scholarly journals The fluorescent protein stability assay: an efficient method for monitoring intracellular protein stability

BioTechniques ◽  
2021 ◽  
Author(s):  
Armelle Roisin ◽  
Samuel Buchsbaum ◽  
Vincent Mocquet ◽  
Pierre Jalinot
Keyword(s):  
Protein Stability ◽  
Efficient Method ◽  
Fluorescent Protein ◽  
Specific Protein ◽  
Intracellular Protein ◽  
Fluorescent Reporter ◽  
Cellular Environment ◽  
Intracellular Proteins ◽  
Reporter Proteins ◽  
The Stability

The stability of intracellular proteins is highly variable, from a few minutes to several hours, and can be tightly regulated to respond to external and internal cellular environment changes. Several techniques can be used to study the stability of a specific protein, including pulse-chase labeling and blocking of translation. Another approach that has gained interest in recent years is fusing a protein of interest to a fluorescent reporter. In this report, the authors present a new version of this approach aimed at optimizing expression and comparison of the two reporter proteins. The authors show that the system works efficiently in various cells and can be useful for studying changes in protein stability and assessing the effects of drugs.

scholarly journals Nonmyofilament-associated troponin T fragments induce apoptosis

2009 ◽  
Vol 297 (1) ◽  
pp. H283-H292 ◽  
Author(s):  
Euy-Myong Jeong ◽  
Xin Wang ◽  
Kun Xu ◽  
M. Moazzem Hossain ◽  
J.-P. Jin
Keyword(s):  
Fluorescent Protein ◽  
Striated Muscle ◽  
Troponin T ◽  
Variable Region ◽  
Nemaline Myopathy ◽  
Specific Protein ◽  
Cellular Protection ◽  
Cellular Environment ◽  
Nonmuscle Cells ◽  
Green Fluorescent

Troponin T (TnT) is a striated muscle-specific protein and an abundant component of the myofilaments. Nonmyofilament-associated TnT is rapidly degraded in myocytes, implying an importance in the maintenance of the cellular environment. However, if the level of nonmyofilament-associated TnT or TnT fragments exceeds the degradation capacity, it may cause cytotoxicity. To investigate this hypothesis, we constructed bicistronic vectors to express different portions of TnT polypeptide chain, together with nonfusion green fluorescent protein as a tracer for the transfection. Cytotoxicity of the TnT fragments was studied through forced expression in C2C12 myoblasts and human embryonic kidney-293 nonmuscle cells and examination of the viability of the transfected cells. The results demonstrated that, in the absence of myofilaments, the conserved COOH-terminal and middle fragments of TnT were highly effective on inducing cell death via apoptosis, whereas the NH2-terminal variable region was not. As combined effects, nonmyofilament-associated intact cardiac TnT and a COOH-terminal truncated slow TnT fragment found in Amish nemaline myopathy exhibited intermediate cytotoxicity. A particular significance of this finding is that peak releases of TnT or TnT fragments from decomposition of a large number of myofibrils in acute myocardial infarction may breach the cellular protection of proteolytic degradation and result in apoptosis as a potential cause for the loss of cardiomyocytes.

scholarly journals Quantitative Analysis of Global Protein Stability Rates in Tissues

2019 ◽  
Author(s):  
Daniel B. McClatchy ◽  
Yu Gao ◽  
Mathieu Lavallée-Adam ◽  
John R. Yates
Keyword(s):  
Protein Stability ◽  
Protein Degradation ◽  
Expression Patterns ◽  
Specific Protein ◽  
Normal Diet ◽  
Cellular Environment ◽  
Degradation Rates ◽  
Mouse Tissues ◽  
Protein Functions ◽  
The Brain

AbstractProtein degradation is an essential mechanism for maintaining homeostasis in response to internal and external perturbations. Disruption of this process is implicated in many human diseases, but quantitation of global stability rates has not yet been achieved in tissues. We have developed QUAD (Quantification of Azidohomoalanine Degradation), a technique to quantitate global protein degradation using mass spectrometry. Azidohomoalanine (AHA) is pulsed into mouse tissues through their diet. The mice are then returned to a normal diet and the decrease of AHA abundance can be quantitated in the proteome. QUAD analysis reveals that protein stability varied within tissues, but discernible trends in the data suggest that cellular environment is a major factor dictating stability. Within a tissue, different organelles, post-translation modifications, and protein functions were enriched with different stability patterns. Surprisingly, subunits of the TRIC molecular chaperonin possessed markedly distinct stability trajectories in the brain. Further investigation revealed that these subunits also possessed different subcellular localization and expression patterns that were uniquely altered with age and in Alzheimer’s disease transgenic mice, indicating a potential non-canonical chaperonin. Finally, QUAD analysis demonstrated that protein stability is enhanced with age in the brain but not in the liver. Overall, QUAD allows the first global quantitation of protein stability rates in tissues, which may lead to new insights and hypotheses in basic and translational research.SummaryProtein degradation is an important component of the proteostasis network, but no techniques are available to globally quantitate degradation rates in tissues. In this study, we demonstrate a new method QUAD (Quantification of Azidohomoalanine Degradation) that can accurately quantitate degradation rates in tissues. QUAD analysis of mouse tissues reveal that unique degradation trends can define different tissue proteomes. Within a tissue, specific protein characteristics are correlated with different levels of protein stability. Further investigation of the TRIC chaperonin with strikingly different subunit stabilities suggests a non-canonical chaperonin in brain tissue. Consistent with the theory that the proteostasis network is compromised with age, we discovered that protein stability is globally enhanced in brains of old mice compared to young mice.

scholarly journals Generation of Transgenic Medaka Oryzias curvinotus (Nichols & Pope, 1927) Carrying a Cyan Fluorescent Protein Gene Driven by Alpha Actin Promoter

2021 ◽  
Vol 34 (1) ◽  
Author(s):  
VY NGUYEN HOANG THUY ◽  
TRUNG MAI NGUYEN THANH ◽  
BINH NGUYEN QUOC ◽  
HOA NGUYEN THI KIEU ◽  
DU NGUYEN VAN ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Fluorescent Protein ◽  
Cyan Fluorescent Protein ◽  
Cell Stage ◽  
Fluorescent Reporter ◽  
Myosin Light Chain 2 ◽  
Oryzias Curvinotus ◽  
The Stability ◽  
Green Fluorescent ◽  
Alpha Actin

The study aimed to produce fluorescent protein transgenic medaka Oryzias curvinotus (Nichols & Pope, 1927) as a novel strain of ornamental fish. These fish were produced by transferring a plasmid consisting of a fluorescent reporter gene and a strong promoter into one-cell stage embryos. For this purpose, myosin light chain 2, but not other promoters, was mainly used. The study also evaluated the stability of the transgenic medaka germline acquiring vivid fluorescent phenotypes via the transgenesis of the cyan fluorescent protein (CFP) gene under the control of O. curvinotus skeletal alpha-actin (OCacta) promoter. The pOCacta-CFP plasmid, containing a OCacta promoter and CFP reporter gene, was transferred into the one-cell stage of O. curvinotus embryos by a microinjection technique. As a result, 36 of 1386 microinjected O. curvinotus embryos exhibited CFP signals in their trunks. The expressed CFP signals in O. curvinotus embryos and adults were detected under a microscope using a green fluorescent protein (GFP) filter (450–490 nm wavelength), and blue LED light (400–450 nm wavelength). Five O. curvinotus founders showing clear CFP signals were selected and crossed with non-transgenic counterparts to produce subsequent generations. Among strains, the frequency of germline transmission from founder to F1 was highly variable. Only two of the five founders successfully pass the transgene to the F1 generation. At present, the progeny of subsequent generations is being produced and tested for the expression of CFP signals, and therefore, stable lines are ongoing.

scholarly journals Construction of Two mCherry Plasmids (pXG-mCherry) for Transgenic Leishmania: Valuable Tools for Future Molecular Analysis

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Andrés Vacas ◽  
Conor Sugden ◽  
Óscar Velasco-Rodriguez ◽  
Miriam Algarabel-Olona ◽  
José Peña-Guerrero ◽  
...  
Keyword(s):  
Drug Screening ◽  
Fluorescent Protein ◽  
Red Fluorescent Protein ◽  
Fluorescent Reporter ◽  
The World ◽  
Colorimetric Assays ◽  
Reporter Proteins ◽  
Indicator Dyes ◽  
Multiple Cloning Sites

Leishmania is the causative agent of leishmaniasis, a neglected tropical disease that affects more than 12 million people around the world. Current treatments are toxic and poorly effective due to the acquisition of resistance within Leishmania populations. Thus, the pursuit for new antileishmanial drugs is a priority. The available methods for drug screening based on colorimetric assays using vital dyes are time-consuming. Currently, the use of fluorescent reporter proteins is replacing the use of viability indicator dyes. We have constructed two plasmids expressing the red fluorescent protein mCherry with multiple cloning sites (MCS), adequate for N- and C-terminal fusion protein constructs. Our results also show that the improved pXG-mCherry plasmid can be employed for drug screening in vitro. The use of the red fluorescent protein, mCherry, is an easier tool for numerous assays, not only to test pharmacological compounds, but also to determine the subcellular localization of proteins.

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

scholarly journals INTRACISTRONIC MAPPING OF ELECTROPHORETIC SITES IN DROSOPHILA MELANOGASTER: FIDELITY OF INFORMATION TRANSFER BY GENE CONVERSION

Genetics ◽  
1974 ◽  
Vol 76 (2) ◽  
pp. 289-299
Author(s):  
Margaret McCarron ◽  
William Gelbart ◽  
Arthur Chovnick
Keyword(s):  
Drosophila Melanogaster ◽  
Information Transfer ◽  
Large Scale ◽  
Genetic Basis ◽  
Xanthine Dehydrogenase ◽  
Specific Protein ◽  
Wild Type ◽  
Electrophoretic Variation ◽  
The Stability ◽  
Recombination Experiments

ABSTRACT A convenient method is described for the intracistronic mapping of genetic sites responsible for electrophoretic variation of a specific protein in Drosophila melanogaster. A number of wild-type isoalleles of the rosy locus have been isolated which are associated with the production of electrophoretically distinguishable xanthine dehydrogenases. Large-scale recombination experiments were carried out involving null enzyme mutants induced on electrophoretically distinct wild-type isoalleles, the genetic basis for which is followed as a nonselective marker in the cross. Additionally, a large-scale recombination experiment was carried out involving null enzyme rosy mutants induced on the same wild-type isoallele. Examination of the electrophoretic character of crossover and convertant products recovered from the latter experiment revealed that all exhibited the same parental electrophoretic character. In addition to documenting the stability of the xanthine dehydrogenase electrophoretic character, this observation argues against a special mutagenesis hypothesis to explain conversions resulting from allele recombination studies.

Abstract 38: Rbp-j Regulates the Genetic Program of the Myo-epithelioid JG Cell

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Ruth M Castellanos Rivera ◽  
Ellen S. Pentz ◽  
Kenneth W. Gross ◽  
Silvia Medrano ◽  
Jing Yu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Fluorescent Protein ◽  
Smooth Muscle Actin ◽  
Artificial Chromosome ◽  
Specific Protein ◽  
Genetic Program ◽  
Cell Phenotype ◽  
Gfp Expression ◽  
Renin Gene

RBP-J , the major downstream effector of Notch signaling, is necessary to maintain the number of juxtaglomerular (JG) cells. In addition, RBP-J regulates the plasticity of arteriolar smooth muscle cells to adopt the renin cell phenotype when homeostasis is threatened. We hypothesized that RBP-J acts as an on/off switch controlling the expression of genes that determine the renin phenotype. To determine whether RBP-J directly affects renin gene expression, we generated mice harboring a bacterial artificial chromosome (BAC) transgene with green fluorescent protein (GFP) under the control of the renin gene carrying a mutation in its RBP-J- binding site (Mut-BAC). Mut-BAC mice had markedly reduced GFP expression to 12.9 % ±0.01 (n=3) of the control (Wt-BAC) and a diminished response to homeostatic challenges: mut-BAC mice had a reduced number of GFP positive JG areas per total number of glomeruli (Wt-BAC: 25.1 % ±3.0, n=3; Mut-BAC: 9.3 % ±1.4, n=2, p<0.02) and no GFP expression along the arterioles. To determine whether the decrease in the number of JG cells in mice lacking RBP-J (cKO) was due to a diminished endowment of renin progenitor cells, we traced the fate of cells derived from the renin lineage by generating mice ( RBP-J fl/fl ; Ren1d +/cre ; R26R +/- ) in which cells lacking RBP-J simultaneously expressed β-galactosidase (β-gal). The pattern of β-gal in cKO and control kidneys was identical, indicating that cells derived from the renin lineage did not die but instead changed their phenotype. Next we investigated the phenotype adopted by the cells derived from the renin lineage. Expression of α-smooth muscle actin and smoothelin (a marker of mature smooth muscle) was significantly decreased to 41 % ±7.0 (n=2) and 44 % ±8.8 (n=2) respectively with respect to controls (p<0.01). In addition, mutant JG cells in vivo did not express genes characteristic of the renin phenotype such as renin, calponin1, Nfat and Akr1b7 expressing instead fibroblast-specific protein 1 indicating the adoption of a fibroblast-like phenotype. Results indicate that RBP-J directly governs a genetic program that controls the dual endocrine-contractile phenotype of the JG cell, which is crucial to maintain blood pressure and fluid-electrolyte homeostasis.

Abstract 23: Characterization of MitoTimer, a Novel Tool for Monitoring Mitochondrial Turnover

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Christine A Thornton ◽  
Allen M Andres ◽  
Genaro Hernandez ◽  
Jon Sin ◽  
Roberta Gottlieb
Keyword(s):  
Protein Import ◽  
Spatial Information ◽  
Fluorescent Protein ◽  
Mitochondrial Dynamics ◽  
Inducible Promoter ◽  
Subcellular Fractionation ◽  
Fluorescent Reporter ◽  
Hek 293 ◽  
Mitochondrial Turnover

Fluorescent Timer, or DsRed1-E5, is a mutant of the red fluorescent protein, dsRed, developed by Terskikh and colleagues. Its fluorescence shifts over time from green to red as the protein matures. This molecular clock gives temporal and spatial information on protein turnover. To visualize mitochondrial turnover, we targeted Timer to the mitochondrial matrix with a mitochondrial targeting sequence (coined “MitoTimer”) and cloned it into a tetracycline-inducible promoter construct to regulate its expression. Here we report characterization of this novel fluorescent reporter for mitochondrial dynamics. Tet-On HEK 293 cells were transfected with pTRE-tight-MitoTimer and induced production with doxycycline. Mitochondrial distribution was demonstrated by fluorescence microscopy and verified by subcellular fractionation and western blot analysis. Doxycycline addition for as little as 1hr was sufficient to label mitochondria. MitoTimer was detected as early as 4hr following doxycycline addition, and persisted in mitochondria for at least 72hr. The color-specific conformation of MitoTimer was stable after fixation with 4% paraformaldehyde. MitoTimer matured to red fluorescence within 48hr, at which time a second pulse of doxycycline induced expression of green (immature) MitoTimer which was selectively incorporated into a subset of mitochondria actively engaged in protein import. The extent of new protein incorporation during a second pulse was increased under conditions of mito-biogenesis and reduced if mitochondrial membrane potential was dissipated. We conclude that MitoTimer can be used to monitor mitophagy and biogenesis.

The yeast UME5 gene regulates the stability of meiotic mRNAs in response to glucose

1994 ◽  
Vol 14 (5) ◽  
pp. 3446-3458
Author(s):  
R T Surosky ◽  
R Strich ◽  
R E Esposito
Keyword(s):  
Vegetative Growth ◽  
Signal Transduction Pathway ◽  
Kinase Domain ◽  
Specific Protein ◽  
Transcript Stability ◽  
The Family ◽  
Two Factors ◽  
Genetic Epistasis ◽  
The Stability ◽  
Acetate Medium

We reported previously that early meiotic transcripts are highly unstable. These mRNAs exhibit half-lives of approximately 3 min when expressed during vegetative growth in glucose medium and are stabilized twofold during sporulation in acetate medium. Two genes, UME2 and UME5, that regulate the stability of meiosis-specific transcripts have been identified. The wild-type UME5 gene, which has been analyzed in detail, decreases the stability of all meiotic mRNAs tested approximately twofold when expressed during vegetative growth but has no effect on the half-lives of a number of vegetative mRNAs examined. The UME5 gene is dispensable for mitotic and meiotic development. Cells in which the entire UME5 gene has been deleted are viable, although the generation time is slightly longer and sporulation is less efficient. The UME5 transcript is constitutively expressed, and its stability is not autoregulated. The UME5 gene encodes a predicted 63-kDa protein with homology to the family of CDC28 serine/threonine-specific protein kinases. The kinase activity appears to be central to the function of the UME5 protein, since alteration of a highly conserved amino acid in the kinase domain results in a phenotype identical to that of a ume5 deletion. Genetic epistasis studies suggest that the UME2 and UME5 gene products act in the same pathway to regulate meiotic transcript stability. This pathway is independent of deadenylation and translation, two factors known to be important in regulating mRNA turnover. Significantly, the UME5-mediated destabilization of meiotic mRNAs occurs in glucose- but not in acetate-containing medium. Thus, the UME5 gene appears to participate in a glucose signal transduction pathway governing message stability.

scholarly journals Microglia is a Key Player in the Reduction of Stroke Damage Promoted by the New Antithrombotic Agent Ticagrelor

2014 ◽  
Vol 34 (6) ◽  
pp. 979-988 ◽  
Author(s):  
Paolo Gelosa ◽  
Davide Lecca ◽  
Marta Fumagalli ◽  
Dorota Wypych ◽  
Alice Pignieri ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Interleukin 1 ◽  
Artery Occlusion ◽  
Cerebral Injury ◽  
Sprague Dawley ◽  
Antithrombotic Agent ◽  
Fluorescent Reporter ◽  
Monocyte Chemoattractant Protein 1 ◽  
Proinflammatory Mediators ◽  
Green Fluorescent

The ADP-responsive P2Y12 receptor is expressed on both platelets and microglia. Clinical data show that ticagrelor, a direct-acting, reversibly binding P2Y12-receptor antagonist, reduces total cardiovascular events, including stroke. In our present study, we investigated the expression of P2Y12 receptors and the effects of ticagrelor on brain injury in Sprague-Dawley rats subjected to a permanent middle cerebral artery occlusion (MCAo). Rats were treated per os with ticagrelor 3 mg/kg or vehicle at 10 minutes, 22, and 36 hours after MCAo and killed after 48 hours. Immunofluorescence analysis showed an ischemia-related modulation of the P2Y12 receptor, which is constitutively expressed in Iba1+ resting microglia. After MCAo, activated microglia was mainly concentrated around the lesion, with fewer cells present inside the ischemic core. Ticagrelor significantly attenuated the evolution of ischemic damage—evaluated by magnetic resonance imaging (MRI) at 2, 24, and 48 hours after MCAo—, the number of infiltrating cells expressing the microglia/monocyte marker ED-1, the cerebral expression of proinflammatory mediators (interleukin 1 (IL-1), monocyte chemoattractant protein 1 (MCP-1), nitric oxide synthase (iNOS)) and the associated neurologic impairment. In transgenic fluorescent reporter CX3CR1-green fluorescent protein (GFP) mice, 72 hours after MCAo, ticagrelor markedly reduced GFP+ microglia and both early and late infiltrating blood-borne cells. Finally, in primary cultured microglia, ticagrelor fully inhibited ADP-induced Chemotaxis ( P<0.01). Our results show that ticagrelor is protective against ischemia-induced cerebral injury and this effect is mediated, at least partly, by inhibition of P2Y12-mediated microglia activation and Chemotaxis.

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