scholarly journals Crosstalk between diverse synthetic protein degradation tags inEscherichia coli

2017 ◽  
Author(s):  
Nicholas C. Butzin ◽  
William H. Mather
Keyword(s):  
Protein Degradation ◽  
Fluorescent Proteins ◽  
Inescherichia Coli ◽  
E Coli ◽  
Synthetic Protein ◽  
Degradation Rates ◽  
Cell Assays ◽  
Synthetic Circuit ◽  
Microplate Reader ◽  
Single Protease

AbstractRecently, a synthetic circuit inE. colidemonstrated that two proteins engineered with LAA tags targeted to the native protease ClpXP are susceptible to crosstalk due to competition for degradation between proteins. To understand proteolytic crosstalk beyond the single protease regime, we investigated inE. colia set of synthetic circuits designed to probe the dynamics of existing and novel degradation tags fused to fluorescent proteins. These circuits were tested using both microplate reader and single-cell assays. We first quantified the degradation rates of each tag in isolation. We then tested if there was crosstalk between two distinguishable fluorescent proteins engineered with identical or different degradation tags. We demonstrated that proteolytic crosstalk was indeed not limited to the LAA degradation tag, but was also apparent between other diverse tags, supporting the complexity of theE. coliprotein degradation system.

scholarly journals High-Throughput Quantitative Assay Technologies for Accelerating the Discovery and Optimization of Targeted Protein Degradation Therapeutics

2021 ◽  
pp. 247255522098504
Author(s):  
Jeffrey R. Simard ◽  
Linda Lee ◽  
Ellen Vieux ◽  
Reina Improgo ◽  
Trang Tieu ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Protein Degradation ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
De Novo ◽  
Rapid Development ◽  
Dependent Manner ◽  
Western Blots ◽  
Advantages And Disadvantages ◽  
Degradation Rates

The aberrant regulation of protein expression and function can drastically alter cellular physiology and lead to numerous pathophysiological conditions such as cancer, inflammatory diseases, and neurodegeneration. The steady-state expression levels of endogenous proteins are controlled by a balance of de novo synthesis rates and degradation rates. Moreover, the levels of activated proteins in signaling cascades can be further modulated by a variety of posttranslational modifications and protein–protein interactions. The field of targeted protein degradation is an emerging area for drug discovery in which small molecules are used to recruit E3 ubiquitin ligases to catalyze the ubiquitination and subsequent degradation of disease-causing target proteins by the proteasome in both a dose- and time-dependent manner. Traditional approaches for quantifying protein level changes in cells, such as Western blots, are typically low throughput with limited quantification, making it hard to drive the rapid development of therapeutics that induce selective, rapid, and sustained protein degradation. In the last decade, a number of techniques and technologies have emerged that have helped to accelerate targeted protein degradation drug discovery efforts, including the use of fluorescent protein fusions and reporter tags, flow cytometry, time-resolved fluorescence energy transfer (TR-FRET), and split luciferase systems. Here we discuss the advantages and disadvantages associated with these technologies and their application to the development and optimization of degraders as therapeutics.

scholarly journals T7 phage factor required for managing RpoS inEscherichia coli

2018 ◽  
Vol 115 (23) ◽  
pp. E5353-E5362 ◽  
Author(s):  
Aline Tabib-Salazar ◽  
Bing Liu ◽  
Declan Barker ◽  
Lynn Burchell ◽  
Udi Qimron ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Stationary Phases ◽  
Inescherichia Coli ◽  
E Coli ◽  
Bacterial Transcription ◽  
Bacterial Rna ◽  
Predominant Form ◽  
Phage Development ◽  
T7 Phage

T7 development inEscherichia colirequires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ70, by two T7 proteins: Gp2 and Gp5.7. Although the biological role of Gp2 is well understood, that of Gp5.7 remains to be fully deciphered. Here, we present results from functional and structural analyses to reveal that Gp5.7 primarily serves to inhibit EσS, the predominant form of the RNAP in the stationary phase of growth, which accumulates in exponentially growingE. colias a consequence of the buildup of guanosine pentaphosphate [(p)ppGpp] during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development inE. colicells in the stationary phase of growth. Our finding represents a paradigm for how some lytic phages have evolved distinct mechanisms to inhibit the bacterial transcription machinery to facilitate phage development in bacteria in the exponential and stationary phases of growth.

scholarly journals Protein degradation during terminal cytodifferentiation. Studies on mammary gland in organ culture

1980 ◽  
Vol 192 (1) ◽  
pp. 311-320 ◽  
Author(s):  
C J Wilde ◽  
N Paskin ◽  
J Saxton ◽  
R J Mayer
Keyword(s):  
Mammary Gland ◽  
Fatty Acid ◽  
Protein Degradation ◽  
Degradation Rate ◽  
Fatty Acid Synthase ◽  
Average Rate ◽  
Cytosol Fraction ◽  
Isotope Method ◽  
Degradation Rates ◽  
Double Isotope

1. In mammary gland explants subjected to experimental manipulation, average rates (during 24 h periods) of degradation of fatty acid synthase, casein and cytosol-fraction proteins were measured by a double-isotope method. Rates of degradation of fatty acid synthase were also computed from measurements of changing enzyme amount and rate of synthesis. 2. During the period of most rapid enzyme accumulation there is a transient decrease in the computed rate of degradation of fatty acid synthase. Removal of hormones produces a rapid increase in the computed rate of degradation of the enzyme. 3. The average rate of degradation of fatty acid synthase measured by the double-isotope method is low in the presence of hormones, and increases on hormone removal. This increase in degradation rate is inhibited by adrenaline and further blocked by insulin. NH4Cl (10 mM) also partially inhibits the increase in protein degradation on hormone removal. 4. The pattern of changes in the average rate of degradation of cytosol-fraction proteins is similar to that for fatty acid synthase alone. There is no relationship between subunit molecular weight and rate of degradation under all experimental conditions. 5. Isotope ratios for resolved cytosol protein mixtures are transformed logarithmically to make the standard deviations an estimate of heterogeneity of degradation rates. By this analysis, in some conditions there appears to be significant measureable heterogeneity of degradation rates. 6. Little degradation of casein is measured in the presence of hormones, but a marked increase in the rate of degradation can be measured when hormones are removed. Whereas at 24-48h NH4Cl (10 mM) has little effect on this enhanced rate of degradation, at 48-72h it causes a large decrease in degradation rate. 7. Results are discussed in terms of a two-component degradation system in mammary gland explants.

scholarly journals Protein degradation rate as affected by plant proteases among fresh samples of perennial ryegrass cultivars (Lolium perenne L.) / Einfluss pflanzlicher Proteasen auf den Proteinabbau bei unterschiedlichen Englischen Raigrass Sorten (Lolium perenne L.)

2016 ◽  
Vol 67 (2) ◽  
pp. 61-68
Author(s):  
Martin Gierus ◽  
Marc Loesche ◽  
Heba Salama ◽  
Antje Herrmann ◽  
Friedhelm Taube
Keyword(s):  
Protein Degradation ◽  
Protein Content ◽  
Lolium Perenne ◽  
Perennial Ryegrass ◽  
Incubation Time ◽  
Large Subunit ◽  
Ethanol Solution ◽  
Bisphosphate Carboxylase ◽  
Lolium Perenne L ◽  
Degradation Rates

Summary The objective of this study was to quantify the proteolytic activity of a set of 10 diploid early intermediate heading cultivars of Lolium perenne under rumenlike conditions. A field experiment was conducted in Northern Germany, where the perennial ryegrass cultivars were grown during two growing seasons. Leaves of the first and second cut were sampled in the field, sterilized with 800 ml. l−1 ethanol solution and incubated for 0, 6, and 24 h under rumenlike conditions (darkness, 39°C, pH 6.5) without the presence of rumen microbes. Results revealed that the leaf protein content declined with increasing incubation time, confirming the involvement of plant-mediated proteolysis in the degradation process. Gel electrophoresis illustrated that the decrease in protein content is probably mainly caused by the loss of the large subunit of Rubisco (ribulose-1, 5-bisphosphate carboxylase/oxygenase), which was entirely degraded during the incubation time. Although differences among harvests and years were evident, genetic variation among the 10 diploid perennial grass samples concerning protein degradation rates and degradation characteristics was not detected.

scholarly journals A miniaturized biotyping system for strain discrimination inEscherichia coli

1993 ◽  
Vol 111 (1) ◽  
pp. 81-88
Author(s):  
P. B. Crichton ◽  
J. M. J. Logan ◽  
D. C. Old
Keyword(s):  
Escherichia Coli ◽  
Efficient Method ◽  
High Index ◽  
Inescherichia Coli ◽  
E Coli

SummaryA two-tier miniaturized scheme of eight tests was devised for biotyping strains ofEscherichia coliin microwell plates. Primary biotypes were defined by positive and negative reactions in tests for fermentation of raffinose, sorbose, dulcitol and 2-deoxy-D-ribose and for decarboxylation of ornithine when read after specified periods of incubation; subtypes were identified within primary biotypes according to results in secondary tests for rhamnose fermentation, lysine decarboxylation and motility. The method gave reproducible results on different occasions of testing.Among 100E. colistrains from various sources, 26 of the 32 possible primary biotypes and 56 full biotypes, as defined by results in both primary and secondary tests, were identified, thus demonstrating a high index of strain discrimination (D = 0·98).The scheme is recommended as a simple, reliable, inexpensive and efficient method of differentiating strains ofE. coli.

Evaluation of Structurally Different Carotenoids inEscherichia coli Transformants as Protectants against UV-B Radiation

1998 ◽  
Vol 64 (5) ◽  
pp. 1972-1974 ◽  
Author(s):  
Gerhard Sandmann ◽  
Silvia Kuhn ◽  
Peter B�ger
Keyword(s):  
Escherichia Coli ◽  
Inescherichia Coli ◽  
Short Term ◽  
E Coli ◽  
Carotenogenic Genes ◽  
Β Carotene

ABSTRACT Escherichia coli cells transformed with several carotenogenic genes to mediate the formation of ζ-carotene, neurosporene, lycopene, β-carotene, and zeaxanthin were exposed to UV-B radiation. Short-term kinetics revealed that endogenous levels of neurosporene and β-carotene protected E. coli against irradiation with UV-B. Zeaxanthin protected against only the photosensitized UV-B treatment. All other carotenoids were ineffective.

scholarly journals Membrane-bound Steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form

Blood ◽  
1995 ◽  
Vol 85 (3) ◽  
pp. 641-649 ◽  
Author(s):  
K Miyazawa ◽  
DA Williams ◽  
A Gotoh ◽  
J Nishimaki ◽  
HE Broxmeyer ◽  
...  
Keyword(s):  
Cell Surface ◽  
Tyrosine Kinase ◽  
Life Span ◽  
Protein Degradation ◽  
Cell Line ◽  
Soluble Form ◽  
Steel Factor ◽  
Degradation Rates ◽  
Membrane Bound ◽  
Kinetics Of

Alternative splicing of exon 6 results in the production of two isoforms of Steel factor (SLF): the membrane-bound and soluble forms. To investigate differences in the kinetics of c-kit tyrosine kinase activated by these two isoforms, we used a stromal cell line (SI/SI4) established from SI/SI homozygous murine embryo fetal liver and its stable transfectants containing either hSCF248 cDNA (including exon 6; secreted form) or hSCF220 cDNA (lacking exon 6; membrane-bound form) as the source of each isoform. Interaction of factor dependent myeloid cell line MO7e with stromal cells producing either isoform resulted in activated c-kit tyrosine kinase and induction of the same series of tyrosine phosphorylated cellular proteins in MO7e cells. However, SI4- h220 (membrane-bound form) induced more persistent activation of c-kit kinase than SI4-h248 (soluble form) did. Flow cytometric analysis and pulse-chase studies using [35S]methionine showed that SI4-h248 induced rapid downmodulation of cell-surface c-kit expression and its protein degradation in MO7e cells, whereas SI4-h220 induced more prolonged life span of c-kit protein. Addition of soluble recombinant human SLF to SI4- h220 cultures enhanced reduction of cell-surface c-kit expression and its protein degradation. Because the kinetics of c-kit inactivation strikingly fits with the protein degradation rates of c-kit under the conditions described above, rapid proteolysis of c-kit protein induced by soluble SLF stimulation may function as a “turn-off switch” for activated c-kit kinase.

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