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      Targeting Nuclear Import Shuttles, Importins/Karyopherins alpha by a Peptide Mimicking the NFκB1/p50 Nuclear Localization Sequence

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          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 β. 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 α. However, selectivity and specificity of NTMs for importins/karyopherins α were undetermined.

          Methods and Results

          We analyzed interaction of the NTM hydrophilic module, N50 peptide, derived from the NLS of NFκB1/p50, with endogenous human importins/karyopherins α to determine the mechanism of NTM modulation of importin α‐mediated nuclear transport. We show that N50 peptide forms stable complexes with multiple importins/karyopherins α. However, only interaction with importin α5 (Imp α5) displayed specific, high‐affinity binding. The 2:1 stoichiometry of the N50‐Imp α5 interaction (K D 1=73 nmol/L, K D 2=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 α5 major NLS binding pocket that may stabilize N50 binding. Imp α5 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 α5, encouraging further refinement of NLS‐derived peptides as new tools to modulate inflammatory disorders.

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          Most cited references44

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          Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes.

          The synthesis of fatty acids and cholesterol, the building blocks of membranes, is regulated by three membrane-bound transcription factors: sterol regulatory element-binding proteins (SREBP)-1a, -1c, and -2. Their function in liver has been characterized in transgenic mice that overexpress each SREBP isoform and in mice that lack all three nuclear SREBPs as a result of gene knockout of SREBP cleavage-activating protein (SCAP), a protein required for nuclear localization of SREBPs. Here, we use oligonucleotide arrays hybridized with RNA from livers of three lines of mice (transgenic for SREBP-1a, transgenic for SREBP-2, and knockout for SCAP) to identify genes that are likely to be direct targets of SREBPs in liver. A total of 1,003 genes showed statistically significant increased expression in livers of transgenic SREBP-1a mice, 505 increased in livers of transgenic SREBP-2 mice, and 343 showed decreased expression in Scap-/- livers. A subset of 33 genes met the stringent combinatorial criteria of induction in both SREBP transgenics and decreased expression in SCAP-deficient mice. Of these 33 genes, 13 were previously identified as direct targets of SREBP action. Of the remaining 20 genes, 13 encode enzymes or carrier proteins involved in cholesterol metabolism, 3 participate in fatty acid metabolism, and 4 have no known connection to lipid metabolism. Through application of stringent combinatorial criteria, the transgenic/knockout approach allows identification of genes whose activities are likely to be controlled directly by one family of transcription factors, in this case the SREBPs.
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            Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway.

            The transcription factor NF-kappa B is sequestered in the cytoplasm by the inhibitor protein I kappa B alpha. Extracellular inducers of NF-kappa B activate signal transduction pathways that result in the phosphorylation and subsequent degradation of I kappa B alpha. At present, the link between phosphorylation of I kappa B alpha and its degradation is not understood. In this report we provide evidence that phosphorylation of serine residues 32 and 36 of I kappa B alpha targets the protein to the ubiquitin-proteasome pathway. I kappa B alpha is ubiquitinated in vivo and in vitro following phosphorylation, and mutations that abolish phosphorylation and degradation of I kappa B alpha in vivo prevent ubiquitination in vitro. Ubiquitinated I kappa B alpha remains associated with NF-kappa B, and the bound I kappa B alpha is degraded by the 26S proteasome. Thus, ubiquitination provides a mechanistic link between phosphorylation and degradation of I kappa B alpha.
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              Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence.

              Point mutagenesis of the nuclear targeting sequence of nucleoplasmin has identified two interdependent basic domains. These are separated by 10 intervening "spacer" amino acids that tolerate point mutations and some insertions. Amino acids in both basic domains are required for nuclear targeting, and the transport defect of a mutation in one domain is amplified by a simultaneous mutation in the other. Therefore, these basic domains are interdependent. A strikingly similar motif of two clusters of basic residues is seen in the nuclear targeting sequence of Xenopus N1. It is also conserved in the related nucleolar protein NO38. Several other short sequences known to be necessary for nuclear targeting fall within a similar motif.
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                Author and article information

                Journal
                J Am Heart Assoc
                J Am Heart Assoc
                ahaoa
                jah3
                Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease
                Blackwell Publishing Ltd
                2047-9980
                October 2013
                25 October 2013
                : 2
                : 5
                : e000386
                Affiliations
                [1 ]Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville, 37232, TN (J.Z., A.A., J.H.)
                [2 ]Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, 37232, TN (J.H.)
                Author notes
                Correspondence to: Jacek Hawiger, MD, PhD, Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, 1161 21st Avenue South, Nashville, TN 37232. E‐mail: jacek.hawiger@ 123456vanderbilt.edu

                Ms. Armitage is currently located at the Northern Virginia Community College, 8333 Little River Turnpike, CS‐116, Annandale, VA.

                Article
                jah3309
                10.1161/JAHA.113.000386
                3835248
                24042087
                b68f82a4-103b-421d-af29-42a27dbdcc6a
                © 2013 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

                This is an Open Access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

                History
                : 21 June 2013
                : 27 August 2013
                Categories
                Original Research
                Vascular Medicine

                Cardiovascular Medicine
                cell‐penetrating peptide,importin,importin α diversity region,karyopherin,nfκb,nuclear import adaptors,nuclear transport,steatohepatitis,transcription factor,vascular inflammation

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