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Using In Vitro Pull-Down and In-Cell Overexpression Assays to Study Protein Interactions with Arrestin.
Perry NA, Zhan X, Gurevich EV, Iverson TM, Gurevich VV
(2019) Methods Mol Biol 1957: 107-120
MeSH Terms: Animals, Arrestin, Biological Assay, COS Cells, Chlorocebus aethiops, HEK293 Cells, Humans, Immobilized Proteins, Mice, Protein Binding, Protein Interaction Mapping, Recombinant Fusion Proteins
Show Abstract · Added April 1, 2019
Nonvisual arrestins (arrestin-2/arrestin-3) interact with hundreds of G protein-coupled receptor (GPCR) subtypes and dozens of non-receptor signaling proteins. Here we describe the methods used to identify the interaction sites of arrestin-binding partners on arrestin-3 and the use of monofunctional individual arrestin-3 elements in cells. Our in vitro pull-down assay with purified proteins demonstrates that relatively few elements in arrestin engage each partner, whereas cell-based functional assays indicate that certain arrestin elements devoid of other functionalities can perform individual functions in living cells.
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12 MeSH Terms
The C-terminal region of A-kinase anchor protein 350 (AKAP350A) enables formation of microtubule-nucleation centers and interacts with pericentriolar proteins.
Kolobova E, Roland JT, Lapierre LA, Williams JA, Mason TA, Goldenring JR
(2017) J Biol Chem 292: 20394-20409
MeSH Terms: A Kinase Anchor Proteins, Biomarkers, Cell Cycle Proteins, Cell Line, Centrosome, Cytoskeletal Proteins, Humans, Imaging, Three-Dimensional, Intracellular Signaling Peptides and Proteins, Luminescent Proteins, Microscopy, Electron, Transmission, Microtubule-Associated Proteins, Microtubule-Organizing Center, Models, Molecular, Nerve Tissue Proteins, Peptide Fragments, Phosphoproteins, Protein Interaction Domains and Motifs, Protein Interaction Mapping, Protein Multimerization, Proteomics, RNA Interference, Recombinant Fusion Proteins, Recombinant Proteins, Two-Hybrid System Techniques
Show Abstract · Added April 3, 2018
Microtubules in animal cells assemble (nucleate) from both the centrosome and the cis-Golgi cisternae. A-kinase anchor protein 350 kDa (AKAP350A, also called AKAP450/CG-NAP/AKAP9) is a large scaffolding protein located at both the centrosome and Golgi apparatus. Previous findings have suggested that AKAP350 is important for microtubule dynamics at both locations, but how this scaffolding protein assembles microtubule nucleation machinery is unclear. Here, we found that overexpression of the C-terminal third of AKAP350A, enhanced GFP-AKAP350A(2691-3907), induces the formation of multiple microtubule-nucleation centers (MTNCs). Nevertheless, these induced MTNCs lacked "true" centriole proteins, such as Cep135. Mapping analysis with AKAP350A truncations demonstrated that AKAP350A contains discrete regions responsible for promoting or inhibiting the formation of multiple MTNCs. Moreover, GFP-AKAP350A(2691-3907) recruited several pericentriolar proteins to MTNCs, including γ-tubulin, pericentrin, Cep68, Cep170, and Cdk5RAP2. Proteomic analysis indicated that Cdk5RAP2 and Cep170 both interact with the microtubule nucleation-promoting region of AKAP350A, whereas Cep68 interacts with the distal C-terminal AKAP350A region. Yeast two-hybrid assays established a direct interaction of Cep170 with AKAP350A. Super-resolution and deconvolution microscopy analyses were performed to define the association of AKAP350A with centrosomes, and these studies disclosed that AKAP350A spans the bridge between centrioles, co-localizing with rootletin and Cep68 in the linker region. siRNA-mediated depletion of AKAP350A caused displacement of both Cep68 and Cep170 from the centrosome. These results suggest that AKAP350A acts as a scaffold for factors involved in microtubule nucleation at the centrosome and coordinates the assembly of protein complexes associating with the intercentriolar bridge.
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25 MeSH Terms
Protocols for Molecular Modeling with Rosetta3 and RosettaScripts.
Bender BJ, Cisneros A, Duran AM, Finn JA, Fu D, Lokits AD, Mueller BK, Sangha AK, Sauer MF, Sevy AM, Sliwoski G, Sheehan JH, DiMaio F, Meiler J, Moretti R
(2016) Biochemistry 55: 4748-63
MeSH Terms: Algorithms, Computational Biology, Internet, Models, Molecular, Protein Binding, Protein Conformation, Protein Folding, Protein Interaction Mapping, Proteins, RNA, Software, User-Computer Interface
Show Abstract · Added April 8, 2017
Previously, we published an article providing an overview of the Rosetta suite of biomacromolecular modeling software and a series of step-by-step tutorials [Kaufmann, K. W., et al. (2010) Biochemistry 49, 2987-2998]. The overwhelming positive response to this publication we received motivates us to here share the next iteration of these tutorials that feature de novo folding, comparative modeling, loop construction, protein docking, small molecule docking, and protein design. This updated and expanded set of tutorials is needed, as since 2010 Rosetta has been fully redesigned into an object-oriented protein modeling program Rosetta3. Notable improvements include a substantially improved energy function, an XML-like language termed "RosettaScripts" for flexibly specifying modeling task, new analysis tools, the addition of the TopologyBroker to control conformational sampling, and support for multiple templates in comparative modeling. Rosetta's ability to model systems with symmetric proteins, membrane proteins, noncanonical amino acids, and RNA has also been greatly expanded and improved.
1 Communities
2 Members
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12 MeSH Terms
XPA: A key scaffold for human nucleotide excision repair.
Sugitani N, Sivley RM, Perry KE, Capra JA, Chazin WJ
(2016) DNA Repair (Amst) 44: 123-135
MeSH Terms: Amino Acid Sequence, Animals, DNA, DNA Damage, DNA Repair, Humans, Models, Molecular, Mutation, Protein Binding, Protein Domains, Protein Interaction Mapping, Protein Structure, Secondary, Severity of Illness Index, Xeroderma Pigmentosum, Xeroderma Pigmentosum Group A Protein
Show Abstract · Added April 18, 2017
Nucleotide excision repair (NER) is essential for removing many types of DNA lesions from the genome, yet the mechanisms of NER in humans remain poorly understood. This review summarizes our current understanding of the structure, biochemistry, interaction partners, mechanisms, and disease-associated mutations of one of the critical NER proteins, XPA.
Copyright © 2016 Elsevier B.V. All rights reserved.
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15 MeSH Terms
A G Protein-biased Designer G Protein-coupled Receptor Useful for Studying the Physiological Relevance of Gq/11-dependent Signaling Pathways.
Hu J, Stern M, Gimenez LE, Wanka L, Zhu L, Rossi M, Meister J, Inoue A, Beck-Sickinger AG, Gurevich VV, Wess J
(2016) J Biol Chem 291: 7809-20
MeSH Terms: Animals, Arrestins, COS Cells, Calcium, Cells, Cultured, Chlorocebus aethiops, GTP-Binding Protein alpha Subunits, Gq-G11, Gene Knockdown Techniques, Glucose, HEK293 Cells, Hepatocytes, Humans, Mice, Inbred C57BL, Protein Interaction Mapping, Protein Interaction Maps, Receptors, G-Protein-Coupled, Signal Transduction, beta-Arrestins
Show Abstract · Added February 15, 2016
Designerreceptorsexclusivelyactivated by adesignerdrug (DREADDs) are clozapine-N-oxide-sensitive designer G protein-coupled receptors (GPCRs) that have emerged as powerful novel chemogenetic tools to study the physiological relevance of GPCR signaling pathways in specific cell types or tissues. Like endogenous GPCRs, clozapine-N-oxide-activated DREADDs do not only activate heterotrimeric G proteins but can also trigger β-arrestin-dependent (G protein-independent) signaling. To dissect the relative physiological relevance of G protein-mediatedversusβ-arrestin-mediated signaling in different cell types or physiological processes, the availability of G protein- and β-arrestin-biased DREADDs would be highly desirable. In this study, we report the development of a mutationally modified version of a non-biased DREADD derived from the M3muscarinic receptor that can activate Gq/11with high efficacy but lacks the ability to interact with β-arrestins. We also demonstrate that this novel DREADD is activein vivoand that cell type-selective expression of this new designer receptor can provide novel insights into the physiological roles of G protein (Gq/11)-dependentversusβ-arrestin-dependent signaling in hepatocytes. Thus, this novel Gq/11-biased DREADD represents a powerful new tool to study the physiological relevance of Gq/11-dependent signaling in distinct tissues and cell types, in the absence of β-arrestin-mediated cellular effects. Such studies should guide the development of novel classes of functionally biased ligands that show high efficacy in various pathophysiological conditions but display a reduced incidence of side effects.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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18 MeSH Terms
Differential role of an NF-κB transcriptional response element in endothelial versus intimal cell VCAM-1 expression.
Milstone DS, Ilyama M, Chen M, O'Donnell P, Davis VM, Plutzky J, Brown JD, Haldar SM, Siu A, Lau AC, Zhu SN, Basheer MF, Collins T, Jongstra-Bilen J, Cybulsky MI
(2015) Circ Res 117: 166-77
MeSH Terms: 5' Untranslated Regions, Animals, Atherosclerosis, Carotid Artery Injuries, Cells, Cultured, Chemotaxis, Leukocyte, Cholesterol, Dietary, E-Selectin, Endothelial Cells, Endothelium, Vascular, Humans, Mice, Mice, Inbred C57BL, Mice, Knockout, Promoter Regions, Genetic, Protein Interaction Mapping, RNA Polymerase II, Receptors, LDL, Response Elements, Transcription Factor RelA, Transcription, Genetic, Tunica Intima, Vascular Cell Adhesion Molecule-1
Show Abstract · Added September 6, 2016
RATIONALE - Human and murine Vcam1 promoters contain 2 adjacent nuclear factor-κB (NF-κB)-binding elements. Both are essential for cytokine-induced transcription of transiently transfected promoter-reporter constructs. However, the relevance of these insights to regulation of the endogenous Vcam1 gene and to pathophysiological processes in vivo remained unknown.
OBJECTIVE - Determine the role of the 5' NF-κB-binding element in expression of the endogenous Vcam1 gene.
METHODS AND RESULTS - Homologous recombination in embryonic stem cells was used to inactivate the 5' NF-κB element in the Vcam1 promoter and alter 3 nucleotides in the 5' untranslated region to allow direct comparison of wild-type versus mutant allele RNA expression and chromatin configuration in heterozygous mice. Systemic treatment with inflammatory cytokines or endotoxin (lipopolysaccharide) induced lower expression of the mutant allele relative to wild-type by endothelial cells in the aorta, heart, and lungs. The mutant allele also showed lower endothelial expression in 2-week atherosclerotic lesions in Vcam1 heterozygous/low-density lipoprotein receptor-deficient mice fed a cholesterol-rich diet. In vivo chromatin immunoprecipitation assays of heart showed diminished lipopolysaccharide-induced association of RNA polymerase 2 and NF-κB p65 with the mutant promoter. In contrast, expression of mutant and wild-type alleles was comparable in intimal cells of wire-injured carotid artery and 4- to 12-week atherosclerotic lesions.
CONCLUSIONS - This study highlights differences between in vivo and in vitro promoter analyses, and reveals a differential role for a NF-κB transcriptional response element in endothelial vascular cell adhesion molecule-1 expression induced by inflammatory cytokines or a cholesterol-rich diet versus intimal cell expression in atherosclerotic lesions and injured arteries.
© 2015 American Heart Association, Inc.
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23 MeSH Terms
Annotation of human cancers with EGFR signaling-associated protein complexes using proximity ligation assays.
Smith MA, Hall R, Fisher K, Haake SM, Khalil F, Schabath MB, Vuaroqueaux V, Fiebig HH, Altiok S, Chen YA, Haura EB
(2015) Sci Signal 8: ra4
MeSH Terms: Animals, Antibodies, Monoclonal, Humanized, Biomarkers, Tumor, Cell Line, Tumor, Cetuximab, ErbB Receptors, GRB2 Adaptor Protein, Heterografts, Humans, Immunoassay, Lung Neoplasms, Mice, Multiprotein Complexes, Protein Interaction Mapping, Signal Transduction
Show Abstract · Added April 18, 2017
Strategies to measure functional signaling-associated protein complexes have the potential to augment current molecular biomarker assays, such as genotyping and expression profiling, used to annotate diseases. Aberrant activation of epidermal growth factor receptor (EGFR) signaling contributes to diverse cancers. We used a proximity ligation assay (PLA) to detect EGFR in a complex with growth factor receptor-bound protein 2 (GRB2), the major signaling adaptor for EGFR. We used multiple lung cancer cell lines to develop and characterize EGFR:GRB2 PLA and correlated this assay with established biochemical measures of EGFR signaling. In a panel of patient-derived xenografts in mice, the intensity of EGFR:GRB2 PLA correlated with the reduction in tumor size in response to the EGFR inhibitor cetuximab. In tumor biopsies from three cohorts of lung cancer patients, positive EGFR:GRB2 PLA was observed in patients with and without EGFR mutations, and the intensity of EGFR:GRB2 PLA was predictive of overall survival in an EGFR inhibitor-treated cohort. Thus, we established the feasibility of using PLA to measure EGFR signaling-associated protein complexes in patient-based materials, suggesting the potential for similar assays for a broader array of receptor tyrosine kinases and other key signaling molecules.
Copyright © 2015, American Association for the Advancement of Science.
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15 MeSH Terms
An efficient fluorescent protein-based multifunctional affinity purification approach in mammalian cells.
Ma H, McLean JR, Gould KL, McCollum D
(2014) Methods Mol Biol 1177: 175-91
MeSH Terms: Animals, Chromatography, Affinity, Cloning, Molecular, Humans, Protein Interaction Mapping, Proteomics, Recombinant Proteins, Tandem Mass Spectrometry
Show Abstract · Added January 20, 2015
Knowledge of an individual protein's modifications, binding partners, and localization is essential for understanding complex biological networks. We recently described a fluorescent protein-based (mVenus) multifunctional affinity purification (MAP) tag that can be used both to purify a given protein and determine its localization (Ma et al., Mol Cell Proteomics 11:501-511, 2012). MAP purified protein complexes can be further analyzed to identify binding partners and posttranslational modifications by LC-MS/MS. The MAP approach offers rapid FACS-selection of stable clonal cell lines based on the expression level/fluorescence of the MAP-protein fusion. The MAP tag is highly efficient and shows little variability between proteins. Here we describe the general MAP purification method in detail, and show how it can be applied to a specific protein using the human Cdc14B phosphatase as an example.
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8 MeSH Terms
NFI transcription factors interact with FOXA1 to regulate prostate-specific gene expression.
Grabowska MM, Elliott AD, DeGraff DJ, Anderson PD, Anumanthan G, Yamashita H, Sun Q, Friedman DB, Hachey DL, Yu X, Sheehan JH, Ahn JM, Raj GV, Piston DW, Gronostajski RM, Matusik RJ
(2014) Mol Endocrinol 28: 949-64
MeSH Terms: Androgen-Binding Protein, Base Sequence, Binding Sites, Consensus Sequence, Enhancer Elements, Genetic, Gene Expression Regulation, HeLa Cells, Hepatocyte Nuclear Factor 3-alpha, Humans, Male, NFI Transcription Factors, Organ Specificity, Promoter Regions, Genetic, Prostate, Protein Binding, Protein Interaction Mapping, Receptors, Androgen, Transcription, Genetic
Show Abstract · Added May 19, 2014
Androgen receptor (AR) action throughout prostate development and in maintenance of the prostatic epithelium is partly controlled by interactions between AR and forkhead box (FOX) transcription factors, particularly FOXA1. We sought to identity additional FOXA1 binding partners that may mediate prostate-specific gene expression. Here we identify the nuclear factor I (NFI) family of transcription factors as novel FOXA1 binding proteins. All four family members (NFIA, NFIB, NFIC, and NFIX) can interact with FOXA1, and knockdown studies in androgen-dependent LNCaP cells determined that modulating expression of NFI family members results in changes in AR target gene expression. This effect is probably mediated by binding of NFI family members to AR target gene promoters, because chromatin immunoprecipitation (ChIP) studies found that NFIB bound to the prostate-specific antigen enhancer. Förster resonance energy transfer studies revealed that FOXA1 is capable of bringing AR and NFIX into proximity, indicating that FOXA1 facilitates the AR and NFI interaction by bridging the complex. To determine the extent to which NFI family members regulate AR/FOXA1 target genes, motif analysis of publicly available data for ChIP followed by sequencing was undertaken. This analysis revealed that 34.4% of peaks bound by AR and FOXA1 contain NFI binding sites. Validation of 8 of these peaks by ChIP revealed that NFI family members can bind 6 of these predicted genomic elements, and 4 of the 8 associated genes undergo gene expression changes as a result of individual NFI knockdown. These observations suggest that NFI regulation of FOXA1/AR action is a frequent event, with individual family members playing distinct roles in AR target gene expression.
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3 Members
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18 MeSH Terms
Integrating genomic, transcriptomic, and interactome data to improve Peptide and protein identification in shotgun proteomics.
Wang X, Zhang B
(2014) J Proteome Res 13: 2715-23
MeSH Terms: Animals, Gene Expression Profiling, Humans, Peptide Mapping, Protein Interaction Mapping, Proteome, Proteomics, Tandem Mass Spectrometry
Show Abstract · Added May 21, 2014
Mass spectrometry (MS)-based shotgun proteomics is an effective technology for global proteome profiling. The ultimate goal is to assign tandem MS spectra to peptides and subsequently infer proteins and their abundance. In addition to database searching and protein assembly algorithms, computational approaches have been developed to integrate genomic, transcriptomic, and interactome information to improve peptide and protein identification. Earlier efforts focus primarily on making databases more comprehensive using publicly available genomic and transcriptomic data. More recently, with the increasing affordability of the Next Generation Sequencing (NGS) technologies, personalized protein databases derived from sample-specific genomic and transcriptomic data have emerged as an attractive strategy. In addition, incorporating interactome data not only improves protein identification but also puts identified proteins into their functional context and thus facilitates data interpretation. In this paper, we survey the major integrative bioinformatics approaches that have been developed during the past decade and discuss their merits and demerits.
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8 MeSH Terms