Karyopherin binding interactions and nuclear import mechanism of nuclear pore complex protein Tpr.

BACKGROUND: Tpr is a large protein with an extended coiled-coil domain that is localized within the nuclear basket of the nuclear pore complex. Previous studies 1 involving antibody microinjection into mammalian cells suggested a role for Tpr in nuclear export of proteins via the CRM1 export receptor. In addition, Tpr was found to co-immunoprecipitate with importins alpha and beta from Xenopus laevis egg extracts 2, although the function of this is unresolved. Yeast Mlp1p and Mlp2p, which are homologous to vertebrate Tpr, have been implicated in mRNA surveillance to retain unspliced mRNAs in the nucleus34. To augment an understanding of the role of Tpr in nucleocytoplasmic trafficking, we explored the interactions of recombinant Tpr with the karyopherins CRM1, importin beta and importin alpha by solid phase binding assays. We also investigated the conditions required for nuclear import of Tpr using an in vitro assay. RESULTS: We found that Tpr binds strongly and specifically to importin alpha, importin beta, and a CRM1 containing trimeric export complex, and that the binding sites for importins alpha and beta are distinct. We also determined that the nuclear import of Tpr is dependent on cytosolic factors and energy and is efficiently mediated by the importin alpha/beta import pathway. CONCLUSION: Based on the binding and nuclear import assays, we propose that Tpr is imported into the nucleus by the importin alpha/beta heterodimer. In addition, we suggest that Tpr can serve as a nucleoporin binding site for importin beta during import of importin beta cargo complexes and/or importin beta recycling. Our finding that Tpr bound preferentially to CRM1 in an export complex strengthens the notion that Tpr is involved in protein export.

In vitro dendritic cell infection by pseudotyped adenoviral vectors does not correlate with their in vivo immunogenicity.

Expression of antigens in dendritic cells (DC) can stimulate protective immunity against both viral infection and tumor growth, making them important targets for gene therapy. In-vitro-generated DC are commonly used in gene delivery studies with the assumption that the results will correlate with in vivo activity. Adenovirus Type 5 (Ad5) vectors have been widely used with DC, but these cells lack the primary receptor (CAR) used by Ad5 and are poorly infected. We investigated the use of Ad5 vector particles pseudotyped with fibers from other Ad serotypes in DC targeting. Several fiber proteins, including those from Ad16 (Subgroup B) and Ad37 (Subgroup D), conferred dramatically increased in vitro infection. Surprisingly, neither dendritic cell infection nor the immune response to an Ad-delivered antigen was improved when the modified viruses were tested in vivo. These results underscore the importance of using appropriate animal models in gene delivery studies.