A Noncanonical Function of Sortase Enables Site-Specific Conjugation of Small Molecules to Lysine Residues in Proteins

2014 ◽  
Vol 127 (2) ◽  
pp. 451-455 ◽  
Author(s):  
Joseph J. Bellucci ◽  
Jayanta Bhattacharyya ◽  
Ashutosh Chilkoti
Keyword(s):  
Small Molecules ◽  
Site Specific ◽  
Lysine Residues

Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification

2018 ◽  
Author(s):  
Daniel D. Brauer ◽  
Emily C. Hartman ◽  
Daniel L.V. Bader ◽  
Zoe N. Merz ◽  
Danielle Tullman-Ercek ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Modification ◽  
Positive Charge ◽  
Specific Protein ◽  
High Yield ◽  
Site Specific ◽  
Protein Cage ◽  
Protein Carriers ◽  
Site Selective ◽  
Targeted Library

<div> <p>Site-specific protein modification is a widely-used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically-hindered N termini, such as virus-like particles like the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.</p> </div>

Aim for the Readers! Bromodomains As New Targets Against Chagas’ Disease

2019 ◽  
Vol 26 (36) ◽  
pp. 6544-6563
Author(s):  
Victoria Lucia Alonso ◽  
Luis Emilio Tavernelli ◽  
Alejandro Pezza ◽  
Pamela Cribb ◽  
Carla Ritagliati ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Small Molecules ◽  
Chagas Disease ◽  
Causative Agent ◽  
Current Knowledge ◽  
Sequence Similarity ◽  
Lysine Acetylation ◽  
Specific Manner ◽  
Lysine Residues ◽  
Antiparasitic Drugs

Bromodomains recognize and bind acetyl-lysine residues present in histone and non-histone proteins in a specific manner. In the last decade they have raised as attractive targets for drug discovery because the miss-regulation of human bromodomains was discovered to be involved in the development of a large spectrum of diseases. However, targeting eukaryotic pathogens bromodomains continues to be almost unexplored. We and others have reported the essentiality of diverse bromodomain- containing proteins in protozoa, offering a new opportunity for the development of antiparasitic drugs, especially for Trypansoma cruzi, the causative agent of Chagas’ disease. Mammalian bromodomains were classified in eight groups based on sequence similarity but parasitic bromodomains are very divergent proteins and are hard to assign them to any of these groups, suggesting that selective inhibitors can be obtained. In this review, we describe the importance of lysine acetylation and bromodomains in T. cruzi as well as the current knowledge on mammalian bromodomains. Also, we summarize the myriad of small-molecules under study to treat different pathologies and which of them have been tested in trypanosomatids and other protozoa. All the information available led us to propose that T. cruzi bromodomains should be considered as important potential targets and the search for smallmolecules to inhibit them should be empowered.

scholarly journals Peptide-tags for site-specific protein labelling in vitro and in vivo

2016 ◽  
Vol 12 (6) ◽  
pp. 1731-1745 ◽  
Author(s):  
Jonathan Lotze ◽  
Ulrike Reinhardt ◽  
Oliver Seitz ◽  
Annette G. Beck-Sickinger
Keyword(s):  
Metal Ions ◽  
Small Molecules ◽  
Protein Localization ◽  
Specific Protein ◽  
Site Specific ◽  
Protein Labelling ◽  
Peptide Interactions ◽  
Peptide Tags

Peptide-tag based labelling can be achieved by (i) enzymes (ii) recognition of metal ions or small molecules and (iii) peptide–peptide interactions and enables site-specific protein visualization to investigate protein localization and trafficking.

scholarly journals Acetylated Thioredoxin Reductase 1 Resists Oxidative Inactivation

2021 ◽  
Vol 9 ◽  
Author(s):  
David. E. Wright ◽  
Nikolaus Panaseiko ◽  
Patrick O’Donoghue
Keyword(s):  
Oxidative Stress ◽  
Thioredoxin Reductase ◽  
Oxygen Species ◽  
Site Specific ◽  
Oxidative Inactivation ◽  
Thioredoxin Reductase 1 ◽  
Lysine Residues ◽  
Canonical Amino Acid ◽  
Genetic Code Expansion ◽  
Low Activity

Thioredoxin Reductase 1 (TrxR1) is an enzyme that protects human cells against reactive oxygen species generated during oxidative stress or in response to chemotherapies. Acetylation of TrxR1 is associated with oxidative stress, but the function of TrxR1 acetylation in oxidizing conditions is unknown. Using genetic code expansion, we produced recombinant and site-specifically acetylated variants of TrxR1 that also contain the non-canonical amino acid, selenocysteine, which is essential for TrxR1 activity. We previously showed site-specific acetylation at three different lysine residues increases TrxR1 activity by reducing the levels of linked dimers and low activity TrxR1 tetramers. Here we use enzymological studies to show that acetylated TrxR1 is resistant to both oxidative inactivation and peroxide-induced multimer formation. To compare the effect of programmed acetylation at specific lysine residues to non-specific acetylation, we produced acetylated TrxR1 using aspirin as a model non-enzymatic acetyl donor. Mass spectrometry confirmed aspirin-induced acetylation at multiple lysine residues in TrxR1. In contrast to unmodified TrxR1, the non-specifically acetylated enzyme showed no loss of activity under increasing and strongly oxidating conditions. Our data suggest that both site-specific and general acetylation of TrxR1 regulate the enzyme’s ability to resist oxidative damage.

scholarly journals Site-selective enzymatic chemistry for polymer conjugation to protein lysine residues: PEGylation of G-CSF at lysine-41

2016 ◽  
Vol 7 (42) ◽  
pp. 6545-6553 ◽  
Author(s):  
A. Mero ◽  
A. Grigoletto ◽  
K. Maso ◽  
H. Yoshioka ◽  
A. Rosato ◽  
...  
Keyword(s):  
Specific Protein ◽  
Microbial Transglutaminase ◽  
Site Specific ◽  
Lysine Residues ◽  
Polymer Conjugation ◽  
Site Selective

Microbial transglutaminase (mTGase) is an enzyme that catalyzes site-specific protein derivatization at specific glutamines and lysines.

scholarly journals Synthetic Elaboration of Native DNA by RASS (SENDR)

2020 ◽  
Author(s):  
Dillon T. Flood ◽  
Kyle W. Knouse ◽  
Julien C. Vantourout ◽  
Brittany Sanchez ◽  
Emily J. Sturgell ◽  
...  
Keyword(s):  
Small Molecules ◽  
Solid Support ◽  
High Yield ◽  
Organic Media ◽  
Two Stage ◽  
Site Specific ◽  
Reversible Adsorption ◽  
Dna Conformations ◽  
Single Coupling ◽  
Stage Process

The controlled, site-specific ligation of molecules to native DNA remains an unanswered challenge. Herein, we report a simple solution to achieve this ligation through the tactical combination of two recently developed technologies: One for the manipulation of DNA in organic media, and another for the chemoselective labeling of alcohols. Reversible Adsorption of Solid Support (RASS) is employed to immobilize DNA and facilitate its transfer into dry acetonitrile. Subsequent ligation with P(V)-based Ψ reagents takes place in high yield with exquisite selectivity for the exposed 3’ or 5’ alcohols on DNA. This two-stage process, dubbed SENDR for Synthetic Elaboration of Native DNA by RASS, can be applied to a multitude of DNA conformations and sequences with a variety of functionalized Ψ reagents to generate useful constructs. Such entities can address numerous longstanding challenges, including the selective single coupling of DNA to proteins, ASOs, and functional small molecules, and also can allow the synthesis of doubly-labeled congeners for novel probe constructs including ones of potential interest to COVID-19 research. Finally, a prototype for the industrialization of SENDR in a kit format is presented.

scholarly journals The TAZ2 domain of CBP/p300 directs acetylation towards H3K27 within chromatin

2020 ◽  
Author(s):  
Thomas W. Sheahan ◽  
Viktoria Major ◽  
Kimberly M. Webb ◽  
Elana Bryan ◽  
Philipp Voigt
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Enzymatic Activity ◽  
Crucial Role ◽  
Regulation Of Gene Expression ◽  
Histone H3 ◽  
Embryonic Stem ◽  
Site Specific ◽  
Lysine Residues

AbstractThe closely related acetyltransferases CBP and p300 are key regulators of gene expression in metazoans. CBP/p300 acetylate several specific lysine residues within nucleosomes, including histone H3 lysine 27 (H3K27), a hallmark of active enhancers and promoters. However, it has remained largely unclear how specificity of CBP/p300 towards H3K27 is achieved. Here we show that the TAZ2 domain of CBP is required for efficient acetylation of H3K27, while curbing activity towards other lysine residues within nucleosomes. We find that TAZ2 is a sequence-independent DNA binding module, promoting interaction between CBP and nucleosomes, thereby enhancing enzymatic activity and regulating substrate specificity of CBP. TAZ2 is further required to stabilize CBP binding to chromatin in mouse embryonic stem cells, facilitating specificity towards H3K27 and modulating gene expression. These findings reveal a crucial role of TAZ2 in regulating H3K27ac, while highlighting the importance of correct site-specific acetylation for proper regulation of gene expression.

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