scholarly journals Transcriptional and Biochemical Characterization of Cytosolic Pyruvate Kinases in Arabidopsis thaliana

Plants ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 353
Author(s):  
Sabine Wulfert ◽  
Sören Schilasky ◽  
Stephan Krueger
Keyword(s):  
Arabidopsis Thaliana ◽  
Pyruvate Kinase ◽  
Expression Pattern ◽  
Biochemical Characterization ◽  
Fluorescent Protein ◽  
Quaternary Structure ◽  
Tricarboxylic Acid Cycle ◽  
Yellow Fluorescent Protein ◽  
Allosteric Effectors ◽  
Pyruvate Kinases

Glycolysis is a central catabolic pathway in every living organism with an essential role in carbohydrate breakdown and ATP synthesis, thereby providing pyruvate to the tricarboxylic acid cycle (TCA cycle). The cytosolic pyruvate kinase (cPK) represents a key glycolytic enzyme by catalyzing phosphate transfer from phosphoenolpyruvate (PEP) to ADP for the synthesis of ATP. Besides its important functions in cellular energy homeostasis, the activity of cytosolic pyruvate kinase underlies tight regulation, for instance by allosteric effectors, that impact stability of its quaternary structure. We determined five cytosol-localized pyruvate kinases, out of the fourteen putative pyruvate kinase genes encoded by the Arabidopsis thaliana genome, by investigation of phylogeny and localization of yellow fluorescent protein (YFP) fusion proteins. Analysis of promoter β-glucuronidase (GUS) reporter lines revealed an isoform-specific expression pattern for the five enzymes, subject to plant tissue and developmental stage. Investigation of the heterologously expressed and purified cytosolic pyruvate kinases revealed that these enzymes are differentially regulated by metabolites, such as citrate, fructose-1,6-bisphosphate (FBP) and ATP. In addition, measured in vitro enzyme activities suggest that pyruvate kinase subunit complexes consisting of cPK2/3 and cPK4/5 isoforms, respectively, bear regulatory properties. In summary, our study indicates that the five identified cytosolic pyruvate kinase isoforms adjust the carbohydrate flux through the glycolytic pathway in Arabidopsis thaliana, by distinct regulatory qualities, such as individual expression pattern as well as dissimilar responsiveness to allosteric effectors and enzyme subgroup association.

scholarly journals Use of Yellow Fluorescent Protein Fluorescence to Track OPR3 Expression in Arabidopsis Thaliana Responses to Insect Herbivory

2019 ◽  
Vol 10 ◽  
Author(s):  
Mélanie J.A. Body ◽  
Dhruveesh F. Dave ◽  
Clayton M. Coffman ◽  
Taylor Y. Paret ◽  
Abraham J. Koo ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Insect Herbivory ◽  
Protein Fluorescence

scholarly journals Subunit composition of an energy-coupling-factor-type biotin transporter analysed in living bacteria

2010 ◽  
Vol 431 (3) ◽  
pp. 373-381 ◽  
Author(s):  
Friedrich Finkenwirth ◽  
Olivia Neubauer ◽  
Julia Gunzenhäuser ◽  
Janna Schoknecht ◽  
Silvia Scolari ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Quaternary Structure ◽  
Transmembrane Protein ◽  
Yellow Fluorescent Protein ◽  
Energy Coupling ◽  
Coupling Factor ◽  
Supramolecular Organization ◽  
High Affinity ◽  
Donor Acceptor ◽  
Living Bacteria

BioMNY, a bacterial high-affinity biotin transporter, is a member of the recently defined class of ECF (energy-coupling factor) transporters. These systems are composed of ABC (ATP-binding-cassette) ATPases (represented by BioM in the case of the biotin transporter), a universally conserved transmembrane protein (BioN) and a core transporter component (BioY), in unknown stoichiometry. The quaternary structure of BioY, which functions as a low-affinity biotin transporter in the absence of BioMN, and of BioMNY was investigated by a FRET (Förster resonance energy transfer) approach using living recombinant Escherichia coli cells. To this end, the donor–acceptor pair, of Cerulean and yellow fluorescent protein respectively, were fused to BioM, BioN and BioY. The fusion proteins were stable and the protein tags did not interfere with transport and ATPase activities. Specific donor–acceptor interactions were characterized by lifetime-based FRET spectroscopy. The results suggest an oligomeric structure for the solitary BioY core transporter and oligomeric forms of BioM and BioY in BioMNY complexes. We surmise that oligomers of BioY are the functional units of the low- and high-affinity biotin transporter in the living cell. Beyond its relevance for clarifying the supramolecular organization of ECF transporters, the results demonstrate the general applicability of lifetime-based FRET studies in living bacteria.

The control of pyruvate kinases of Escherichia coli: Further studies of the enzyme activated by ribose-5-phosphate

1978 ◽  
Vol 56 (6) ◽  
pp. 647-653 ◽  
Author(s):  
John S. Mort ◽  
B. D. Sanwal
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Kinase ◽  
Quaternary Structure ◽  
Native Enzyme ◽  
Open Structure ◽  
Allosteric Inhibitor ◽  
Pyruvate Kinases ◽  
Allosteric Activator

The pyruvate kinase activated by ribose-5-phosphate from Escherichia coli has been purified to homogeneity, taking advantage of the stabilization of the enzyme by its inhibitor phosphate and by thiol reagents. The native enzyme has a tetrameric quaternary structure which, while prone to dissociation under many conditions, remains intact in the presence of the above reagents.The enzyme was found to reactivate on dilution out of 8 M urea. Interestingly, the recovery of activity is greatly increased by phosphate, an allosteric inhibitor, but markedly reduced by the allosteric activator, ribose-5-phosphate, implying that it is harder for the enzyme to refold to a 'relaxed state.' Proteolysis studies indicate a more open structure in the presence of the activator.

scholarly journals Diethylstilbestrol, a Novel ANO1 Inhibitor, Exerts an Anticancer Effect on Non-Small Cell Lung Cancer via Inhibition of ANO1

2021 ◽  
Vol 22 (13) ◽  
pp. 7100
Author(s):  
Yohan Seo ◽  
Sung Baek Jeong ◽  
Joo Han Woo ◽  
Oh-Bin Kwon ◽  
Sion Lee ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Cell ◽  
Channel Activity ◽  
Small Cell Lung ◽  
Protein Levels

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.

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