Proteasome substrate receptor hRpn13 is a promising anti-cancer target. By integrated in silico and biophysical screening, we identified a chemical scaffold that binds hRpn13 with non-covalent ...interactions that mimic the proteasome and a weak electrophile for Michael addition. hRpn13 Pru domain binds proteasomes and ubiquitin whereas its DEUBAD domain binds deubiquitinating enzyme UCHL5. NMR revealed lead compound XL5 to interdigitate into a hydrophobic pocket created by lateral movement of a Pru β-hairpin with an exposed end for Proteolysis Targeting Chimeras (PROTACs). Implementing XL5-PROTACs as chemical probes identified a DEUBAD-lacking hRpn13 species (hRpn13
) present naturally with cell type-dependent abundance. XL5-PROTACs preferentially target hRpn13
, causing its ubiquitination. Gene-editing and rescue experiments established hRpn13 requirement for XL5-PROTAC-triggered apoptosis. These data establish hRpn13 as an anti-cancer target for multiple myeloma and introduce an hRpn13-targeting scaffold that can be optimized for preclinical trials against hRpn13
-producing cancer types.
Intratumoral heterogeneity in bladder cancer is a barrier to accurate molecular sub-classification and treatment efficacy. However, individual cellular and mechanistic contributions to tumor ...heterogeneity are controversial. We examined potential mechanisms of FOXA1 and PTEN inactivation in bladder cancer and their contribution to tumor heterogeneity. These analyses were complemented with inactivation of FOXA1 and PTEN in intermediate and luminal mouse urothelium. We show inactivation and reduced expression of FOXA1 and PTEN is prevalent in human disease, where PTEN and FOXA1 are downregulated by allelic loss and site-specific DNA hypermethylation, respectively. Conditional inactivation of both Foxa1 and Pten in intermediate/luminal cells in mice results in development of bladder cancer exhibiting squamous features as well as enhanced sensitivity to a bladder-specific carcinogen. In addition, FOXA1 is hypermethylated in basal bladder cancer cell lines, and this is reversed by treatment with DNA methyltransferase inhibitors. By integrating human correlative and in vivo studies, we define a critical role for PTEN loss and epigenetic silencing of FOXA1 in heterogeneous human disease and show genetic targeting of luminal/intermediate cells in mice drives squamous differentiation.
The ubiquitin-proteasome system (UPS) is critical for protein quality control and regulating protein lifespans. Following ubiquitination, UPS substrates bind multidomain receptors that, in addition ...to ubiquitin-binding sites, contain functional domains that bind to deubiquitinating enzymes (DUBs) or the E3 ligase E6AP/UBE3A. We provide an overview of the proteasome, focusing on its receptors and DUBs. We highlight the key role of dynamics and importance of the substrate receptors having domains for both binding and processing ubiquitin chains. The UPS is rich with therapeutic opportunities, with proteasome inhibitors used clinically and ongoing development of small molecule proteolysis targeting chimeras (PROTACs) for the degradation of disease-associated proteins. We discuss the therapeutic potential of proteasome receptors, including hRpn13, for which PROTACs have been developed.
Inhibitors against the proteasome core particle (CP) treat hematological cancers, and new therapies that target the proteasome continue to be developed.At the proteasome, substrate receptors (Rpn1, Rpn10, and Rpn13) have their own intrinsic dynamics and bind to ubiquitin chains dynamically, aiding in substrate capture and processing.Proteasome substrate receptors are multimodular, with distinct domains for binding to substrates through ubiquitin chains and to DUBs USP14 and UCHL5 or ubiquitin E3 ligase E6AP.Inducing proteasomal degradation is done naturally by pathogens that generate neo-substrates through Rpn10 and E6AP and a PROTAC has been developed against Rpn13.
We talk to Vasty Osei Amponsa and Kylie J. Walters about their paper “hRpn13 shapes the proteome and transcriptome through epigenetic factors HDAC8, PADI4, and transcription factor NF-κB p50”, their ...journeys across continents leading them to the NCI, and how Kylie tries to foster curiosity and a sense of belonging in her lab.
Discrete bladder cancer molecular subtypes exhibit differential clinical aggressiveness and therapeutic response, which may have significant implications for identifying novel treatments for this ...common malignancy. However, research is hindered by the lack of suitable models to study each subtype. To address this limitation, we classified bladder cancer cell lines into molecular subtypes using publically available data in the Cancer Cell Line Encyclopedia (CCLE), guided by genomic characterization of bladder cancer by The Cancer Genome Atlas (TCGA). This identified a panel of bladder cancer cell lines which exhibit genetic alterations and gene expression patterns consistent with luminal and basal molecular subtypes of human disease. A subset of bladder cancer cell lines exhibit in vivo histomorphologic patterns consistent with luminal and basal subtypes, including papillary architecture and squamous differentiation. Using the molecular subtype assignments, and our own RNA-seq analysis, we found overexpression of GATA3 and FOXA1 cooperate with PPARɣ activation to drive transdifferentiation of a basal bladder cancer cells to a luminial phenotype. In summary, our analysis identified a set of human cell lines suitable for the study of molecular subtypes in bladder cancer, and furthermore indicates a cooperative regulatory network consisting of GATA3, FOXA1, and PPARɣ drive luminal cell fate.
Regulated protein degradation in eukaryotes is performed by the 26S proteasome, which contains a 19-subunit regulatory particle (RP) that binds, processes, and translocates substrates to a 28-subunit ...hollow core particle (CP) where proteolysis occurs. In addition to its intrinsic subunits, myriad proteins interact with the proteasome transiently, including factors that assist and/or regulate its degradative activities. Efforts to identify proteasome-interacting components and/or to solve its structure have relied on over-expression of a tagged plasmid, establishing stable cell lines, or laborious purification protocols to isolate native proteasomes from cells. Here, we describe an engineered human cell line, derived from colon cancer HCT116 cells, with a biotin handle on the RP subunit hRpn1/PSMD2 (proteasome 26S subunit, non-ATPase 2) for purification of 26S proteasomes. A 75-residue sequence from Propionibacterium shermanii that is biotinylated in mammalian cells was added following a tobacco etch virus protease cut site at the C terminus of hRpn1. We tested and found that 26S proteasomes can be isolated from this modified HCT116 cell line by using a simple purification protocol. More specifically, biotinylated proteasomes were purified from the cell lysates by using neutravidin agarose resin and released from the resin following incubation with tobacco etch virus protease. The purified proteasomes had equivalent activity in degrading a model ubiquitinated substrate, namely ubiquitinated p53, compared to commercially available bovine proteasomes that were purified by fractionation. In conclusion, advantages of this approach to obtain 26S proteasomes over others is the simple purification protocol and that all cellular proteins, including the tagged hRpn1 subunit, remain at endogenous stoichiometry.
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.
Preservation of fine cellular details of semi-adherent or suspension cells for imaging by immunofluorescence is challenging. This protocol enables staining of floating cells with minimal ...morphological distortions, as we demonstrate with the semi-adherent multiple myeloma cell line RPMI 8226. We describe steps to better preserve structural details by fixing, permeabilizing, and staining cells in solution, while minimizing the number of centrifugation steps and centrifugation g-force.
For complete details on the use and execution of this protocol, please refer to Osei-Amponsa et al.1
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•Protocol for immunofluorescence microscopy of semi-adherent cells•Steps for preparing multiple myeloma RPMI 8226 cells for immunostaining•Approach for minimal distortion of cell morphology by reducing centrifugation
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Preservation of fine cellular details of semi-adherent or suspension cells for imaging by immunofluorescence is challenging. This protocol enables staining of floating cells with minimal morphological distortions, as we demonstrate with the semi-adherent multiple myeloma cell line RPMI 8226. We describe steps to better preserve structural details by fixing, permeabilizing, and staining cells in solution, while minimizing the number of centrifugation steps and centrifugation g-force.
The anti-cancer target hRpn13 is a proteasome substrate receptor. However, hRpn13-targeting molecules do not impair its interaction with proteasomes or ubiquitin, suggesting other critical cellular ...activities. We find that hRpn13 depletion causes correlated proteomic and transcriptomic changes, with pronounced effects in myeloma cells for cytoskeletal and immune response proteins and bone-marrow-specific arginine deiminase PADI4. Moreover, a PROTAC against hRpn13 co-depletes PADI4, histone deacetylase HDAC8, and DNA methyltransferase MGMT. PADI4 binds and citrullinates hRpn13 and proteasomes, and proteasomes from PADI4-inhibited myeloma cells exhibit reduced peptidase activity. When off proteasomes, hRpn13 can bind HDAC8, and this interaction inhibits HDAC8 activity. Further linking hRpn13 to transcription, its loss reduces nuclear factor κB (NF-κB) transcription factor p50, which proteasomes generate by cleaving its precursor protein. NF-κB inhibition depletes hRpn13 interactors PADI4 and HDAC8. Altogether, we find that hRpn13 acts dually in protein degradation and expression and that proteasome constituency and, in turn, regulation varies by cell type.
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•hRpn13 loss alters expression of cytoskeletal, epigenetic, and immune response factors•PADI4 binds and citrullinates hRpn13, which, when off proteasomes, can also bind HDAC8•PADI4 binds, citrullinates, and affects the peptidase activity of proteasomes•hRpn13 loss reduces NF-κB p50, the inhibition of which reduces PADI4 and HDAC8
Osei-Amponsa et al. discover hRpn13 interaction with epigenetic factors physically and functionally both on and off proteasomes and contribution to NF-κB p50 production. These and additionally identified hRpn13 transcriptional requirements provide insights into its cellular impact beyond its known role at proteasomes and rationale for anti-cancer activities of hRpn13 PROTACs.
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