Protein degradation through covalent inhibitor-based PROTACs

2020 ◽  
Vol 56 (10) ◽  
pp. 1521-1524 ◽  
Author(s):  
Gang Xue ◽  
Jiahui Chen ◽  
Lihong Liu ◽  
Danli Zhou ◽  
Yingying Zuo ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Live Cells ◽  
Target Proteins ◽  
Covalent Inhibitor ◽  
Application Scope

Covalent inhibitor-based PROTACs were successfully developed for the degradation of target proteins in live cells to further extend the application scope of PROTACs.

scholarly journals Assay Systems for Profiling Deubiquitinating Activity

2020 ◽  
Vol 21 (16) ◽  
pp. 5638
Author(s):  
Jinhong Cho ◽  
Jinyoung Park ◽  
Eunice EunKyeong Kim ◽  
Eun Joo Song
Keyword(s):  
Protein Degradation ◽  
Protein Interactions ◽  
Live Cells ◽  
Deubiquitinating Enzyme ◽  
Protein Protein Interactions ◽  
Deubiquitinating Enzymes ◽  
Cell Lysates ◽  
Cellular Processes

Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein–protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo.

A cascading reaction sequence involving ligand-directed azaelectrocyclization and autooxidation-induced fluorescence recovery enables visualization of target proteins on the surfaces of live cells

2014 ◽  
Vol 12 (9) ◽  
pp. 1412-1418 ◽  
Author(s):  
Katsunori Tanaka ◽  
Masataka Kitadani ◽  
Ayumi Tsutsui ◽  
Ambara R. Pradipta ◽  
Rie Imamaki ◽  
...  
Keyword(s):  
Target Protein ◽  
Fluorescence Recovery ◽  
Live Cells ◽  
Reaction Sequence ◽  
Target Proteins

A general probe designed to induce a cascading sequence of reactions on a target protein was efficiently synthesized.

scholarly journals Rapid Degradation of Caenorhabditis elegans Proteins at Single-Cell Resolution with a Synthetic Auxin

2019 ◽  
Vol 10 (1) ◽  
pp. 267-280 ◽  
Author(s):  
Michael A. Q. Martinez ◽  
Brian A. Kinney ◽  
Taylor N. Medwig-Kinney ◽  
Guinevere Ashley ◽  
James M. Ragle ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Protein Degradation ◽  
Single Cell ◽  
Water Soluble ◽  
Growth Media ◽  
Synthetic Analog ◽  
Rapid Degradation ◽  
Target Proteins ◽  
C Elegans ◽  
Link Type

As developmental biologists in the age of genome editing, we now have access to an ever-increasing array of tools to manipulate endogenous gene expression. The auxin-inducible degradation system allows for spatial and temporal control of protein degradation via a hormone-inducible Arabidopsis F-box protein, transport inhibitor response 1 (TIR1). In the presence of auxin, TIR1 serves as a substrate-recognition component of the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), ubiquitinating auxin-inducible degron (AID)-tagged proteins for proteasomal degradation. Here, we optimize the Caenorhabditis elegans AID system by utilizing 1-naphthaleneacetic acid (NAA), an indole-free synthetic analog of the natural auxin indole-3-acetic acid (IAA). We take advantage of the photostability of NAA to demonstrate via quantitative high-resolution microscopy that rapid degradation of target proteins can be detected in single cells within 30 min of exposure. Additionally, we show that NAA works robustly in both standard growth media and physiological buffer. We also demonstrate that K-NAA, the water-soluble, potassium salt of NAA, can be combined with microfluidics for targeted protein degradation in C. elegans larvae. We provide insight into how the AID system functions in C. elegans by determining that TIR1 depends on C. elegansSKR-1/2, CUL-1, and RBX-1 to degrade target proteins. Finally, we present highly penetrant defects from NAA-mediated degradation of the FTZ-F1 nuclear hormone receptor, NHR-25, during C. elegans uterine-vulval development. Together, this work improves our use and understanding of the AID system for dissecting gene function at the single-cell level during C. elegans development.

Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells

2016 ◽  
Vol 34 (1) ◽  
pp. 161-174 ◽  
Author(s):  
Ruth Röder ◽  
Jonas Helma ◽  
Tobias Preiß ◽  
Joachim O. Rädler ◽  
Heinrich Leonhardt ◽  
...  
Keyword(s):  
Intracellular Delivery ◽  
Live Cells ◽  
Target Proteins

scholarly journals Opportunities and Challenges of Small Molecule Induced Targeted Protein Degradation

2021 ◽  
Vol 9 ◽  
Author(s):  
Ming He ◽  
Wenxing Lv ◽  
Yu Rao
Keyword(s):  
Rna Interference ◽  
Protein Degradation ◽  
Small Molecules ◽  
Small Molecule ◽  
Gene Editing ◽  
Small Molecule Inhibitors ◽  
Tumor Resistance ◽  
Target Proteins ◽  
Enzymatic Function ◽  
New Type

Proteolysis targeting chimeras (PROTAC) represents a new type of small molecule induced protein degradation technology that has emerged in recent years. PROTAC uses bifunctional small molecules to induce ubiquitination of target proteins and utilizes intracellular proteasomes for chemical knockdown. It complements the gene editing and RNA interference for protein knockdown. Compared with small molecule inhibitors, PROTAC has shown great advantages in overcoming tumor resistance, affecting the non-enzymatic function of target proteins, degrading undruggable targets, and providing new rapid and reversible chemical knockout tools. At the same time, its challenges and problems also need to be resolved as a fast-developing newchemical biology technology.

Photo-triggered fluorescent labelling of recombinant proteins in live cells

2015 ◽  
Vol 51 (47) ◽  
pp. 9670-9673 ◽  
Author(s):  
Deokho Jung ◽  
Kohei Sato ◽  
Kyoungmi Min ◽  
Akira Shigenaga ◽  
Juyeon Jung ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Live Cells ◽  
Fluorescent Labelling ◽  
Target Proteins ◽  
Spatiotemporal Control

A method to photo-chemically trigger fluorescent labelling of proteins in live cells is developed for background-free fluorescent labelling of target proteins with the necessary spatiotemporal control.

scholarly journals Development of an Acrylamide-Based Inhibitor of Protein S-Acylation

2021 ◽  
Author(s):  
Saara-Anne Azizi ◽  
Tong Lan ◽  
Clémence Delalande ◽  
Rahul Kathayat ◽  
Bryan Dickinson
Keyword(s):  
Cell Signaling ◽  
Human Disease ◽  
Cellular Signaling ◽  
Protein S ◽  
Live Cells ◽  
Synthesis And Characterization ◽  
Post Translational Modification ◽  
Target Proteins ◽  
Cellular Events

<div><div><div><p>Protein S-acylation is a dynamic lipid post-translational modification that can modulate the localization and activity of target proteins. In humans, the installation of the lipid onto target proteins is catalyzed by a family of 23 Asp-His-His-Cys domain-containing protein acyltransferases (DHHC-PATs). DHHCs are increasingly recognized as critical players in cellular signaling events and in human disease. However, progress elucidating the functions and mechanisms of DHHC “writers” has been hampered by a lack of chemical tools to perturb their activity in live cells. Herein, we report the synthesis and characterization of PATi, a pan- DHHC inhibitor more potent than 2-bromopalmitate (2BP), the most commonly used DHHC inhibitor in the field. Possessing an acrylamide warhead, PATi pairs its gain in potency with decreases in both toxicity and inhibition of the S-acylation eraser enzymes – two of the major weaknesses of 2BP. Our studies show that PATi engages with DHHC family proteins in cells, inhibits protein S-acylation, and disrupts DHHC-regulated cellular events. PATi represents an improved chemical tool for untangling the complexities of DHHC-mediated cell signaling by protein S-acylation.</p></div></div></div>

scholarly journals A novel auxin-inducible degron system for rapid, cell cycle-specific targeted proteolysis

2021 ◽  
Author(s):  
Marina Capece ◽  
Anna Tessari ◽  
Joseph Mills ◽  
Gian Luca Rampioni Vinciguerra ◽  
Chenyu Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Degradation ◽  
Cell Population ◽  
Target Proteins ◽  
Endogenous Gene ◽  
Protein Depletion ◽  
On Demand ◽  
Cell Synchronization ◽  
Cell Cycle Status ◽  
The Way

AbstractThe OsTIR1/auxin-inducible degron (AID) system allows “on demand” selective and reversible protein degradation upon exposure to the phytohormone auxin. In the current format, this technology does not allow to study the effect of acute protein depletion selectively in one phase of the cell cycle, as auxin similarly affects all the treated cells irrespectively of their proliferation status. Therefore, the AID system requires coupling with cell synchronization techniques, which can alter the basal biological status of the studied cell population. Here, we introduce a new AID system to Regulate OsTIR1 Levels based on the Cell Cycle Status (ROLECCS system), which induces proteolysis of both exogenously transfected and endogenous gene-edited targets in specific phases of the cell cycle. This new tool paves the way to studying the differential roles that target proteins may have in specific phases of the cell cycle.

scholarly journals Emerging branches of the N-end rule pathways are revealing the sequence complexities of N-termini dependent protein degradation

2018 ◽  
Vol 96 (3) ◽  
pp. 289-294 ◽  
Author(s):  
Mohamed A. Eldeeb ◽  
Luana C.A. Leitao ◽  
Richard P. Fahlman
Keyword(s):  
Protein Degradation ◽  
Target Proteins ◽  
Molecular Machinery ◽  
Dependent Protein ◽  
Cellular Machinery ◽  
Additional Sequence ◽  
Terminal Amino ◽  
Protein Instability ◽  
Sequence Requirements

The N-end rule links the identity of the N-terminal amino acid of a protein to its in vivo half-life, as some N-terminal residues confer metabolic instability to a protein via their recognition by the cellular machinery that targets them for degradation. Since its discovery, the N-end rule has generally been defined as set of rules of whether an N-terminal residue is stabilizing or not. However, recent studies are revealing that the N-terminal code of amino acids conferring protein instability is more complex than previously appreciated, as recent investigations are revealing that the identity of adjoining downstream residues can also influence the metabolic stability of N-end rule substrate. This is exemplified by the recent discovery of a new branch of N-end rule pathways that target proteins bearing N-terminal proline. In addition, recent investigations are demonstrating that the molecular machinery in N-termini dependent protein degradation may also target proteins for lysosomal degradation, in addition to proteasome-dependent degradation. Herein, we describe some of the recent advances in N-end rule pathways and discuss some of the implications regarding the emerging additional sequence requirements.

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