Faculty Opinions recommendation of Conditional control of fluorescent protein degradation by an auxin-dependent nanobody.

2020 ◽  
Author(s):  
Mary Goll
Keyword(s):  
Protein Degradation ◽  
Fluorescent Protein ◽  
Conditional Control

scholarly journals Conditional control of fluorescent protein degradation by an auxin-dependent nanobody

2018 ◽  
Author(s):  
Katrin Daniel ◽  
Jaroslav Icha ◽  
Cindy Horenburg ◽  
Doris Müller ◽  
Caren Norden ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Experimental Models ◽  
Anaphase Promoting Complex ◽  
Reversible Inactivation ◽  
Ubiquitin E3 Ligase ◽  
Conditional Control ◽  
Protein Functions ◽  
Vertebrate Model

AbstractThe conditional and reversible depletion of proteins by auxin-mediated degradation is a powerful tool to investigate protein functions in cells and whole organisms. However, its wider applications require fusing the auxin-inducible degron (AID) to individual target proteins. Thus, establishing the auxin system for multiple proteins can be challenging. Another approach for directed protein degradation are anti-GFP nanobodies, which can be applied to GFP stock collections that are readily available in different experimental models. Here, we combine the advantages of auxin and nanobody-based degradation technologies creating an AID-nanobody to degrade GFP-tagged proteins at different cellular structures in a conditional and reversible manner in human cells. We demonstrate efficient and reversible inactivation of the anaphase promoting complex/cyclosome (APC/C) and thus provide new means to study the functions of this essential ubiquitin E3 ligase. Further, we establish auxin degradation in a vertebrate model organism by employing AID-nanobodies in zebrafish.

scholarly journals Conditional control of fluorescent protein degradation by an auxin-dependent nanobody

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Katrin Daniel ◽  
Jaroslav Icha ◽  
Cindy Horenburg ◽  
Doris Müller ◽  
Caren Norden ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Fluorescent Protein ◽  
Conditional Control

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 A Promising Intracellular Protein-Degradation Strategy: TRIMbody-Away Technique Based on Nanobody Fragment

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1512
Author(s):  
Gang Chen ◽  
Yu Kong ◽  
You Li ◽  
Ailing Huang ◽  
Chunyu Wang ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Intracellular Protein ◽  
Full Size ◽  
Intracellular Protein Degradation ◽  
Potential Applications ◽  
Ubiquitin Proteasome ◽  
Green Fluorescent

Most recently, a technology termed TRIM-Away has allowed acute and rapid destruction of endogenous target proteins in cultured cells using specific antibodies and endogenous/exogenous tripartite motif 21 (TRIM21). However, the relatively large size of the full-size mAbs (150 kDa) results in correspondingly low tissue penetration and inaccessibility of some sterically hindered epitopes, which limits the target protein degradation. In addition, exogenous introduction of TRIM21 may cause side effects for treated cells. To tackle these limitations, we sought to replace full-size mAbs with the smaller format of antibodies, a nanobody (VHH, 15 kDa), and construct a new type of fusion protein named TRIMbody by fusing the nanobody and RBCC motif of TRIM21. Next, we introduced enhanced green fluorescent protein (EGFP) as a model substrate and generated αEGFP TRIMbody using a bispecific anti-EGFP (αEGFP) nanobody. Remarkably, inducible expression of αEGFP TRIMbody could specifically degrade intracellular EGFP in HEK293T cells in a time-dependent manner. By treating cells with inhibitors, we found that intracellular EGFP degradation by αEGFP TRIMbody relies on both ubiquitin–proteasome and autophagy–lysosome pathways. Taken together, these results suggested that TRIMbody-Away technology could be utilized to specifically degrade intracellular protein and could expand the potential applications of degrader technologies.

scholarly journals Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts

2007 ◽  
Vol 71A (10) ◽  
pp. 827-834 ◽  
Author(s):  
Michael Halter ◽  
Alex Tona ◽  
Kiran Bhadriraju ◽  
Anne L. Plant ◽  
John T. Elliott
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Degradation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Green Fluorescent

Chloroplast proteotoxic stress-induced autophagy is involved in the degradation of chloroplast proteins in Chlamydomonas reinhardtii

2021 ◽  
Author(s):  
Nan Li ◽  
Sakuya Nakamura ◽  
Silvia Ramundo ◽  
Yoshiki Nishimura ◽  
Shinya Hagihara ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Chlamydomonas Reinhardtii ◽  
Fluorescent Protein ◽  
Large Subunit ◽  
Yellow Fluorescent Protein ◽  
Model Organism ◽  
Small Subunit ◽  
Proteotoxic Stress ◽  
Chloroplast Protein ◽  
Biochemical Detection

Abstract Intraorganellar proteases and cytoplasmic proteolytic systems such as autophagy orchestrate the degradation of organellar proteins to ensure organelle homeostasis in eukaryotic cells. The green alga Chlamydomonas reinhardtii is an ideal unicellular model organism for elucidating the mechanisms maintaining proteostasis in chloroplasts. However, the autophagic pathways targeting the photosynthetic organelles of these algae have not been clearly elucidated. Here, we explored the role of autophagy in chloroplast protein degradation in Chlamydomonas cells. We labeled the chloroplast protein Rubisco small subunit (RBCS) with the yellow fluorescent protein Venus in a Chlamydomonas strain in which expression of the chloroplast gene clpP1, encoding a major catalytic subunit of the chloroplast Clp protease, can be conditionally repressed to selectively perturb chloroplast protein homeostasis. We observed transport of both nucleus-encoded RBCS-Venus fusion protein and chloroplast-encoded Rubisco large subunit (rbcL) from the chloroplast to the vacuoles in response to chloroplast proteotoxic stress induced by clpP1 inhibition. This process was retarded by the addition of autophagy inhibitors. Biochemical detection of lytic cleavage of RBCS-Venus supported the notion that Rubisco is degraded in the vacuoles via autophagy. Electron microscopy revealed vacuolar accumulation of autophagic vesicles and exposed their ultrastructure during repression of clpP1 expression. Treatment with an autophagy activator also induced chloroplast autophagy. These results indicate that autophagy contributes to chloroplast protein degradation in Chlamydomonas cells.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

Conditional Control of Spatial Information Provided by a Landmark

2014 ◽  
Author(s):  
Kenneth Leising ◽  
Joshua E. Wolf ◽  
Chad M. Ruprecht
Keyword(s):  
Spatial Information ◽  
Conditional Control
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