scholarly journals Ultrafast and Selective Labeling of Endogenous Proteins Using Affinity-based Benzotriazole Chemistry

2021 ◽  
Author(s):  
Xiaoyi Xin ◽  
Yu Zhang ◽  
Massimiliano Gaetani ◽  
Susanna Lundström ◽  
Roman Zubarev ◽  
...  
Keyword(s):  
Protein Function ◽  
Target Identification ◽  
Reaction Rate Constant ◽  
Live Cells ◽  
Selective Labeling ◽  
Systems Chemistry ◽  
Covalent Inhibitors ◽  
Chemical Modification Of Proteins ◽  
Endogenous Proteins

Chemical modification of proteins is enormously useful for characterizing protein function in complex biological systems and for drug development. Selective labeling of native or endogenous proteins is challenging owing to the existence of distinct functional groups in proteins and in living systems. Chemistry for rapid and selective labeling of proteins remains in high demand. Here we have developed novel affinity labeling probes using benzotriazole (BTA) chemistry. We showed that affinity-based BTA probes selectively and covalently label a lysine residue in the vicinity of the ligand binding site of a target protein with a reaction half-time of 28-42 s. The reaction rate constant is comparable to the fastest biorthogonal chemistry. This approach was used to selectively label different cytosolic and membrane proteins in vitro and in live cells. BTA chemistry could be widely useful for labeling of native/endogenous proteins, target identification and development of covalent inhibitors.

scholarly journals Interaction between Acetylated MyoD and the Bromodomain of CBP and/or p300

2001 ◽  
Vol 21 (16) ◽  
pp. 5312-5320 ◽  
Author(s):  
Anna Polesskaya ◽  
Irina Naguibneva ◽  
Arnaud Duquet ◽  
Eyal Bengal ◽  
Philippe Robin ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Protein Function ◽  
Live Cells ◽  
Regulation Of Transcription ◽  
Muscle Creatine Kinase ◽  
Nonhistone Protein ◽  
Myogenic Factor ◽  
First Time

ABSTRACT Acetylation is emerging as a posttranslational modification of nuclear proteins that is essential to the regulation of transcription and that modifies transcription factor affinity for binding sites on DNA, stability, and/or nuclear localization. Here, we present both in vitro and in vivo evidence that acetylation increases the affinity of myogenic factor MyoD for acetyltransferases CBP and p300. In myogenic cells, the fraction of endogenous MyoD that is acetylated was found associated with CBP or p300. In vitro, the interaction between MyoD and CBP was more resistant to high salt concentrations and was detected with lower doses of MyoD when MyoD was acetylated. Interestingly, an analysis of CBP mutants revealed that the interaction with acetylated MyoD involves the bromodomain of CBP. In live cells, MyoD mutants that cannot be acetylated did not associate with CBP or p300 and were strongly impaired in their ability to cooperate with CBP for transcriptional activation of a muscle creatine kinase-luciferase construct. Taken together, our data suggest a new mechanism for activation of protein function by acetylation and demonstrate for the first time an acetylation-dependent interaction between the bromodomain of CBP and a nonhistone protein.

scholarly journals Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells

2014 ◽  
Vol 25 (22) ◽  
pp. 3610-3618 ◽  
Author(s):  
Robert Mahen ◽  
Birgit Koch ◽  
Malte Wachsmuth ◽  
Antonio Z. Politi ◽  
Alexis Perez-Gonzalez ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Quantitative Imaging ◽  
Live Cells ◽  
Single Molecule Spectroscopy ◽  
Mitotic Kinase ◽  
Endogenous Proteins

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

scholarly journals A Versatile Vector System for the Fast Generation of Knock-in Cell Lines with CRISPR

2020 ◽  
Author(s):  
Oscar Perez-Leal ◽  
Jonathon Nixon-Abell ◽  
Carlos A. Barrero ◽  
John Gordon ◽  
Mario C. Rico
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Protein Function ◽  
Vector System ◽  
Live Cells ◽  
Modular Construction ◽  
New Era ◽  
Recombination System ◽  
Endogenous Genes ◽  
Endogenous Proteins

ABSTRACTUntil recent advancements in genome editing via CRISPR/Cas9 technology, understanding protein function typically involved artificially overexpressing proteins of interest. Despite that CRISPR/Cas9 has ushered in a new era of possibilities for modifying endogenous genes with labeling tags (knock-in) to more accurately study proteins under physiological conditions, the technique is largely underutilized due to its tedious, multi-step process. Here we outline a homologous recombination system (FAST-HDR) to be used in combination with CRISPR/Cas9 that significantly simplifies and accelerates this process while introducing multiplexing to allow live-cell studies of 3 endogenous proteins within the same cell line. Furthermore, the recombination vectors are assembled in a single reaction that is enhanced for eliminating false positives and reduces the overall creation time for the knockin cell line from ~8 weeks to <15 days. Finally, the system utilizes a modular construction to allow for seamlessly swapping labeling tags to ensure flexibility according to the area under study. We validated this new methodology by developing advanced cell lines with 3 fluorescent-labeled endogenous proteins that support high-content phenotypic drug screening without using antibodies or exogenous staining. Therefore, Fast-HDR cell lines provide a robust alternative for studying multiple proteins of interest in live cells without artificially overexpressing labeled proteins.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

Elucidating the anti-biofilm and anti-quorum sensing potential of selenocystine against respiratory tract infections causing bacteria: in vitro and in silico studies

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Bharti Patel ◽  
Subrata Mishra ◽  
Indira K. Priyadarsini ◽  
Sirisha L. Vavilala
Keyword(s):  
Quorum Sensing ◽  
Respiratory Tract ◽  
Protein Function ◽  
Docking Studies ◽  
Klebsiella Pneumonia ◽  
Potential Candidate ◽  
In Silico Studies ◽  
Specific Proteins ◽  
Tract Infections

Abstract Bacteria are increasingly relying on biofilms to develop resistance to antibiotics thereby resulting in their failure in treating many infections. In spite of continuous research on many synthetic and natural compounds, ideal anti-biofilm molecule is still not found thereby warranting search for new class of molecules. The current study focuses on exploring anti-biofilm potential of selenocystine against respiratory tract infection (RTI)-causing bacteria. Anti-bacterial and anti-biofilm assays demonstrated that selenocystine inhibits the growth of bacteria in their planktonic state, and formation of biofilms while eradicating preformed-biofilm effectively. Selenocystine at a MIC50 as low as 42 and 28 μg/mL effectively inhibited the growth of Klebsiella pneumonia and Pseudomonas aeruginosa. The antibacterial effect is further reconfirmed by agar cup diffusion assay and growth-kill assay. Selenocystine showed 30–60% inhibition of biofilm formation in K. pneumonia, and 44–70% in P. aeruginosa respectively. It also distorted the preformed-biofilms by degrading the eDNA component of the Extracellular Polymeric Substance matrix. Molecular docking studies of selenocystine with quorum sensing specific proteins clearly showed that through the carboxylic acid moiety it interacts and inhibits the protein function, thereby confirming its anti-biofilm potential. With further validation selenocystine can be explored as a potential candidate for the treatment of RTIs.

The Use of the Vitality and Chemotaxis of Human Polymorphonuclear Leukocytes for the In Vitro Estimation of the Acute Toxicity of the First Ten Chemicals from the MEIC List

1993 ◽  
Vol 21 (1) ◽  
pp. 73-80
Author(s):  
Matteo Valentino ◽  
Francesca Monaco ◽  
Maria Antonietta Pizzichini ◽  
Mario Governa
Keyword(s):  
Ethidium Bromide ◽  
Polymorphonuclear Leukocytes ◽  
Rank Correlation ◽  
Fluorescein Diacetate ◽  
Live Cells ◽  
In Vitro Toxicity ◽  
Ic50 Values ◽  
Acute Cytotoxicity ◽  
Human Polymorphonuclear Leukocytes

The acute cytotoxicity of the first ten MEIC chemicals has been estimated by others in various cell lines. In the present investigation, isolated human polymorphonuclear leukocytes (PMN) from ten healthy non-smoking laboratory personnel were used to assess in vitro toxicity of the same chemicals. The cells were treated with different concentrations of the respective chemicals for three hours and their vitality and chemotaxis were tested. Vitality was measured by fluorescence microscopy after the addition of fluorescein diacetate and ethidium bromide. Living cells which took up and hydrolysed fluorescein diacetate, and dead cells, stained by ethidium bromide, were counted and the percentage of live cells was calculated. Locomotion stimulated by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (F-MLP), was measured in blind-well Boyden chambers and a chemotactic index was calculated. The results were mathematically transformed to produce a linear curve, and then fitted by the linear least squares procedure, from which LC50 and IC50 values were obtained by interpolation. All the chemicals decreased the vitality and inhibited the chemotaxis of the PMN. Obviously the chemotactic test was more sensitive than the vitality one. A correlation (r = 0.933) was found between vitality and chemotaxis inhibition. Spearman rank correlation analysis revealed significant correlations between our results and those from in vitro experiments conducted in other laboratories, as well as with data concerning mouse, rat and human lethal doses.

scholarly journals Insights in Behavior of Variably Formulated Alginate-Based Microcapsules for Cell Transplantation

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Pia Montanucci ◽  
Silvia Terenzi ◽  
Claudio Santi ◽  
Ilaria Pennoni ◽  
Vittorio Bini ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Chemical Properties ◽  
Live Cells ◽  
High Performing ◽  
Physical Chemical ◽  
Degenerative Disorders ◽  
Selection Of ◽  
Better Than

Alginate-based microencapsulation of live cells may offer the opportunity to treat chronic and degenerative disorders. So far, a thorough assessment of physical-chemical behavior of alginate-based microbeads remains cloudy. A disputed issue is which divalent cation to choose for a high performing alginate gelling process. Having selected, in our system, high mannuronic (M) enriched alginates, we studied different gelling cations and their combinations to determine their eventual influence on physical-chemical properties of the final microcapsules preparation,in vitroandin vivo. We have shown that used of ultrapure alginate allows for high biocompatibility of the formed microcapsules, regardless of gelation agents, while use of different gelling cations is associated with corresponding variable effects on the capsules’ basic architecture, as originally reported in this work. However, only the final application which the capsules are destined to will ultimately guide the selection of the ideal, specific gelling divalent cations, since in principle there are no capsules that are better than others.

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