Year-end Sale % OFF 
Abstract
Proteasomes hydrolyze most cellular proteins. The standard reaction to determine proteasome activity in cellular lysate or elsewhere contains AMC-conjugated peptide substrate, ATP, Mg2+, and DTT. ATP and Mg2+ are included to maintain 26S proteasome functionality. However, most cellular proteasomes are 20S proteasomes, and the effects of ATP on the turnover of fluorogenic substrates by 20S complexes are largely unknown. Here, we evaluated the effect of ATP alone or in combination with Mg2+ on the degradation of AMC-conjugated fluorogenic substrates by purified 20S proteasomes. Degradation of substrates used to determine chymotrypsin-, caspase- and trypsin-like proteasome activities was gradually decreased with the rise of ATP concentration from 0.25 to 10 mM. These effects were not associated with the blockage of the proteasome catalytic subunit active sites or unspecific alterations of AMC fluorescence by the ATP. However, ATP-induced peptide degradation slowdown was rescued by the addition of Mg2+. Moreover, the substrate degradation efficacy was proportional to the Mg2+/ATP ratio, being equal to control values when equimolar concentrations of the molecules were used. The obtained results indicate that when proteasome activity is assessed, the reciprocal effects of ATP and Mg2+ on the hydrolysis of AMC-conjugated fluorogenic substrates by the 20S proteasomes should be considered.
Highlights
Cells have two systems responsible for the destruction of organelles and proteins
Entire organelles and long-lived proteins are degraded by autophagy, while most intracellular short-lived proteins are hydrolyzed by the ubiquitin–proteasome system (UPS) [1]
The assay based on the degradation of short peptides conjugated to 7-amido-4methylcoumarin (AMC) has several limitations [12]; such peptides are the most widely used substrates for proteasome activity measurements [13]
Summary
Cells have two systems responsible for the destruction of organelles and proteins. Entire organelles and long-lived proteins are degraded by autophagy, while most intracellular short-lived proteins are hydrolyzed by the ubiquitin–proteasome system (UPS) [1]. Within the UPS, the proteasomes perform degradation of protein substrates. The 26S proteasome is capable of selective recognition and degradation of ubiquitinated proteins. It is composed of a 19S regulator and a 20S core particle. The 19S complex allows specific recognition and ATP-dependent unfolding and translocation of the substrates into the 20S proteasome, where the protein hydrolysis takes place [2]. The proteasome has caspase-, trypsin- and chymotrypsin-like proteolytic activities. The classical assay mixture is a buffered solution containing short fluorogenic peptide substrates, DTT, ATP, and Mg2+ , which are necessary to maintain 26S proteasome function and integrity [4]. We investigated the effects of ATP on the degradation of short fluorogenic substrates by purified 20S proteasomes
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
