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

Ebola Virus Entry: From Molecular Characterization to Drug Discovery

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections, characterized by a high fatality rate, and caused by a member of the Filoviridae family. The recent large outbreak of EVD in Western Africa (2013\u207b2016) highlighted the worldwide threat represented by the disease and its impact on global public health and the economy. The development of highly needed anti-Ebola virus antivirals has been so far hampered by the shortage of tools to study their life cycle in vitro, allowing to screen for potential active compounds outside a biosafety level-4 (BSL-4) containment. Importantly, the development of surrogate models to study Ebola virus entry in a BSL-2 setting, such as viral pseudotypes and Ebola virus-like particles, tremendously boosted both our knowledge of the viral life cycle and the identification of promising antiviral compounds interfering with viral entry. In this context, the combination of such surrogate systems with large-scale small molecule compounds and haploid genetic screenings, as well as rational drug design and drug repurposing approaches will prove priceless in our quest for the development of a treatment for EVD

Induced pluripotent stem cells line (UNIPDi003-A) from a patient affected by EEC syndrome carrying the R279H mutation in TP63 gene.

Abstract Oral mucosa epithelial stem cells from a patient affected by Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome carrying the R279H mutation in the TP63 gene were reprogrammed into human induced pluripotent stem cells (hiPSCs) with episomal vectors. The generated UNIPDi003-A-hPSC line retained the mutation of the parental cells and showed a normal karyotype upon long term culture. Analysis of residual transgenes expression showed that the episomal vectors were eliminated from the cell line. UNIPDi003-A-hiPSCs expressed the undifferentiated state marker alkaline phosphatase along with a panel of pluripotency markers, and formed embryoid bodies capable of expressing markers belonging to all the three germ layers

Generation of a transgene-free induced pluripotent stem cells line (UNIPDi002-A) from oral mucosa epithelial stem cells carrying the R304Q mutation in TP63 gene.

Abstract Transgene free UNIPDi002-A-human induced pluripotent stem cell (hiPSC) line was generated by Sendai Virus Vectors reprogramming from human oral mucosal epithelial stem cells (hOMESCs) of a patient affected by ectrodactyly-ectodermal dysplasia-clefting (EEC)-syndrome, carrying a mutation in exon 8 of the TP63 gene (R304Q). The UNIPDi002-A-hiPSC line retained the mutation of the parental R304Q-hOMESCs and displayed a normal karyotype. No residual expression of transgenes nor Sendai virus vector sequences were detected in the line at passage 8. UNIPDi002-A-hiPSC expressed a panel of pluripotency-associated markers and could form embryoid bodies expressing markers belonging to the three germ layers ectoderm, endoderm and mesoderm

Generation of a transgene-free human induced pluripotent stem cell line (UNIPDi001-A) from oral mucosa epithelial stem cells.

Abstract Human oral mucosa epithelial stem cells (hOMESCs) were obtained from a fresh oral biopsy collected from a healthy subject at the Fondazione Banca degli Occhi del Veneto (FBOV). An integration-free reprogramming protocol was applied exploiting episomal plasmids transfected into cells using a Nucleofector device. Around day 20 post transfection, several human induced pluripotent stem cell (hiPSC) colonies were manually picked and expanded. One of these (UNIPDi001-A-hiPSCs) expressed undifferentiated state marker alkaline phosphatase along with a panel of pluripotency state markers and was able to differentiate into the derivatives of all the three germ layers

New Frontiers of Corneal Gene Therapy

Corneal diseases are among the most prevalent causes of blindness worldwide. The transparency and clarity of the cornea are guaranteed by a delicate physiological, anatomic, and functional balance. For this reason, all the disorders, including those of genetic origin, that compromise this state of harmony can lead to opacity and eventually vision loss. Many corneal disorders have a genetic etiology, and some are associated with rather rare and complex syndromes. Conventional treatments, such as corneal transplantation, are often ineffective, and to date, many of these disorders are still incurable. Gene therapy carries the promise of being a potential cure for many of these diseases, with solutions and strategies that did not seem possible until a few years ago. With its potential to treat genetic disease by means of deletion, replacement, or editing of a defective gene, the challenge can also be extended to corneal disorders in order to achieve long-term, if not definitive, relief. The aim of this paper is to review the state of the art of the different gene therapy approaches as potential treatments for corneal diseases and the future perspectives for the development of personalized gene-based medicine

The central region of the msp gene of Treponema denticola has sequence heterogeneity among clinical samples, obtained from patients with periodontitis

<p>Abstract</p> <p>Background</p> <p><it>Treponema denticola </it>is an oral spirochete involved in the pathogenesis and progression of periodontal disease. Of its virulence factors, the major surface protein (MSP) plays a role in the interaction between the treponeme and host. To understand the possible evolution of this protein, we analyzed the sequence of the <it>msp </it>gene in 17 <it>T. denticola </it>positive clinical samples.</p> <p>Methods</p> <p>Nucleotide and amino acid sequence of MSP have been determined by PCR amplification and sequencing in seventeen <it>T. denticola </it>clinical specimens to evaluate the genetic variability and the philogenetic relationship of the <it>T. denticola msp </it>gene among the different amplified sequence of positive samples. In silico antigenic analysis was performed on each MSP sequences to determined possible antigenic variation.</p> <p>Results</p> <p>The <it>msp </it>sequences showed two highly conserved 5' and 3' ends and a central region that varies substantially. Phylogenetic analysis categorized the 17 specimens into 2 principal groups, suggesting a low rate of evolutionary variability and an elevated degree of conservation of <it>msp </it>in clinically derived genetic material. Analysis of the predicted antigenic variability between isolates, demonstrated that the major differences lay between amino acids 200 and 300.</p> <p>Conclusion</p> <p>These findings showed for the first time, the nucleotide and amino acids variation of the <it>msp </it>gene in infecting <it>T. denticola</it>, <it>in vivo</it>. This data suggested that the antigenic variability found in to the MSP molecule, may be an important factor involved in immune evasion by <it>T. denticola</it>.</p

Reprogramming the host: Modification of cell functions upon viral infection

Viruses and their hosts have co-evolved for million years. In order to successfully replicate their genome, viruses need to usurp the biosynthetic machinery of the host cell. Depending on the complexity and the nature of the genome, replication might involve or not a relatively large subset of viral products, in addition to a number of host cell factors, and take place in several subcellular compartments, including the nucleus, the cytoplasm, as well as virus-induced, rearranged membranes. Therefore viruses need to ensure the correct subcellular localization of their effectors and to be capable of disguising from the cellular defensive mechanisms. In addition, viruses are capable of exploiting host cell activities, by modulating their post-translational modification apparatus, resulting in profound modifications in the function of cellular and viral products. Not surprisingly infection of host cells by these parasites can lead to alterations of cellular differentiation and growing properties, with important pathogenic consequences. In the present hot topic highlight entitled “Reprogramming the host: modification of cell functions upon viral infection”, a number of leading virologists and cell biologist thoroughly describe recent advances in our understanding of how viruses modulate cellular functions to achieve successful replication and propagation at the expenses of human cells

Comment on Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights by Rona et al. (2013)

no abstract availabl

Comment on Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights by Rona et al., (2013)

no abstract availabl