An ubiquitin-binding molecule can work as an inhibitor of ubiquitin processing enzymes and ubiquitin receptors

Abstract

The ubiquitin pathway plays a critical role in regulating diverse biological processes, and its dysregulation is associated with various diseases. Therefore, it is important to have a tool that can control the ubiquitin pathway in order to improve understanding of this pathway and to develop therapeutics against relevant diseases. We found that Chicago Sky Blue 6B binds directly to the β-groove, a major interacting surface of ubiquitin. Hence, it could successfully inhibit the enzymatic activity of ubiquitin processing enzymes and the binding of ubiquitin to the CXCR4, a cell surface ubiquitin receptor. Furthermore, we demonstrated that this ubiquitin binding chemical could effectively suppress the ubiquitin induced cancer cell migration by blocking ubiquitin-CXCR4 interaction. Current results suggest that ubiquitin binding molecules can be developed as inhibitors of ubiquitin-protein interactions, which will have the value not only in unveiling the biological role of ubiquitin but also in treating related diseases.

Functional and structural consequences of chemokine (C-X-C motif) receptor 4 activation with cognate and non-cognate agonists

“…β-arrestin 1 and 2 have been described to be involved in CXCL12 mediated chemotaxis and in CXCR4 internalization upon CXCL12 binding [28–30]. As several lines of evidence suggest that ubiquitin binding to CXCR4 expressing cells also leads to co-internalization of the ligand-receptor complex [4, 10–12, 14], the mechanisms underlying CXCR4 internalization upon ubiquitin and CXCL12 binding may be different. Nevertheless, the findings of the present study, in combination with our previous observation that ERK1/2 phosphorylation upon ubiquitin activation of CXCR4 was rapid and transient, whereas ERK1/2 phosphorylation upon CXCL12 activation was persistent, suggest that ubiquitin functions as a biased CXCR4 agonist that preferentially signals via G protein-mediated pathways [5, 31].…”

“…Recently, ubiquitin has been identified as a non-cognate non-chemokine agonist of CXCR4 [4]. While previous studies suggest that ubiquitin and CXCL12 bind to distinct contact sites on CXCR4 [5], multiple lines of evidence indicate that both agonists induce Gα i -mediated signaling, receptor internalization and regulate cell movements via CXCR4 in various cell types [4–14]. Chemotactic activities of ubiquitin in primary human cells, however, are poorly characterized.…”

The small molecule Chicago Sky Blue promotes heart repair following myocardial infarction in mice

“…CSB is an azo-dye used as a counterstain for reducing background in immunofluorescence staining. CSB was shown to interact with several proteins, including the vesicular glutamate transporter (VGLUT) (37), macrophage migration inhibitory factor (MIF) (38), RAD1 (39), and ubiquitin (40). To validate the proliferative effect observed in the screen we first repeated the screen assay several times.…”

Suppression of the Ubiquitin Pathway by Small Molecule Binding to Ubiquitin Enhances Doxorubicin Sensitivity of the Cancer Cells

“…In 2016, we described a new inhibitor in this pathway, which bound to the ubiquitin polypeptide [35]. Chicago Sky Blue 6B (CSB6B), an azo dye compound, was identified as an inhibitor to a deubiquitinase in a high throughput screening, but later revealed not to bind to deubiquitinase, but to ubiquitin.…”

“…Chicago Sky Blue 6B (CSB6B), an azo dye compound, was identified as an inhibitor to a deubiquitinase in a high throughput screening, but later revealed not to bind to deubiquitinase, but to ubiquitin. It was subsequently shown that CSB6B shut down all the pathways involving ubiquitin, including ubiquitination, deubiquitination and ligand-receptor interaction [35]. As CSB6B is a highly sulfated molecule, it could not penetrate the cell membrane, but instead, successfully inhibited the activity of extracellular ubiquitin.…”