Characterization of the nuclear import pathway for HIV-1 integrase

The karyophilic properties of the human immunodeficiency virus, type I (HIV-1) pre-integration complex (PIC) allow the virus to infect non-dividing cells. To better understand the mechanisms responsible for nuclear translocation of the PIC, we investigated nuclear import of HIV-1 integrase (IN), a PIC-associated viral enzyme involved in the integration of the viral genome in the host cell DNA. Accumulation of HIV-1 IN into nuclei of digitonin-permeabilized cells does not result from passive diffusion but rather from an active transport that occurs through the nuclear pore complexes. HIV-1 IN is imported by a saturable mechanism, implying that a limiting cellular factor is responsible for this process. Although IN has been previously proposed to contain classical basic nuclear localization signals, we found that nuclear accumulation of IN does not involve karyopherins alpha, beta1, and beta2-mediated pathways. Neither the non-hydrolyzable GTP analog, guanosine 5'-O-(thiotriphosphate), nor the GTP hydrolysis-deficient Ran mutant, RanQ69L, significantly affects nuclear import of IN, which depends instead on ATP hydrolysis. Therefore these results support the idea that IN import is not mediated by members of the karyopherin beta family. More generally, in vitro nuclear import of IN does not require addition of cytosolic factors, suggesting that cellular factor(s) involved in this active but atypical pathway process probably remain associated with the nuclear compartment or the nuclear pore complexes from permeabilized cells.

Structure of importin-a from a filamentous fungus in complex with a classical nuclear localization signal

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

Deciphering the Nuclear Import Pathway for the Cytoskeletal Red Cell Protein 4.1R

The erythroid membrane cytoskeletal protein 4.1 is the prototypical member of a genetically and topologically complex family that is generated by combinatorial alternative splicing pathways and is localized at diverse intracellular sites including the nucleus. To explore the molecular determinants for nuclear localization, we transfected COS-7 cells with epitope-tagged versions of natural red cell protein 4.1 (4.1R) isoforms as well as mutagenized and truncated derivatives. Two distant topological sorting signals were required for efficient nuclear import of the 4.1R80 isoform: a basic peptide, KKKRER, encoded by alternative exon 16 and acting as a weak core nuclear localization signal (4.1R NLS), and an acidic peptide, EED, encoded by alternative exon 5. 4.1R80 isoforms lacking either of these two exons showed decreased nuclear import. Fusion of various 4.1R80 constructs to the cytoplasmic reporter protein pyruvate kinase confirmed a requirement for both motifs for full NLS function. 4.1R80 was efficiently imported in the nuclei of digitonin-permeabilized COS-7 cells in the presence of recombinant Rch1 (human importin α2), importin β, and GTPase Ran. Quantitative analysis of protein–protein interactions using a resonant mirror detection technique showed that 4.1R80 bound to Rch1 in vitro with high affinity (KD = 30 nM). The affinity decreased at least 7- and 20-fold, respectively, if the EED motif in exon 5 or if 4.1R NLS in exon 16 was lacking or mutated, confirming that both motifs were required for efficient importin-mediated nuclear import of 4.1R80.

Nuclear Import Mechanisms of STAT and NF-kB Transcription Factors

The eukaryotic cell nucleoplasm is separated from the cytoplasm by the nuclear envelope. This compartmentation of eukaryotic cells requires that all nuclear proteins must be transported from the cytoplasm into the nucleus. Transport of macromolecules between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs). Proteins to be targeted into the nucleus by the classical nuclear import system contain nuclear localization signals (NLSs), which are recognized by importin alpha, the NLS receptor. Importin alpha binds to importin beta, which docks the importin-cargo complex on the cytoplasmic side of the NPC and mediates the movement of the complex into the nucleus. Presently six human importin alpha isoforms have been identified. Transcription factors are among the most important regulators of gene expression in eukaryotic organisms. Transcription factors bind to specific DNA sequences on target genes and modulate the activity of the target gene. Many transcription factors, including signal transducers and activators of transcription (STAT) and nuclear factor kB (NF-kB), reside in the cytoplasm in an inactive form, and upon activation they are rapidly transported into the nucleus. In the nucleus STATs and NF-kB regulate the activity of genes whose products are critical in controlling numerous cellular and organismal processes, such as inflammatory and immune responses, cell growth, differentiation and survival. The aim of this study was to investigate the nuclear import mechanisms of STAT and NF-kB transcription factors. This work shows that STAT1 homodimers and STAT1/STAT2 heterodimers bind specifically and directly to importin alpha5 molecule via unconventional dimer-specific NLSs. Importin alpha molecules have two regions, which have been shown to directly interact with the amino acids in the NLS of the cargo molecule. The Arm repeats 2-4 comprise the N-terminal NLS binding site and Arm repeats 7-8 the C-terminal NLS binding site. In this work it is shown that the binding site for STAT1 homodimers and STAT1/STAT2 heterodimers is composed of Arm repeats 8 and 9 of importin alpha5 molecule. This work demonstrates that all NF-kB proteins are transported into the nucleus by importin alpha molecules. In addition, NLS was identified in RelB protein. The interactions between NF-kB proteins and importin alpha molecules were found to be directly mediated by the NLSs of NF-kB proteins. Moreover, we found that p50 binds to the N-terminal and p65 to the C-terminal NLS binding site of importin alpha3. The results from this thesis work identify previously uncharacterized mechanisms in nuclear import of STAT and NF-kB. These findings provide new insights into the molecular mechanisms regulating the signalling cascades of these important transcription factors from the cytoplasm into the nucleus to the target genes.

Estudos estruturais com a importina-α do fungo Neurospora crassa e sequências de localização nuclear

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

Biophysical characterization of the recombinant importin-α from neurospora crassa

Neurospora crassa has been widely used as a model organism and contributed to the development of biochemistry and molecular biology by allowing the identification of many metabolic pathways and mechanisms responsible for gene regulation. Nuclear proteins are synthesized in the cytoplasm and need to be translocated to the nucleus to exert their functions which the importin-α receptor has a key role for the classical nuclear import pathway. In an attempt to get structural information of the nuclear transport process in N. crassa, we present herein the cloning, expression, purification and structural studies with N-terminally truncated IMPα from N. crassa (IMPα-Nc). Circular dichroism analysis revealed that the IMPα-Nc obtained is correctly folded and presents a high structural conservation compared to other importins-α. Dynamic light scattering, analytical size-exclusion chromatography experiments and molecular dynamics simulations indicated that the IMPα-Nc unbound to any ligand may present low stability in solution. The IMPα-Nc theoretical model displayed high similarity of its inner concave surface, which binds the cargo proteins containing the nuclear localization sequences, among IMPα from different species. However, the presence of non-conserved amino acids relatively close to the NLS binding region may influence the binding specificity of IMPα-Nc to cargo proteins. Copyright © 2012 Bentham Science Publishers. All Rights Reserved.

Biophysical Characterization of the Recombinant Importin-alpha from Neurospora crassa

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

Two Nuclear Localization Signals in USP1 Mediate Nuclear Import of the USP1/UAF1 Complex

9 p. : il.

Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1.

Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence-containing protein, binding to the alpha adaptor subunit of the importin-alpha/beta heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-beta that is distinct from that used to bind importin-alpha.

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

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

Structural Basis of Importin-alpha-Mediated Nuclear Transport for Ku70 and Ku80

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

Biophysical Characterization of Interactions Involving Importin-α during Nuclear Import

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-α/β heterodimer. Importin-α contains the NLS binding site, whereas importin-β mediates the translocation through the nuclear pore. We characterized the interactions involving importin-α during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-α is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-β (stoichiometry, 1:1; K D = 1.1 × 10 -8 M) increases the affinity for NLSs; the importin-α/β complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K D = 3.5 × 10 -8 M and 4.8 × 10 -8 M, respectively) comparable with those of a truncated importin-α lacking the autoinhibitory domain (T-antigen NLS, K D = 1.7 × 10 -8 M; nucleoplasmin NLS, K D = 1.4 × 10 -8 M). The autoinhibitory domain (as a separate peptide) binds the truncated importin-α, and the crystal structure of the complex resembles the structure of full-length importin-α. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-α and provide a quantitative description of the binding and regulatory steps during nuclear import.

Two domains of the V protein of virulent canine distemper virus selectively inhibit STAT1 and STAT2 nuclear import

Canine distemper virus (CDV) causes in dogs a severe systemic infection, with a high frequency of demyelinating encephalitis. Among the six genes transcribed by CDV, the P gene encodes the polymerase cofactor protein (P) as well as two additional nonstructural proteins, C and V; of these V was shown to act as a virulence factor. We investigated the molecular mechanisms by which the P gene products of the neurovirulent CDV A75/17 strain disrupt type I interferon (IFN-alpha/beta)-induced signaling that results in the establishment of the antiviral state. Using recombinant knockout A75/17 viruses, the V protein was identified as the main antagonist of IFN-alpha/beta-mediated signaling. Importantly, immunofluorescence analysis illustrated that the inhibition of IFN-alpha/beta-mediated signaling correlated with impaired STAT1/STAT2 nuclear import, whereas the phosphorylation state of these proteins was not affected. Coimmunoprecipitation assays identified the N-terminal region of V (VNT) responsible for STAT1 targeting, which correlated with its ability to inhibit the activity of the IFN-alpha/beta-mediated antiviral state. Conversely, while the C-terminal domain of V (VCT) could not function autonomously, when fused to VNT it optimally interacted with STAT2 and subsequently efficiently suppressed the IFN-alpha/beta-mediated signaling pathway. The latter result was further supported by a single mutation at position 110 within the VNT domain of CDV V protein, resulting in a mutant that lost STAT1 binding while retaining a partial STAT2 association. Taken together, our results identified the CDV VNT and VCT as two essential modules that complement each other to interfere with the antiviral state induced by IFN-alpha/beta-mediated signaling. Hence, our experiments reveal a novel mechanism of IFN-alpha/beta evasion among the morbilliviruses.

Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium

Import of DNA into mammalian nuclei is generally inefficient. Therefore, one of the current challenges in human gene therapy is the development of efficient DNA delivery systems. Here we tested whether bacterial proteins could be used to target DNA to mammalian cells. Agrobacterium tumefaciens, a plant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells. Agrobacterium-mediated T-DNA transfer to plant cells is the only known example for interkingdom DNA transfer and is widely used for plant transformation. Agrobacterium virulence proteins VirD2 and VirE2 perform important functions in this process. We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA) in vitro. These complexes were tested for import into HeLa cell nuclei. Import of ssDNA required both VirD2 and VirE2 proteins. A VirD2 mutant lacking its C-terminal nuclear localization signal was deficient in import of the ssDNA–protein complexes into nuclei. Import of VirD2–ssDNA–VirE2 complexes was fast and efficient, and was shown to depended on importin α, Ran, and an energy source. We report here that the bacterium-derived and plant-adapted protein–DNA complex, made in vitro, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway. This demonstrates the potential of our approach to enhance gene transfer to animal cells.

Karyopherin β2 mediates nuclear import of a mRNA binding protein

We have cloned and sequenced cDNA for human karyopherin β2, also known as transportin. In a solution binding assay, recombinant β2 bound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1’s previously characterized M9 nuclear localization sequence (NLS), but not by a peptide representing a classical NLS. As previously shown for karyopherin β1, karyopherin β2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both β1 and β2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, β2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of β2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of β2 there was marked stimulation of import by Ran. Import was inhibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ agglutinin. Consistent with the solution binding results, karyopherin β2 inhibited karyopherin α/β1-mediated import of a classical NLS containing substrate and, vice versa, β1 inhibited β2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of β1 and β2 to repeat nucleoporins.