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1.
J Biol Chem ; 289(8): 5109-21, 2014 Feb 21.
Article in English | MEDLINE | ID: mdl-24375443

ABSTRACT

γ-Secretase generates amyloid ß-protein (Aß), a pathogenic molecule in Alzheimer disease, through the intramembrane cleavage of the ß-carboxyl-terminal fragment (ßCTF) of ß-amyloid precursor protein. We previously showed the framework of the γ-secretase cleavage, i.e. the stepwise successive processing of ßCTF at every three (or four) amino acids. However, the membrane integrity of γ-secretase was not taken into consideration because of the use of the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized reconstituted γ-secretase system. Here, we sought to address how the membrane-integrated γ-secretase cleaves ßCTF by using γ-secretase associated with lipid rafts. Quantitative analyses using liquid chromatography-tandem mass spectrometry of the ßCTF transmembrane domain-derived peptides released along with Aß generation revealed that the raft-associated γ-secretase cleaves ßCTF in a stepwise sequential manner, but novel penta- and hexapeptides as well as tri- and tetrapeptides are released. The cropping of these peptides links the two major tripeptide-cleaving pathways generating Aß40 and Aß42 at several points, implying that there are multiple interactive pathways for the stepwise cleavages of ßCTF. It should be noted that Aß38 and Aß43 are generated through three routes, and γ-secretase modulator 1 enhances all the three routes generating Aß38, which results in decreases in Aß42 and Aß43 and an increase in Aß38. These observations indicate that multiple interactive pathways for stepwise successive processing by γ-secretase define the species and quantity of Aß produced.


Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/metabolism , Membrane Microdomains/metabolism , Protein Processing, Post-Translational , Signal Transduction , Amyloid Precursor Protein Secretases/chemistry , Animals , Brain/metabolism , CHO Cells , Cricetinae , Cricetulus , Models, Biological , Oligopeptides/metabolism , Rats , Rats, Wistar , Time Factors
2.
PLoS One ; 7(6): e38878, 2012.
Article in English | MEDLINE | ID: mdl-22761710

ABSTRACT

Despite the introduction of newly developed drugs such as lenalidomide and bortezomib, patients with multiple myeloma are still difficult to treat and have a poor prognosis. In order to find novel drugs that are effective for multiple myeloma, we tested the antitumor activity of 29 phthalimide derivatives against several multiple myeloma cell lines. Among these derivatives, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3- dione (TC11) was found to be a potent inhibitor of tumor cell proliferation and an inducer of apoptosis via activation of caspase-3, 8 and 9. This compound also showed in vivo activity against multiple myeloma cell line KMS34 tumor xenografts in ICR/SCID mice. By means of mRNA display selection on a microfluidic chip, the target protein of TC11 was identified as nucleophosmin 1 (NPM). Binding of TC11 and NPM monomer was confirmed by surface plasmon resonance. Immunofluorescence and NPM knockdown studies in HeLa cells suggested that TC11 inhibits centrosomal clustering by inhibiting the centrosomal-regulatory function of NPM, thereby inducing multipolar mitotic cells, which undergo apoptosis. NPM may become a novel target for development of antitumor drugs active against multiple myeloma.


Subject(s)
Apoptosis , Cell Proliferation , Centrosome/drug effects , Multiple Myeloma/drug therapy , Nuclear Proteins/metabolism , Phthalimides/pharmacology , Amino Acid Sequence , Animals , Blotting, Western , Centrosome/metabolism , Centrosome/pathology , Flow Cytometry , Fluorescent Antibody Technique , Humans , Male , Mice , Mice, Inbred ICR , Mice, SCID , Microfluidics , Molecular Sequence Data , Molecular Structure , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Nucleophosmin , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Surface Plasmon Resonance , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
3.
PLoS One ; 7(1): e30084, 2012.
Article in English | MEDLINE | ID: mdl-22253889

ABSTRACT

The G protein-coupled receptors (GPCRs), which form the largest group of transmembrane proteins involved in signal transduction, are major targets of currently available drugs. Thus, the search for cognate and surrogate peptide ligands for GPCRs is of both basic and therapeutic interest. Here we describe the application of an in vitro DNA display technology to screening libraries of peptide ligands for full-length GPCRs expressed on whole cells. We used human angiotensin II (Ang II) type-1 receptor (hAT1R) as a model GPCR. Under improved selection conditions using hAT1R-expressing Chinese hamster ovary (CHO)-K1 cells as bait, we confirmed that Ang II gene could be enriched more than 10,000-fold after four rounds of selection. Further, we successfully selected diverse Ang II-like peptides from randomized peptide libraries. The results provide more precise information on the sequence-function relationships of hAT1R ligands than can be obtained by conventional alanine-scanning mutagenesis. Completely in vitro DNA display can overcome the limitations of current display technologies and is expected to prove widely useful for screening diverse libraries of mutant peptide and protein ligands for receptors that can be expressed functionally on the surface of CHO-K1 cells.


Subject(s)
DNA/metabolism , Peptide Library , Peptides/metabolism , Receptor, Angiotensin, Type 1/metabolism , Amino Acid Sequence , Angiotensin II/chemistry , Angiotensin II/genetics , Animals , Base Sequence , Binding, Competitive , CHO Cells , Calcium/metabolism , Cricetinae , Cricetulus , Humans , Ligands , Molecular Sequence Data , Peptides/chemistry , Recombinant Proteins/metabolism
4.
FASEB J ; 24(7): 2201-10, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20181936

ABSTRACT

Bcl-X(L), an antiapoptotic member of the Bcl-2 family, is a mitochondrial protein that inhibits activation of Bax and Bak, which commit the cell to apoptosis, and it therefore represents a potential target for drug discovery. Peptides have potential as therapeutic molecules because they can be designed to engage a larger portion of the target protein with higher specificity. In the present study, we selected 16-mer peptides that interact with Bcl-X(L) from random and degenerate peptide libraries using mRNA display. The selected peptides have sequence similarity with the Bcl-2 family BH3 domains, and one of them has higher affinity (IC(50)=0.9 microM) than Bak BH3 (IC(50)=11.8 microM) for Bcl-X(L) in vitro. We also found that GFP fusions of the selected peptides specifically interact with Bcl-X(L), localize in mitochondria, and induce cell death. Further, a chimeric molecule, in which the BH3 domain of Bak protein was replaced with a selected peptide, retained the ability to bind specifically to Bcl-X(L). These results demonstrate that this selected peptide specifically antagonizes the function of Bcl-X(L) and overcomes the effects of Bcl-X(L) in intact cells. We suggest that mRNA display is a powerful technique to identify peptide inhibitors with high affinity and specificity for disease-related proteins.


Subject(s)
Peptides/pharmacology , bcl-X Protein/antagonists & inhibitors , Cell Death/drug effects , Gene Library , Humans , Mitochondria , Peptide Library , Peptides/metabolism , Protein Binding , RNA, Messenger , bcl-X Protein/genetics , bcl-X Protein/metabolism
5.
J Biol Chem ; 284(1): 478-485, 2009 Jan 02.
Article in English | MEDLINE | ID: mdl-19001369

ABSTRACT

The importin alpha/beta pathway mediates nuclear import of proteins containing the classical nuclear localization signals (NLSs). Although the consensus sequences of the classical NLSs have been defined, there are still many NLSs that do not match the consensus rule and many nonfunctional sequences that match the consensus. We report here six different NLS classes that specifically bind to distinct binding pockets of importin alpha. By screening of random peptide libraries using an mRNA display, we selected peptides bound by importin alpha and identified six classes of NLSs, including three novel classes. Two noncanonical classes (class 3 and class 4) specifically bound the minor binding pocket of importin alpha, whereas the classical monopartite NLSs (class 1 and class 2) bound to the major binding pocket. Using a newly developed universal green fluorescent protein expression system, we found that these NLS classes, including plant-specific class 5 NLSs and bipartite NLSs, fundamentally require the regions outside the core basic residues for their activity and have specific residues or patterns that confer the activities differently between yeast, plants, and mammals. Furthermore, amino acid replacement analyses revealed that the consensus basic patterns of the classical NLSs are not essential for activity, thereby generating more unconventional patterns, including redox-sensitive NLSs. These results explain the causes of the NLS diversity. The defined consensus patterns and properties of importin alpha-dependent NLSs provide useful information for identifying NLSs.


Subject(s)
Nicotiana/metabolism , Nuclear Localization Signals/metabolism , Plant Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , alpha Karyopherins/metabolism , Animals , Gene Expression Profiling/methods , HeLa Cells , Humans , Mice , NIH 3T3 Cells , Nuclear Localization Signals/classification , Nuclear Localization Signals/genetics , Plant Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Nicotiana/genetics , alpha Karyopherins/genetics
6.
Nucleic Acids Res ; 32(21): e169, 2004 Dec 02.
Article in English | MEDLINE | ID: mdl-15576676

ABSTRACT

Although yeast two-hybrid assay and biochemical methods combined with mass spectrometry have been successfully employed for the analyses of protein-protein interactions in the field of proteomics, these methods encounter various difficulties arising from the usage of living cells, including inability to analyze toxic proteins and restriction of testable interaction conditions. Totally in vitro display technologies such as ribosome display and mRNA display are expected to circumvent these difficulties. In this study, we applied an mRNA display technique to screening for interactions of a basic leucine zipper domain of Jun protein in a mouse brain cDNA library. By performing iterative affinity selection and sequence analyses, we selected 16 novel Jun-associated protein candidates in addition to four known interactors. By means of real-time PCR and pull-down assay, 10 of the 16 newly discovered candidates were confirmed to be direct interactors with Jun in vitro. Furthermore, interaction of 6 of the 10 proteins with Jun was observed in cultured cells by means of co-immunoprecipitation and observation of subcellular localization. These results demonstrate that this in vitro display technology is effective for the discovery of novel protein-protein interactions and can contribute to the comprehensive mapping of protein-protein interactions.


Subject(s)
Protein Biosynthesis , Protein Interaction Mapping/methods , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , Animals , Cell Line , Gene Library , Immunoprecipitation , Leucine Zippers , Mice , Polymerase Chain Reaction , Proto-Oncogene Proteins c-jun/chemistry , RNA, Messenger/metabolism , Transcription, Genetic
7.
Nucleic Acids Res ; 32(12): e95, 2004 Jul 06.
Article in English | MEDLINE | ID: mdl-15247328

ABSTRACT

Restriction endonucleases are widely used in laboratory applications from recombinant DNA technology to diagnostics, but engineering of restriction enzymes by structure-guided design and in vivo directed evolution is at an early stage. Here, we report the use of an in vitro compartmentalization system for completely in vitro selection of restriction enzymes. Compartmentalization of a single gene in a rabbit reticulocyte in vitro transcription/translation system serves to isolate individually synthesized enzymes from each other. In each compartment, an active enzyme cleaves only its own encoding gene, whereas genes encoding inactive enzymes remain intact. Affinity selection of the cleaved DNA encoding active restriction endonucleases was accomplished by the use of streptavidin-immobilized beads and dUTP-biotin, which was efficiently incorporated into the cohesive end of the cleaved DNA using a DNA polymerase. We confirmed that genes encoding active restriction endonuclease FokI could be selected from a randomized library. This method overcomes the limitations of current in vivo technologies and should prove useful for rapid screening and evolution of novel restriction enzymes from diverse mutant libraries, as well as for studies of catalytic and evolutionary mechanisms of restriction enzymes.


Subject(s)
DNA Restriction Enzymes/genetics , Directed Molecular Evolution/methods , Amino Acid Sequence , Base Sequence , Catalysis , Cloning, Molecular , Deoxyribonucleases, Type II Site-Specific/chemistry , Deoxyribonucleases, Type II Site-Specific/genetics , Deoxyribonucleases, Type II Site-Specific/metabolism , Gene Library , Models, Molecular , Molecular Sequence Data , Mutation
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