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      Nuclear localization of amyloid-β precursor protein-binding protein Fe65 is dependent on regulated intramembrane proteolysis

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          Abstract

          Fe65 is an adaptor protein involved in both processing and signaling of the Alzheimer-associated amyloid-β precursor protein, APP. Here, the subcellular localization was further investigated using TAP-tagged Fe65 constructs expressed in human neuroblastoma cells. Our results indicate that PTB2 rather than the WW domain is important for the nuclear localization of Fe65. Electrophoretic mobility shift of Fe65 caused by phosphorylation was not detected in the nuclear fraction, suggesting that phosphorylation could restrict nuclear localization of Fe65. Furthermore, both ADAM10 and γ-secretase inhibitors decreased nuclear Fe65 in a similar way indicating an important role also of α-secretase in regulating nuclear translocation.

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          A generic protein purification method for protein complex characterization and proteome exploration.

          We have developed a generic procedure to purify proteins expressed at their natural level under native conditions using a novel tandem affinity purification (TAP) tag. The TAP tag allows the rapid purification of complexes from a relatively small number of cells without prior knowledge of the complex composition, activity, or function. Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a given target protein. The TAP method has been tested in yeast but should be applicable to other cells or organisms.
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            The tandem affinity purification (TAP) method: a general procedure of protein complex purification.

            Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level. Prior knowledge of complex composition or function is not required. The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented. The TAP method was initially developed in yeast but can be successfully adapted to various organisms. Its simplicity, high yield, and wide applicability make the TAP method a very useful procedure for protein purification and proteome exploration. Copyright 2001 Academic Press.
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              Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans.

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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                21 March 2017
                2017
                : 12
                : 3
                : e0173888
                Affiliations
                [001]Stockholm University, Department of Neurochemistry, Stockholm, Sweden
                National Center for Geriatrics and Gerontology, JAPAN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: NAK KI.

                • Data curation: NAK.

                • Formal analysis: NAK EVI.

                • Funding acquisition: NAK KI.

                • Investigation: NAK AKE PKM EVI SB.

                • Methodology: NAK KI.

                • Project administration: NAK KI.

                • Resources: KI.

                • Supervision: NAK KI.

                • Validation: NAK.

                • Visualization: NAK EVI KI.

                • Writing – original draft: NAK KI.

                • Writing – review & editing: NAK AKE PKM EVI SB.

                Author information
                http://orcid.org/0000-0002-0308-1964
                Article
                PONE-D-16-37230
                10.1371/journal.pone.0173888
                5360310
                28323844
                8fd5d83c-4876-4485-9ada-4080289e0eb9
                © 2017 Koistinen et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 16 September 2016
                : 28 February 2017
                Page count
                Figures: 4, Tables: 0, Pages: 14
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100004359, Vetenskapsrådet;
                Award ID: 521-2012-2367
                Award Recipient :
                This work was supported by the Swedish Research Couuncil (to K.I.; 521-2012-2367), and by the Foundation Olle Enkvist Byggmästare (to N.A.K.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Proteins
                Post-Translational Modification
                Phosphorylation
                Biology and Life Sciences
                Biochemistry
                Enzymology
                Enzymes
                Phosphatases
                Biology and Life Sciences
                Biochemistry
                Proteins
                Enzymes
                Phosphatases
                Biology and Life Sciences
                Biochemistry
                Proteins
                Protein Domains
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Gene Mapping
                Restriction Fragment Mapping
                Electrophoretic Mobility Shift Assay
                Research and Analysis Methods
                Molecular Biology Techniques
                Gene Mapping
                Restriction Fragment Mapping
                Electrophoretic Mobility Shift Assay
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cytoplasm
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Mutagenesis and Gene Deletion Techniques
                Deletion Mutagenesis
                Research and Analysis Methods
                Molecular Biology Techniques
                Mutagenesis and Gene Deletion Techniques
                Deletion Mutagenesis
                Research and Analysis Methods
                Separation Processes
                Fractionation
                Subcellular Fractionation
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cell Membranes
                Custom metadata
                All relevant data are within the paper.

                Uncategorized
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