scholarly journals High-affinity free ubiquitin sensors as quantitative probes of ubiquitin homeostasis and deubiquitination

2019 ◽  
Author(s):  
Yun-Seok Choi ◽  
Sarah A. Bollinger ◽  
Luisa F. Prada ◽  
Francesco Scavone ◽  
Tingting Yao ◽  
...  
Keyword(s):  
Real Time ◽  
Fluorescent Sensors ◽  
Ongoing Study ◽  
Post Translational Modification ◽  
Free Protein ◽  
Cell Lysates ◽  
High Affinity ◽  
Robust Techniques ◽  
Major Impediment ◽  
Free Ubiquitin

AbstractUbiquitin (Ub) conjugation is an essential post-translational modification that affects nearly all proteins in eukaryotes. The functions and mechanisms of ubiquitination are areas of extensive and ongoing study, and yet the dynamics and regulation of even free (i.e., unconjugated) Ub are poorly understood. A major impediment has been the lack of simple and robust techniques to quantify Ub levels in cells and to monitor Ub release from conjugates. Here we describe the development of avidity-based fluorescent sensors that address this need. The sensors bind specifically to free Ub, have Kd values down to 60 pM, and, in concert with a newly developed workflow, allow us to distinguish and quantify the pools of free, protein-conjugated, and thioesterified forms of Ub from cell lysates. Alternatively, free Ub in fixed cells can be visualized microscopically by staining with a sensor. Real-time assays using the sensors afford unprecedented flexibility and precision to measure deubiquitination of virtually any (poly)Ub conjugate.

Real-time monitoring of cell-free protein synthesis on a surface plasmon resonance chip

2007 ◽  
Vol 366 (2) ◽  
pp. 170-174 ◽  
Author(s):  
Kyung-Ho Lee ◽  
Hyou-Arm Joung ◽  
Jin-Ho Ahn ◽  
Kyeong-Ohn Kim ◽  
In-Seok Oh ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Surface Plasmon Resonance ◽  
Real Time ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Real Time Monitoring ◽  
Free Protein ◽  
Cell Free Protein Synthesis

Performing Security on Digital Images

2019 ◽  
pp. 211-246 ◽  
Author(s):  
Abdallah Soualmi ◽  
Lamri Laouamer ◽  
Adel Alti
Keyword(s):  
Real Time ◽  
Digital Images ◽  
Image Watermarking ◽  
Surveillance Systems ◽  
Original Image ◽  
Robust Techniques ◽  
Monitoring And Surveillance ◽  
Robust Image ◽  
Broadcast Monitoring

In image watermarking, information is embedded in the original image for many reasons, such as ownership proofing, alteration detection, and/or fingerprinting, but it can also be used for real-time services such as e-payment, broadcast monitoring, and surveillance systems. For these, the data embedded must be extractable even if the image is manipulated intentionally or unintentionally. In contrast, robust techniques are the kind of watermarking that could assure the authenticity and protect the copyright. Many robust image watermarking approaches have been proposed in the last few years, and the purpose of this chapter is to provide a survey about recent relevant robust image watermarking methods existing in the literature.

scholarly journals Isoleucine 44 Hydrophobic Patch Controls Toxicity of Unanchored, Linear Ubiquitin Chains through NF-κB Signaling

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1519 ◽  
Author(s):  
Jessica R. Blount ◽  
Kozeta Libohova ◽  
Gustavo M. Silva ◽  
Sokol V. Todi
Keyword(s):  
Drosophila Melanogaster ◽  
Fruit Fly ◽  
Post Translational Modification ◽  
Ubiquitin Chain ◽  
Interaction Site ◽  
Cellular Processes ◽  
Protein Adduct ◽  
Linear Chains ◽  
Linear Poly ◽  
Free Ubiquitin

Ubiquitination is a post-translational modification that regulates cellular processes by altering the interactions of proteins to which ubiquitin, a small protein adduct, is conjugated. Ubiquitination yields various products, including mono- and poly-ubiquitinated substrates, as well as unanchored poly-ubiquitin chains whose accumulation is considered toxic. We previously showed that transgenic, unanchored poly-ubiquitin is not problematic in Drosophila melanogaster. In the fruit fly, free chains exist in various lengths and topologies and are degraded by the proteasome; they are also conjugated onto other proteins as one unit, eliminating them from the free ubiquitin chain pool. Here, to further explore the notion of unanchored chain toxicity, we examined when free poly-ubiquitin might become problematic. We found that unanchored chains can be highly toxic if they resemble linear poly-ubiquitin that cannot be modified into other topologies. These species upregulate NF-κB signaling, and modulation of the levels of NF-κB components reduces toxicity. In additional studies, we show that toxicity from untethered, linear chains is regulated by isoleucine 44, which anchors a key interaction site for ubiquitin. We conclude that free ubiquitin chains can be toxic, but only in uncommon circumstances, such as when the ability of cells to modify and regulate them is markedly restricted.

Highly sensitive conjugated polymer fluorescent sensors based on benzochalcogendiazole for nickel ions in real-time detection

2014 ◽  
Vol 2 (35) ◽  
pp. 7402-7410 ◽  
Author(s):  
Yunxiang Lei ◽  
Hui Li ◽  
Wenxia Gao ◽  
Miaochang Liu ◽  
Jiuxi Chen ◽  
...  
Keyword(s):  
Real Time ◽  
Conjugated Polymer ◽  
Fluorescent Sensors ◽  
Sensitive Detection ◽  
Nickel Ions ◽  
Highly Sensitive ◽  
Real Time Detection ◽  
Highly Sensitive Detection

Two conjugated polymer fluorescent sensors using a benzochalcogendiazole unit and a triazole unit as cooperative receptors were synthesized for the highly sensitive detection of nickel ions.

scholarly journals High-affinity free ubiquitin sensors for quantifying ubiquitin homeostasis and deubiquitination

2019 ◽  
Vol 16 (8) ◽  
pp. 771-777 ◽  
Author(s):  
Yun-Seok Choi ◽  
Sarah A. Bollinger ◽  
Luisa F. Prada ◽  
Francesco Scavone ◽  
Tingting Yao ◽  
...  
Keyword(s):  
High Affinity ◽  
Free Ubiquitin

Real-time, Label-free Protein Binding Detection with a One Dimensional Photonic Crystal Sensor

2009 ◽  
Author(s):  
Yunbo Guo ◽  
Thommey P. Thomas ◽  
Jing Yong Ye ◽  
Andrzej Myc ◽  
James R. Baker ◽  
...  
Keyword(s):  
Photonic Crystal ◽  
Protein Binding ◽  
Real Time ◽  
Label Free ◽  
One Dimensional ◽  
Free Protein ◽  
Dimensional Photonic Crystal

scholarly journals Cell-Free Protein Synthesis Enhancement from Real-Time NMR Metabolite Kinetics: Redirecting Energy Fluxes in Hybrid RRL Systems

2017 ◽  
Vol 7 (1) ◽  
pp. 218-226 ◽  
Author(s):  
Baptiste Panthu ◽  
Théophile Ohlmann ◽  
Johan Perrier ◽  
Uwe Schlattner ◽  
Pierre Jalinot ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Real Time ◽  
Metabolite Kinetics ◽  
Energy Fluxes ◽  
Free Protein ◽  
Cell Free Protein Synthesis

The Detection of Large Deletions of PROS1 - a Common Cause of Protein S Deficiency

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4101-4101
Author(s):  
Ruth B Wheeler ◽  
Jacqueline A Cutler ◽  
Mike Mitchell
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Protein S ◽  
Protein S Deficiency ◽  
Phenotypic Data ◽  
Exon 2 ◽  
Common Cause ◽  
Free Protein ◽  
Large Deletions ◽  
S Deficiency

Abstract Protein S is a vitamin-K dependent plasma glycoprotein which regulates coagulation by acting as a non-enzymatic co-factor for activated protein C during the inactivation of FVa and FVIIIa. Protein S circulates in the plasma as either free protein S or in association with C4b binding protein but only free protein S has anticoagulant properties. Protein S deficiency is an autosomal dominant disorder associated with an increased risk of venous thrombosis. The PROS1 gene is located on the long arm of chromosome 3 near the centromere, in close association with a highly homologous pseudogene PROSP. More than 200 PROS1 mutations have been described to date, but a large cohort of patients exists in whom no mutations have been detected. Several recent studies have suggested that large deletions of PROS1 are common in protein S deficient patients and should be considered when conventional methodologies fail to identify the causative mutation. Therefore there is a requirement for robust and reliable tests capable of rapidly detecting and defining deletions which can be used in a diagnostic setting. We have developed an approach which combines haplotype analysis at several SNPs and simple sequence repeats located throughout the PROS1 genomic sequence which runs in parallel with conventional mutation analysis, followed by confirmation of DNA copy number by quantitative PCR using an ABI7500 Real Time PCR System and more recently multiplex ligation-dependent probe amplification (MLPA) using the SALSA MLPA P112 Kit (MRC Holland). We have used this approach to identify and define large PROS1 deletions in three of six patients referred with protein S deficiency, and in whom mutations had not been detected. Patient 1 presented with recurrent thrombosis and was found to have a severe type I protein S deficiency. PROS1 sequence was normal, but analysis of parental DNA at Pro626 identified the presence of a null allele. Real-time PCR and MLPA revealed that one whole copy of the gene was deleted in this patient. A homozygous mutation in exon 2 was identified in patient 2 who presented with severe protein S deficiency. Analysis of DNA from his son, who also presented with protein S deficiency, showed exon 2 to be normal indicating that this sequence change was hemizygous. A partial deletion involving exons 1 to 8 was subsequently detected in this family. No phenotypic data was available for family 3, which comprised two sisters, but our combined approach identified a deletion of over 50Kb involving the 5′UTR and exons 1 and 2. This highlights the usefulness of this methodology for mutation screening when phenotypic data is unavailable, which is often the case when patients are on continuous anticoagulant therapy. None of the remaining three patients had a family history of protein S deficiency and phenotypic data was inconclusive. Analysis failed to identify any genetic abnormalities in these patients. Our analyses confirm that deletions of PROS1 are a common cause of thrombosis. Further when deletions are not detected using this methodology the information may be useful as supporting evidence for clinicians when reaching a diagnostic decision as to the cause of thrombosis. In summary our approach offers a rapid and reliable approach to the detection of large protein S deletions which would otherwise be missed by routine technologies.

Histological, Spectroscopic, and Surface Analysis of Microdamage in Bone:  Toward Real-Time Analysis Using Fluorescent Sensors

2007 ◽  
Vol 19 (7) ◽  
pp. 1656-1663 ◽  
Author(s):  
Raman Parkesh ◽  
Sahar Mohsin ◽  
T. Clive Lee ◽  
Thorfinnur Gunnlaugsson
Keyword(s):  
Real Time ◽  
Surface Analysis ◽  
Fluorescent Sensors ◽  
Time Analysis ◽  
Real Time Analysis

scholarly journals Phosphorylation of Kindlins and the Control of Integrin Function

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 825
Author(s):  
Katarzyna Bialkowska ◽  
Jun Qin ◽  
Edward F. Plow
Keyword(s):  
Beta Subunits ◽  
Post Translational Modification ◽  
Integrin Activation ◽  
High Affinity ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Cytoplasmic Tails ◽  
Cell Matrix Interactions ◽  
Integrin Regulation

Integrins serve as conduits for the transmission of information between cells and their extracellular environment. Signaling across integrins is bidirectional, transducing both inside-out and outside-signaling. Integrin activation, a transition from a low affinity/avidity state to a high affinity/avidity state for cognate ligands, is an outcome of inside-signaling. Such activation is particularly important for the recognition of soluble ligands by blood cells but also influences cell-cell and cell-matrix interactions. Integrin activation depends on a complex series of interactions, which both accelerate and inhibit their interconversion from the low to the high affinity/avidity state. There are three components regarded as being most proximately involved in integrin activation: the integrin cytoplasmic tails, talins and kindlins. The participation of each of these molecules in integrin activation is highly regulated by post-translation modifications. The importance of targeted phosphorylation of integrin cytoplasmic tails and talins in integrin activation is well-established, but much less is known about the role of post-translational modification of kindlins. The kindlins, a three-member family of 4.1-ezrin-radixin-moesin (FERM)-domain proteins in mammals, bind directly to the cytoplasmic tails of integrin beta subunits. This commentary provides a synopsis of the emerging evidence for the role of kindlin phosphorylation in integrin regulation.

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