Supra-blot: an accurate and reliable assay for detecting target proteins with a synthetic host molecule–enzyme hybrid

2020 ◽  
Vol 56 (10) ◽  
pp. 1549-1552 ◽  
Author(s):  
Gihyun Sung ◽  
Song-Yi Lee ◽  
Myeong-Gyun Kang ◽  
Kyung Lock Kim ◽  
Jaeyeon An ◽  
...  
Keyword(s):  
Biological Samples ◽  
Mammalian Cells ◽  
Host Molecule ◽  
Target Proteins ◽  
High Affinity

A new way to detect target proteins is developed using a high-affinity host–guest interaction for a wide variety of biological samples including bacteria and mammalian cells.

scholarly journals Molecular cloning, expression, and mapping of the high affinity actin-capping domain of chicken cardiac tensin.

1995 ◽  
Vol 128 (6) ◽  
pp. 1095-1109 ◽  
Author(s):  
J Z Chuang ◽  
D C Lin ◽  
S Lin
Keyword(s):  
Mammalian Cells ◽  
Actin Polymerization ◽  
Critical Concentration ◽  
Focal Adhesions ◽  
Inhibitory Effect ◽  
Complete Inhibition ◽  
Fusion Construct ◽  
Chicken Gizzard ◽  
High Affinity ◽  
Actin Capping

Tensin, an actin filament capping protein first purified from chicken gizzard, is localized to various types of adherens junctions in muscle and nonmuscle cells. In this paper, we describe the isolation and sequencing of tensin cDNA from a chicken cardiac library. The 6.3-kb chicken cardiac tensin cDNA encodes an open reading frame of 1,792 amino acids. Mammalian cells transfected with the chicken tensin cDNA expressed a polypeptide of approximately 200 kD recognizable by antibodies to chicken gizzard tensin. The expressed protein was incorporated into focal adhesions and other actin-containing structures in the transfected cells. To map the domain associated with tensin's high affinity, barbed-end F-actin-capping activity, bacterially expressed recombinant fusion proteins containing various segments of tensin were prepared and assayed for activity. The results of these experiments show that the high affinity capping domain (kD = 1.3 nM) lies within amino acid residues R1037-V1169. Additional studies on a shorter construct, S1061-H1145, showed that these 85 residues were sufficient for producing complete inhibition of actin polymerization and depolymerization. While this active domain is located within that of the "insertin" sequence (Weigt, C., A. Gaertner, A. Wegner, H. Korte, and H. E. Meyer. 1992. J. Mol. Biol. 227:593-595), our data showing complete inhibition of polymerization and shift in critical concentration are consistent with a simple barbed-end capping mechanism rather than the "insertin model." Our results also differ from those of a recent report (Lo, S. H., P. A. Janmey, J. H. Hartwig, and L. B. Chen. 1994. J. Cell Biol. 125:1067-1075), which concluded that their recombinant tensin has an "insertin-like" inhibitory effect on barbed-end actin polymerization, and that this activity is attributed to residues T936-R1037 (residues 888-989 in their numbering system). In our study, a fusion construct (N790-K1060) encompassing T936-R1037 had no significant effect on actin polymerization and depolymerization, even at high concentrations.

scholarly journals Conserved Mode of Interaction between Yeast Bro1 Family V Domains and YP(X) n L Motif-Containing Target Proteins

2015 ◽  
Vol 14 (10) ◽  
pp. 976-982 ◽  
Author(s):  
Yoko Kimura ◽  
Mirai Tanigawa ◽  
Junko Kawawaki ◽  
Kenji Takagi ◽  
Tsunehiro Mizushima ◽  
...  
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Mammalian Cells ◽  
Tyrosine Phosphatase ◽  
Binding Specificity ◽  
Target Protein ◽  
Target Proteins ◽  
Protein Tyrosine ◽  
Hydrophobic Groove ◽  
Domain Protein ◽  
Common Architecture

ABSTRACT Yeast Bro1 and Rim20 belong to a family of proteins which possess a common architecture of Bro1 and V domains. Alix and His domain protein tyrosine phosphatase (HD-PTP), mammalian Bro1 family proteins, bind YP(X) n L ( n = 1 to 3) motifs in their target proteins through their V domains. In Alix, the Phe residue, which is located in the hydrophobic groove of the V domain, is critical for binding to the YP(X) n L motif. Although the overall sequences are not highly conserved between mammalian and yeast V domains, we show that the conserved Phe residue in the yeast Bro1 V domain is important for binding to its YP(X) n L-containing target protein, Rfu1. Furthermore, we show that Rim20 binds to its target protein Rim101 through the interaction between the V domain of Rim20 and the YPIKL motif of Rim101. The mutation of either the critical Phe residue in the Rim20 V domain or the YPIKL motif of Rim101 affected the Rim20-mediated processing of Rim101. These results suggest that the interactions between V domains and YP(X) n L motif-containing proteins are conserved from yeast to mammalian cells. Moreover, the specificities of each V domain to their target protein suggest that unidentified elements determine the binding specificity.

scholarly journals A novel mass assay to quantify the bioactive lipid PtdIns3P in various biological samples

2012 ◽  
Vol 447 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Gaëtan Chicanne ◽  
Sonia Severin ◽  
Cécile Boscheron ◽  
Anne-Dominique Terrisse ◽  
Marie-Pierre Gratacap ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Liquid Chromatography ◽  
Biological Samples ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Human Platelets ◽  
Bhk Cells ◽  
Early Endosomes ◽  
Important Player ◽  
Mass Level

PtdIns3P is recognized as an important player in the control of the endocytotic pathway and in autophagy. Recent data also suggest that PtdIns3P contributes to molecular mechanisms taking place at the plasma membrane and at the midbody during cytokinesis. This lipid is present in low amounts in mammalian cells and remains difficult to quantify either by traditional techniques based on radiolabelling followed by HPLC to separate the different phosphatidylinositol monophosphates, or by high-sensitive liquid chromatography coupled to MS, which is still under development. In the present study, we describe a mass assay to quantify this lipid from various biological samples using the recombinant PtdIns3P 5-kinase, PIKfyve. Using this assay, we show an increase in the mass level of PtdIns3P in mouse and human platelets following stimulation, loss of this lipid in Vps34-deficient yeasts and its relative enrichment in early endosomes isolated from BHK cells.

scholarly journals A heparin-binding activity on Leishmania amastigotes which mediates adhesion to cellular proteoglycans.

1993 ◽  
Vol 123 (3) ◽  
pp. 759-766 ◽  
Author(s):  
D C Love ◽  
J D Esko ◽  
D M Mosser
Keyword(s):  
Heparan Sulfate ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Binding Activity ◽  
Heparan Sulfate Proteoglycans ◽  
Heparin Binding ◽  
Wild Type ◽  
Amastigote Form ◽  
High Affinity ◽  
Leishmania Amastigotes

The intracellular amastigote form of leishmania is responsible for the cell-to-cell spread of leishmania infection in the mammalian host. In this report, we identify a high-affinity, heparin-binding activity on the surface of the amastigote form of leishmania. Amastigotes of Leishmania amazonensis bound approximately 120,000 molecules of heparin per cell, with a Kd of 8.8 x 10(-8) M. This heparin-binding activity mediates the adhesion of amastigotes to mammalian cells via heparan sulfate proteoglycans, which are expressed on the surface of mammalian cells. Amastigotes bound efficiently to a variety of adherent cells which express cell-surface proteoglycans. Unlike wild-type CHO cells, which bound amastigotes avidly, CHO cells with genetic deficiencies in heparan sulfate proteoglycan biosynthesis or cells treated with heparitinase failed to bind amastigotes even at high parasite-input dosages. Cells which express normal levels of undersulfated heparan bound amastigotes nearly as efficiently as did wild-type cells. The adhesion of amastigotes to wild-type nonmyeloid cells was almost completely inhibited by the addition of micromolar amounts of soluble heparin or heparan sulfate but not by the addition of other sulfated polysaccharides.l Binding of amastigotes to macrophages, however, was inhibited by only 60% after pretreatment of amastigotes with heparin, suggesting that macrophages have an additional mechanism for recognizing amastigotes. These results suggest that leishmania amastigotes express a high-affinity, heparin-binding activity on their surface which can interact with heparan sulfate proteoglycans on mammalian cells. This interaction may represent an important first step in the invasion of host cells by amastigotes.

scholarly journals A Chimera of Interleukin 2 and a Binding Variant of Aerolysin Is Selectively Toxic to Cells Displaying the Interleukin 2 Receptor

2007 ◽  
Vol 283 (3) ◽  
pp. 1572-1579 ◽  
Author(s):  
Milan Osusky ◽  
Lisa Teschke ◽  
Xiaoying Wang ◽  
Kevin Wong ◽  
J. Thomas Buckley
Keyword(s):  
Cell Death ◽  
Mammalian Cells ◽  
Therapeutic Agent ◽  
Interleukin 2 ◽  
Bacterial Toxin ◽  
Hybrid Protein ◽  
High Affinity ◽  
Interleukin 2 Receptor ◽  
Hybrid Molecules ◽  
N Terminus

Aerolysin is a bacterial toxin that binds to glycosylphosphatidylinositol-anchored proteins (GPI-AP) on mammalian cells and oligomerizes, inserting into the target membranes and forming channels that cause cell death. We have made a variant of aerolysin, R336A, that has greatly reduced the ability to bind to GPI-AP, and as a result it is only very weakly active. Fusion of interleukin 2 (IL2) to the N terminus of R336A-aerolysin results in a hybrid that has little or no activity against cells that do not have an IL2 receptor because it cannot bind to the GPI-AP on the cells. Strikingly, the presence of the IL2 moiety allows this hybrid to bind to cells displaying high affinity IL2 receptors. Once bound, the hybrid molecules form insertion-competent oligomers. Cell death occurs at picomolar concentrations of the hybrid, whereas the same cells are insensitive to much higher concentrations of R336A-aerolysin lacking the IL2 domain. The targeted channel-forming hybrid protein may have important advantages as a therapeutic agent.

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