice (Jizera Mountains, northern Czech Republic). Two GIS-based rainfall-runoff models will simulate over 10 years of runoff in streams based on rain and snowfall measurements, and further meteorological variables. The models use information on the geographical settings of the catchments together with knowledge of the hydrological processes of runoff generation from rainfall, looking particularly at the relationship between spring snowmelt and summer droughts. These processes include snow accumulation and melt, evapotranspiration, groundwater recharge in spring that contributes to (the) summer runoff, and will be studied by means of the environmental isotopes 18O and 2H. Knowledge about the isotopic composition of the different water sources will allow to identify the flow paths and estimate the residence time of snow meltwater in the subsurface and its contribution to the stream. The application of the models in different nested or neighbouring catchments will explore their potential for further development and allow a better early prediction of low-flow periods in various mountainous zones across Europe. The paper presents the planned activities including a first analysis of already available dataset of environmental isotopes, discharge, snow water equivalent and modelling experiments of the (already) available datasets

Cost-effectiveness of healthy eating and/or physical activity promotion in pregnant women at increased risk of gestational diabetes mellitus: Economic evaluation alongside the DALI study, a European multicenter randomized controlled trial

Background: Gestational diabetes mellitus (GDM) is associated with perinatal health risks to both mother and offspring, and represents a large economic burden. The DALI study is a multicenter randomized controlled trial, undertaken to add to the knowledge base on the effectiveness of interventions for pregnant women at increased risk for GDM. The purpose of this study was to evaluate the cost-effectiveness of the healthy eating and/or physical activity promotion intervention compared to usual care among pregnant women at increased risk of GDM from a societal perspective. Methods: An economic evaluation was performed alongside a European multicenter-randomized controlled trial. A total of 435 pregnant women at increased risk of GDM in primary and secondary care settings in nine European countries, were recruited and randomly allocated to a healthy eating and physical activity promotion intervention (HE + PA intervention), a healthy eating promotion intervention (HE intervention), or a physical activity promotion intervention (PA intervention). Main outcome measures were gestational weight gain, fasting glucose, insulin resistance (HOMA-IR), quality adjusted life years (QALYs), and societal costs. Results: Between-group total cost and effect differences were not significant, besides significantly less gestational weight gain in the HE + PA group compared with the usual care group at 35-37 weeks ( 2.3;95%CI:-3.7;-0.9). Cost-effectiveness acceptability curves indicated that the HE + PA intervention was the preferred intervention strategy. At 35-37 weeks, it depends on the decision-makers\u27 willingness to pay per kilogram reduction in gestational weight gain whether the HE + PA intervention is cost-effective for gestational weight gain, whereas it was not cost-effective for fasting glucose and HOMA-IR. After delivery, the HE + PA intervention was cost-effective for QALYs, which was predominantly caused by a large reduction in delivery-related costs. Conclusions: Healthy eating and physical activity promotion was found to be the preferred strategy for limiting gestational weight gain. As this intervention was cost-effective for QALYs after delivery, this study lends support for broad implementation