scholarly journals Regulation of β cell glucokinase by S-nitrosylation and association with nitric oxide synthase

2003 ◽  
Vol 161 (2) ◽  
pp. 243-248 ◽  
Author(s):  
Mark A. Rizzo ◽  
David W. Piston
Keyword(s):  
Nitric Oxide ◽  
Fluorescent Proteins ◽  
Signal Sequence ◽  
Secretory Granules ◽  
Quantitative Imaging ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
No Production ◽  
Β Cells ◽  
Pancreatic Β Cells

Glucokinase (GK) activity plays a key role in glucose-stimulated insulin secretion from pancreatic β cells. Insulin regulates GK activity by modulating its association with secretory granules, although little is known about the mechanisms involved in regulating this association. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO). Our results in cultured β cells show that insulin stimulates NO production and leads to S-nitrosylation of GK. Furthermore, inhibition of NO synthase (NOS) activity blocks insulin-stimulated changes in both GK association with secretory granules and GK conformation. Mutation of cysteine 371 to serine blocks S-nitrosylation of GK and causes GK to remain tightly bound to secretory granules. GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer. Finally, attachment of a nuclear localization signal sequence to NOS drives GK to the nucleus in addition to its normal cytoplasmic and granule targeting. Together, these data suggest that the regulation of GK localization and activity in pancreatic β cells is directly related to NO production and that the association of GK with secretory granules occurs through its interaction with NOS.

scholarly journals Nitric oxide interacts with cholinoceptors to modulate insulin secretion by pancreatic β cells

2020 ◽  
Vol 472 (10) ◽  
pp. 1469-1480
Author(s):  
Bashair M. Mussa ◽  
Ankita Srivastava ◽  
Abdul Khader Mohammed ◽  
Anthony J. M. Verberne
Keyword(s):  
Nitric Oxide ◽  
Insulin Secretion ◽  
Cell Viability ◽  
Combined Treatment ◽  
No Production ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Tnf Α ◽  
Ifn Γ

Abstract Dysfunction of the pancreatic β cells leads to several chronic disorders including diabetes mellitus. Several mediators and mechanisms are known to be involved in the regulation of β cell secretory function. In this study, we propose that cytokine-induced nitric oxide (NO) production interacts with cholinergic mechanisms to modulate insulin secretion from pancreatic β cells. Using a rat insulinoma cell line INS-1, we demonstrated that β cell viability decreases significantly in the presence of SNAP (NO donor) in a concentration- and time-dependent manner. Cell viability was also found to be decreased in the presence of a combined treatment of SNAP with SMN (muscarinic receptor antagonist). We then investigated the impact of these findings on insulin secretion and found a significant reduction in glucose uptake by INS-1 cells in the presence of SNAP and SMN as compared with control. Nitric oxide synthase 3 gene expression was found to be significantly reduced in response to combined treatment with SNAP and SMN suggesting an interaction between the cholinergic and nitrergic systems. The analysis of gene and protein expression further pin-pointed the involvement of M3 muscarinic receptors in the cholinergic pathway. Upon treatment with cytokines, reduced cell viability was observed in the presence of TNF-α and IFN-γ. A significant reduction in insulin secretion was also noted after treatment with TNF-α and IFN-γ and IL1-β. The findings of the present study have shown for the first time that the inhibition of the excitatory effects of cholinergic pathways on glucose-induced insulin secretion may cause β cell injury and dysfunction of insulin secretion in response to cytokine-induced NO production.

Quantum Dots and FRET-Nanobeads for Probing Genes, Proteins, and Drug Targets in Single Cells

2003 ◽  
Author(s):  
Amit Agrawal ◽  
Xiaohu Gao ◽  
Nitin Nitin ◽  
Gang Bao ◽  
Shuming Nie
Keyword(s):  
Quantum Dots ◽  
Drug Targets ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Quantitative Imaging ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Length Scale ◽  
Imaging And Spectroscopy

Quantum dots are tiny light-emitting particles on the length scale of 2–10 nm, and FRET-nanobeads for fluorophore-embedded nanoparticles on the length scale of 40–200 nm based on the phenomenon of fluorescence resonance energy transfer (FRET). These materials are emerging as a new class of biological labels with properties and applications that are not available with traditional organic dyes and fluorescent proteins. In this ASME contribution, we report new developments in using semiconductor quantum dots for quantitative imaging and spectroscopy of single cancer cells. We also show results from intracellular staining of actin filaments using FRET-nanobeads. These results raise new possibilities in disease diagnostics, drug and biochemical discovery, cancer imaging, molecular profiling, and disease staging.

scholarly journals Pancreatic β-cells express hepcidin, an iron-uptake regulatory peptide

2008 ◽  
Vol 197 (2) ◽  
pp. 241-249 ◽  
Author(s):  
Hasan Kulaksiz ◽  
Evelyn Fein ◽  
Peter Redecker ◽  
Wolfgang Stremmel ◽  
Guido Adler ◽  
...  
Keyword(s):  
Iron Uptake ◽  
Secretory Granules ◽  
Rinm5f Cells ◽  
Β Cells ◽  
Additional Source ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Vital Functions ◽  
Immunohistochemical Studies ◽  
Insight Into

Body iron is involved in various vital functions. Its uptake in the intestine is regulated by hepcidin, a bioactive peptide originally identified in plasma and urine and subsequently in the liver. In the present study, we provide evidence at the transcriptional and translational levels that hepcidin is also expressed in the pancreas of rat and man. Immunohistochemical studies localized the peptide exclusively to β-cells of the islets of Langerhans. Immunoelectron microscopical analyses revealed that hepcidin is confined to the insulin-storing β-cell secretory granules. As demonstrated in insulinoma-derived RINm5F cells, the expression of hepcidin in β-cells is regulated by iron. Based on the present findings we conclude that pancreatic islets are an additional source of the peptide hepcidin. The localization of this peptide to β-cells suggests that pancreatic β-cells may be involved in iron metabolism in addition to their genuine function in blood glucose regulation. In view of the various linked iron/glucose disorders in the pancreas, the present findings may provide an insight into the phenomenology of intriguing mutual relationships between iron and glucose metabolisms.

PPARγ is not required for the inhibitory actions of PGJ2 on cytokine signaling in pancreatic β-cells

2004 ◽  
Vol 286 (3) ◽  
pp. E329-E336 ◽  
Author(s):  
Sarah M. Weber ◽  
Anna L. Scarim ◽  
John A. Corbett
Keyword(s):  
Gene Expression ◽  
Nitric Oxide ◽  
Cytokine Signaling ◽  
Rinm5f Cells ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Inos Gene ◽  
Ifn Γ ◽  
Anti Inflammatory ◽  
Inos Gene Expression

Peroxisome proliferator-activated receptor (PPAR)γ agonists, such as 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2) and troglitazone, have been shown to elicit anti-inflammatory effects in pancreatic β-cells that include inhibition of cytokine-stimulated inducible nitric oxide synthase (iNOS) gene expression and production of nitric oxide. In addition, these ligands impair IL-1-induced NF-κB and MAPK as well as IFN-γ-stimulated signal transducer and activator of transcription (STAT)1 activation in β-cells. The purpose of this study was to determine if PPARγ activation participates in the anti-inflammatory actions of PGJ2 in β-cells. Pretreatment of RINm5F cells for 6 h with PGJ2 results in inhibition of IL-1-stimulated IκB degradation and IFN-γ-stimulated STAT1 phosphorylation. Overexpression of a dominant-negative (dn) PPARγ mutant or treatment with the PPARγ antagonist GW-9662 does not modulate the inhibitory actions of PGJ2 on cytokine signaling in RINm5F cells. Although these agents fail to attenuate the inhibitory actions of PGJ2 on cytokine signaling, they do inhibit PGJ2-stimulated PPARγ response element reporter activity. Consistent with the inability to attenuate the inhibitory actions of PGJ2 on cytokine signaling, neither dnPPARγ nor GW-9662 prevents the inhibitory actions of PGJ2 on IL-1-stimulated iNOS gene expression or nitric oxide production by RINm5F cells. These findings support a PPARγ-independent mechanism by which PPARγ ligands impair cytokine signaling and iNOS expression by islets.

scholarly journals Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3105 ◽  
Author(s):  
Henning Höfig ◽  
Michele Cerminara ◽  
Ilona Ritter ◽  
Antonie Schöne ◽  
Martina Pohl ◽  
...  
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Strong Increase ◽  
Single Molecule Level ◽  
Labeling Scheme

Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor.

scholarly journals Proteasomal conformation controls unfolding ability

2021 ◽  
Vol 118 (25) ◽  
pp. e2101004118
Author(s):  
Julianna R. Cresti ◽  
Abramo J. Manfredonia ◽  
Christopher E. Bragança ◽  
Joseph A. Boscia ◽  
Christina M. Hurley ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Conformational State ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Ubiquitinated Protein ◽  
Equilibrium Conditions ◽  
Substrate Processing

The 26S proteasome is the macromolecular machine responsible for the bulk of protein degradation in eukaryotic cells. As it degrades a ubiquitinated protein, the proteasome transitions from a substrate-accepting conformation (s1) to a set of substrate-processing conformations (s3 like), each stabilized by different intramolecular contacts. Tools to study these conformational changes remain limited, and although several interactions have been proposed to be important for stabilizing the proteasome’s various conformations, it has been difficult to test these directly under equilibrium conditions. Here, we describe a conformationally sensitive Förster resonance energy transfer assay, in which fluorescent proteins are fused to Sem1 and Rpn6, which are nearer each other in substrate-processing conformations than in the substrate-accepting conformation. Using this assay, we find that two sets of interactions, one involving Rpn5 and another involving Rpn2, are both important for stabilizing substrate-processing conformations. Mutations that disrupt these interactions both destabilize substrate-processing conformations relative to the substrate-accepting conformation and diminish the proteasome’s ability to successfully unfold and degrade hard-to-unfold substrates, providing a link between the proteasome’s conformational state and its unfolding ability.

scholarly journals Development of FRET-based indicators for visualizing homophilic trans interaction of a clustered protocadherin

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takashi Kanadome ◽  
Natsumi Hoshino ◽  
Takeharu Nagai ◽  
Tomoki Matsuda ◽  
Takeshi Yagi
Keyword(s):  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Direct Visualization ◽  
Binding Properties ◽  
New Approach ◽  
Self Recognition ◽  
Highly Sensitive ◽  
Donor And Acceptor ◽  
Clustered Protocadherins

AbstractClustered protocadherins (Pcdhs), which are cell adhesion molecules, play a fundamental role in self-recognition and non-self-discrimination by conferring diversity on the cell surface. Although systematic cell-based aggregation assays provide information regarding the binding properties of Pcdhs, direct visualization of Pcdh trans interactions across cells remains challenging. Here, we present Förster resonance energy transfer (FRET)-based indicators for directly visualizing Pcdh trans interactions. We developed the indicators by individually inserting FRET donor and acceptor fluorescent proteins (FPs) into the ectodomain of Pcdh molecules. They enabled successful visualization of specific trans interactions of Pcdh and revealed that the Pcdh trans interaction is highly sensitive to changes in extracellular Ca2+ levels. We expect that FRET-based indicators for visualizing Pcdh trans interactions will provide a new approach for investigating the roles of Pcdh in self-recognition and non-self-discrimination processes.

scholarly journals FRET Microscopy in Yeast

Biosensors ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 122 ◽  
Author(s):  
Skruzny ◽  
Pohl ◽  
Abella
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Bioactive Molecules ◽  
Biophysical Processes ◽  
Microscopy Method ◽  
Fret Biosensors

Förster resonance energy transfer (FRET) microscopy is a powerful fluorescence microscopy method to study the nanoscale organization of multiprotein assemblies in vivo. Moreover, many biochemical and biophysical processes can be followed by employing sophisticated FRET biosensors directly in living cells. Here, we summarize existing FRET experiments and biosensors applied in yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, two important models of fundamental biomedical research and efficient platforms for analyses of bioactive molecules. We aim to provide a practical guide on suitable FRET techniques, fluorescent proteins, and experimental setups available for successful FRET experiments in yeasts.

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.

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