153 research outputs found
Structural basis for RanGTP independent entry of spliceosomal U snRNPs into the nucleus
No full textThe nuclear import of assembled spliceosomal subunits, the uridine rich small nuclear ribonucleoprotein particles U snRNPs , is mediated by a nuclear import receptor adaptor couple of importin amp; 946; Imp amp; 946; and snurportin1 SPN1 . In contrast to any other characterized active nuclear import, the Imp amp; 946; SPN1 U snRNP complex does not require RanGTP for the terminal release from the nuclear basket of the nuclear pore complex NPC . The crystal structure of Imp amp; 946; 127 876 in complex with the Imp amp; 946; binding IBB domain of SPN1 1 65 at 2.8 resolution reveals that Imp amp; 946; adopts an open conformation, which is unique for a functional Imp amp; 946; cargo complex, and rather surprisingly, it resembles the conformation of the Imp amp; 946; RanGTP complex. As binding of RanGTP to Imp amp; 946; usually triggers the release of import complexes from the NPC, we propose that by already mimicking a conformation similar to Imp amp; 946; RanGTP the independent dissociation of Imp amp; 946; SPN1 from the nuclear basket is energetically aide
Structure analysis of the conserved methyltransferase domain of human trimethylguanosine synthase TGS1
No full textMethyltransferases play an important role in the post-transcriptional maturation of most ribonucleic acids. The modification of spliceosomal UsnRNAs includes N2-dimethylation of the m(7)G cap catalyzed by trimethylguanosine synthase 1 (TGS1). This 5'-cap hypermethylation occurs during the biogenesis of UsnRNPs as it initiates the m(3)G cap-dependent nuclear import of UsnRNPs. The conserved methyltransferase domain of human TGS1 has been purified, crystallized and the crystal structure of this domain with bound substrate m(7)GpppA was solved by means of multiple-wavelength anomalous dispersion. Crystal structure analysis revealed that m(7)GpppA binds via its adenosine moiety to the structurally conserved adenosylmethionine-binding pocket, while the m(7) guanosine remains unbound. This unexpected binding only occurs in the absence of AdoMet and suggests an incomplete binding pocket for the m(7)G cap which is caused by the N-terminal truncation of the protein. These structural data are consistent with the finding that the crystallized fragment of human TGS1 is catalytically inactive, while a fragment that is 17 amino acids longer exhibits activity
Thermodynamic analysis of H1 nuclear import - Receptor tuning of importin beta/importin7
No full textThe nuclear import of H1 linker histones is mediated by a heterodimer of transport receptors, known as importin beta and importin7. Interestingly, both importins separately interact with H1, but only as a dimer they facilitate the translocation through the nuclear pore. We identified the H1 binding site of importin7, comprising two extended acidic loops near the C terminus of importin7. The analysis of the H1 import complex assembly by means of isothermal titration calorimetry revealed that the formation of a receptor heterodimer in vitro is an enthalpy-driven process, whereas subsequent binding of H1 to the heterodimer is entropy-driven. Furthermore, we show that the importin beta binding domain of importin7 plays a key role in the activation of importin7 by importin beta. This process is allosterically regulated by importin beta and accounts for a specific tuning of the activity of the importin beta center dot importin7 heterodimer. The results presented here provide new insights into cellular strategies to even energy balances in nuclear import and point toward a general regulation of importin beta-related nuclear import processes
Author comment on RC4: 'Review of Strasser et al.: Rofental catchment', Adam Winstral, 02 Oct 2017
No full textThe New Paternalism
No full textThe author argues that the belief that patient autonomy has great
moral value has justified a new form of medical paternalism which can have
effects similar to those of the old rejected form. He cites the argument that
"all illness represents a state of diminished autonomy" and that therefore
autonomy is not overridden when physicians make all decisions. Another view
is that, in some situations, withholding information may prevent patient
deterioration and loss of autonomy. Abridgement of present autonomy, then, is
permissible if it promotes future autonomy. Strasser also rejects physician
decision making based on patients' previously communicated values or on the
theory that patient values are important but not decisive. He concludes that
if we "allow paternalistic practices, then we should admit that we are denying
autonomy in light of some other good rather than claim that, somehow, we are
respecting autonomy by abridging it." (KIE abstract
Structural basis for m(3)G-cap-mediated nuclear import of spliceosomal UsnRNPs by snurportin1
No full textIn higher eukaryotes the biogenesis of spliceosomal UsnRNPs involves a nucleocytoplasmic shuttling cycle. After the m(7) G-cap-dependent export of the snRNAs U1, U2, U4 and U5 to the cytoplasm, each of these snRNAs associates with seven Sm proteins. Subsequently, the m(7)G-cap is hypermethylated to the 2,2,7-trimethylguanosine (m(3)G)-cap. The import adaptor snurportin1 recognises the m(3)G-cap and facilitates the nuclear import of the UsnRNPs by binding to importin-beta. Here we report the crystal structure of the m(3)G-cap-binding domain of snurportin1 with bound m(3)GpppG at 2.4 angstrom resolution, revealing a structural similarity to the mRNA-guanylytransferase. Snurportin1 binds both the hypermethylated cap and the first nucleotide of the RNA in a stacked conformation. This binding mode differs significantly from that of the m(7)G-cap-binding proteins Cap-binding protein 20 (CBP20), eukaryotic initiation factor 4E (eIF4E) and viral protein 39 (VP39). The specificity of the m(3)G-cap recognition by snurportin1 was evaluated by fluorescence spectroscopy, demonstrating the importance of a highly solvent exposed tryptophan for the discrimination of m(7)G-capped RNAs. The critical role of this tryptophan and as well of a tryptophan continuing the RNA base stack was confirmed by nuclear import assays and cap-binding activity tests using several snurportin1 mutants
Phenylalanine-containing hydroxamic acids as selective inhibitors of class IIb histone deacetylases (HDACs)
No full textWe synthesized biarylalanine-containing hydroxamic acids and tested them on immunoprecipitated HDAC1 and HDAC6 and show a subtype selectivity for HDAC6 that was confirmed in cells by Western blot (tubulin vs histones). We obtained an X-ray structure with a HDAC6-selective inhibitor with the bacterial deacetylase HDAH. Docking studies were carried out using HDAC1 and HDAC6 protein models. Antiproliferative activity was shown on cancer cells for selected compounds. (c) 2007 Elsevier Ltd. All rights reserved
The covariance structure of conditional maximum likelihood estimates
No full textIn this paper we consider conditional maximum likelihood (cml) estimates for
item parameters in the Rasch model under random subject parameters. We give
a simple approximation for the asymptotic covariance matrix of the cml-estimates.
The approximation is stated as a limit theorem when the number of item parameters
goes to infinity. The results contain precise mathematical information on the order
of approximation.
The results enable the analysis of the covariance structure of cml-estimates when
the number of items is large. Let us give a rough picture. The covariance matrix has
a dominating main diagonal containing the asymptotic variances of the estimators.
These variances are almost equal to the efficient variances under ml-estimation when
the distribution of the subject parameter is known. Apart from very small numbers
n of item parameters the variances are almost not affected by the number n. The
covariances are more or less negligible when the number of item parameters is large.
Although this picture intuitively is not surprising it has to be established in precise
mathematical terms. This has been done in the present paper.
The paper is based on previous results [5] of the author concerning conditional
distributions of non-identical replications of Bernoulli trials. The mathematical background
are Edgeworth expansions for the central limit theorem. These previous results
are the basis of approximations for the Fisher information matrices of cmlestimates.
The main results of the present paper are concerned with the approximation
of the covariance matrices.
Numerical illustrations of the results and numerical experiments based on the
results are presented in Strasser, [6]. (author's abstract
Structural basis for mm3G-Cap-mediated nuclear import of spliceosomal UsnRNPs by snurportin1
No full textWährend der Biogenese spleißosomaler Untereinheiten (U snRNPs) wird die U snRNA nach der Transkription ins Zytoplasma exportiert, wo die Zusammenlagerung mit sieben Sm-Proteinen erfolgt und nachfolgend das 7-Methyl-Guanosin (m7G)-Cap zu einem 2,2,7-Trimethyl-Guanosin (m3G)-Cap umgewandelt wird. Das hypermethylierte m3G-Cap stellt ein Kernimportsignal der snRNPs U1, U2, U4 und U5 dar. Snurportin1 (SPN1) vermittelt als Importadaptor die Interaktion zwischen den m3G-Cap-tragenden snRNPs und dem Kernimportrezeptor Importin beta, der die Kernporenpassage mediiert. SPN1 besteht aus einer N-terminalen Importin beta-bindenden (IBB)-Domäne und einer C-terminalen m3G-Cap-bindenden Domäne, die keinerlei Sequenzähnlichkeit zu anderen Proteinen aufweist.In der vorliegenden Arbeit wurde die Struktur der m3G-Cap-bindende Domäne von SPN1 im Komplex mit m3GpppG aufgeklärt. SPN1 bindet das hypermethylierte m3GpppG-Cap mit gestapelten Basen zwischen Trp 276 und Leu 104. Diese Form der Wechselwirkung der positiv geladenen Cap-Base mit den pi-Elektronensystemen des benachbartenTrp 276 und der unmethylierten snRNA-Base nennt sich Kationen-pi-Interaktion. Trp 107 liegt senkrecht über dem Basenstapel und geht zusätzlich van-der-Waals Interaktionen mit den Methylgruppen an Position N2 der Cap-Base ein. In allen strukturell analysierten m7G-Cap-bindenden Proteinen wird lediglich die methylierte Cap-Base zwischen zwei aromatischen Aminosäureresten gestapelt und ein aromatischer Rest senkrecht zum Basenstapel fehlt. Zur funktionellen Analyse der Cap-Bindung durch SPN1 wurden sowohl Dissoziationskonstanten mono- und trimethylierter Cap-Analoga bestimmt, als auch das Bindungsverhalten von Tryptophan-Mutanten (W276A und W107A) über Fluoreszenzbindungsassays und deren Importaktivität über einen in vitro Kernimportassay untersucht.Durch die strukturelle Analyse der m3G-Cap-bindenden Domäne von SPN1 konnte eine neue Form der Cap-Bindung im Vergleich zu strukturell untersuchten m7G-Cap-bindenden Proteinen (CBC, eIF4E und VP39) gezeigt werden. Des Weiteren wurden Unterschiede in den Bindungsaffinitäten von SPN1 zu mono- und trimethylierten Cap-Analoga gefunden und der Bindungsbeitrag der mit dem m3GpppG-Cap interagierenden Tryptophan-Reste von SPN1 wurde quantifiziert.During snRNP biogenesis the snRNAs are exported into the cytoplasm, where the assembly with the common Sm proteins occurs and the 5´cap nucleotide is modified from an 7-methyl-guanosine (m7G-) to a 2,2,7-trimethyl-guanosine (m3G-) cap. The hypermethylated m3G-cap represents one of the nuclear localisation signals of the snRNPs U1, U2, U4 and U5. As an import adaptor snurportin1 bridges the interaction between the m3G-cap containing snRNPs and the nuclear import receptor importin beta, which mediates the passage through the nuclear pore complex. Snurportin1 contains a N-terminal importin beta-binding (IBB) domain and a C-terminal m3G-cap-binding region, which shows no similarity to other import factors.This work presents the crystal structure of the m3G-cap binding domain of SPN1 in complex with an m3GpppG cap dinucleotide. SPN1 binds both of the bases of the dinucleotide in a stacked conformation between Trp 276 and Leu 104. The methylated, positively charged guanine base interacts with the pi-electron systems of Trp 276 and the nonmethylated base. This kind of interaction is called cation-pi-interaction. Trp 107 is located perpendicular to the stack and forms van-der-Waals contacts to the methyl groups on N2. Stacking of both bases and an aromatic residue perpendicular to the stack were not observed in other m7G-cap binding proteins. These proteins only bind the methylated cap base between two aromatic side chains in a stacked conformation. For the functional characterisation of cap-binding by SPN1, dissociation constants for mono- and trimethylated caps were defined and the binding affinities of tryptophane mutants for m3GpppG were investigated by fluorescence assays and their import activity was quantified by an in vitro nuclear import assay.The presented crystal structure of SPN1 in complex with m3GpppG shows a novel cap-binding mode in contrast to m7G-cap-binding proteins structurally investigated (CBC, eIF4E and VP39). Furthermore differences in cap-binding affinities of SPN1 for mono- and trimethylated cap-oligonucleotides were demonstrated and binding contributions of the interacting tryptophane residues were quantified
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