scholarly journals The Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide Is Hydrolyzed and Its Signaling Properties Are Altered by Directly Binding the Calpain Small Subunit

Endocrinology ◽  
2005 ◽  
Vol 146 (5) ◽  
pp. 2336-2344 ◽  
Author(s):  
Masako Shimada ◽  
Matthew J. Mahon ◽  
Peter A. Greer ◽  
Gino V. Segre
Keyword(s):  
Parathyroid Hormone ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Subunit ◽  
Hek293 Cells ◽  
Calpain Inhibitor ◽  
Dependent Manner ◽  
Intact Cells ◽  
Calcium Dependent

Abstract We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes ΔNt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of ΔNt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.

scholarly journals MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro

2013 ◽  
Vol 304 (8) ◽  
pp. L511-L518 ◽  
Author(s):  
Shijing Fang ◽  
Anne L. Crews ◽  
Wei Chen ◽  
Joungjoa Park ◽  
Qi Yin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Airway Epithelial Cells ◽  
Dependent Manner ◽  
Airway Epithelial ◽  
Mucin Secretion ◽  
Concentration Dependent Manner

Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation.

scholarly journals The Molecular Chaperone DNAJB6, but Not DNAJB1, Suppresses the Seeded Aggregation of Alpha-Synuclein in Cells

2019 ◽  
Vol 20 (18) ◽  
pp. 4495 ◽  
Author(s):  
Natasja Deshayes ◽  
Sertan Arkan ◽  
Christian Hansen
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Lewy Bodies ◽  
Resonance Energy ◽  
Alpha Synuclein ◽  
Hek293 Cells ◽  
Induced Aggregation ◽  
In Cells

Alpha-synuclein (α-Syn) can misfold and aggregate, causing the degeneration of dopaminergic neurons, as seen in Parkinson’s disease (PD). We recently demonstrated that DNAJB6, a co-chaperone found in Lewy bodies (LB), suppresses the aggregation of α-Syn in cells and in vitro. In this study, we compared the capacities of DNAJB1 and DNAJB6 to suppress the seeded α-Syn aggregation in HEK293 cells expressing α-Syn tagged with cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). The aggregation of α-Syn was seeded by the transfection of the cells with recombinant α-Syn pre-formed fibrils (PFFs), following the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated knockout (KO) of these two genes, respectively. We quantified the α-Syn aggregation by fluorescence microscopy and fluorescence resonance energy transfer (FRET) analysis. We detected significantly more aggregates in the DNAJB6 KO cells compared with the parental cells, whereas the DNAJB1 KO had no effect on the α-Syn aggregation. This is the first evidence that DNAJB6 can suppress α-Syn aggregation, induced by exogenous α-Syn seeds, in cells. Next, we explored whether this mechanism could be dependent on protein degradation pathways. We observed that the increase in the α-Syn PFF-induced aggregation in the DNAJB6 KO cells compared with the parental cells was strongly diminished upon the incubation of the cells with the proteasomal inhibitor MG132. These results consolidate that DNAJB6 is a suppressor of α-Syn aggregation, and suggest that DNAJB6 may target misfolded and/or aggregated α-Syn for proteasomal degradation.

scholarly journals The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 3024
Author(s):  
Martin Fogtmann Berthelsen ◽  
Maria Riedel ◽  
Huiqiang Cai ◽  
Søren H. Skaarup ◽  
Aage K. O. Alstrup ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Somatic Cells ◽  
Genetic Alterations ◽  
Lung Epithelial Cells ◽  
Target Cells ◽  
Multiple Gene ◽  
Conditional Expression ◽  
Gene Alterations

The generation of large transgenic animals is impeded by complex cloning, long maturation and gastrulation times. An introduction of multiple gene alterations increases the complexity. We have cloned a transgenic Cas9 minipig to introduce multiple mutations by CRISPR in somatic cells. Transgenic Cas9 pigs were generated by somatic cell nuclear transfer and were backcrossed to Göttingen Minipigs for two generations. Cas9 expression was controlled by FlpO-mediated recombination and was visualized by translation from red to yellow fluorescent protein. In vitro analyses in primary fibroblasts, keratinocytes and lung epithelial cells confirmed the genetic alterations executed by the viral delivery of single guide RNAs (sgRNA) to the target cells. Moreover, multiple gene alterations could be introduced simultaneously in a cell by viral delivery of sgRNAs. Cells with loss of TP53, PTEN and gain-of-function mutation in KRASG12D showed increased proliferation, confirming a transformation of the primary cells. An in vivo activation of Cas9 expression could be induced by viral delivery to the skin. Overall, we have generated a minipig with conditional expression of Cas9, where multiple gene alterations can be introduced to somatic cells by viral delivery of sgRNA. The development of a transgenic Cas9 minipig facilitates the creation of complex pre-clinical models for cancer research.

scholarly journals Calcium-sensitivity of inositol 1,4,5-trisphosphate metabolism in exocrine cells from the avian salt gland

1992 ◽  
Vol 282 (3) ◽  
pp. 703-710 ◽  
Author(s):  
J P Hildebrandt ◽  
T J Shuttleworth
Keyword(s):  
Substrate Concentration ◽  
Inositol Phosphates ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Salt Glands ◽  
Concentration Dependent Manner ◽  
Intact Cells ◽  
Exocrine Cells ◽  
Tissue Homogenates

The generation of inositol phosphates upon muscarinic-receptor activation was studied in [3H]inositol-loaded exocrine cells from the nasal salt glands of the duck Anas platyrhynchos, and the metabolism of different inositol phosphates in vitro was studied in tissue homogenates, with particular reference to the possible interaction of changes in intracellular [Ca2+] ([Ca2+]i) with the metabolic processes. In intact cells, there was a rapid (within 15 s) generation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, followed by an accumulation of their breakdown products, Ins(1,3,4)P3 and inositol bis- and monophosphates. Ca(2+)-sensitivity of the Ins(1,4,5)P3 3-kinase was demonstrated in tissue homogenates, with the rate of phosphorylation increasing 2-fold at free Ca2+ concentrations greater than 1 microM. However, addition of calmodulin or the presence of the calmodulin inhibitor W-7 (up to 100 microM) had no effect. 3-Kinase activity increased proportionally with the initial Ins(1,4,5)P3 concentration up to 1 microM, but a 10-fold higher substrate concentration produced only a doubling in the phosphorylation rate. Ins(1,3,4,5)P4 was dephosphorylated to Ins(1,3,4)P3, which accumulated in the homogenate assays as well as in intact cells. Depending on its concentration, Ins(1,3,4)P3 was phosphorylated [in part to Ins(1,3,4,6)P4] or dephosphorylated. To investigate the Ca(2+)-sensitivity of the 3-kinase in intact cells, excess quin2 was used to buffer the receptor-mediated transient changes in [Ca2+]i in [3H]inositol-loaded cells. These experiments revealed that increasing [Ca2+]i from less than 100 to approx. 400 nM (i.e. within the physiological range) has no effect on the partitioning of Ins(1,4,5)P3 metabolism (phosphorylation versus dephosphorylation) and on the accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. This indicates that activation of the 3-kinase by physiologically relevant Ca2+ concentrations may not play a major role in the generation of Ins(1,3,4,5)P4 signals upon receptor activation in these cells. The latter are mainly achieved by the receptor-mediated increase in Ins(1,4,5)P3 in the cell and its phosphorylation by the 3-kinase in a substrate-concentration-dependent manner.

Direct effect of parathyroid hormone on insulin secretion from pancreatic islets

1990 ◽  
Vol 258 (6) ◽  
pp. E975-E984 ◽  
Author(s):  
G. Z. Fadda ◽  
M. Akmal ◽  
L. G. Lipson ◽  
S. G. Massry
Keyword(s):  
Insulin Secretion ◽  
Parathyroid Hormone ◽  
Pancreatic Islets ◽  
Insulin Release ◽  
Direct Effect ◽  
Indirect Evidence ◽  
Cytosolic Calcium ◽  
Dependent Manner ◽  
Stimulation Of

Indirect evidence indicates that parathyroid hormone (PTH) interacts with pancreatic islets and modulates their insulin secretion. This property of PTH has been implicated in the genesis of impaired insulin release in chronic renal failure. We examined the direct effect of PTH-(1-84) and PTH-(1-34) on insulin release using in vitro static incubation and dynamic perifusion of pancreatic islets from normal rats. Both moieties of the hormone stimulated in a dose-dependent manner glucose-induced insulin release but higher doses inhibited glucose-induced insulin release. This action of PTH was modulated by the calcium concentration in the media. The stimulatory effect of PTH was abolished by its inactivation and blocked by its antagonist [Tyr-34]bPTH-(7-34)NH2. PTH also augmented phorbol ester (TPA)-induced insulin release, stimulated adenosine 3',5'-cyclic monophosphate (cAMP) generation by pancreatic islets, and significantly increased (+50 +/- 2.7%, P less than 0.01) their cytosolic calcium. Verapamil inhibited the stimulatory effect of PTH on insulin release. The data show that 1) pancreatic islets are a PTH target and may have PTH receptors, 2) stimulation of glucose-induced insulin release by PTH is mediated by a rise in cytosolic calcium, 3) stimulation of cAMP production by PTH and a potential indirect activation of protein kinase C by PTH may also contribute to the stimulatory effect on glucose-induced insulin release, and 4) this action of PTH requires calcium in incubation or perifusion media.

Identity of the renin cell is mediated by cAMP and chromatin remodeling: an in vitro model for studying cell recruitment and plasticity

2008 ◽  
Vol 294 (2) ◽  
pp. H699-H707 ◽  
Author(s):  
Ellen Steward Pentz ◽  
Maria Luisa S. Sequeira Lopez ◽  
Magali Cordaillat ◽  
R. Ariel Gomez
Keyword(s):  
Chromatin Remodeling ◽  
In Vitro Model ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Histone H4 Acetylation ◽  
Renin Gene ◽  
Vitro Model

The renin-angiotensin system (RAS) regulates blood pressure and fluid-electrolyte homeostasis. A key step in the RAS cascade is the regulation of renin synthesis and release by the kidney. We and others have shown that a major mechanism to control renin availability is the regulation of the number of cells capable of making renin. The kidney possesses a pool of cells, mainly in its vasculature but also in the glomeruli, capable of switching from smooth muscle to endocrine renin-producing cells when homeostasis is threatened. The molecular mechanisms governing the ability of these cells to turn the renin phenotype on and off have been very difficult to study in vivo. We, therefore, developed an in vitro model in which cells of the renin lineage are labeled with cyan fluorescent protein and cells actively making renin mRNA are labeled with yellow fluorescent protein. The model allowed us to determine that it is possible to culture cells of the renin lineage for numerous passages and that the memory to express the renin gene is maintained in culture and can be reenacted by cAMP and chromatin remodeling (histone H4 acetylation) at the cAMP-responsive element in the renin gene.

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

scholarly journals A hyperthermoactive-Cas9 editing tool reveals the role of a unique arsenite methyltransferase in the arsenic resistance system of Thermus thermophilus HB27

2021 ◽  
Author(s):  
Giovanni Gallo ◽  
Ioannis Mougiakos ◽  
Mauricio Bianco ◽  
Miriam Carbonaro ◽  
Andrea Carpentieri ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Fluorescent Protein ◽  
Efflux Pump ◽  
Thermus Thermophilus ◽  
Yellow Fluorescent Protein ◽  
Arsenic Resistance ◽  
Wide Range ◽  
Resistance System

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pull-down assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosylmethionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyse SAM-dependent arsenite methylation. In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome, and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus ΔTtarsM genome was substituted by a gene, encoding a stabilised yellow fluorescent protein (sYFP), to create a sensitive genome-based bioreporter system for the detection of arsenic ions.

scholarly journals Extracellular Signal-Regulated Kinases Phosphorylate Mitogen-Activated Protein Kinase Phosphatase 3/DUSP6 at Serines 159 and 197, Two Sites Critical for Its Proteasomal Degradation

2005 ◽  
Vol 25 (2) ◽  
pp. 854-864 ◽  
Author(s):  
Sandrine Marchetti ◽  
Clotilde Gimond ◽  
Jean-Claude Chambard ◽  
Thomas Touboul ◽  
Danièle Roux ◽  
...  
Keyword(s):  
Map Kinases ◽  
Fluorescent Protein ◽  
Proteasomal Degradation ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Double Mutation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.

Characterization of a cDNA encoding a novel band 4.1-like protein in zebrafish

1997 ◽  
Vol 75 (5) ◽  
pp. 623-632 ◽  
Author(s):  
Gregory M Kelly ◽  
Bruno Reversade
Keyword(s):  
Cell Communication ◽  
Membrane Skeleton ◽  
Adult Brain ◽  
Dependent Manner ◽  
Midblastula Transition ◽  
Protein 4.1 ◽  
Calcium Dependent ◽  
Calcium Calmodulin Dependent ◽  
Integral Role

Membrane skeleton protein 4.1 and other members of a family of proteins that link the cytoskeleton to the plasma membrane may play an integral role in cell communication during development. The polymerase chain reaction and degenerate oligodeoxynucleotide primers to consensus sequences in the putative membrane-binding domain of the protein 4.1 superfamily were used to isolate cDNAs encoding members of the zebrafish protein 4.1 family. Zebrafish stage- and tissue-specific first strand cDNA was used in the PCR. After the reaction, amplicons of the predicted size were sequenced to confirm their relationship to the protein 4.1 superfamily. One cDNA, with a high degree of similarity to a mouse novel band 4.1-like cDNA, was used to probe a zebrafish adult brain library. A 2.4-kb cDNA was isolated and found to encode a 619 amino acid polypeptide homologous to mouse novel band 4.1-like protein 4. Zebrafish nbl4 mRNA is maternally supplied and is expressed throughout embryogenesis. In adults, nbl4 is found in the ovary, eye, heart, and brain, but not in gut or skeletal muscle. When synthetic nbl4 mRNA is translated in vitro it binds calmodulin in a calcium-dependent manner. These data indicate that zebrafish nbl4 is a maternal transcript owing to its presence before the midblastula transition, and it is present later on in specific adult structures. The ability to bind calmodulin would suggest that the function of nbl4 protein may be potentially regulated via a calcium-calmodulin dependent mechanism.

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