Crystallization and preliminary X-ray diffraction analysis of importin-alpha acomplexed with NLS peptidomimetics

Importin-alpha is the nuclear import receptor that recognizes cargo proteins with nuclear localization sequences (NLSs). Tile study of NLS peptidomimetics can provide a better understanding of the requirements for the molecular recognition of cargo proteins by importin-alpha, and potentially engender a large number of applications in medicine. Importin-a was crystallized with a set of six NLS peptidomimetics, and X-ray diffraction data were collected in the range 2.1-2.5 angstrom resolution. Preliminary electron density calculations show that the ligands are present in the crystals. (c) 2005 Elsevier B.V All rights reserved.

Enhancement of non-viral transfection efficiency with nuclear localization signal peptides

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Nuclear localization of RelB is associated with effective antigen-presenting cell function

Dendritic cells (DC) are potent APCs that enter resting tissues as precursors and, after Ag exposure, differentiate and migrate to draining lymph nodes. The phenotype of RelB knockout mice implicates this member of the NF kappa B/Rel family in DC differentiation. To further elucidate the role of RelB in DC differentiation, mRNA, intracellular protein expression, and DNA binding activity of RelB were examined in immature and differentiated human DC, as well as other PB mononuclear cell populations. RelB protein and mRNA were detected constitutively in lymphocytes and in activated monocytes, differentiated DC, and monocyte-derived DC. Immunohistochemical staining demonstrated RelB within the differentiated lymph node interdigitating DC and follicular DC, but not undifferentiated DC in normal skin. Active nuclear RelB was detected by supershift assay only in differentiated DC derived from either PB precursors or monocytes and in activated B cells. These RelB+ APC were potent stimulators of the MLR. The data indicate that RelB expression is regulated both transcriptionally and post-translationally in myeloid cells. Within the nucleus, RelB may specifically transactivate genes that are critical for APC function.

Nuclear localization of RelB is associated with effective antigen-presenting cell function

Dendritic cells (DC) are potent APCs that enter resting tissues as precursors and, after Ag exposure, differentiate and migrate to draining lymph nodes. The phenotype of RelB knockout mice implicates this member of the NF kappa B/Rel family in DC differentiation. To further elucidate the role of RelB in DC differentiation, mRNA, intracellular protein expression, and DNA binding activity of RelB were examined in immature and differentiated human DC, as well as other PB mononuclear cell populations. RelB protein and mRNA were detected constitutively in lymphocytes and in activated monocytes, differentiated DC, and monocyte-derived DC. Immunohistochemical staining demonstrated RelB within the differentiated lymph node interdigitating DC and follicular DC, but not undifferentiated DC in normal skin. Active nuclear RelB was detected by supershift assay only in differentiated DC derived from either PB precursors or monocytes and in activated B cells. These RelB(+) APC were potent stimulators of the MLR. The data indicate that RelB expression is regulated both transcriptionally and post-translationally in myeloid cells. Within the nucleus, RelB may specifically transactivate genes that are critical for APC function.

Cytoplasmic and nuclear localization of cadherin in honey bee (Apis mellifera L.) gonads

Cadherins are crucial molecules mediating cell-cell interactions between somatic and germline cells in insect and mammalian male and female gonads. We analysed the presence and localization of cadherins in ovaries of honeybee queens and in testes of drones. Transcripts representing two classical cadherins, E-cadherin (shotgun) and N-cadherin, as well as three protocadherins (Starry night, Fat and Fat-like) were detected in gonads of both sexes. Pan-cadherin antibodies, which most probably detect a honeybee N-cadherin, were used in immunolocalization analyses. In the germarium of ovarioles, cadherin-IR (cadherin immunoreactivity) was evidenced as homogeneously distributed in the cytoplasm and as nuclear foci, in both germline and somatic cells. It was also detected in polyfusomes and ring canals. In testiolar tubules, cadherin-IR showed a cytoplasmic and nuclear distributon alike in ovaries. The unexpected nuclear localization and cytoplasmic distribution in ovaries and testes were corroborated by immunogold electron microscopy, which revealed cadherin aggregates associated with electron-dense nuclear structures. With respect to cadherin localization, the honeybee differs from Drosophila, the model for gametogenesis in insects, raising the question as to how differences among solitary and social species may be built into and generated from the general architecture of polytrophic meroistic ovaries. It also indicates the possibility of divergent roles for cadherin in the functional architecture of insect gonads, in general, especially in taxa with high reproductive output.

HCF-1 self-association via an interdigitated Fn3 structure facilitates transcriptional regulatory complex formation.

Host-cell factor 1 (HCF-1) is an unusual transcriptional regulator that undergoes a process of proteolytic maturation to generate N- (HCF-1(N)) and C- (HCF-1(C)) terminal subunits noncovalently associated via self-association sequence elements. Here, we present the crystal structure of the self-association sequence 1 (SAS1) including the adjacent C-terminal HCF-1 nuclear localization signal (NLS). SAS1 elements from each of the HCF-1(N) and HCF-1(C) subunits form an interdigitated fibronectin type 3 (Fn3) tandem repeat structure. We show that the C-terminal NLS recruited by the interdigitated SAS1 structure is required for effective formation of a transcriptional regulatory complex: the herpes simplex virus VP16-induced complex. Thus, HCF-1(N)-HCF-1(C) association via an integrated Fn3 structure permits an NLS to facilitate formation of a transcriptional regulatory complex.

Relationships of Scincid Lizards (Mabuya spp; Reptilia: Scincidae) from the Cape Verde Islands Based on Mitochondrial and Nuclear DNA Sequences

Partial DNA sequences from two mitochondrial (mt) and one nuclear gene (cytochrome b, 12S rRNA, and C-mos) were used to estimate the phylogenetic relationships among the six extant species of skinks endemic to the Cape Verde Archipelago. The species form a monophyletic unit, indicating a single colonization of the islands, probably from West Africa. Mabuya vaillanti and M. delalandii are sister taxa, as indicated by morphological characters. Mabuya fogoensis and M. stangeri are closely related, but the former is probably paraphyletic. Mabuya spinalis and M. salensis are also probably paraphyletic. Within species, samples from separate islands always form monophyletic groups. Some colonization events can be hypothesized, which are in line with the age of the islands. C-mos variation is concordant with the topology derived from mtDNA.

Calpastatin-mediated inhibition of calpains in the mouse brain prevents mutant ataxin 3 proteolysis, nuclear localization and aggregation, relieving Machado-Joseph disease.

Machado-Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-repetition of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within the ataxin 3 protein, which upon proteolysis may trigger Machado-Joseph disease. We investigated the role of calpains in the generation of toxic ataxin 3 fragments and pathogenesis of Machado-Joseph disease. For this purpose, we inhibited calpain activity in mouse models of Machado-Joseph disease by overexpressing the endogenous calpain-inhibitor calpastatin. Calpain blockage reduced the size and number of mutant ataxin 3 inclusions, neuronal dysfunction and neurodegeneration. By reducing fragmentation of ataxin 3, calpastatin overexpression modified the subcellular localization of mutant ataxin 3 restraining the protein in the cytoplasm, reducing aggregation and nuclear toxicity and overcoming calpastatin depletion observed upon mutant ataxin 3 expression. Our findings are the first in vivo proof that mutant ataxin 3 proteolysis by calpains mediates its translocation to the nucleus, aggregation and toxicity and that inhibition of calpains may provide an effective therapy for Machado-Joseph disease.

Phylogenetic analysis of plastid and nuclear DNA sequences reveals a rapid late Miocene radiation of the temperate bamboo tribe Arundinarieae (Poaceae, Bambusoideae)

Background: Arundinarieae are a large tribe of temperate woody bamboos for which phylogenetics are poorly understood because of limited taxon sampling and lack of informative characters. Aims: This study assessed phylogenetic relationships, origins and classification of Arundinarieae. Methods: DNA sequences (plastid trnL-F; nuclear ITS) were used for parsimony and Bayesian inference including 41 woody bamboo taxa. Divergence dates were estimated using a relaxed Bayesian clock. Results: Arundinarieae were monophyletic but their molecular divergence was low compared to the tropical Bambuseae. Ancestors of the Arundinarieae lineage were estimated to have diverged from the other bamboos 23 (15-30) million years ago (Mya). However, the Arundinarieae radiation occurred 10 (6-16) Mya compared to 18 (11-25) Mya for the tropical Bambuseae. Some groups could be defined within Arundinarieae, but they do not correspond to recognised subtribes such as Arundinariinae or Shibataeinae. Conclusions: Arundinarieae are a relatively ancient bambusoid lineage that underwent a rapid radiation in the late Miocene. The radiation coincides with the continental collision of the Indo-Australian and Eurasian Plates. Arundinarieae are distributed primarily in East Asia and the Himalayas to northern Southeast Asia. It is unknown whether they were present in Asia long before their radiation, but we believe recent dispersal is a more likely scenario. Keywords: Arundinarieae; Bambuseae; internal transcribed spacer (ITS); molecular clock; phylogenetics; radiation; temperate bamboos; Thamnocalaminae; trnL-F

Ligand-induced endocytosis and nuclear localization of angiotensin II receptors expressed in CHO cells

A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization.

A mechanism for co-transcriptional recruitment of mRNA localization factor on nascent mRNAs in budding yeast

Le transport et la localisation des ARN messagers permettent de réguler l’expression spatiale et temporelle de facteurs spécifiques impliqués dans la détermination du destin cellulaire, la plasticité synaptique, la polarité cellulaire et la division asymétrique des cellules. Chez S.cerevisiæ, plus de trente transcrits sont transportés activement vers le bourgeon cellulaire. Parmi ces transcrits, l’ARNm ASH1 (asymetric synthesis of HO) est localisé à l’extrémité du bourgeon pendant l’anaphase. Ce processus va entrainer une localisation asymétrique de la protéine Ash1p, qui sera importée uniquement dans le noyau de la cellule fille, où elle entraine le changement de type sexuel. La localisation asymétrique de l’ARNm ASH1, et donc de Ash1p, implique la présence de différents facteurs de localisation. Parmi ces facteurs, les protéines She (She1p/Myo4p, She2p et She3p) et les répresseurs traductionnels (Puf6p, Loc1p et Khd1p) participent à ce mécanisme. La protéine navette She2p est capable de lier l’ARNm ASH1 et va entrainer le ciblage de cet ARNm vers l’extrémité du bourgeon en recrutant le complexe She3p-Myo4p. Des répresseurs traductionnels régulent la traduction de cet ARNm et évitent l’expression ectopique de la protéine Ash1p pendant son transport. Alors que la fonction cytoplasmique de She2p sur la localisation des ARNm est connue, sa fonction nucléaire est encore inconnue. Nous avons montré que She2p contient une séquence de localisation nucléaire non classique qui est essentielle à son import nucléaire médié par l’importine α (Srp1p). L’exclusion de She2p du noyau par mutation de son NLS empêche la liaison de Loc1p et Puf6p sur l’ARNm ASH1, entrainant un défaut de localisation de l’ARNm et de la protéine. Pour étudier plus en détail l’assemblage de la machinerie de localisation des ARNm dans le noyau, nous avons utilisé des techniques d’immunoprécipitation de chromatine afin de suivre le recrutement des facteurs de localisation et des répresseurs traductionnels sur les ARNm naissants. Nous avons montré que She2p est recruté sur le gène ASH1 pendant sa transcription, via son interaction avec l’ARNm ASH1 naissant. Puf6p est également recruté sur ASH1, mais d’une manière dépendante de la présence de She2p. De façon intéressante, nous avons détecté une interaction entre She2p et la plus grande sous-unité de l’ARN polymérase II (Rpb1p). Cette interaction est détectée avec la forme active en élongation de l’ARN polymérase II. Nous avons également démontré que She2p interagit avec le complexe d’élongation de la transcription Spt4p/Spt5p. Une délétion de SPT4 ou une mutation dans SPT5 (Ts spt5) à température restrictive empêche l’interaction entre She2p et Rpb1p, et diminue le recrutement de She2p au gène ASH1, entrainant un défaut de localisation de l’ARNm et un défaut de localisation asymétrique de la protéine Ash1p. De manière globale, nos résultats montrent que les facteurs impliqués dans la localisation cytoplasmique des ARNm et dans leur contrôle traductionnel sont recrutés de façon co-transcriptionnelle sur les ARNm naissants via leur interaction avec la machinerie de transcription, suggèrant un rôle important de la machinerie transcriptionelle dans la localisation des ARNm.

Study of the subcellular localization of cell cycle regulator Cks1 and its impact on cancer

La progression dans le cycle cellulaire est contrôlée par de vagues oscillantes de cyclines et des kinases cycline-dépendantes (Cdk). Ces kinases sont régulées positivement par l’association des sous-unités cyclines régulatrices et négativement en se liant aux inhibiteurs de Cdk. Parmi ces derniers, p27 inhibe tous les complexes cycline-Cdk quelle que soit la phase cellulaire et agit en tant que régulateur négatif principal de la prolifération cellulaire dans une variété de cellules et de tissus. Intrinsèquement, p27 phosphorylé est ubiquitiné et dégradé par le complexe SCFSkp2-Cks1. Des études génétiques de la souris, ainsi que des examens cliniques chez l’homme, ont montré que p27 est un important suppresseur de tumeur. Le gène est rarement muté. Cependant, p27 est fréquemment réprimé dans les cancers humains en raison d’une augmentation de l’expression de Skp2 et de Cks1 dans le noyau, ce qui est généralement associée à un mauvais pronostic. La localisation subcellulaire de Cks1 est donc d'une importance primordiale dans le contrôle de la prolifération cellulaire. Les résultats récents de notre laboratoire ont montré une interaction entre Cks1 et les protéines de transport nucléaire importine α1 et β3. Aussi, l’analyse de la séquence primaire de Cks1 a également révélé un signal de localisation nucléaire classique (NLS) à son extrémité C-terminale. Des mutations ont été effectuées sur le NLS suspect pour déterminer si oui ou non l'import nucléaire de Cks1 était contrôlé par cette séquence. Un inhibiteur synthétique de l’importine β a également été utilisé pour étudier l’import de Cks1 dans le noyau. Les résultats indiquent que l’extrémité C-terminale de Cks1 est en effet un NLS puisque les mutations de Cks1 et l'inhibition de l’importine β conduisent, tous deux, à l'accumulation de Cks1 dans le cytoplasme. Ces résultats ont été utiles pour mieux comprendre le mécanisme régulant la localisation de Cks1. Toutefois, des travaux futurs sont nécessaires pour mieux comprendre l'impact de la séquestration cytoplasmique de Cks1 sur le cancer et ainsi espérer aboutir à l'identification de nouvelles cibles pharmacologiques impliqués dans la prolifération cellulaire.

Evolutionary patterns in neotropical Helieae (Gentianaceae): evidence from morphology, chloroplast and nuclear DNA sequences

Parsimony-based phylogenetic analyses of the neotropical tribe Helieae (Gentianaceae) are presented, including 22 of the 23 genera and 60 species. This study is based on data from morphology, palynology, and seed micromorphology (127 structural characters), and DNA sequences (matK, trnL intron, ITS). Phylogenetic reconstructions based on ITS and morphology provided the greatest resolution, morphological data further helping to tentatively place several taxa for which DNA was not available (Celiantha, Lagenanthus, Rogersonanthus, Roraimaea, Senaea, Sipapoantha, Zonanthus). Celiantha, Prepusa and Senaea together appear as the sister clade to the rest of Helieae. The remainder of Helieae is largely divided into two large subclades, the Macrocarpaea subclade and the Symbolanthus subclade. The first subclade includes Macrocarpaea, sister to Chorisepalum, Tochia, and Zonanthus. Irlbachia and Neblinantha are placed as sisters to the Symbolanthus subclade, which includes Aripuana, Calolisianthus, Chelonanthus, Helia, Lagenanthus, Lehmanniella, Purdieanthus, Rogersonanthus, Roraimaea, Sipapoantha, and symbolanthus. Generic-level polyphyly is detected in Chelonanthus and Irlbachia. Evolution of morphological characters is discussed, and new pollen and seed characters are evaluated for the first time in a combined morphological-molecular phylogenetic analysis.

Structural basis of nuclear import of flap endonuclease 1 (FEN1)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)