Cell growth selection system to detect extracellular and transmembrane protein interactions

2003 ◽  
Vol 1622 (2) ◽  
pp. 117-127 ◽  
Author(s):  
David M. Urech ◽  
Peter Lichtlen ◽  
Alcide Barberis
Keyword(s):  
Cell Growth ◽  
Protein Interactions ◽  
Transmembrane Protein ◽  
Selection System ◽  
Growth Selection

scholarly journals Identification of Novel Benzoylformate Decarboxylases by Growth Selection

2006 ◽  
Vol 72 (12) ◽  
pp. 7510-7517 ◽  
Author(s):  
Helge Henning ◽  
Christian Leggewie ◽  
Martina Pohl ◽  
Michael Müller ◽  
Thorsten Eggert ◽  
...  
Keyword(s):  
Environmental Dna ◽  
Selective Medium ◽  
The Novel ◽  
Selection System ◽  
Dna Library ◽  
Benzoylformate Decarboxylase ◽  
Growth Deficiency ◽  
Growth Selection ◽  
Genes Encoding ◽  
Encoding Genes

ABSTRACT A growth selection system was established using Pseudomonas putida, which can grow on benzaldehyde as the sole carbon source. These bacteria presumably metabolize benzaldehyde via the β-ketoadipate pathway and were unable to grow in benzoylformate-containing selective medium, but the growth deficiency could be restored by expression in trans of genes encoding benzoylformate decarboxylases. The selection system was used to identify three novel benzoylformate decarboxylases, two of them originating from a chromosomal library of P. putida ATCC 12633 and the third from an environmental-DNA library. The novel P. putida enzymes BfdB and BfdC exhibited 83% homology to the benzoylformate decarboxylase from P. aeruginosa and 63% to the enzyme MdlC from P. putida ATCC 12633, whereas the metagenomic BfdM exhibited 72% homology to a putative benzoylformate decarboxylase from Polaromonas naphthalenivorans. BfdC was overexpressed in Escherichia coli, and the enzymatic activity was determined to be 22 U/ml using benzoylformate as the substrate. Our results clearly demonstrate that P. putida KT2440 is an appropriate selection host strain suitable to identify novel benzoylformate decarboxylase-encoding genes. In principle, this system is also applicable to identify a broad range of different industrially important enzymes, such as benzaldehyde lyases, benzoylformate decarboxylases, and hydroxynitrile lyases, which all catalyze the formation of benzaldehyde.

Live-cell visualization of transmembrane protein oligomerization and membrane fusion using two-fragment haptoEGFP methodology

2012 ◽  
Vol 32 (3) ◽  
pp. 333-343 ◽  
Author(s):  
Derek J. Quinn ◽  
Neil V. McFerran ◽  
John Nelson ◽  
W. Paul Duprex
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
Transmembrane Protein ◽  
Bimolecular Fluorescence Complementation ◽  
Type I ◽  
Protein Oligomerization ◽  
Fluorescent Signal ◽  
Enveloped Virus

Protein interactions play key roles throughout all subcellular compartments. In the present paper, we report the visualization of protein interactions throughout living mammalian cells using two oligomerizing MV (measles virus) transmembrane glycoproteins, the H (haemagglutinin) and the F (fusion) glycoproteins, which mediate MV entry into permissive cells. BiFC (bimolecular fluorescence complementation) has been used to examine the dimerization of these viral glycoproteins. The H glycoprotein is a type II membrane-receptor-binding homodimeric glycoprotein and the F glycoprotein is a type I disulfide-linked membrane glycoprotein which homotrimerizes. Together they co-operate to allow the enveloped virus to enter a cell by fusing the viral and cellular membranes. We generated a pair of chimaeric H glycoproteins linked to complementary fragments of EGFP (enhanced green fluorescent protein) – haptoEGFPs – which, on association, generate fluorescence. Homodimerization of H glycoproteins specifically drives this association, leading to the generation of a fluorescent signal in the ER (endoplasmic reticulum), the Golgi and at the plasma membrane. Similarly, the generation of a pair of corresponding F glycoprotein–haptoEGFP chimaeras also produced a comparable fluorescent signal. Co-expression of H and F glycoprotein chimaeras linked to complementary haptoEGFPs led to the formation of fluorescent fusion complexes at the cell surface which retained their biological activity as evidenced by cell-to-cell fusion.

On the role of junctin in cardiac Ca2+ handling, contractility, and heart failure

2007 ◽  
Vol 293 (1) ◽  
pp. H728-H734 ◽  
Author(s):  
Ulrich Gergs ◽  
Tobias Berndt ◽  
Jan Buskase ◽  
Larry R. Jones ◽  
Uwe Kirchhefer ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Myocytes ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Recombinant Adenovirus ◽  
Transmembrane Protein ◽  
Compensatory Mechanism ◽  
Human Heart Failure ◽  
Release Channel

Junctin is a transmembrane protein located at the cardiac junctional sarcoplasmic reticulum (SR) and forms a quaternary complex with the Ca2+ release channel, triadin and calsequestrin. Impaired protein interactions within this complex may alter the Ca2+ sensitivity of the Ca2+ release channel and may lead to cardiac dysfunction, including hypertrophy, depressed contractility, and abnormal Ca2+ transients. To study the expression of junctin and, for comparison, triadin, in heart failure, we measured the levels of these proteins in SR from normal and failing human hearts. Junctin was below our level of detection in SR membranes from failing human hearts, and triadin was downregulated by 22%. To better understand the role of junctin in the regulation of Ca2+ homeostasis and contraction of cardiac myocytes, we used an adenoviral approach to overexpress junctin in isolated rat cardiac myocytes. A recombinant adenovirus encoding the green fluorescent protein served as a control. Infection of myocytes with the junctin-expressing virus resulted in an increased RNA and protein expression of junctin. Ca2+ transients showed a decreased maximum Ca2+ amplitude, and contractility of myocytes was depressed. Our results demonstrate that an increased expression of junctin is associated with an impaired Ca2+ homeostasis. Downregulation of junctin in human heart failure may thus be a compensatory mechanism.

scholarly journals Mapping transmembrane binding partners for E-cadherin ectodomains

2020 ◽  
Author(s):  
Omer Shafraz ◽  
Bin Xie ◽  
Soichiro Yamada ◽  
Sanjeevi Sivasankar
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Transmembrane Protein ◽  
Binding Assays ◽  
Binding Interactions ◽  
Adhesion Protein ◽  
Bait Protein ◽  
Binding Partners ◽  
Extracellular Region ◽  
E Cadherin

ABSTRACTWe combine proximity labeling and single molecule binding assays, to discover novel transmembrane protein interactions in cells. We first screen for candidate binding partners by tagging the extracellular and cytoplasmic regions of a bait protein with TurboID biotin ligase, and identify proximal proteins that are biotin-tagged on both their extracellular and intracellular regions. We then test direct binding interactions between the proximal proteins and the bait, using single molecule Atomic Force Microscope binding assays. Using this approach, we identify novel binding partners for the extracellular region of E-cadherin, an essential cell-cell adhesion protein. We show that the desmosomal proteins desmoglein-2 and desmocollin-3, the focal adhesion protein integrin-α2β1, and the receptor tyrosine kinase ligand ephrin-B1, all directly interact with E-cadherin ectodomains. Our discovery of previously unknown heterophilic E-cadherin binding interactions, suggest the existence of novel cadherin cross-talk in epithelial cells.

scholarly journals Evaluation of the Phosphomannose Isomerase-Based Selection System for Genetic Transformation of Sweetpotato

2015 ◽  
Vol 9 (3) ◽  
pp. 46-53 ◽  
Author(s):  
Wilton Mbinda ◽  
Sylvia Nawiri ◽  
Makenzi Nzaro ◽  
Benson Kinyagia ◽  
Allan Mgutu ◽  
...  
Keyword(s):  
Cell Growth ◽  
Genetic Transformation ◽  
Life Sciences ◽  
Stem Explants ◽  
Selection System ◽  
Phosphomannose Isomerase ◽  
Public Concern ◽  
Effective Selection ◽  
Minimal Concentration ◽  
Selection Agent

In response to increased public concern on antibiotic or herbicide resistance genes usage in genetically modified plants, mannose was evaluated as selectable agent for the genetic transformation of sweetpotato. Nontransformed sweetpotato stem explants of cv. KSP36 were cultured on media containing various combinations and concentrations sucrose and mannose ranging from 0 to 30 g/l. Likewise, efficacy of hygromycin and kanamycin as selection agents for transformation of sweetpotato was evaluated on media containing varying concentrations of antibiotics ranging 0 to 100 mg/l. Selection agent effectiveness was determined by detecting the minimal concentration of the selection agent required to fully inhibit sweetpotato calli growth. Hygromycin was the most effective selection agent as it inhibited cell growth at concentrations above 20 mg/l. Kanamycin was moderately effective as it inhibited cell growth at 60 mg/l. Sweetpotato calli were able to grow and even produce embryos even when mannose was the only source of carbohydrates.DOI: http://dx.doi.org/10.3126/ijls.v9i3.12466 International Journal of Life Sciences 9 (3): 2015; 46-53

scholarly journals Atg9 antagonizes TOR signaling to regulate intestinal cell growth and epithelial homeostasis in Drosophila

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jung-Kun Wen ◽  
Yi-Ting Wang ◽  
Chih-Chiang Chan ◽  
Cheng-Wen Hsieh ◽  
Hsiao-Man Liao ◽  
...  
Keyword(s):  
Cell Growth ◽  
Marked Decrease ◽  
Transmembrane Protein ◽  
Intestinal Cell ◽  
Tor Signaling ◽  
Animal Development ◽  
Physiological Importance ◽  
Epithelial Homeostasis ◽  
Junction Protein ◽  
Increased Susceptibility

Autophagy is essential for maintaining cellular homeostasis and survival under various stress conditions. Autophagy-related gene 9 (Atg9) encodes a multipass transmembrane protein thought to act as a membrane carrier for forming autophagosomes. However, the molecular regulation and physiological importance of Atg9 in animal development remain largely unclear. Here, we generated Atg9 null mutant flies and found that loss of Atg9 led to shortened lifespan, locomotor defects, and increased susceptibility to stress. Atg9 loss also resulted in aberrant adult midgut morphology with dramatically enlarged enterocytes. Interestingly, inhibiting the TOR signaling pathway rescued the midgut defects of the Atg9 mutants. In addition, Atg9 interacted with PALS1-associated tight junction protein (Patj), which associates with TSC2 to regulate TOR activity. Depletion of Atg9 caused a marked decrease in TSC2 levels. Our findings revealed an antagonistic relationship between Atg9 and TOR signaling in the regulation of cell growth and tissue homeostasis.

A growth factor-responsive gene of murine BALB/c 3T3 cells encodes a protein homologous to human tissue factor

1989 ◽  
Vol 9 (6) ◽  
pp. 2567-2573
Author(s):  
S Hartzell ◽  
K Ryder ◽  
A Lanahan ◽  
L F Lau ◽  
D Nathan
Keyword(s):  
Growth Factors ◽  
Cell Growth ◽  
Tissue Factor ◽  
Human Tissue ◽  
Blood Clotting ◽  
Transmembrane Protein ◽  
3T3 Cells ◽  
Proteolytic Activation ◽  
Polypeptide Growth Factors ◽  
Mediate Cell

Polypeptide growth factors rapidly induce the transcription of a set of genes that appear to mediate cell growth. We report that one of the genes induced in BALB/c mouse 3T3 cells encodes a transmembrane protein (mTF) homologous to human tissue factor, which is involved in the proteolytic activation of blood clotting. mTF mRNA is present in many murine tissues and cell lines. Our results raise the possibility that mTF may also play a role in cell growth.

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