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

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. trRpn13 cells demonstrate reduced levels of proteasome-bound ubiquitinated proteins, indicating that the loss of hRpn13 function at proteasomes cannot be fully compensated for by the two other dedicated substrate receptors (hRpn1 and hRpn10). Previous studies indicated that the loss of full-length hRpn13 causes a corresponding reduction of UCHL5. We find UCHL5 levels unaltered in trRpn13 cells, but hRpn11 is elevated in ΔhRpn13 and trRpn13 cells, 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 anticancer molecule RA190, which binds covalently to hRpn13 and UCHL5, to require hRpn13 Pru and not UCHL5 for cytotoxicity.

Structure of E3 ligase E6AP with a proteasome-binding site provided by substrate receptor hRpn10.

Regulated proteolysis by proteasomes involves ~800 enzymes for substrate modification with ubiquitin, including ~600 E3 ligases. We report here that E6AP/UBE3A is distinguished from other E3 ligases by having a 12 nM binding site at the proteasome contributed by substrate receptor hRpn10/PSMD4/S5a. Intrinsically disordered by itself, and previously uncharacterized, the E6AP-binding domain in hRpn10 locks into a well-defined helical structure to form an intermolecular 4-helix bundle with the E6AP AZUL, which is unique to this E3. We thus name the hRpn10 AZUL-binding domain RAZUL. We further find in human cells that loss of RAZUL by CRISPR-based gene editing leads to loss of E6AP at proteasomes. Moreover, proteasome-associated ubiquitin is reduced following E6AP knockdown or displacement from proteasomes, suggesting that E6AP ubiquitinates substrates at or for the proteasome. Altogether, our findings indicate E6AP to be a privileged E3 for the proteasome, with a dedicated, high affinity binding site contributed by hRpn10.

Prognosis, Biology, and Targeting of TP53 Dysregulation in Multiple Myeloma.

Multiple myeloma (MM) is the second most common hematological cancer and is characterized by genetic features including translocations, chromosomal copy number aberrations, and mutations in key oncogene and tumor suppressor genes. Dysregulation of the tumor suppressor TP53 is important in the pathogenesis of many cancers, including MM. In newly-diagnosed MM patients, TP53 dysregulation occurs in three subsets: monoallelic deletion as part of deletion of chromosome 17p (del17p) (~8%), monoallelic mutations (~6%), and biallelic inactivation (~4%). Del17p is an established high-risk feature in MM and is included in current disease staging criteria. Biallelic inactivation and mutation have also been reported in MM patients but are not yet included in disease staging criteria for high-risk disease. Emerging clinical and genomics data suggest that the biology of high-risk disease is complex, and so far, traditional drug development efforts to target dysregulated TP53 have not been successful. Here we review the TP53 dysregulation literature in cancer and in MM, including the three segments of TP53 dysregulation observed in MM patients. We propose a reverse translational approach to identify novel targets and disease drivers from TP53 dysregulated patients to address the unmet medical need in this setting.

Structure of hRpn10 Bound to UBQLN2 UBL Illustrates Basis for Complementarity between Shuttle Factors and Substrates at the Proteasome.

The 26S proteasome is a highly complex 2.5-MDa molecular machine responsible for regulated protein degradation. Proteasome substrates are typically marked by ubiquitination for recognition at receptor sites contributed by Rpn1/S2/PSMD2, Rpn10/S5a, and Rpn13/Adrm1. Each receptor site can bind substrates directly by engaging conjugated ubiquitin chains or indirectly by binding to shuttle factors Rad23/HR23, Dsk2/PLIC/UBQLN, or Ddi1, which contain a ubiquitin-like domain (UBL) that adopts the ubiquitin fold. Previous structural studies have defined how each of the proteasome receptor sites binds to ubiquitin chains as well as some of the interactions that occur with the shuttle factors. Here, we define how hRpn10 binds to the UBQLN2 UBL domain, solving the structure of this complex by NMR, and determine affinities for each UIM region by a titration experiment. UBQLN2 UBL exhibits 25-fold stronger affinity for the N-terminal UIM-1 over UIM-2 of hRpn10. Moreover, we discover that UBQLN2 UBL is fine-tuned for the hRpn10 UIM-1 site over the UIM-2 site by taking advantage of the additional contacts made available through the longer UIM-1 helix. We also test hRpn10 versatility for the various ubiquitin chains to find less specificity for any particular linkage type compared to hRpn1 and hRpn13, as expected from the flexible linker region that connects the two UIMs; nonetheless, hRpn10 does exhibit some preference for K48 and K11 linkages. Altogether, these results provide new insights into the highly complex and complementary roles of the proteasome receptor sites and shuttle factors.

Structure of the Rpn13-Rpn2 complex provides insights for Rpn13 and Uch37 as anticancer targets.

Proteasome-ubiquitin receptor hRpn13/Adrm1 binds and activates deubiquitinating enzyme Uch37/UCHL5 and is targeted by bis-benzylidine piperidone RA190, which restricts cancer growth in mice xenografts. Here, we solve the structure of hRpn13 with a segment of hRpn2 that serves as its proteasome docking site; a proline-rich C-terminal hRpn2 extension stretches across a narrow canyon of the ubiquitin-binding hRpn13 Pru domain blocking an RA190-binding surface. Biophysical analyses in combination with cell-based assays indicate that hRpn13 binds preferentially to hRpn2 and proteasomes over RA190. hRpn13 also exists outside of proteasomes where it may be RA190 sensitive. RA190 does not affect hRpn13 interaction with Uch37, but rather directly binds and inactivates Uch37. hRpn13 deletion from HCT116 cells abrogates RA190-induced accumulation of substrates at proteasomes. We propose that RA190 targets hRpn13 and Uch37 through parallel mechanisms and at proteasomes, RA190-inactivated Uch37 cannot disassemble hRpn13-bound ubiquitin chains.

SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis.

Polo-like kinase 1 (Plk1)-interacting checkpoint helicase (PICH) localizes at the centromere and is critical for proper chromosome segregation during mitosis. However, the precise molecular mechanism of PICH's centromeric localization and function at the centromere is not yet fully understood. Recently, using Xenopus egg extract assays, we showed that PICH is a promiscuous SUMO binding protein. To further determine the molecular consequence of PICH/SUMO interaction on PICH function, we identified 3 SUMO-interacting motifs (SIMs) on PICH and generated a SIM-deficient PICH mutant. Using the conditional expression of PICH in cells, we found distinct roles of PICH SIMs during mitosis. Although all SIMs are dispensable for PICH's localization on ultrafine anaphase DNA bridges, only SIM3 (third SIM, close to the C-terminus end of PICH) is critical for its centromeric localization. Intriguingly, the other 2 SIMs function in chromatin bridge prevention. With these results, we propose a novel SUMO-dependent regulation of PICH's function on mitotic centromeres.

SUMOylation of the C-terminal domain of DNA topoisomerase IIα regulates the centromeric localization of Claspin.

DNA topoisomerase II (TopoII) regulates DNA topology by its strand passaging reaction, which is required for genome maintenance by resolving tangled genomic DNA. In addition, TopoII contributes to the structural integrity of mitotic chromosomes and to the activation of cell cycle checkpoints in mitosis. Post-translational modification of TopoII is one of the key mechanisms by which its broad functions are regulated during mitosis. SUMOylation of TopoII is conserved in eukaryotes and plays a critical role in chromosome segregation. Using Xenopus laevis egg extract, we demonstrated previously that TopoIIα is modified by SUMO on mitotic chromosomes and that its activity is modulated via SUMOylation of its lysine at 660. However, both biochemical and genetic analyses indicated that TopoII has multiple SUMOylation sites in addition to Lys660, and the functions of the other SUMOylation sites were not clearly determined. In this study, we identified the SUMOylation sites on the C-terminal domain (CTD) of TopoIIα. CTD SUMOylation did not affect TopoIIα activity, indicating that its function is distinct from that of Lys660 SUMOylation. We found that CTD SUMOylation promotes protein binding and that Claspin, a well-established cell cycle checkpoint mediator, is one of the SUMOylation-dependent binding proteins. Claspin harbors 2 SUMO-interacting motifs (SIMs), and its robust association to mitotic chromosomes requires both the SIMs and TopoIIα-CTD SUMOylation. Claspin localizes to the mitotic centromeres depending on mitotic SUMOylation, suggesting that TopoIIα-CTD SUMOylation regulates the centromeric localization of Claspin. Our findings provide a novel mechanistic insight regarding how TopoIIα-CTD SUMOylation contributes to mitotic centromere activity.

SUMOylation regulates polo-like kinase 1-interacting checkpoint helicase (PICH) during mitosis.

Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres.