Impact of Losing hRpn13 Pru or UCHL5 on Proteasome Clearance of Ubiquitinated Proteins and RA190 Cytotoxicity

Abstract

<jats:p>hRpn13/ADRM1 links substrate recruitment with deubiquitination at the proteasome through its proteasome- and ubiquitin-binding Pru domain and DEUBAD domain, which binds and activates deubiquitinating enzyme (DUB) UCHL5/Uch37. Here, we edit the HCT116 colorectal cancer cell line to delete part of the hRpn13 Pru, producing cells that express truncated hRpn13 (trRpn13), which is competent for UCHL5 binding, but defective for proteasome interaction. <jats:italic>trRpn13</jats:italic> cells demonstrates reduced levels of proteasome-bound ubiquitinated proteins, indicating that loss of hRpn13 function at proteasomes cannot be fully compensated for by the two other dedicated substrate receptors (hRpn1 and hRpn10). Previous studies indicate that loss of full-length hRpn13 causes corresponding reduction of UCHL5. We find UCHL5 levels unaltered in <jats:italic>trRpn13</jats:italic> cells, but that hRpn11 is elevated in <jats:italic>ΔhRpn13</jats:italic> and <jats:italic>trRpn13</jats:italic>, perhaps from cell stress. Despite the ∼90 DUBs in human cells, including two others in addition to UCHL5 at the proteasome, we found deletion of UCHL5 from HCT116 cells to cause increased levels of ubiquitinated proteins in whole cell extract and at proteasomes, suggesting that UCHL5 activity cannot be fully assumed by other DUBs. We also report anti-cancer molecule RA190, which binds covalently to hRpn13 and UCHL5, to require hRpn13 Pru and not UCHL5 for cytotoxicity.</jats:p>

Structure-guided bifunctional molecules hit a DEUBAD-lacking hRpn13 species upregulated in multiple myeloma

“…6a). It was not noticed in our previous studies with HCT116 cells 30,35 due to its low abundance in this cell line (Fig 5e, 1s versus 5 min exposure for hRpn13). The higher expression level in RPMI 8226 cells coupled with the invocation of XL5-PROTACs enabled its identification.…”

“…4c and 4f). Although we do not know how We previously found UCHL5 to be targeted by RA190 35 and multiple studies have demonstrated the cellular abundance of UCHL5 to be dependent on hRpn13 15,20,30,35 .…”

“…4c). HCT116 and RPMI 8226 trRpn13 cells express an hRpn13 protein product that spans M109 to D407 with molecular weight of ~30 kDa 30 . This truncated hRpn13 protein is slightly larger than hRpn13 Pru (Fig.…”

“…CRISPR-based gene editing indicated hRpn13-binding compounds (RA190 and RA183) 27,28 to induce apoptosis in an hRpn13-dependent manner 29,30 , albeit knockdown experiments suggest little dependency 31 , including for an hRpn13-binding peptoid 32,33 .…”

Proteasome interaction with ubiquitinated substrates: from mechanisms to therapies

“…Each of these hRpn13-targeting approaches were found to be synergistic with inhibition of the proteasome CP [163,164]. RA190-induced accumulation of ubiquitinated proteins at the proteasome is lost upon deletion of hRpn13 [62] or its Pru domain [165].…”