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The ubiquitin-proteasome system (UPS) is a regulated mechanism of intracellular protein degradation and turnover, and its dysfunction is associated with various diseases including cancer. UBR5, an E3 ubiquitin ligase, is emerging as an important regulator of the UPS in cancers, but its role in pancreatic cancer is poorly understood. Here, we show that UBR5 is significantly upregulated in pancreatic cancer tissues. High UBR5 expression is correlated with increased lymph node metastasis and poor survival of patients. The loss-of-function and gain-of-function studies demonstrated that UBR5 substantially enhanced the in vitro migratory and invasive ability of pancreatic cancer cells. UBR5 knockdown also markedly inhibited in vivo cancer metastasis in the liver metastatic model of pancreatic cancer in nude mice, suggesting UBR5 as a potent metastatic promoter in pancreatic cancer. Furthermore, using co-immunoprecipitation combined with mass spectrometry analyses, CAPZA1, a member of F-actin capping protein α subunit family, was identified as a novel substrate of UBR5. UBR5 overexpression could promote the degradation of CAPZA1 via the UPS and induce the accumulation of F-actin, which has been described as an essential molecular event during the process of CAPZA1 deficiency-induced cancer cells migration and invasion. UBR5 knockdown significantly increased the intracellular level of CAPZA1 and CAPZA1 downregulation largely reversed the UBR5 knockdown-induced suppression of cell migration and invasion in pancreatic cancer cells. Collectively, our findings unveil UBR5 as a novel and critical regulator of pancreatic cancer metastasis and highlight the potential for UBR5-CAPZA1 axis as a therapeutic target for preventing metastasis in pancreatic cancer patients, especially in those with increased UBR5 expression.

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway., Author summary Ubiquitin ligases modify proteins post-translationally which is essential for a variety of cellular processes. UBR5 is an E3 ubiquitin ligase and in Drosophila is a regulator of Hedgehog signaling. In mammals, the Hedgehog (HH) signalling pathway, among many other roles, plays an essential role in tissue maintenance, a process called homeostasis. A murine genetic system was developed to specifically eliminate UBR5 function from embryonic limb tissue that subsequently forms bone and connective tissue (ligaments and tendons). This approach revealed that UBR5 operates as a potent suppressor of excessive growth of normal cartilage and bone and prevents formation of bone in ectopic sites in connective tissue near the knees and ankle joints. In contrast to abnormal growth, UBR5 inhibits degradation of the articular cartilage that cushions the knee joint leading to extensive exposure of underlying bone. Furthermore, Ubr5 interacts with smoothened, a component of the HH pathway, identifying UBR5 as a regulator of mammalian HH signaling in the postnatal musculoskeletal system. In summary, this work shows that UBR5 interacts with the HH pathway to regulate skeletal homeostasis in and around joints of the legs and identifies targets that may be harnessed for biomedical engineering and clinical applications.

Summary The ubiquitin-proteasome system facilitates the degradation of unstable or damaged proteins. UBR1–7, which are members of hundreds of E3 ubiquitin ligases, recognize and regulate the half-life of specific proteins on the basis of their N-terminal sequences (“N-end rule”). In seven individuals with intellectual disability, epilepsy, ptosis, hypothyroidism, and genital anomalies, we uncovered bi-allelic variants in UBR7. Their phenotype differs significantly from that of Johanson-Blizzard syndrome (JBS), which is caused by bi-allelic variants in UBR1, notably by the presence of epilepsy and the absence of exocrine pancreatic insufficiency and hypoplasia of nasal alae. While the mechanistic etiology of JBS remains uncertain, mutation of both Ubr1 and Ubr2 in the mouse or of the C. elegans UBR5 ortholog results in Notch signaling defects. Consistent with a potential role in Notch signaling, C. elegans ubr-7 expression partially overlaps with that of ubr-5, including in neurons, as well as the distal tip cell that plays a crucial role in signaling to germline stem cells via the Notch signaling pathway. Analysis of ubr-5 and ubr-7 single mutants and double mutants revealed genetic interactions with the Notch receptor gene glp-1 that influenced development and embryo formation. Collectively, our findings further implicate the UBR protein family and the Notch signaling pathway in a neurodevelopmental syndrome with epilepsy, ptosis, and hypothyroidism that differs from JBS. Further studies exploring a potential role in histone regulation are warranted given clinical overlap with KAT6B disorders and the interaction of UBR7 and UBR5 with histones.

BackgroundN-end rule ubiquitination pathway is known to be disrupted in many diseases, including cancer. UBR5, an E3 ubiquitin ligase, is mutated and/or overexpressed in human lung cancer cells suggesting its pathological role in cancer.MethodsWe determined expression of UBR5 protein in multiple lung cancer cell lines and human patient samples. Using immunoprecipitation followed by mass spectrometry we determined the UBR5 interacting proteins. The impact of loss of UBR5 for lung adenocarcinoma cell lines was analyzed using cell viability, clonogenic assays and in vivo xenograft models in nude mice. Additional Western blot analysis was performed to assess the loss of UBR5 on downstream signaling. Statistical analysis was done by one-way ANOVA for in vitro studies and Wilcoxon paired t-test for tumor volumes in vivo.ResultsWe show variability of UBR5 expression levels in lung adenocarcinoma cell lines and in primary human patient samples. To gain better insight into the role that UBR5 may play in lung cancer progression we performed unbiased interactome analyses for UBR5. Data indicate that UBR5 has a wide range of interacting protein partners that are known to be involved in critical cellular processes such as DNA damage, proliferation and cell cycle regulation. We have demonstrated that shRNA-mediated loss of UBR5 decreases cell viability and clonogenic potential of lung adenocarcinoma cell lines. In addition, we found decreased levels of activated AKT signaling after the loss of UBR5 in lung adenocarcinoma cell lines using multiple means of UBR5 knockdown/knockout. Furthermore, we demonstrated that loss of UBR5 in lung adenocarcinoma cells results in significant reduction of tumor volume in nude mice.ConclusionsThese findings demonstrate that deregulation of the N-end rule ubiquitination pathway plays a crucial role in the etiology of some human cancers, and blocking this pathway via UBR5-specific inhibitors, may represent a unique therapeutic target for human cancers.

UBR5 is a nuclear phosphoprotein of obscure functions. Clinical analyses reveal that UBR5 amplifications and overexpression occur in over 20% cases of human breast cancers. Breast cancer patients carrying UBR5 genetic lesions with overexpression have significantly reduced survival. Experimental work in vitro and in vivo demonstrates that UBR5, functioning as an oncoprotein, plays a profound role in breast cancer growth and metastasis. UBR5 drives tumor growth largely through paracrine interactions with the immune system, particularly through inhibiting the cytotoxic response mediated by CD8+ T lymphocytes, whereas it facilitates metastasis in a tumor cell-autonomous manner via its transcriptional control of key regulators of the epithelial–mesenchymal transition, ID1 and ID3. Furthermore, simultaneous targeting of UBR5 and PD-L1 yields strong therapeutic benefit to tumor-bearing hosts. This work significantly expands our scarce understanding of the pathophysiology and immunobiology of a fundamentally important molecule and has strong implications for the development of novel immunotherapy to treat highly aggressive breast cancers that resist conventional treatment.

The E3 ubiquitin protein UBR5 has been implicated in the regulation of multiple biological functions and has recently emerged as a key regulator of the ubiquitin-proteasome system (UPS) in cancer. However, the clinical significance and biological function of UBR5 in colorectal cancer (CRC) are poorly understood. In this study, we compared the expression pattern of UBR5 between CRC and adjacent normal tissues and found that UBR5 expression was frequently elevated in CRC, possibly through chromosomal gains. Using three CRC patient cohorts, we found that patients with high UBR5 mRNA levels, UBR5 gene amplification, or high nuclear UBR5 protein levels had poor prognoses. Multivariate analysis showed that the alterations in UBR5 were independent predictors of CRC prognosis with the TNM stage as a confounding factor. Furthermore, knockdown of UBR5 prevented the proliferation, colony formation, migration, and invasion of CRC cells in cell culture models. An in vivo animal model further confirmed that UBR5 knockdown reduced the growth of CRC tumors. In conclusion, our study is the first to systematically investigate the clinical and biological significance of UBR5 and to conclude that an elevated UBR5 level plays an oncogenic role and may be a potential prognostic marker in CRC.

Human T-cell leukemia virus type 1 (HTLV-1) encodes a protein derived from the antisense strand of the proviral genome designated HBZ (HTLV-1 basic leucine zipper factor). HBZ is the only viral gene consistently expressed in infected patients and adult T-cell leukemia/lymphoma (ATL) tumor cell lines. It functions to antagonize many activities of the Tax viral transcriptional activator, suppresses apoptosis, and supports proliferation of ATL cells. Factors that regulate the stability of HBZ are thus important to the pathophysiology of ATL development. Using affinity-tagged protein and shotgun proteomics, we identified UBR5 as a novel HBZ-binding partner. UBR5 is an E3 ubiquitin-protein ligase that functions as a key regulator of the ubiquitin proteasome system in both cancer and developmental biology. Herein, we investigated the role of UBR5 in HTLV-1-mediated T-cell transformation and leukemia/lymphoma development. The UBR5/HBZ interaction was verified in vivo using over-expression constructs, as well as endogenously in T-cells. shRNA-mediated knockdown of UBR5 enhanced HBZ steady-state levels by stabilizing the HBZ protein. Interestingly, the related HTLV-2 antisense-derived protein, APH-2, also interacted with UBR5 in vivo. However, knockdown of UBR5 did not affect APH-2 protein stability. Co-immunoprecipitation assays identified ubiquitination of HBZ and knockdown of UBR5 resulted in a decrease in HBZ ubiquitination. MS/MS analysis identified seven ubiquitinated lysines in HBZ. Interestingly, UBR5 expression was upregulated in established T lymphocytic leukemia/lymphoma cell lines and the later stage of T-cell transformation in vitro. Finally, we demonstrated loss of UBR5 decreased cellular proliferation in transformed T-cell lines. Overall, our study provides evidence for UBR5 as a host cell E3 ubiquitin-protein ligase responsible for regulating HBZ protein stability. Additionally, our data suggests UBR5 plays an important role in maintaining the proliferative phenotype of transformed T-cell lines.

Using a conditional Ubr5 mutant (Ubr5mt) mouse the Ditzel lab identified a novel role for the ubiquitin-protein ligase UBR5 in maintaining articular cartilage (AC) homeostasis, suppressing osteoarthritis (OA) and controlling heterotopic tendon ossification (HO), potentially through influencing the Hedgehog signalling pathway. Histological observations revealed aberrant chondrocyte behaviour in both the AC and ossifying tendons. Interestingly, application of the Hedgehog pathway inhibitor cyclopamine enhanced the Ubr5mt OA and HO phenotypes. Based on these observations I hypothesised that UBR5 utilises Hh signalling to control AC and tendon homeostasis through influencing AC- or tendon-resident stem/progenitor cell function. I planned to address this hypothesis by designing an in vitro cell-based model to investigate the chondrogenic potential of UBR5-deficient stem/progenitor cells and by investigating the Hh signalling expression pattern in tendons collected from normal and Ubr5mt animals. To address the molecular and cellular defects on Ubr5mt cells I established an in vitro system to culture mesenchymal stem cells (MSCs) and promote their differentiation into chondrocytes. First, I optimised the experimental conditions for the extraction and culture of bone marrow derived cells from C57BL/6 mice. The identity of the isolated cells was confirmed by functional chondrogenesis assays and expression of specific MSCs markers. Once I was confident about the isolation and culture techniques I aimed to culture cells derived from conditionally Ubr5mt animals. Unfortunately, these Cre-recombinase bearing MSCs proved difficult to culture, which severely hampered experimentation and made the proposed experimental approach unfeasible. To investigate the Hh signalling expression and cellular pattern in tendons affected by HO, I compared the presence of various markers in patellar tendons collected from normal and Ubr5mt mice. It turned out that the level of IHH and PTCH1, proteins involved in Hh signalling pathway, was much higher in the latter. Overall, I have identified differential expression of Hedgehog pathway components in control and Ubr5mt tendons as well as successfully isolated and cultured MSC. These findings and achievement have contributed to a manuscript and will form the basis of a grant proposal.

Despite recent advances, breast cancer remains a major burden on the healthcare system in Australia and abroad. Of particular concern are cancer subtypes that currently lack targetted therapeutics. As such, the continued development of targetted therapeutics remains a high priority. The ubiquitin system offers a fresh avenue for the development of targetted therapeutics. Ubiquitin tagging (ubiquitylation) is a very common posttranslational modification of proteins that signals degradation, changes in protein interactions and other outcomes. UBR5 is a protein originally identified as a tumour suppressor in Drosophila that catalyses attachment of ubiquitin to substrate proteins. UBR5 is amplified in a significant proportion of cancers, particularly in breast and ovarian. Intriguingly, UBR5 amplification correlates with a significantly poorer survival for breast cancer patients. Although UBR5 has well characterised roles in development and orchestrating a cellular response to DNA damage, it is unknown how UBR5 amplification may contribute to a poorer patient outcome. This study aims to comprehensively characterise the role of UBR5 in breast cancer biology using cellular and animal models. UBR5 does not fit into a classical binary definition of either oncogene or tumour suppressor, as initial studies manipulating UBR5 expression did not reveal a significant role for UBR5 in basic processes such as increased proliferation or invasion/metastasis. This study identified a novel role for UBR5 primary cilia formation (ciliogenesis). Primary cilia are antennae like structures important for cellular signalling in most cell types. Functional studies showed UBR5 to act upstream of cilia formation on granular structures known as centriolar satellites. Further work described a mechanism whereby UBR5 ubiquitylates the cilia protein CSPP1 via a non-degrading lysine 63-linked ubiquitin chain, altering sub-cellular localisation. This work represents a major step forward in understanding the biology of primary cilia, with implications for not only cancer, but also a rare group of disorders known as ciliopathies that are characterised by cilia dysfunction. To the best of our knowledge aberrations in UBR5 has not been studied in this context, and future experiments should examine a genetic link between UBR5 and cilia dysfunction in cohorts of ciliopathies.

Ubiquitylation is one of the most abundant protein modifications in cellular signaling, controlling numerous cellular pathways such as transcription, translation, vesicle transport and apoptosis. Ubiquitinlabels substrate proteins via a highly ordered multi-step enzymatic cascade, with specific differences in the length and topology of poly-Ub chains signalling a range of signalling outcomes or proteolytic degradation via the proteasome. Identification of E3 ubiquitin ligase substrates is key to defining their biological function and understanding their roles in disease. However, even with advances in proteomics and in vitro assays, substrate identification remains a significant challenge. An integrated approach was developed to define the E3 ligase substrates, combining orthogonal proteomics approaches to identify interacting proteins and ubiquitylation targets, combined with BiFC to validate substrates in situ. This approach was applied to identify substrates of the UBR5 ligase, which has been implicated in cancer progression and chemoresistance. UBR5-interacting proteins were isolated using GFP-Trap affinity purification followed by nanoLCMS/MS identification and label-free quantitation. This approach identified ~ 450 UBR5-interacting proteins, many dependent on the UBR and HECT functional domains for binding. Analysis of differentially ubiquitylated proteins in breast cancer cells depleted of UBR5 by shRNA identified ~1500 proteins with altered ubiquitylation when UBR5 is depleted. Intriguingly, a large number ofubiquitin-proteasome system components have altered ubiquitylation in UBR5-depleted cells, suggesting co-ordinate regulation of this system. Integration of the UBR5-interactome and ubiquitome generated a set of 171 high-confidence ligase substrates. A number of these were validated using BiFC and the role of UBR5 functional domains in mediating these interactions using disease-specific mutants was mapped. Importantly, a novel functional domain in UBR5 was identified and was found to be important for binding to some UBR5-interacting proteins. These orthogonal but complementary approaches provided interesting new insights into the function of UBR5, suggesting a role in mediating crosstalk between DNA damage response and transcriptional regulation, and the regulation of H2B post-translational modification cross talk.

The terminal step in the ubiquitin modification system relies on an E3 ubiquitin ligase to facilitate transfer of ubiquitin to a protein substrate. The substrate recognition and ubiquitin transfer activities of the E3 ligase may be mediated by a single polypeptide or may rely on separate subunits. The latter organization is particularly prevalent among members of largest class of E3 ligases, the RING family, although examples of this type of arrangement have also been reported among members of the smaller HECT family of E3 ligases. This review describes recent discoveries that reveal the surprising and distinctive ability of VprBP (DCAF1) to serve as a substrate recognition subunit for a member of both major classes of E3 ligase, the RING-type CRL4 ligase and the HECT-type EDD/UBR5 ligase. The cellular processes normally regulated by VprBP-associated E3 ligases, and their targeting and subversion by viral accessory proteins are also discussed. Taken together, these studies provide important insights and raise interesting new questions regarding the mechanisms that regulate or subvert VprBP function in the context of both the CRL4 and EDD/UBR5 E3 ligases.