Structural basis for the specificity of bipartite nuclear localization sequence binding by importin-alpha

Importin-alpha is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-alpha, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-alpha and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX10-12KRRK) that can be used to identify novel nuclear proteins.

Probing the Specificity of Binding to the Major Nuclear Localization Sequence-binding Site of Importin-alpha Using Oriented Peptide Library Screening

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

Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-alpha

The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser(112) in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-alpha (Impalpha), we co-crystallized non-autoinhibited Impalpha with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impalpha. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impalpha. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impalpha, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impalpha.

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.

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

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

Nucleotide sequence, genomic organization and chromosome localization of 5S rDNA in two species of Curimatidae (Teleostei, Characiformes)

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

Nucleotide sequence of 5S rDNA and localization of the ribosomal RNA genes to metaphase chromosomes of the Tilapiine cichlid fish, Oreochromis niloticus

In this study, we report the cloning and nucleotide sequence of PCR-generated 5S rDNA from the Tilapiine cichlid fish, Oreochromis niloticus. Two types of 5S rDNA were detected that differed by insertions and/or deletions and base substitutions within the non-transcribed spacer (NTS). Two 5S rDNA loci were observed by fluorescent in situ hybridization (FISH) in metaphase spreads of tilapia chromosomes. FISH using an 18S rDNA probe and silver nitrate sequential staining of 5S-FISH slides showed three 18S rDNA loci that are not syntenic to the 5S rDNA loci.

Molecular characterization and chromosomal localization of two families of satellite DNA in Prochilodus lineatus (Pisces, Prochilodontidae), a species with B chromosomes

Prochilodus lineatus, an abundant species in the Mogi-Guaçu river basin, represents a large part of the region's fishing potential. Karyotypic analyses based on classic cytogenetic techniques have revealed the presence of 54 metasubmetacentric type chromosomes, together with the occurrence of small supernumerary chromosomes with intra and interindividual variations. This paper describes the genomic organization of two families of satellite DNA in the P. lineatus genome. The chromosomal localization these two repetitive DNA families through fluorescence in situ hybridization (FISH) demonstrated that the SATH1 satellite DNA family, composed of approximately 900 bp, was located in the pericentromeric region of a group of chromosomes of the standard complement, as well as on all the B chromosomes. The SATH2 satellite family has a monomeric unit of 441 bp and was located in the pericentromeric regions of some chromosomes of the standard complement, but was absent in the B chromosomes. Double FISH analyses showed that these two families participate jointly in the pericentromeric organization of several chromosomes of this species. The data obtained in this study support the hypothesis that the B chromosomes derive from chromosomes of the standard complement, which are carriers of the SATH1 satellite DNA.

New insights into telomeric DNA sequence (TTAGGG)n location in bat chromosomes

Molossidae species, Cynomops abrasus (2n = 34, fundamental number, FN = 64), Eumops auripendulus (2n = 42, FN = 62), Molossus rufus (2n = 48, FN = 64), Molossops temminckii (2n = 48, FN = 64), and Nyctinomops laticaudatus (2n = 48, FN = 64), and Phyllostomidae species, Phyllostomus discolor (2n = 32, FN = 60), have karyotypes with different chromosome and fundamental numbers, different localization of constitutive heterochromatin, and different numbers and location of nucleolar organizer regions (NORs). Fluorescence in situ hybridization with a human probe of the telomeric sequence (TTAGGG)n produced fluorescent signals in telomeric regions of the six bat species' chromosomes; in E. auripendulus, pericentromeric signals were also observed in the acrocentric and subtelocentric chromosomes. A relationship between telomeric sequences and NORs, and between telomeric sequences and constitutive heterochromatin was detected in chromosomes bearing NORs in C. abrasus, M. temminckii, N. laticaudatus, and P. discolor. No interstitial signal was observed in the meta- or submetacentric chromosomes of these species. ©FUNPEC-RP.

Paracoccidoides brasiliensis 30 kDa Adhesin: Identification as a 14-3-3 Protein, Cloning and Subcellular Localization in Infection Models

Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections) and 30 days (chronic infection). An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis. © 2013 Silva et al.

General characterization of Tityus fasciolatus scorpion venom. Molecular identification of toxins and localization of linear B-cell epitopes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)