Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates

Abstract

Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell-based high-throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin–proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell-based reporters, detection of global ubiquitination status, and proteasome-mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell-based IκBα degradation assay, Thsp is a slow inhibitor and 4 hrs of treatment achieves the same effects as MG-132 at 30 min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes in vitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together, our results uncover an important role of Thsp in 19S proteasome inhibition.

Biological Evaluation of Naproxen–Dehydrodipeptide Conjugates with Self-Hydrogelation Capacity as Dual LOX/COX Inhibitors

“…The dehydrodipeptides generally elicited only modest effects on the production of • NO. IC 50 values of 64.7 µM and 84.4 µM were determined for the most active compounds, 3 and 8, respectively, in line with the IC 50 value of 79.3 µM, previously reported for compound 1 [20].…”

“…The dehydrodipeptides generally elicited only modest effects on the production of • NO. IC50 values of 64.7 µ M and 84.4 µ M were determined for the most active compounds, 3 and 8, respectively, in line with the IC50 value of 79.3 µ M, previously reported for compound 1 [20]. The compounds which were shown to be non-toxic to rat macrophages (3, 4, 6 and 8) were tested for their ability to inhibit LPS-dependent • NO production in rat macrophages (Figure 4).…”

“…Dehydroamino acid residues are commonly encountered in drug discovery, with plinabulin, thiostrepton, imipenem and cilastatin being medicinally important molecules [19][20][21]. In our laboratory, we have investigated peptides containing dehydroamino acids (dehydrophenylalanine-∆Phe, dehydroalanine-∆Ala, dehydroaminobutyric acid-∆Abu) as alternatives to D-amino acids, not only to provide proteolytic stability but also for the reduced conformational flexibility of the peptide backbone.…”

YcaO-Dependent Posttranslational Amide Activation: Biosynthesis, Structure, and Function

“…528 Thiostrepton covalently binds the RP via Cys adducts to its dehydroamino acids, which dysregulates proteolysis. 529 In an example of overlapping mode of action, proteosomal inhibition by thiostrepton stabilizes a negative regulator of FOXM1 (Hsp70). 530 It appears that thiostrepton can also directly bind FOXM1 and blocks its ability to activate gene transcription.…”

Thiostrepton degrades mutant p53 by eliciting an autophagic response in SW480 cells

“…Potentiality of THSP to inhibit 20S proteasome has also been thoroughly investigated (Schoof et al, ). THSP has the ability to directly bind to certain proteins and to block their entry into the 20S proteasome subunit (Sandu et al, ). Our results in SW480 cells showed the ability of THSP to decrease mutant p53 protein by transcriptional and translational mechanisms.…”