Bifunctional Fusion Proteins of Calmodulin and Protein A as Affinity Ligands in Protein Purification and in the Study of Protein−Protein Interactions

1996 ◽  
Vol 68 (22) ◽  
pp. 3939-3944 ◽  
Author(s):  
Nathaniel G. Hentz ◽  
Sylvia Daunert
Keyword(s):  
Protein Purification ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Protein A ◽  
Protein Protein Interactions ◽  
Affinity Ligands

scholarly journals Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

2006 ◽  
Vol 4 (1) ◽  
pp. nrs.04021 ◽  
Author(s):  
Kristen L. Koterba ◽  
Brian G. Rowan
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Renilla Luciferase ◽  
Receptor Protein ◽  
Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

scholarly journals Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles

2004 ◽  
Vol 167 (6) ◽  
pp. 1087-1098 ◽  
Author(s):  
Rutilio A. Fratti ◽  
Youngsoo Jun ◽  
Alexey J. Merz ◽  
Nathan Margolis ◽  
William Wickner
Keyword(s):  
Membrane Fusion ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Membrane Microdomains ◽  
Rab Gtpase ◽  
Protein Protein Interactions ◽  
Px Domain ◽  
Protein Partitioning ◽  
Membrane Fusion Proteins ◽  
Rab Effector

Membrane microdomains are assembled by lipid partitioning (e.g., rafts) or by protein–protein interactions (e.g., coated vesicles). During docking, yeast vacuoles assemble “vertex” ring-shaped microdomains around the periphery of their apposed membranes. Vertices are selectively enriched in the Rab GTPase Ypt7p, the homotypic fusion and vacuole protein sorting complex (HOPS)–VpsC Rab effector complex, SNAREs, and actin. Membrane fusion initiates at vertex microdomains. We now find that the “regulatory lipids” ergosterol, diacylglycerol and 3- and 4-phosphoinositides accumulate at vertices in a mutually interdependent manner. Regulatory lipids are also required for the vertex enrichment of SNAREs, Ypt7p, and HOPS. Conversely, SNAREs and actin regulate phosphatidylinositol 3-phosphate vertex enrichment. Though the PX domain of the SNARE Vam7p has direct affinity for only 3-phosphoinositides, all the regulatory lipids which are needed for vertex assembly affect Vam7p association with vacuoles. Thus, the assembly of the vacuole vertex ring microdomain arises from interdependent lipid and protein partitioning and binding rather than either lipid partitioning or protein interactions alone.

scholarly journals Detecting Protein-Protein Interactions Using Renilla Luciferase Fusion Proteins

BioTechniques ◽  
2002 ◽  
Vol 33 (5) ◽  
pp. 1044-1050 ◽  
Author(s):  
Peter D. Burbelo ◽  
Adam E. Kisailus ◽  
Jeremy W. Peck
Keyword(s):  
Protein Interactions ◽  
Fusion Proteins ◽  
Renilla Luciferase ◽  
Protein Protein Interactions

scholarly journals A Computational Approach for Protein-Protein Interactions of Bacterial Surface Layer Protein with Human Erb3 and αIIB-β3 Receptors

2021 ◽  
Vol 12 (1) ◽  
pp. 420-430
Keyword(s):  
Surface Layer ◽  
Molecular Docking ◽  
Protein Interactions ◽  
Protein A ◽  
Computational Approach ◽  
Microbial Interactions ◽  
Protein Protein Interactions ◽  
Epithelial Surface ◽  
Surface Receptors ◽  
Surface Layer Protein

Host microbial interactions had significant factors in maintains homeostasis and immune-related activity. One such interaction made by Lactobacillus sp. with Surface layer proteins (Slps) had been studied through a computational approach. Erb3 and αIIB-β3, which are epithelial surface layer receptors, are subjected to interact with the Slp homology model. Both cell surface receptors were subjected to interact through computational docking, followed by molecular dynamics simulations through the coarse-grain method to explore the conformational stability. Through the implementation of the molecular docking for the surface layer protein A, we have shown the surface layer protein A, protein-protein interactions are higher in cellular receptors with epidermal growth factor receptor at an -34.45 ΔG and -51.19 ΔG through molecular docking with Erb3 and αIIB-β3. This study shows the unique interaction of Slp with the epithelial surface receptors like Erb3 and αIIB-β3, which are multipurpose applications in microbial-based drug therapeutics.

Direct Monitoring of the Inhibition of Protein-Protein Interactions in Cells by Translocation of PKCδ Fusion Proteins

2011 ◽  
Vol 50 (6) ◽  
pp. 1314-1317 ◽  
Author(s):  
Kyung-Bok Lee ◽  
Jung Me Hwang ◽  
Insung S. Choi ◽  
Jaerang Rho ◽  
Jong-Soon Choi ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fusion Proteins ◽  
Protein Protein Interactions ◽  
In Cells

MLL Translocation Partners AF4, AF5q31, and ENL Associate in a Higher Order Protein Complex with CDK9 and Cyclin T1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 99-99
Author(s):  
Min Lin ◽  
Michael L. Cleary
Keyword(s):  
Western Blotting ◽  
Protein Interactions ◽  
Protein Complex ◽  
Fusion Proteins ◽  
Higher Order ◽  
Chromosomal Translocations ◽  
Transcriptional Elongation ◽  
Protein Protein Interactions ◽  
Methyltransferase Activity ◽  
Cyclin T1

Abstract The Mixed Lineage Leukemia (MLL) gene is frequently involved in chromosomal translocations that cause acute leukemia. More than 40 different genes have been identified as MLL translocation partners, with the expression of corresponding MLL fusion proteins. The MLL protein has histone methyltransferase activity and is required for embryonic development and hematopoiesis. Several proteins have been demonstrated to associate with MLL in a macromolecular complex, which is believed to have chromatin remodeling function. However, the C-terminal SET domain of MLL, which carries the histone methyltransferase activity, is lost in all MLL fusion proteins, thus making the biochemical functions of the fusion proteins unclear. Moreover, the promiscuity of MLL translocation partners, most of them with no known functions, further complicates an understanding of MLL leukemogenic mechanisms. In this study, we purified a protein complex containing AF4, the most common MLL translocation partner, using a combination of conventional column chromatography and immunoaffinity techniques. The AF4 protein complex contains AF5q31 and ENL, two other MLL translocation partners, as well as CDK9 and Cyclin T1, a heterodimer that regulates transcriptional elongation. Gel filtration confirmed that these five proteins co-fractionate with an estimated overall size of 0.8 MDa. All protein-protein interactions were further confirmed by immunoprecipitation-western blotting from K562 cell nuclear extract. To investigate whether these protein-protein interactions are retained in corresponding MLL fusion proteins, immunoprecipitation-western blotting assays were carried out in human leukemia cell lines harboring MLL chromosomal translocations. We found that MLL-AF4, MLL-AF5q31, MLL-ENL and MLL-AF9 each associate with wild type AF4 complex components, including CDK9 and Cyclin T1. In contrast, MLL-AF6 does not associate with any of the AF4 complex components. We propose that the four nuclear MLL translocation partner proteins (AF4, AF5q31, ENL/AF9), whose translocations are found in over 75% of MLL leukemias, associate in a higher order protein complex with CDK9 and Cyclin T1 and thus function in part to regulate transcriptional elongation. The association of CDK9 and Cyclin T1 with the four MLL fusion proteins suggests a common leukemogenic mechanism that may involve transcriptional elongation, which we are currently investigating. Conversely, MLL-cytosolic fusions, e.g. MLL-AF6, appear to function independently of association with the AF4 protein complex, possibly through a homo-dimerization pathway.

scholarly journals Structure of PDE3A–SLFN12 complex and structure-based design for a potent apoptosis inducer of tumor cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jie Chen ◽  
Nan Liu ◽  
Yinpin Huang ◽  
Yuanxun Wang ◽  
Yuxing Sun ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Catalytic Domain ◽  
Cultured Cells ◽  
Hydrophobic Interactions ◽  
Complex Structure ◽  
Protein A ◽  
Protein Translation ◽  
Protein Protein Interactions ◽  
Cryo Electron Microscopy ◽  
Apoptosis Inducer

AbstractMolecular glues are a class of small molecular drugs that mediate protein-protein interactions, that induce either the degradation or stabilization of target protein. A structurally diverse group of chemicals, including 17-β-estradiol (E2), anagrelide, nauclefine, and DNMDP, induces apoptosis by forming complexes with phosphodiesterase 3A (PDE3A) and Schlafen 12 protein (SLFN12). They do so by binding to the PDE3A enzymatic pocket that allows the compound-bound PDE3A to recruit and stabilize SLFN12, which in turn blocks protein translation, leading to apoptosis. In this work, we report the high-resolution cryo-electron microscopy structure of PDE3A-SLFN12 complexes isolated from cultured HeLa cells pre-treated with either anagrelide, or nauclefine, or DNMDP. The PDE3A-SLFN12 complexes exhibit a butterfly-like shape, forming a heterotetramer with these small molecules, which are packed in a shallow pocket in the catalytic domain of PDE3A. The resulting small molecule-modified interface binds to the short helix (E552-I558) of SLFN12 through hydrophobic interactions, thus “gluing” the two proteins together. Based on the complex structure, we designed and synthesized analogs of anagrelide, a known drug used for the treatment of thrombocytosis, to enhance their interactions with SLFN12, and achieved superior efficacy in inducing apoptosis in cultured cells as well as in tumor xenografts.

scholarly journals ProtFus: A Comprehensive Method Characterizing Protein-Protein Interactions of Fusion Proteins

2019 ◽  
Vol 15 (8) ◽  
pp. e1007239 ◽  
Author(s):  
Somnath Tagore ◽  
Alessandro Gorohovski ◽  
Lars Juhl Jensen ◽  
Milana Frenkel-Morgenstern
Keyword(s):  
Protein Interactions ◽  
Fusion Proteins ◽  
Protein Protein Interactions ◽  
Comprehensive Method
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