scholarly journals Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP

Sensors ◽  
2019 ◽  
Vol 19 (15) ◽  
pp. 3253 ◽  
Author(s):  
Keelan J. Trull ◽  
Piper Miller ◽  
Kiet Tat ◽  
S. Ashley Varney ◽  
Jason M. Conley ◽  
...  
Keyword(s):  
Optical Sensors ◽  
Stress Responses ◽  
Fluorescent Sensor ◽  
Purinergic Signaling ◽  
Osmotic Shock ◽  
Resonance Energy ◽  
Model Systems ◽  
High Affinity ◽  
Solution Studies ◽  
Nucleotide Release

Purinergic signals, such as extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP), mediate intercellular communication and stress responses throughout mammalian tissues, but the dynamics of their release and clearance are still not well understood. Although physiochemical methods provide important insight into physiology, genetically encoded optical sensors have proven particularly powerful in the quantification of signaling in live specimens. Indeed, genetically encoded luminescent and fluorescent sensors provide new insights into ATP-mediated purinergic signaling. However, new tools to detect extracellular ADP are still required. To this end, in this study, we use protein engineering to generate a new genetically encoded sensor that employs a high-affinity bacterial ADP-binding protein and reports a change in occupancy with a change in the Förster-type resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. We characterize the sensor in both protein solution studies, as well as live-cell microscopy. This new sensor responds to nanomolar and micromolar concentrations of ADP and ATP in solution, respectively, and in principle it is the first fully-genetically encoded sensor with sufficiently high affinity for ADP to detect low levels of extracellular ADP. Furthermore, we demonstrate that tethering the sensor to the cell surface enables the detection of physiologically relevant nucleotide release induced by hypoosmotic shock as a model of tissue edema. Thus, we provide a new tool to study purinergic signaling that can be used across genetically tractable model systems.

scholarly journals Role of Chiral Configuration in the Photoinduced Interaction of D- and L-Tryptophan with Optical Isomers of Ketoprofen in Linked Systems

2021 ◽  
Vol 22 (12) ◽  
pp. 6198
Author(s):  
Aleksandra A. Ageeva ◽  
Ilya M. Magin ◽  
Alexander B. Doktorov ◽  
Victor F. Plyusnin ◽  
Polina S. Kuznetsova ◽  
...  
Keyword(s):  
Amino Acid ◽  
Excited States ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dipole Interaction ◽  
The Body ◽  
Model Systems ◽  
Optical Isomers ◽  
Amino Acid Properties ◽  
Parkinson’S Diseases

The study of the L- and D-amino acid properties in proteins and peptides has attracted considerable attention in recent years, as the replacement of even one L-amino acid by its D-analogue due to aging of the body is resulted in a number of pathological conditions, including Alzheimer’s and Parkinson’s diseases. A recent trend is using short model systems to study the peculiarities of proteins with D-amino acids. In this report, the comparison of the excited states quenching of L- and D-tryptophan (Trp) in a model donor–acceptor dyad with (R)- and (S)-ketoprofen (KP-Trp) was carried out by photochemically induced dynamic nuclear polarization (CIDNP) and fluorescence spectroscopy. Quenching of the Trp excited states, which occurs via two mechanisms: prevailing resonance energy transfer (RET) and electron transfer (ET), indeed demonstrates some peculiarities for all three studied configurations of the dyad: (R,S)-, (S,R)-, and (S,S)-. Thus, the ET efficiency is identical for (S,R)- and (R,S)-enantiomers, while RET differs by 1.6 times. For (S,S)-, the CIDNP coefficient is almost an order of magnitude greater than for (R,S)- and (S,R)-. To understand the source of this difference, hyperpolarization of (S,S)-and (R,S)- has been calculated using theory involving the electron dipole–dipole interaction in the secular equation.

scholarly journals The Craterostigma plantagineum protein kinase CpWAK1 interacts with pectin and integrates different environmental signals in the cell wall

Planta ◽  
2021 ◽  
Vol 253 (5) ◽  
Author(s):  
Peilei Chen ◽  
Valentino Giarola ◽  
Dorothea Bartels
Keyword(s):  
Cell Wall ◽  
Stress Responses ◽  
Expression Profiles ◽  
Phylogenetic Analyses ◽  
Cell Wall Protein ◽  
Biological Processes ◽  
Environmental Signals ◽  
High Affinity ◽  
Craterostigma Plantagineum ◽  
Receptor Kinases

Abstract Main conclusion The cell wall protein CpWAK1 interacts with pectin, participates in decoding cell wall signals, and induces different downstream responses. Abstract Cell wall-associated protein kinases (WAKs) are transmembrane receptor kinases. In the desiccation-tolerant resurrection plant Craterostigma plantagineum, CpWAK1 has been shown to be involved in stress responses and cell expansion by forming a complex with the C. plantagineum glycine-rich protein1 (CpGRP1). This prompted us to extend the studies of WAK genes in C. plantagineum. The phylogenetic analyses of WAKs from C. plantagineum and from other species suggest that these genes have been duplicated after species divergence. Expression profiles indicate that CpWAKs are involved in various biological processes, including dehydration-induced responses and SA- and JA-related reactions to pathogens and wounding. CpWAK1 shows a high affinity for “egg-box” pectin structures. ELISA assays revealed that the binding of CpWAKs to pectins is modulated by CpGRP1 and it depends on the apoplastic pH. The formation of CpWAK multimers is the prerequisite for the CpWAK–pectin binding. Different pectin extracts lead to opposite trends of CpWAK–pectin binding in the presence of Ca2+ at pH 8. These observations demonstrate that CpWAKs can potentially discriminate and integrate cell wall signals generated by diverse stimuli, in concert with other elements, such as CpGRP1, pHapo, Ca2+[apo], and via the formation of CpWAK multimers.

scholarly journals Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose

2020 ◽  
Vol 21 (14) ◽  
pp. 5004
Author(s):  
Ekaterina O. Serebrovskaya ◽  
Nadezda M. Podvalnaya ◽  
Varvara V. Dudenkova ◽  
Anna S. Efremova ◽  
Nadya G. Gurskaya ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Fluorescent Sensor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Post Translational Modification ◽  
Cellular Processes ◽  
Hydrogen Peroxide Treatment ◽  
Damage Response

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.

scholarly journals Identifying protein–protein interactions in somatic hypermutation

2005 ◽  
Vol 201 (4) ◽  
pp. 493-496 ◽  
Author(s):  
Myron F. Goodman ◽  
Matthew D. Scharff
Keyword(s):  
Mouse Model ◽  
Protein Interactions ◽  
Somatic Hypermutation ◽  
Model Systems ◽  
Antigen Binding ◽  
Immunoglobulin Genes ◽  
Protein Protein Interactions ◽  
Cell Systems ◽  
High Affinity ◽  
Key Players

Somatic hypermutation (SHM) in immunoglobulin genes is required for high affinity antibody–antigen binding. Cultured cell systems, mouse model systems, and human genetic deficiencies have been the key players in identifying likely SHM pathways, whereas “pure” biochemical approaches have been far less prominent, but change appears imminent. Here we comment on how, when, and why biochemistry is likely to emerge from the shadows and into the spotlight to elucidate how the somatic mutation of antibody variable (V) regions is generated.

scholarly journals FRET-based cyclic GMP biosensors measure low cGMP concentrations in cardiomyocytes and neurons

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Gaia Calamera ◽  
Dan Li ◽  
Andrea Hembre Ulsund ◽  
Jeong Joo Kim ◽  
Oliver C. Neely ◽  
...  
Keyword(s):  
Guanylyl Cyclase ◽  
Intracellular Signaling ◽  
Stellate Ganglion ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Soluble Guanylyl Cyclase ◽  
High Affinity ◽  
Dependent Protein ◽  
Ganglion Neurons ◽  
Intracellular Detection

Abstract Several FRET (fluorescence resonance energy transfer)-based biosensors for intracellular detection of cyclic nucleotides have been designed in the past decade. However, few such biosensors are available for cGMP, and even fewer that detect low nanomolar cGMP concentrations. Our aim was to develop a FRET-based cGMP biosensor with high affinity for cGMP as a tool for intracellular signaling studies. We used the carboxyl-terminal cyclic nucleotide binding domain of Plasmodium falciparum cGMP-dependent protein kinase (PKG) flanked by different FRET pairs to generate two cGMP biosensors (Yellow PfPKG and Red PfPKG). Here, we report that these cGMP biosensors display high affinity for cGMP (EC50 of 23 ± 3 nM) and detect cGMP produced through soluble guanylyl cyclase and guanylyl cyclase A in stellate ganglion neurons and guanylyl cyclase B in cardiomyocytes. These biosensors are therefore optimal tools for real-time measurements of low concentrations of cGMP in living cells.

scholarly journals An Nd3+-Sensitized Upconversion Fluorescent Sensor for Epirubicin Detection

Nanomaterials ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 1700
Author(s):  
Jingwen Mo ◽  
Long Shen ◽  
Qian Xu ◽  
Jiaying Zeng ◽  
Jingjie Sha ◽  
...  
Keyword(s):  
Laser Excitation ◽  
Fluorescent Sensor ◽  
Low Cost ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorometric Detection ◽  
Energy Transfer Mechanism ◽  
Dynamic Quenching ◽  
Light Penetration

We describe here an Nd3+-sensitized upconversion fluorescent sensor for epirubicin (EPI) detection in aqueous solutions under 808 nm laser excitation. The upconversion fluorescence of nanoparticles is effectively quenched in the presence of EPI via a fluorescence resonance energy transfer mechanism. The dynamic quenching constant was 2.10 × 104 M−1. Normalized fluorescence intensity increased linearly as the EPI concentration was raised from 0.09 μM to 189.66 μM and the fluorometric detection limit was 0.05 μM. The sensing method was simple, fast, and low-cost and was able to be applied to determine the levels of EPI in urine with spike recoveries from 97.5% to 102.6%. Another important feature of the proposed fluorescent sensor is that it holds a promising potential for in vivo imaging and detection due to its distinctive properties such as weak autofluorescence, low heating effect, and high light penetration depth.

scholarly journals Flow-Induced [Ca2+]i Increase Depends on Nucleotide Release and Subsequent Purinergic Signaling in the Intact Nephron

2007 ◽  
Vol 18 (7) ◽  
pp. 2062-2070 ◽  
Author(s):  
Mikkel Erik Juul Jensen ◽  
Elvin Odgaard ◽  
Mette Høgh Christensen ◽  
Helle A. Praetorius ◽  
Jens Leipziger
Keyword(s):  
Purinergic Signaling ◽  
Nucleotide Release

scholarly journals Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation.

1995 ◽  
Vol 129 (6) ◽  
pp. 1543-1558 ◽  
Author(s):  
T W Gadella ◽  
T M Jovin
Keyword(s):  
Energy Transfer ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
A431 Cells ◽  
High Affinity ◽  
Fret Efficiency ◽  
Epidermal Growth

The aggregation states of the epidermal growth factor receptor (EGFR) on single A431 human epidermoid carcinoma cells were assessed with two new techniques for determining fluorescence resonance energy transfer: donor photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence lifetime imaging microscopy (FLIM). Fluorescein-(donor) and rhodamine-(acceptor) labeled EGF were bound to the cells and the extent of oligomerization was monitored by the spatially resolved FRET efficiency as a function of the donor/acceptor ratio and treatment conditions. An average FRET efficiency of 5% was determined after a low temperature (4 degrees C) incubation with the fluorescent EGF analogs for 40 min. A subsequent elevation of the temperature for 5 min caused a substantial increase of the average FRET efficiency to 14% at 20 degrees C and 31% at 37 degrees C. In the context of a two-state (monomer/dimer) model for the EGFR, these FRET efficiencies were consistent with minimal average receptor dimerizations of 13, 36, and 69% at 4, 20, and 37 degrees C, respectively. A431 cells were pretreated with the monoclonal antibody mAb 2E9 that specifically blocks EGF binding to the predominant population of low affinity EGFR (15). The average FRET efficiency increased dramatically to 28% at 4 degrees C, indicative of a minimal receptor dimerization of 65% for the subpopulation of high affinity receptors. These results are in accordance with prior studies indicating that binding of EGF leads to a fast and temperature-dependent microclustering of EGFR, but suggest in addition that the high affinity functional subclass of receptors on quiescent A431 cells are present in a predimerized or oligomerized state. We propose that the transmission of the external ligand-binding signal to the cytoplasmic domain is effected by a concerted relative rotational rearrangement of the monomeric units comprising the dimeric receptor, thereby potentiating a mutual activation of the tyrosine kinase domains.

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