Human TAP is implicated in mRNA nuclear export and is used by simian type D retroviruses to export their unspliced genomic RNA to the cytoplasm of the host cell. We have determined the crystal structure of the minimal TAP fragment that binds the constitutive transport element (CTE) of retroviral RNAs. Unexpectedly, we find the fragment consists of a ribonucleoprotein (RNP) domain, which is not identifiable by its sequence, and a leucine-rich repeat (LRR) domain. The non-canonical RNP domain functions as the general RNA-binding portion of the fragment. The LRR domain is required in cis to the RNP domain for CTE RNA binding. The structural and biochemical properties of the domains point to a remarkable similarity with the U2B"(RNP)-U2A'(LRR) spliceosomal heterodimer. Our in vitro and in vivo functional studies using structure-based mutants suggest that a phylogenetically conserved surface of the LRR domain of TAP may have different roles in the export of viral and cellular RNAs.

The targeting of the tumor suppressor PTEN protein to distinct subcellular compartments is a major regulatory mechanism of PTEN function, by controlling its access to substrates and effector proteins. Here, we investigated the molecular basis and functional consequences of PTEN nuclear/cytoplasmic distribution. PTEN accumulated in the nucleus of cells treated with apoptotic stimuli. Nuclear accumulation of PTEN was enhanced by mutations targeting motifs in distinct PTEN domains, and it was dependent on an N-terminal nuclear localization domain. Coexpression of a dominant negative Ran GTPase protein blocked PTEN accumulation in the nucleus, which was also affected by coexpression of importin ␣ proteins. The lipid-and protein-phosphatase activity of PTEN differentially modulated PTEN nuclear accumulation. Furthermore, catalytically active nuclear PTEN enhanced cell apoptotic responses. Our findings indicate that multiple nuclear exclusion motifs and a nuclear localization domain control PTEN nuclear localization by a Ran-dependent mechanism and suggest a proapoptotic role for PTEN in the cell nucleus. INTRODUCTIONPTEN is a tumor suppressor phosphatase involved in the control of cell growth and cell cycle traverse, apoptosis, cell size, and cell migration Waite and Eng, 2002;Leslie and Downes, 2004;Parsons, 2004;Sansal and Sellers, 2004). The PTEN gene is mutated or lost in a wide variety of human tumors, including malignant glioblastomas, an aggressive tumor of the CNS in humans (Bonneau and Longy, 2000;Eng, 2003). The major tumor suppressor function of PTEN is mediated by the dephosphorylation of phosphatidylinositol-3,4,5-triphosphate (PIP3; Maehama and Dixon, 1998). Through this lipid phosphatase activity, PTEN counteracts the action of the prosurvival proto-oncogenes, the phosphatidylinositol 3-kinases, and protein kinase B/Akt, which control the function of key downstream effectors of this pathway, including cell cycle regulators (such as p27Kip1, cyclin D1, and CHK1) and transcription factors (such as NF-B and FKHR;Li and Sun, 1998;Nakamura et al., 2000;Gustin et al., 2001;Weng et al., 2001;Radu et al., 2003;Puc et al., 2005). To control the PIP3 levels at the plasma membrane, PTEN possesses, in addition to its N-terminal phosphatase catalytic domain (residues 14 -185), a C-terminal phospholipidbinding C2 domain (residues 186 -350), which is critical for optimal binding to membranes and PIP3 dephosphorylation (Lee et al., 1999). In addition, the N-terminus of PTEN contains a phosphatidylinositol-4,5-diphosphate (PIP2) binding motif (residues 6 -15), which is also essential for PTEN membrane binding and activity Funamoto et al., 2002;Iijima and Devreotes, 2002;Campbell et al., 2003;McConnachie et al., 2003;Iijima et al., 2004;Walker et al., 2004;Vazquez et al., 2006). On the other hand, PTEN possesses a C-terminal tail (residues 350 -403) that plays a major role in the stabilization of the molecule and that is the target of posttranslational modifications, including phosphorylation by the protein kinase CK2 ...

The transport of macromolecules between the nucleus and cytoplasm of eukaryotic cells is largely mediated by a single family of transport factors, the karyopherin or importin ␤-like family. Structural and biochemical evidence suggests conformational flexibility of these modular HEAT-repeat proteins is crucial for their regulation. Here we use small angle x-ray scattering to assess the extent of conformational variation within a set of nuclear import and export factors. The study reveals that importin ␤, transportin, and the exportin Xpo-t share a similar S-like superhelical conformation in their unbound state. There are no obvious differences in the overall structures that might generally distinguish nuclear export from nuclear import mediators. Two other members of the family, the exportins Cse1 and Xpo1, possess a significantly more globular conformation, indicating that the extended S-like architecture is not a hallmark of all karyopherins. Binding of RanGTP/cargo to importin ␤, transportin, and Xpo-t triggers distinct conformational responses, suggesting that even closely related karyopherins employ different mechanisms of conformational regulation and that cargo and nuclear pore-interacting surfaces of the different receptors may be unique.Nucleo-cytoplasmic transport factors of the karyopherin ␤ family shuttle through nuclear pore complexes spanning the nuclear envelope and mediate the nuclear import and/or export of specific protein or RNA cargoes (for review, see Refs.

Cse1 mediates nuclear export of importin alpha, the nuclear localization signal (NLS) import adaptor. We report the 3.1 A resolution structure of cargo-free Cse1, representing this HEAT repeat protein in its cytosolic state. Cse1 is compact, consisting of N- and C-terminal arches that interact to form a ring. Comparison with the structure of cargo-bound Cse1 shows a major conformational change leading to opening of the structure upon cargo binding. The largest structural changes occur within a hinge region centered at HEAT repeat 8. This repeat contains a conserved insertion that connects the RanGTP and importin alpha contact sites and that is essential for binding. In the cargo-free state, the RanGTP binding sites are occluded and the importin alpha sites are distorted. Mutations that destabilize the N- to C-terminal interaction uncouple importin alpha and Ran binding, suggesting that the closed conformation prevents association with importin alpha.

Non-viral gene delivery vectors have lagged far behind viral ones in the current pipeline of clinical trials of gene therapy nanomedicines. Even when non-viral nanovectors pose less safety risks than do viruses, their efficacy is much lower. Since the early studies to deliver pDNA, chitosan has been regarded as a highly attractive biopolymer to deliver nucleic acids intracellularly and induce a transgenic response resulting in either upregulation of protein expression (for pDNA, mRNA) or its downregulation (for siRNA or microRNA). This is explained as the consequence of a multi-step process involving condensation of nucleic acids, protection against degradation, stabilization in physiological conditions, cellular internalization, release from the endolysosome (“proton sponge” effect), unpacking and enabling the trafficking of pDNA to the nucleus or the siRNA to the RNA interference silencing complex (RISC). Given the multiple steps and complexity involved in the gene transfection process, there is a dearth of understanding of the role of chitosan’s structural features (Mw and degree of acetylation, DA%) on each step that dictates the net transfection efficiency and its kinetics. The use of fully characterized chitosan samples along with the utilization of complementary biophysical and biological techniques is key to bridging this gap of knowledge and identifying the optimal chitosans for delivering a specific gene. Other aspects such as cell type and administration route are also at play. At the same time, the role of chitosan structural features on the morphology, size and surface composition of synthetic virus-like particles has barely been addressed. The ongoing revolution brought about by the recent discovery of CRISPR-Cas9 technology will undoubtedly be a game changer in this field in the short term. In the field of rare diseases, gene therapy is perhaps where the greatest potential lies and we anticipate that chitosans will be key players in the translation of research to the clinic.