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Targeting nuclear import shuttles, importins/karyopherins alpha by a peptide mimicking the NF?B1/p50 nuclear localization sequence.

ABSTRACT: BACKGROUND:We recently reported that a bifunctional nuclear transport modifier (NTM), cSN50.1 peptide, reduced atherosclerosis, plasma cholesterol, triglycerides, and glucose along with liver fat and inflammatory markers, in a murine model of familial hypercholesterolemia. We determined that cSN50.1 improved lipid homeostasis by modulating nuclear transport of sterol regulatory element-binding proteins through interaction with importin ?. Previous studies established that cSN50.1 and related NTMs also modulate nuclear transport of proinflammatory transcription factors mediated by binding of their nuclear localization sequences (NLSs) to importins/karyopherins ?. However, selectivity and specificity of NTMs for importins/karyopherins ? were undetermined. METHODS AND RESULTS:We analyzed interaction of the NTM hydrophilic module, N50 peptide, derived from the NLS of NF?B1/p50, with endogenous human importins/karyopherins ? to determine the mechanism of NTM modulation of importin ?-mediated nuclear transport. We show that N50 peptide forms stable complexes with multiple importins/karyopherins ?. However, only interaction with importin ?5 (Imp ?5) displayed specific, high-affinity binding. The 2:1 stoichiometry of the N50-Imp ?5 interaction (KD1 = 73 nmol/L, KD2 = 140 nmol/L) indicated occupancy of both major and minor NLS binding pockets. Utilizing in silico 3-dimensional (3-D) docking models and comparative structural analysis, we identified a structural component of the Imp ?5 major NLS binding pocket that may stabilize N50 binding. Imp ?5 also displayed rapid stimulus-induced turnover, which could influence its availability for nuclear transport during the inflammatory response. CONCLUSIONS:These results provide direct evidence that N50 peptide selectively targets Imp ?5, encouraging further refinement of NLS-derived peptides as new tools to modulate inflammatory disorders.

SUBMITTER: Zienkiewicz J

PROVIDER: S-EPMC3835248 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Targeting nuclear import shuttles, importins/karyopherins alpha by a peptide mimicking the NFκB1/p50 nuclear localization sequence.

Zienkiewicz Jozef J Armitage Amy A Hawiger Jacek J

Journal of the American Heart Association 20130916 5

<h4>Background</h4>We recently reported that a bifunctional nuclear transport modifier (NTM), cSN50.1 peptide, reduced atherosclerosis, plasma cholesterol, triglycerides, and glucose along with liver fat and inflammatory markers, in a murine model of familial hypercholesterolemia. We determined that cSN50.1 improved lipid homeostasis by modulating nuclear transport of sterol regulatory element-binding proteins through interaction with importin β. Previous studies established that cSN50.1 and rel ...[more]

PMID: 24042087

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Project description:Histone deacetylase 5 (HDAC5), a class IIa deacetylase, is a prominent regulator of cellular and epigenetic processes that underlie the progression of human disease, ranging from cardiac hypertrophy to cancer. Although it is established that phosphorylation mediates 14-3-3 protein binding and provides the essential link between HDAC5 nucleo-cytoplasmic shuttling and transcriptional repression, thus far only four phospho-acceptor sites have been functionally characterized. Here, using a combinatorial proteomics approach and phosphomutant screening, we present the first evidence that HDAC5 has at least 17 in vivo phosphorylation sites within functional domains, including Ser278 and Ser279 within the nuclear localization signal (NLS), Ser1108 within the nuclear export signal, and Ser755 in deacetylase domain. Global and targeted MS/MS analyses of NLS peptides demonstrated the presence of single (Ser278 and Ser279) and double (Ser278/Ser279) phosphorylations. The double S278/279A mutation showed reduced association with HDAC3, slightly decreased deacetylation activity, and significantly increased cytoplasmic localization compared with wild type HDAC5, whereas the S278A and S1108A phosphomutants were not altered. Live cell imaging revealed a deficiency in nuclear import of S278/279A HDAC5. Phosphomutant stable cell lines confirmed the cellular redistribution of NLS mutants and revealed a more pronounced cytoplasmic localization for the single S279A mutant. Proteomic analysis of immunoisolated S278/279A, S279A, and S259/498A mutants linked altered cellular localization to changes in protein interactions. S278/279A and S279A HDAC5 showed reduced association with the NCoR-HDAC3 nuclear corepressor complex as well as protein kinase D enzymes, which were potentiated in the S259/498A mutant. These results provide the first link between phosphorylation outside the known 14-3-3 sites and downstream changes in protein interactions. Together these studies identify Ser279 as a critical phosphorylation within the NLS involved in the nuclear import of HDAC5, providing a regulatory point in nucleo-cytoplasmic shuttling that may be conserved in other class IIa HDACs-HDAC4 and HDAC9.

Dataset Information

Targeting nuclear import shuttles, importins/karyopherins alpha by a peptide mimicking the NF?B1/p50 nuclear localization sequence.

Publications

Targeting nuclear import shuttles, importins/karyopherins alpha by a peptide mimicking the NFκB1/p50 nuclear localization sequence.

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