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Importin alpha protein acts as a negative regulator for Snail protein nuclear import.

ABSTRACT: Snail, a zinc finger-containing transcriptional regulator, migrates into the nucleus where it controls gene expression. We demonstrated previously that importin ?1 directly recognizes the zinc finger domain of Snail and transports it into the nucleus. Here, using in vitro and in vivo assays, we show that importin ?, an adaptor protein for importin ?1, negatively regulates the nuclear import of Snail mediated by importin ?1. In vitro binding assays indicated that importin ? interacted with the zinc finger domain of Snail to compete with the binding of importin ?1 and that Snail did not form a ternary complex with importin ?/importin ?1. Overexpression of importin ? in A549 cells reduced the endogenous Snail protein level, which was restored by inhibitors of the proteasome and glycogen synthase kinase 3?. Furthermore, knockdown of importin ? by siRNA treatment increased the endogenous Snail protein level in several cancer cell lines. This study provides a novel regulatory mechanism of the nuclear protein import process by importin ? and gives an implication to control Snail activity by inhibiting its nuclear localization.

SUBMITTER: Sekimoto T

PROVIDER: S-EPMC3083189 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Importin alpha protein acts as a negative regulator for Snail protein nuclear import.

Sekimoto Toshihiro T Miyamoto Yoshimi Y Arai Shouko S Yoneda Yoshihiro Y

The Journal of biological chemistry 20110317 17

Snail, a zinc finger-containing transcriptional regulator, migrates into the nucleus where it controls gene expression. We demonstrated previously that importin β1 directly recognizes the zinc finger domain of Snail and transports it into the nucleus. Here, using in vitro and in vivo assays, we show that importin α, an adaptor protein for importin β1, negatively regulates the nuclear import of Snail mediated by importin β1. In vitro binding assays indicated that importin α interacted with the zi ...[more]

PMID: 21454664

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Project description:Human adenoviruses (HAdV) are ubiquitous within the human population and comprise a significant burden of respiratory illnesses worldwide. Pediatric and immunocompromised individuals are at particular risk for developing severe disease; however, no approved antiviral therapies specific to HAdV exist. Ivermectin is an FDA-approved broad-spectrum antiparasitic drug that also exhibits antiviral properties against a diverse range of viruses. Its proposed function is inhibiting the classical protein nuclear import pathway mediated by importin-α (Imp-α) and -β1 (Imp-β1). Many viruses, including HAdV, rely on this host pathway for transport of viral proteins across the nuclear envelope. In this study, we show that ivermectin inhibits HAdV-C5 early gene transcription, early and late protein expression, genome replication, and production of infectious viral progeny. Similarly, ivermectin inhibits genome replication of HAdV-B3, a clinically important pathogen responsible for numerous recent outbreaks. Mechanistically, we show that ivermectin disrupts binding of the viral E1A protein to Imp-α without affecting the interaction between Imp-α and Imp-β1. Our results further extend ivermectin's broad antiviral activity and provide a mechanistic underpinning for its mode of action as an inhibitor of cellular Imp-α/β1-mediated nuclear import.IMPORTANCE Human adenoviruses (HAdVs) represent a ubiquitous and clinically important pathogen without an effective antiviral treatment. HAdV infections typically cause mild symptoms; however, individuals such as children, those with underlying conditions, and those with compromised immune systems can develop severe disseminated disease. Our results demonstrate that ivermectin, an FDA-approved antiparasitic agent, is effective at inhibiting replication of several HAdV types in vitro This is in agreement with the growing body of literature suggesting ivermectin has broad antiviral activity. This study expands our mechanistic knowledge of ivermectin by showing that ivermectin targets the ability of importin-α (Imp-α) to recognize nuclear localization sequences, without effecting the Imp-α/β1 interaction. These data also exemplify the applicability of targeting host factors upon which viruses rely as a viable antiviral strategy.

Project description:Porcine parvovirus (PPV) NS1, the major nonstructural protein of this virus, plays an important role in PPV replication. We show, for the first time, that NS1 dynamically shuttles between the nucleus and cytoplasm, although its subcellular localization is predominantly nuclear. NS1 contains two nuclear export signals (NESs) at amino acids 283 to 291 (designated NES2) and amino acids 602 to 608 (designated NES1). NES1 and NES2 are both functional and transferable NESs, and their nuclear export activity is blocked by leptomycin B (LMB), suggesting that the export of NS1 from the nucleus is dependent upon the chromosome region maintenance 1 (CRM1) pathway. Deletion and site-directed mutational analyses showed that NS1 contains a bipartite nuclear localization signal (NLS) at amino acids 256 to 274. Coimmunoprecipitation assays showed that NS1 interacts with importins α5 and α7 through its NLS. The overexpression of CRM1 and importins α5 and α7 significantly promoted PPV replication, whereas the inhibition of CRM1- and importin α/β-mediated transport by specific inhibitors (LMB, importazole, and ivermectin) clearly blocked PPV replication. The mutant viruses with deletions of the NESs or NLS motif of NS1 by using reverse genetics could not be rescued, suggesting that the NESs and NLS are essential for PPV replication. Collectively, these findings suggest that NS1 shuttles between the nucleus and cytoplasm, mediated by its functional NESs and NLS, via the CRM1-dependent nuclear export pathway and the importin α/β-mediated nuclear import pathway, and PPV proliferation was inhibited by blocking NS1 nuclear import or export. IMPORTANCE PPV replicates in the nucleus, and the nuclear envelope is a barrier to its entry into and egress from the nucleus. PPV NS1 is a nucleus-targeting protein that is important for viral DNA replication. Because the NS1 molecule is large (>50 kDa), it cannot pass through the nuclear pore complex by diffusion alone and requires specific transport receptors to permit its nucleocytoplasmic shuttling. In this study, the two functional NESs in the NS1 protein were identified, and their dependence on the CRM1 pathway for nuclear export was demonstrated. The nuclear import of NS1 utilizes importins α5 and α7 in the importin α/β nuclear import pathway.

Dataset Information

Importin alpha protein acts as a negative regulator for Snail protein nuclear import.

Publications

Importin alpha protein acts as a negative regulator for Snail protein nuclear import.

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