Effects of the Fc-III tag on activity and stability of green fluorescent protein and human muscle creatine kinase.
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ABSTRACT:
SUBMITTER: Feng S
PROVIDER: S-EPMC3719094 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundHundreds of genes, including muscle creatine kinase (MCK), are differentially expressed in fast- and slow-twitch muscle fibers, but the fiber type-specific regulatory mechanisms are not well understood.ResultsModulatory region 1 (MR1) is a 1-kb regulatory region within MCK intron 1 that is highly active in terminally differentiating skeletal myocytes in vitro. A MCK small intronic enhancer (MCK-SIE) containing a paired E-box/myocyte enhancer factor 2 (MEF2) regulatory motif resides within MR1. The SIE's transcriptional activity equals that of the extensively characterized 206-bp MCK 5'-enhancer, but the MCK-SIE is flanked by regions that can repress its activity via the individual and combined effects of about 15 different but highly conserved 9- to 24-bp sequences. ChIP and ChIP-Seq analyses indicate that the SIE and the MCK 5'-enhancer are occupied by MyoD, myogenin and MEF2. Many other E-boxes located within or immediately adjacent to intron 1 are not occupied by MyoD or myogenin. Transgenic analysis of a 6.5-kb MCK genomic fragment containing the 5'-enhancer and proximal promoter plus the 3.2-kb intron 1, with and without MR1, indicates that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers (types I and IIa, respectively), but is not required for expression in fast-twitch muscle fibers (types IIb and IId).ConclusionsIn this study, we discovered that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers and that MR1's positive transcriptional activity depends on a paired E-box MEF2 site motif within a SIE. This is the first study to delineate the DNA controls for MCK expression in different skeletal muscle fiber types.
| S-EPMC3157005 | biostudies-literature
Project description:Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity.
| S-EPMC8448711 | biostudies-literature
Project description:Calmodulin (CaM) is a ubiquitous Ca2+ sensing protein that binds to and modulates numerous target proteins and enzymes during cellular signaling processes. A large number of CaM-target complexes have been identified and structurally characterized, revealing a wide diversity of CaM-binding modes. A newly identified target is creatine kinase (CK), a central enzyme in cellular energy homeostasis. This study reports two high-resolution X-ray structures, determined to 1.24 Å and 1.43 Å resolution, of calmodulin in complex with peptides from human brain and muscle CK, respectively. Both complexes adopt a rare extended binding mode with an observed stoichiometry of 1:2 CaM:peptide, confirmed by isothermal titration calorimetry, suggesting that each CaM domain independently binds one CK peptide in a Ca2+-depended manner. While the overall binding mode is similar between the structures with muscle or brain-type CK peptides, the most significant difference is the opposite binding orientation of the peptides in the N-terminal domain. This may extrapolate into distinct binding modes and regulation of the full-length CK isoforms. The structural insights gained in this study strengthen the link between cellular energy homeostasis and Ca2+-mediated cell signaling and may shed light on ways by which cells can 'fine tune' their energy levels to match the spatial and temporal demands.
| S-EPMC8244255 | biostudies-literature
Project description:The low photostability of conventional organic dyes and the toxicity of cadmium-based luminescent quantum dots have prompted the development of novel probes for in vitro and in vivo labelling. Here, a new fluorescent lanthanide probe based on silica nanoparticles is fabricated and investigated for optically traceable in vitro translocator protein (TSPO) targeting. The targeting and detection of TSPO receptor, overexpressed in several pathological states, including neurodegenerative diseases and cancers, may provide valuable information for the early diagnosis and therapy of human disorders. Green fluorescent terbium(III)-calix[4]arene derivative complexes are encapsulated within silica nanoparticles and surface functionalized amine groups are conjugated with selective TSPO ligands based on a 2-phenylimidazo[1,2-a]pyridine acetamide structure containing derivatizable carboxylic groups. The photophysical properties of the terbium complex, promising for biological labelling, are demonstrated to be successfully conveyed to the realized nanoarchitectures. In addition, the high degree of biocompatibility, assessed by cell viability assay and the selectivity towards TSPO mitochondrial membrane receptors, proven by subcellular fractional studies, highlight targeting potential of this nanostructure for in vitro labelling of mitochondria.
| S-EPMC6651519 | biostudies-literature
Project description:Muscle ring finger-1 (MuRF1) is a muscle-specific protein implicated in the regulation of cardiac myocyte size and contractility. MuRF2, a closely related family member, redundantly interacts with protein substrates, and hetero-dimerizes with MuRF1. Mice lacking either MuRF1 or MuRF2 are phenotypically normal whereas mice lacking both proteins develop a spontaneous cardiac and skeletal muscle hypertrophy indicating cooperative control of muscle mass by MuRF1 and MuRF2. In order to identify the role that MuRF1 plays in regulating cardiac hypertrophy in vivo, we created transgenic mice expressing increased amounts of cardiac MuRF1. Adult MuRF1 transgenic (Tg+) hearts exhibited a non-progressive thinning of the left ventricular wall and a concomitant decrease in cardiac function. Experimental induction of cardiac hypertrophy by trans-aortic constriction (TAC) induced rapid failure of MuRF1 Tg+ hearts. Microarray analysis identified that the levels of genes associated with metabolism (and in particular mitochondrial processes) were significantly altered in MuRF1 Tg+ hearts, both at baseline and during the development of cardiac hypertrophy. Surprisingly, ATP levels in MuRF1 Tg+ mice did not differ from wild type mice despite the depressed contractility following TAC. To explain this discrepancy between the ongoing heart failure and maintained ATP levels in MuRF1 Tg+ hearts, we compared the level and activity of creatine kinase (CK) between wild type and MuRF1 Tg+ hearts. Although mCK and CK-M/B protein levels were unaffected in MuRF1 Tg+ hearts, total CK activity was significantly inhibited. We conclude that MuRF1’s inhibition of CK activity leads to increased susceptibility to heart failure following TAC, demonstrating for the first time that MuRF1 regulates cardiac energetics in vivo. Keywords: Genetic modification, physiological manipulation. Three-condition experiment, MuRF1 Tg+ vs. WT mice. Biological replicates: 4 WT baseline, 3 MuRF1 Tg+ baseline, cardiac overpressure hypertrophy induced by trans-aortic banding at 1 week (3 WT, 3 MurF Tg) and 4 weeks (3 WT, 3 MurF Tg), hearts harvested. One replicate per array.
2009-06-03 | E-GEOD-11661 | biostudies-arrayexpress
Project description:Morphine is an analgesic alkaloid used to relieve severe pain, and irreversible binding of morphine to specific unknown proteins has been previously observed. In the brain, changes in the expression of energy metabolism enzymes contribute to behavioral abnormalities during chronic morphine treatment. Creatine kinase B (CK-B) is a key enzyme involved in brain energy metabolism. CK-B also corresponds to the imidazoline-binding protein I2 which binds dopamine (a precursor of morphine biosynthesis) irreversibly. Using biochemical approaches, we show that recombinant mouse CK-B possesses a ?M affinity for morphine and binds to morphine in vitro. The complex formed by CK-B and morphine is resistant to detergents, reducing agents, heat treatment and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). CK-B-derived peptides CK-B1-75 and CK-B184-258 were identified as two specific morphine binding-peptides. In vitro, morphine (1-100 ?M) significantly reduces recombinant CK-B enzymatic activity. Accordingly, in vivo morphine administration (7.5 mg/kg, i.p.) to mice significantly decreased brain extract CK-B activity compared to saline-treated animals. Together, these results show that morphine strongly binds CK-B and inhibits its activity in vitro and in vivo.
| S-EPMC6286964 | biostudies-literature
Project description:Muscle ring finger-1 (MuRF1) is a muscle-specific protein implicated in the regulation of cardiac myocyte size and contractility. MuRF2, a closely related family member, redundantly interacts with protein substrates, and hetero-dimerizes with MuRF1. Mice lacking either MuRF1 or MuRF2 are phenotypically normal whereas mice lacking both proteins develop a spontaneous cardiac and skeletal muscle hypertrophy indicating cooperative control of muscle mass by MuRF1 and MuRF2. In order to identify the role that MuRF1 plays in regulating cardiac hypertrophy in vivo, we created transgenic mice expressing increased amounts of cardiac MuRF1. Adult MuRF1 transgenic (Tg+) hearts exhibited a non-progressive thinning of the left ventricular wall and a concomitant decrease in cardiac function. Experimental induction of cardiac hypertrophy by trans-aortic constriction (TAC) induced rapid failure of MuRF1 Tg+ hearts. Microarray analysis identified that the levels of genes associated with metabolism (and in particular mitochondrial processes) were significantly altered in MuRF1 Tg+ hearts, both at baseline and during the development of cardiac hypertrophy. Surprisingly, ATP levels in MuRF1 Tg+ mice did not differ from wild type mice despite the depressed contractility following TAC. To explain this discrepancy between the ongoing heart failure and maintained ATP levels in MuRF1 Tg+ hearts, we compared the level and activity of creatine kinase (CK) between wild type and MuRF1 Tg+ hearts. Although mCK and CK-M/B protein levels were unaffected in MuRF1 Tg+ hearts, total CK activity was significantly inhibited. We conclude that MuRF1’s inhibition of CK activity leads to increased susceptibility to heart failure following TAC, demonstrating for the first time that MuRF1 regulates cardiac energetics in vivo. Keywords: Genetic modification, physiological manipulation.
2009-06-03 | GSE11661 | GEO
Project description:In the directed evolution experiments of EGFP or StayGold, the objective was to blue-shift the excitation light spectrum of GFP towards shorter wavelengths, resulting in an enhancement of fluorescence intensity upon 405 nm laser excitation. This facilitates the advancement of novel fluorescent proteins. These experiments represent a category of experiments that utilize REPLACE to achieve discontinuous directed evolution through FACS sorting (screening).
2024-09-30 | GSE271876 | GEO
Project description:BackgroundGenetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming.Methodology/principal findingsHere we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8xHis), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8xHis and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry.Conclusions and significanceThe multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.
| S-EPMC2585475 | biostudies-literature
Project description:Creatine kinase catalyzes the reversible transfer of the gamma phosphate from ATP to creatine forming the high energy compound creatine phosphate. Muscle creatine kinase (CKm) activity maintains energetic homeostasis as variations in energy requirements dictate that ATP be readily available. Recent studies suggest that CKm activity is altered during aging. Proteomic analyses have shown that CKm is 3-nitrotyrosine (3-NT) modified and carbonylated in aged rodent skeletal muscle. However, it remains unknown if these modifications affect its structure and activity. To address this we characterized oxidatively modified CKm from the quadriceps of young, middle-aged, and aged mice. Our data indicate that 3-NT modified and carbonylated CKm are found predominantly in aged muscle and that it exists in high molecular weight oligomers and insoluble protein aggregates. CKm from middle-aged and aged mouse quadriceps also exhibits structural instability that may account for its reduction in function. These structural and functional changes correlate with the differential protein modifications. Interestingly, the majority of the age-related changes in enzyme activity and protein stability occurred by middle age. Our studies indicate that the age-associated oxidative and nitrative modification of CKm results in a decrease in its activity and may cause structural changes that promote oligomerization and aggregation.
| S-EPMC2830079 | biostudies-literature