Effect of Proteasome Inhibitors With Different Chemical Structures on the Ubiquitin–Proteasome System In Vitro

Abstract

Proteasome inhibitor therapeutics (PITs) have the potential to cause peripheral neuropathy. In a mouse model of PIT-induced peripheral neuropathy, the authors demonstrated that ubiquitin-positive multifocal protein aggregates with nuclear displacement appear in dorsal root ganglion cells of animals that subsequently develop nerve injuries. This peripheral-nerve effect in nonclinical models has generally been recognized as the correlate of grade 3 neuropathy in clinical testing. In differentiated PC12 cells, the authors demonstrated perturbations correlative with the development of neuropathy in vivo, including ubiquitinated protein aggregate (UPA) formation and/or nuclear displacement associated with the degree of proteasome inhibition. They compared 7 proteasome inhibitors of 3 chemical scaffolds (peptide boronate, peptide epoxyketone, and lactacystin analog) to determine if PIT-induced peripheral neuropathy is modulated by inhibition of the proteasome (ie, a mechanism-based effect) or due to effects independent of proteasome inhibition (ie, an off target or chemical-structure-based effect). The appearance of UPAs was assayed at IC(90) +/- 5% (90% inhibition concentration +/- 5%) for 20S proteasome inhibition. Results show that each of the investigated proteasome inhibitors induced identical proteasome-inhibitor-specific ubiquitin-positive immunostaining and nuclear displacement in PC12 cells. Other agents--such as paclitaxel, cisplatin, and thalidomide, which cause neuropathy by other mechanisms--did not cause UPAs or nuclear displacement, demonstrating that the effect was specific to proteasome inhibitors. In conclusion, PIT-induced neuronal cell UPA formation and nuclear displacement are mechanism based and independent of the proteasome inhibitor scaffold. These data indicate that attempts to modulate the neuropathy associated with PIT may not benefit from changing scaffolds.

Proteasome regulates turnover of toxic human amylin in pancreatic cells

“…Synthetic hA efficiently competed with 20S specific fluorogenic substrate for binding to the complex as evident by a marked loss in fluorescent signal over time (Figure 8A), thus confirming the IP studies and formation of hA/20S complex (Figures 5B and 5D). These in vitro studies revealed that peptides interact more avidly with 20S proteasome complex (IC 50 ~ 100 nM, Figures 8B and 8C) as compared with Lac (IC 50 ~ 1 μ M, Figure 8D), in agreement with recent complementary study [67]. Thus, amylin directly interacts with the 20S complex and efficiently modulates its proteolytic activity even in the absence of ubiquitination.…”

Toxic Effects of Bortezomib on Primary Sensory Neurons and Schwann Cells of Adult Mice

“…However, how bortezomib is affecting axonal transport is not clear yet. Impairment of the proteasome ubiquitin system facilitates aggresome formation (Csizmadia et al 2010). In fact, we already detected aggresomes in cultured sensory neurons exposed to low concentrations of bortezomib.…”

Proteasome inhibitors bortezomib and carfilzomib used for the treatment of multiple myeloma do not inhibit the serine protease HtrA2/Omi

“…Although the mechanism of bortezomib-associated peripheral neuropathy is unknown, we have previously demonstrated that it is related to the drug's mechanism of action. 3 Peptide boronic acids are known to inhibit proteases other than the proteasome, such as leukocyte elastase, pancreatic elastase, cathepsin G, and chymotrypsin, but the role of these proteases in the homeostasis of sensory neurons is not known. 4…”