Fluorescent Probes with Unnatural Amino Acids to Monitor Proteasome Activity in Real-Time

<div> <div> <div> <p>The proteasome is an essential protein complex that, when dysregulated, can result in various diseases in eukaryotic cells. As such, understanding the enzymatic activity of the proteasome and what can alter it is crucial to elucidating its roles in these diseases. This can be done effectively by using activity-based fluorescent substrate probes, of which there are many commercially available that target the individual protease-like subunits in the 20S CP of the proteasome. Unfortunately, these probes have not displayed appropriate characteristics for their use in live cell-based assays. In the work presented here, we have developed a set of probes which have shown improved fluorescence properties and selectivity towards the proteasome compared to other cellular proteases. By including unnatural amino acids, we have found probes which can be utilized in various applications, including monitoring the effects of small molecule stimulators of the proteasome in live cells and comparing the relative proteasome activity across different cancer cell types. In future studies, we expect the fluorescent probes presented here will serve as tools to support the discovery and characterization of small molecule modulators of proteasome activity. </p> </div> </div> </div>

Download Full-text

Fluorescent Probes with Unnatural Amino Acids to Monitor Proteasome Activity in Real-Time

10.26434/chemrxiv.12043101.v1 ◽

2020 ◽

Author(s):

Breanna L. Zerfas ◽

Rachel A. Coleman ◽

Andres Salazar Chaparro ◽

Nathaniel J. Macatangay ◽

Darci Trader

Keyword(s):

Amino Acids ◽

Small Molecule ◽

Fluorescent Probes ◽

Unnatural Amino Acids ◽

Cell Types ◽

Proteasome Activity ◽

Live Cells ◽

Fluorescent Substrate ◽

The Individual ◽

Small Molecule Modulators

<div> <div> <div> <p>The proteasome is an essential protein complex that, when dysregulated, can result in various diseases in eukaryotic cells. As such, understanding the enzymatic activity of the proteasome and what can alter it is crucial to elucidating its roles in these diseases. This can be done effectively by using activity-based fluorescent substrate probes, of which there are many commercially available that target the individual protease-like subunits in the 20S CP of the proteasome. Unfortunately, these probes have not displayed appropriate characteristics for their use in live cell-based assays. In the work presented here, we have developed a set of probes which have shown improved fluorescence properties and selectivity towards the proteasome compared to other cellular proteases. By including unnatural amino acids, we have found probes which can be utilized in various applications, including monitoring the effects of small molecule stimulators of the proteasome in live cells and comparing the relative proteasome activity across different cancer cell types. In future studies, we expect the fluorescent probes presented here will serve as tools to support the discovery and characterization of small molecule modulators of proteasome activity. </p> </div> </div> </div>

Download Full-text

Live Confocal Analysis of Fertilization and Early Development

Microscopy and Microanalysis ◽

10.1017/s1431927600031135 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 1012-1013

Author(s):

Uyen Tram ◽

William Sullivan

Keyword(s):

Drosophila Melanogaster ◽

Embryonic Development ◽

Real Time ◽

Early Development ◽

Fluorescent Probes ◽

Primary Defect ◽

Fluorescent Analysis ◽

Dynamic Event ◽

Enormous Amount ◽

Fluorescent Studies

Embryonic development is a dynamic event and is best studied in live animals in real time. Much of our knowledge of the early events of embryogenesis, however, comes from immunofluourescent analysis of fixed embryos. While these studies provide an enormous amount of information about the organization of different structures during development, they can give only a static glimpse of a very dynamic event. More recently real-time fluorescent studies of living embryos have become much more routine and have given new insights to how different structures and organelles (chromosomes, centrosomes, cytoskeleton, etc.) are coordinately regulated. This is in large part due to the development of commercially available fluorescent probes, GFP technology, and newly developed sensitive fluorescent microscopes. For example, live confocal fluorescent analysis proved essential in determining the primary defect in mutations that disrupt early nuclear divisions in Drosophila melanogaster. For organisms in which GPF transgenics is not available, fluorescent probes that label DNA, microtubules, and actin are available for microinjection.

Download Full-text