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

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

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

A clathrin/dynamin- and mannose-6-phosphate receptor–independent pathway for granzyme B–induced cell death

The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B–mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin–overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR-null L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis

Respiratory Syncytial Virus Matrix Protein-Chromatin Association Is Key to Transcriptional Inhibition in Infected Cells

The morbidity and mortality caused by the globally prevalent human respiratory pathogen respiratory syncytial virus (RSV) approaches that world-wide of influenza. We previously demonstrated that the RSV matrix (M) protein shuttles, in signal-dependent fashion, between host cell nucleus and cytoplasm, and that this trafficking is central to RSV replication and assembly. Here we analyze in detail the nuclear role of M for the first time using a range of novel approaches, including quantitative analysis of de novo cell transcription in situ in the presence or absence of RSV infection or M ectopic expression, as well as in situ DNA binding. We show that M, dependent on amino acids 110–183, inhibits host cell transcription in RSV-infected cells as well as cells transfected to express M, with a clear correlation between nuclear levels of M and the degree of transcriptional inhibition. Analysis of bacterially expressed M protein and derivatives thereof mutated in key residues within M’s RNA binding domain indicates that M can bind to DNA as well as RNA in a cell-free system. Parallel results for point-mutated M derivatives implicate arginine 170 and lysine 172, in contrast to other basic residues such as lysine 121 and 130, as critically important residues for inhibition of transcription and DNA binding both in situ and in vitro. Importantly, recombinant RSV carrying arginine 170/lysine 172 mutations shows attenuated infectivity in cultured cells and in an animal model, concomitant with altered inflammatory responses. These findings define an RSV M-chromatin interface critical for host transcriptional inhibition in infection, with important implications for anti-RSV therapeutic development

HIV-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splicing and virus production in macrophages

BACKGROUND: Macrophages are important targets and long-lived reservoirs of HIV-1, which are not cleared of infection by currently available treatments. In the primary monocyte-derived macrophage model of infection, replication is initially productive followed by a decline in virion output over ensuing weeks, coincident with a decrease in the levels of the essential viral transactivator protein Tat. We investigated two possible mechanisms in macrophages for regulation of viral replication, which appears to be primarily regulated at the level of tat mRNA: 1) differential mRNA stability, used by cells and some viruses for the rapid regulation of gene expression and 2) control of HIV-1 alternative splicing, which is essential for optimal viral replication. RESULTS: Following termination of transcription at increasing times after infection in macrophages, we found that tat mRNA did indeed decay more rapidly than rev or nef mRNA, but with similar kinetics throughout infection. In addition, tat mRNA decayed at least as rapidly in peripheral blood lymphocytes. Expression of cellular splicing factors in uninfected and infected macrophage cultures from the same donor showed an inverse pattern over time between enhancing factors (members of the SR family of RNA binding proteins) and inhibitory factors (members of the hnRNP family). While levels of the SR protein SC35 were greatly up-regulated in the first week or two after infection, hnRNPs of the A/B and H groups were down-regulated. Around the peak of virus production in each culture, SC35 expression declined to levels in uninfected cells or lower, while the hnRNPs increased to control levels or above. We also found evidence for increased cytoplasmic expression of SC35 following long-term infection. CONCLUSION: While no evidence of differential regulation of tat mRNA decay was found in macrophages following HIV-1 infection, changes in the balance of cellular splicing factors which regulate alternative viral pre-mRNA splicing were observed. These changes correlated with changes in Tat expression and virus production and could play an important role in viral persistence in macrophages. This mechanism could provide a novel target for control of infection in this critical cell type, which would be necessary for eventual eradication of the virus from infected individuals