Ubiquitin-dependent Proteolysis Research Articles

Thioredoxin-interacting protein (TXNIP) is a substrate of the NEDD4-like E3 ubiquitin-protein ligase WWP1 in cellular redox state regulation of acute myeloid leukemia cells.

The HECT-type E3 ubiquitin WWP1 (also known as NEDD4-like E3 ubiquitin-protein ligase WWP1) acts as an oncogenic factor in acute myeloid leukemia (AML) cells. WWP1 overexpression in AML confers a proliferative advantage to leukemic blasts (abnormal immature white bloodcells) and counteracts apoptotic cell death and differentiation. In aneffort to elucidate the molecular basis of WWP1 oncogenic activities, we identified WWP1 as a previously unknown negative regulator of thioredoxin-interacting protein (TXNIP)-mediated reactive oxygen species (ROS) production in AML cells. TXNIP inhibits the disulfide reductase enzymatic activity of thioredoxin (Trx), impairing its antioxidant function and, ultimately, leading to the disruption of cellular redox homeostasis. In addition, TXNIP restricts cell growth and survival by blocking glucose uptake and metabolism. Here, we found that WWP1 directly interacts with TXNIP, thus promoting its ubiquitin-dependent proteasomal proteolysis. As a result, accumulation of TXNIP in response to WWP1 inactivation inAML blasts reduces Trx activity and increases ROS production, henceinducing cellular oxidative stress. Increased ROS generation in WWP1-depleted cells culminates in DNA strand breaks and subsequent apoptosis. Coherently with TXNIP stabilization following WWP1 inactivation, we also observed an impairment of both glucose up-take and consumption. Hence, a contribution to the increased cell death observed in WWP1-depleted cells also possibly arises from the attenuation of glucose up-take and glycolytic flux resulting from TXNIP accumulation. Future studies are needed to establish whether TXNIP-dependent deregulation of redox homeostasis in WWP1-overexpressing blasts may affect the response of leukemic cells to chemotherapeutic drugs.

Melatonin Prevents Thioacetamide-Induced Gut Leakiness and Liver Fibrosis Through the Gut-Liver Axis via Modulating Sirt1-Related Deacetylation of Gut Junctional Complex and Hepatic Proteins.

Intestinal barrier dysfunction with high serum endotoxin is common in patients with liver fibrosis, but the mechanisms underlying liver fibrosis remain unclear. Melatonin is a well-recognized antioxidant and an anti-inflammatory agent that benefits multiple organs. However, the beneficial effects of melatonin on gut leakiness-associated liver fibrosis have not been systemically studied. Here, we investigated the protective mechanisms of melatonin against thioacetamide (TAA)-induced gut barrier dysfunction and hepatic fibrosis by focusing on posttranslational protein modifications through the gut-liver axis. Our results showed that gut leakiness markers, including decreased gut tight/adherens junction proteins (TJ/AJs) with increased intestinal deformation, apoptosis, and serum endotoxin, were observed early at 1 week after TAA exposure. Liver injury, apoptosis, and fibrosis were prominent at 2 and 4 weeks. Mechanistically, we found that gut TJ/AJs were hyper-acetylated, followed by ubiquitin-dependent proteolysis, leading to their degradation and gut leakiness. Gut dysbiosis, hepatic protein hyper-acetylation, and SIRT1 downregulation were also observed. Consistently, intestinal Sirt1 deficiency greatly enhanced protein hyper-acetylation, gut leakiness, endotoxemia, and liver fibrosis. Pretreatment with melatonin prevented or improved all these changes in both the gut and liver. Furthermore, melatonin blunted protein acetylation and injury in TAA-exposed T84 human intestinal and AML12 mouse liver cells. Overall, this study demonstrated novel mechanisms by which melatonin prevents gut leakiness and liver fibrosis through the gut-liver axis by attenuating the acetylation of intestinal and hepatic proteins. Thus, melatonin consumption can become a potentially safe supplement for liver fibrosis patients by preventing protein hyper-acetylation and gut leakiness.

Autophagy and ubiquitin-dependent proteolysis processes in left ventricular mass loss in pulmonary arterial hypertension

The goal of this study was to evaluate the intensity of autophagy and ubiquitin-dependent proteolysis processes occurring in myocardium of left ventricle (LV) in subsequent stages of pulmonary arterial hypertension (PAH) to determine mechanisms responsible for LV mass loss in a monocrotaline-induced PAH rat model. LV myocardium samples collected from 32 Wistar rats were analyzed in an early PAH group (n = 8), controls time-paired (n = 8), an end-stage PAH group (n = 8), and their controls (n = 8). Samples were subjected to histological analyses with immunofluorescence staining, autophagy assessment by western blotting, and evaluation of ubiquitin-dependent proteolysis in the LV by immunoprecipitation of ubiquitinated proteins. Echocardiographic, hemodynamic, and heart morphometric parameters were assessed regularly throughout the experiment. Considerable morphological and hemodynamic remodeling of the LV was observed over the course of PAH. The end-stage PAH was associated with significantly impaired LV systolic function and a decrease in LV mass. The LC3B-II expression in the LV was significantly higher in the end-stage PAH group compared to the early PAH group (p = 0.040). The measured LC3B-II/LC3B-I ratios in the end-stage PAH group were significantly elevated compared to the controls (p = 0.039). Immunofluorescence staining showed a significant increase in the abundance of LC3 puncta in the end-stage PAH group compared to the matched controls. There were no statistically significant differences in the levels of expression of all ubiquitinated proteins when comparing both PAH groups and matched controls. Autophagy may be considered as the mechanism behind the LV mass loss at the end stage of PAH.

Foodborne toxin aflatoxin B1 induced glomerular podocyte inflammation through proteolysis of RelA, downregulation of miR-9 and CXCR4/TXNIP/NLRP3 pathway

Aflatoxin B1 (AFB1) is a naturally-occurring mycotoxin and recognized as the most toxic foodborne toxin, particularly causing damages to kidney. Glomerular podocytes are terminally differentiated epithelial cells. AFB1 induces podocyte inflammation, proteinuria and renal dysfunction. Studying the mechanism of AFB1-induced podocyte inflammation and murine kidney dysfunction, we detected that AFB1 increased ubiquitin-dependent degradation of the transcription factor RelA through enhanced interaction of RelA with E3 ubiquitin ligase tripartite motif containing 7 (TRIM7) in mouse podocyte clone-5 (MPC-5) and mouse glomeruli. Reduction of RelA resulted in decreasing microRNA-9 (miR-9) and activating the chemokine receptor 4 (CXCR4), thioredoxin interacting protein (TXNIP), and NOD-like receptor pyrin domain-containing 3 (NLRP3) signaling axis (CXCR4/TXNIP/NLRP3 pathway), leading to podocyte inflammation. We also determined that downregulation of miR-9 led to CXCR4 expression and the downstream TXNIP/NLRP3 pathway activation. Overexpression of miR-9 or deletion of CXCR4 suppressed AFB1-induced CXCR4/TXNIP/NLRP3 pathway, resulting in alleviating podocyte inflammation and kidney dysfunction. Our findings indicated that ubiquitin-dependent proteolysis of RelA, downregulation of miR-9, and activation of CXCR4/TXNIP/NLRP3 pathway played an essential role in AFB1-induced glomerular podocyte inflammation. Our study revealed a novel mechanism, via RelA, for the control of AFB1’s nephrotoxicity, leading to an effective protection of food safety and public health.

Depressing Ubiquitin-Dependent Proteolysis in a Hibernator Whose Body Temperature Is Normally Variable: The Common Tenrec

In a canonical mammalian hibernator like a ground squirrel, the cold body temperatures (Tb) of the torpid squirrel depresses protein degradation at the level of the 26S proteasome. Ubiquitylation per se is less temperature sensitive as ubiquitylation rates of colder temperatures continue at 30% to 60% of the maximal rates of warmer temperatures. This leads to an accumulation of ubiquitin-conjugated proteins of 2-3 fold in torpid squirrels as compared to active squirrels. The common tenrec is a bizarre Afrotherian placental mammal with many plesiomorphic traits such as a cloaca, internal testes, indeterminate growth, and the smallest brain of all extant mammals with very little neocortex. Interestingly, these tenrecs may be fully active with a Tb of 12°C or hibernating at a Tb of 28°C. Moreover, oxygen consumption rates may vary 25 fold in active tenrecs. We found expected decreases of 26S proteolytic activity at low temperatures. Capacitance for proteolysis varied by state and there was no evidence that torpor per se would arrest protein degradation. Indeed, in sharp contrast to the squirrel, ubiquitylation rates were essentially unaffected by temperature and ranged at colder temperatures from 80% to 90% of the maximal ubiquitylation rates. In other words, the only in vitro evidence for the control of ubiquitin-dependent proteolysis that we found would only be available to very cold tenrecs independent of if they were active or torpid. However, ubiquitin-conjugate concentrations appear low but unaffected by state. All in all, we hypothesize a ‘Life in the Slow Lane’ model for tenrecs wherein an inability to effectively regulate proteolysis led to a modest rate of degradation that is not as affected by changes incurred by torpor state or changes in Tb. NSF and Department of Education (GAANN). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 6587: Discovery of molecular glue of FBXW7:MYC interaction using small molecule microarray

Abstract MYC deregulation is a key driver and feature of many human cancers. Yet, despite its importance in cancer progression, MYC is considered “undruggable” by small molecules due to its disordered conformation. Therefore, interfering with its interactions with protein partners to modulate MYC activity appears viable. FBXW7 is an E3 ligase, which serves as a constituent of the SCF (Skp1-Cul1-F box) ubiquitin ligase complex. It mediates the degradation of both c-Myc and Cyclin E through ubiquitin-dependent proteolysis. FBXW7 mutations are found in various human tumor types, establishing its role as a general human tumor suppressor. Specifically, FBXW7 down-regulation increases MYC level, and its expression correlates to lethal outcomes of breast epithelial cells, poor prognosis in glioblastoma, and T cell acute lymphoblastic leukemia remission. Therefore, our project aims to discover potent small molecule glues that would enhance the interaction between FBXW7 and MYC, leading to MYC degradation through proteolysis and eventually suppression of various cancers. Taking advantage of our high throughput screening technology, Small Molecule Microarrays (SMMs), we successfully identified 29 compounds that potently bind to both c-Myc and FBXW7 after screening our 65,000 ‘drug-like’ small molecule library. We then performed secondary assays using a mCherry-GFP MYC reporter cell line to identify our lead compound, KI-FBX-001, which decreases MYC’s expression:transcription ratio by over 50% compared to control. Additionally, this down-regulation can be rescued by protease inhibitor MG132, indicating that the effect is achieved via proteolysis. In vitro dose-response and time-course treatment revealed an optimal drugging concentration at 15uM in K562 cells, with inhibition reaching its peak at the 4-hour time point at over 60%. Using the Qiagen MYC reporter assay, the in vitro IC50 of KI-FBX-001 is found at 7.42uM. Beyond the protein level, the qPCR result also reveals a decrease in MYC mRNA level at over 70%. Surface Plasmon Resonance (SPR) is used to confirm direct binding between FBXW7 and KI-FBX-001, which showed promising binding with kd=0.17µM. Proximity Ligation Assay further confirms KI-FBX-001’s effect in enhancing FBXW7:MYC interaction in the cell. In summary, our findings proved that KI-FBX-001 is a promising molecular glue in the FBXW7:MYC interaction, and can effectively modulate the MYC level for future therapeutic purposes. Citation Format: Shenghao Guo, Morgan Stilgenbauer, Bocheng Wu, Yatin Mankan, Angela N. Koehler. Discovery of molecular glue of FBXW7:MYC interaction using small molecule microarray [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6587.

Lysine Methylation-Dependent Proteolysis by the Malignant Brain Tumor (MBT) Domain Proteins.

Lysine methylation is a major post-translational protein modification that occurs in both histones and non-histone proteins. Emerging studies show that the methylated lysine residues in non-histone proteins provide a proteolytic signal for ubiquitin-dependent proteolysis. The SET7 (SETD7) methyltransferase specifically transfers a methyl group from S-Adenosyl methionine to a specific lysine residue located in a methylation degron motif of a protein substrate to mark the methylated protein for ubiquitin-dependent proteolysis. LSD1 (Kdm1a) serves as a demethylase to dynamically remove the methyl group from the modified protein. The methylated lysine residue is specifically recognized by L3MBTL3, a methyl-lysine reader that contains the malignant brain tumor domain, to target the methylated proteins for proteolysis by the CRL4DCAF5 ubiquitin ligase complex. The methylated lysine residues are also recognized by PHF20L1 to protect the methylated proteins from proteolysis. The lysine methylation-mediated proteolysis regulates embryonic development, maintains pluripotency and self-renewal of embryonic stem cells and other stem cells such as neural stem cells and hematopoietic stem cells, and controls other biological processes. Dysregulation of the lysine methylation-dependent proteolysis is associated with various diseases, including cancers. Characterization of lysine methylation should reveal novel insights into how development and related diseases are regulated.

A Natural Small Molecule Mitigates Kidney Fibrosis by Targeting Cdc42-mediated GSK-3β/β-catenin Signaling.

Kidney fibrosis is a common fate of chronic kidney diseases (CKDs), eventually leading to renal dysfunction. Yet, no effective treatment for this pathological process has been achieved. During the bioassay-guided chemical investigation of the medicinal plant Wikstroemia chamaedaphne, a daphne diterpenoid, daphnepedunin A (DA), is characterized as a promising anti-renal fibrotic lead. DA shows significant anti-kidney fibrosis effects in cultured renal fibroblasts and unilateral ureteral obstructed mice, being more potent than the clinical trial drug pirfenidone. Leveraging the thermal proteome profiling strategy, cell division cycle 42 (Cdc42) is identified as the direct target of DA. Mechanistically, DA targets to reduce Cdc42 activity and down-regulates its downstream phospho-protein kinase Cζ(p-PKCζ)/phospho-glycogen synthase kinase-3β (p-GSK-3β), thereby promoting β-catenin Ser33/37/Thr41 phosphorylation and ubiquitin-dependent proteolysis to block classical pro-fibrotic β-catenin signaling. These findings suggest that Cdc42 is a promising therapeutic target for kidney fibrosis, and highlight DA as a potent Cdc42 inhibitor for combating CKDs.

Lysine deserts and cullin-RING ligase receptors: Navigating untrodden paths in proteostasis

The ubiquitin-proteasome system (UPS) governs the degradation of proteins by ubiquitinating their lysine residues. Our study focuses on lysine deserts - regions in proteins conspicuously low in lysine residues- in averting ubiquitin-dependent proteolysis. We spotlight the prevalence of lysine deserts among bacteria leveraging the pupylation-dependent proteasomal degradation, and in the UPS of eukaryotes. To further scrutinize this phenomenon, we focused on human receptors VHL and SOCS1 to ascertain if lysine deserts could limit their ubiquitination within the cullin-RING ligase (CRL) complex. Our data indicate that the wild-type and lysine-free variants of VHL and SOCS1 maintain consistent turnover rates, unaltered by CRL-mediated ubiquitination, hinting at a protective mechanism facilitated by lysine deserts. Nonetheless, we noted their ubiquitination at non-lysine sites, alluding to alternative regulation by the UPS. Our research underscores the role of lysine deserts in limiting CRL-mediated ubiquitin tagging while promoting non-lysine ubiquitination, thereby advancing our understanding of proteostasis.

Integrated metabolomic and transcriptomic responses to heat stress in a high-altitude fish, Triplophysa siluroides

Species in Triplophysa display strong adaptability to the extreme environment of the plateau, thus offering an ideal model to study the molecular mechanism of fish adaptation to environmental stress. In the present study, we conducted integrated analysis of the transcriptome and metabolism of liver tissue in Triplophysa siluroides under heat stress (28 °C) and control (10 °C) conditions to identify heat stress-induced genes, metabolites and pathways. RNA-Seq identified 2373 differentially expressed genes, which consisted of 1360 upregulated genes and 1013 downregulated genes, in the heat stress group vs. the control group. Genes in the heat shock protein (Hsp) family, including Hsp40, Hsp70, Hsp90 and other Hsps, were strongly upregulated by heat stress. Pathway enrichment analysis revealed that the PI3K/AKT/mTOR and protein processing in the endoplasmic reticulum (ER) pathways were significantly affected by heat stress. Metabolism sequencing identified a total of 155 differentially abundant metabolites, including 118 significantly upregulated metabolites and 37 downregulated metabolites. Combined analysis of the transcriptome and metabolism results showed that ubiquitin-dependent proteolysis and purine metabolism pathways were enhanced in response to acute heat stress to protect cells from damage under stress conditions. The results of this study may contribute to our understanding of the underlying molecular mechanism of the heat stress response in cold-water fish.

DLBCL-associated NOTCH2 mutations escape ubiquitin-dependent degradation and promote chemoresistance

AbstractDiffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Up to 40% of patients with DLBCL display refractory disease or relapse after standard chemotherapy treatment (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone [R-CHOP]), leading to significant morbidity and mortality. The molecular mechanisms of chemoresistance in DLBCL remain incompletely understood. Using a cullin–really interesting new gene (RING) ligase-based CRISPR-Cas9 library, we identify that inactivation of the E3 ubiquitin ligase KLHL6 promotes DLBCL chemoresistance. Furthermore, proteomic approaches helped identify KLHL6 as a novel master regulator of plasma membrane–associated NOTCH2 via proteasome-dependent degradation. In CHOP-resistant DLBCL tumors, mutations of NOTCH2 result in a protein that escapes the mechanism of ubiquitin-dependent proteolysis, leading to protein stabilization and activation of the oncogenic RAS signaling pathway. Targeting CHOP-resistant DLBCL tumors with the phase 3 clinical trial molecules nirogacestat, a selective γ-secretase inhibitor, and ipatasertib, a pan-AKT inhibitor, synergistically promotes DLBCL destruction. These findings establish the rationale for therapeutic strategies aimed at targeting the oncogenic pathway activated in KLHL6- or NOTCH2-mutated DLBCL.

Protein arginine methyltransferase 5-mediated arginine methylation stabilizes Kruppel-like factor 4 to accelerate neointimal formation.

Accumulating evidence supports the indispensable role of protein arginine methyltransferase 5 (PRMT5) in the pathological progression of several human cancers. As an important enzyme-regulating protein methylation, how PRMT5 participates in vascular remodelling remains unknown. The aim of this study was to investigate the role and underlying mechanism of PRMT5 in neointimal formation and to evaluate its potential as an effective therapeutic target for the condition. Aberrant PRMT5 overexpression was positively correlated with clinical carotid arterial stenosis. Vascular smooth muscle cell (SMC)-specific PRMT5 knockout inhibited intimal hyperplasia with an enhanced expression of contractile markers in mice. Conversely, PRMT5 overexpression inhibited SMC contractile markers and promoted intimal hyperplasia. Furthermore, we showed that PRMT5 promoted SMC phenotypic switching by stabilizing Kruppel-like factor 4 (KLF4). Mechanistically, PRMT5-mediated KLF4 methylation inhibited ubiquitin-dependent proteolysis of KLF4, leading to a disruption of myocardin (MYOCD)-serum response factor (SRF) interaction and MYOCD-SRF-mediated the transcription of SMC contractile markers. Our data demonstrated that PRMT5 critically mediated vascular remodelling by promoting KLF4-mediated SMC phenotypic conversion and consequently the progression of intimal hyperplasia. Therefore, PRMT5 may represent a potential therapeutic target for intimal hyperplasia-associated vascular diseases.

Abstract 6107: Novel roles for AMBRA1 in regulating DNA double-strand breaks

Abstract AMBRA1 (Activating Molecule in Beclin-1-Regulated Autophagy) is a tumor suppressor protein whose expression is downregulated in several human malignancies. AMBRA1 was recently found to function as a substrate receptor for the cullin-4-based ubiquitin ligase (CRL4AMBRA1) that promotes the ubiquitin-dependent proteolysis of D-type cyclins (cyclin D1, D2 and D3); this serves to guard against premature S-phase entry and replication stress, providing a potential mechanism for its tumor suppressive activity. Cyclin D1 protein, which is stabilized, along with cyclin D2 and D3, in AMBRA1-deficient cells, has been shown to promote homologous recombination (HR) repair of DNA double-strand breaks (DSBs), suggesting that AMBRA1 may be additionally involved in the repair of DSBs. Using our recently published CRISPR-Cas9-based dual fluorescent reporter assay system, we show that while the loss of AMBRA1 increased D-type cyclins and Rb hyperphosphorylation as expected, it did, surprisingly, inhibit HR-mediated repair and stimulated error-free non-homologous end-joining (NHEJ), and that both of these new activities are independent of cyclin D1 or RB1. High-throughput sequencing of DSB repair junctions confirm these results and shows significant depletion of indels with reduction in microhomology-mediated end-joining in AMBRA1-deficient cells, suggesting that AMBRA1 plays a role in promoting resection at DSBs. In support of this hypothesis, we found that the loss of AMBRA1 reduces RAD51 recruitment to DSBs in cells exposed to ionizing radiation. Consistent with its role in promoting HR, we found that AMBRA1-deficient cells are significantly more sensitive to PARP inhibitors compared to AMBRA1-proficient cells. In summary, we show that AMBRA1 is a novel regulator of DSBs, and that its downregulation in cancer cells and tumors render cancer cells susceptible to inhibition by PARP inhibitors. Citation Format: Yuning Jiang, Tarek Abbas. Novel roles for AMBRA1 in regulating DNA double-strand breaks. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6107.

Structure-based drug design of potential inhibitors of FBXW8, the substrate recognition component of Cullin-RING ligase 7.

FBXW8 plays an irreplaceable role in the substrate recognition of ubiquitin-dependent proteolysis, which further regulates cell cycle progression and signal transduction. However, the abnormal expression of FBXW8 triggers malignancy, inflammation, and autophagy irregulation. FBXW8 is considered as an effective therapeutic target for Cullin-RING ligase 7 (CRL7)-related cancers. Still, the lack of selective inhibitors hinders further therapeutic development and limits the exploration of its biological mechanism. This study constructed an integrated protocol that combines pharmacophore modeling, structure-based virtual screening, and Molecular Dynamic Simulation. It was then used as a screening query to identify hit compounds targeted at the substrate recognition site of FBXW8 from a large-scale compound database including 120 million compounds. Then, ten lead compounds were retrieved by using molecular docking analysis and ADMET prediction. Finally, MD simulations were performed to validate the binding stability of selected drug candidates. The result indicated that three newly obtained compounds, namely ZINC96179876, ZINC72174069, and ZINC97730272, might be potent FBXW8 inhibitors against CRL7-related cancers such as endometrial cancer.

Thermosensation in Caenorhabditis elegans is linked to ubiquitin-dependent protein turnover via insulin and calcineurin signalling

Organismal physiology and survival are influenced by environmental conditions and linked to protein quality control. Proteome integrity is achieved by maintaining an intricate balance between protein folding and degradation. In Caenorhabditis elegans, acute heat stress determines cell non-autonomous regulation of chaperone levels. However, how the perception of environmental changes, including physiological temperature, affects protein degradation remains largely unexplored. Here, we show that loss-of-function of dyf-1 in Caenorhabditis elegans associated with dysfunctional sensory neurons leads to defects in both temperature perception and thermal adaptation of the ubiquitin/proteasome system centered on thermosensory AFD neurons. Impaired perception of moderate temperature changes worsens ubiquitin-dependent proteolysis in intestinal cells. Brain-gut communication regulating protein turnover is mediated by upregulation of the insulin-like peptide INS-5 and inhibition of the calcineurin-regulated forkhead-box transcription factor DAF-16/FOXO. Our data indicate that perception of ambient temperature and its neuronal integration is important for the control of proteome integrity in complex organisms.

Discovery of E3 Ligase Ligands for Target Protein Degradation.

Target protein degradation has emerged as a promising strategy for the discovery of novel therapeutics during the last decade. Proteolysis-targeting chimera (PROTAC) harnesses a cellular ubiquitin-dependent proteolysis system for the efficient degradation of a protein of interest. PROTAC consists of a target protein ligand and an E3 ligase ligand so that it enables the target protein degradation owing to the induced proximity with ubiquitin ligases. Although a great number of PROTACs has been developed so far using previously reported ligands of proteins for their degradation, E3 ligase ligands have been mostly limited to either CRBN or VHL ligands. Those PROTACs showed their limitation due to the cell type specific expression of E3 ligases and recently reported resistance toward PROTACs with CRBN ligands or VHL ligands. To overcome these hurdles, the discovery of various E3 ligase ligands has been spotlighted to improve the current PROTAC technology. This review focuses on currently reported E3 ligase ligands and their application in the development of PROTACs.

The Role of the APC/C and Its Coactivators Cdh1 and Cdc20 in Cancer Development and Therapy.

To sustain genomic stability by correct DNA replication and mitosis, cell cycle progression is tightly controlled by the cyclic activity of cyclin-dependent kinases, their binding to cyclins in the respective phase and the regulation of cyclin levels by ubiquitin-dependent proteolysis. The spindle assembly checkpoint plays an important role at the metaphase-anaphase transition to ensure a correct separation of sister chromatids before cytokinesis and to initiate mitotic exit, as an incorrect chromosome distribution may lead to genetically unstable cells and tumorigenesis. The ubiquitin ligase anaphase-promoting complex or cyclosome (APC/C) is essential for these processes by mediating the proteasomal destruction of cyclins and other important cell cycle regulators. To this end, it interacts with the two regulatory subunits Cdh1 and Cdc20. Both play a role in tumorigenesis with Cdh1 being a tumor suppressor and Cdc20 an oncogene. In this review, we summarize the current knowledge about the APC/C-regulators Cdh1 and Cdc20 in tumorigenesis and potential targeted therapeutic approaches.

Transcriptome, metabolome and suppressor analysis reveal an essential role for the ubiquitin-proteasome system in seedling chloroplast development

BackgroundMany regulatory circuits in plants contain steps of targeted proteolysis, with the ubiquitin proteasome system (UPS) as the mediator of these proteolytic events. In order to decrease ubiquitin-dependent proteolysis, we inducibly expressed a ubiquitin variant with Arg at position 48 instead of Lys (ubK48R). This variant acts as an inhibitor of proteolysis via the UPS, and allowed us to uncover processes that are particularly sensitive to UPS perturbation.ResultsExpression of ubK48R during germination leads to seedling death. We analyzed the seedling transcriptome, proteome and metabolome 24 h post ubK48R induction and confirmed defects in chloroplast development. We found that mutations in single genes can suppress seedling lethality, indicating that a single process in seedlings is critically sensitive to decreased performance of the UPS. Suppressor mutations in phototropin 2 (PHOT2) suggest that a contribution of PHOT2 to chloroplast protection is compromised by proteolysis inhibition.ConclusionsOverall, the results reveal protein turnover as an integral part of a signal transduction chain that protects chloroplasts during development.

The 20S as a stand-alone proteasome in cells can degrade the ubiquitin tag

The proteasome, the primary protease for ubiquitin-dependent proteolysis in eukaryotes, is usually found as a mixture of 30S, 26S, and 20S complexes. These complexes have common catalytic sites, which makes it challenging to determine their distinctive roles in intracellular proteolysis. Here, we chemically synthesize a panel of homogenous ubiquitinated proteins, and use them to compare 20S and 26S proteasomes with respect to substrate selection and peptide-product generation. We show that 20S proteasomes can degrade the ubiquitin tag along with the conjugated substrate. Ubiquitin remnants on branched peptide products identified by LC-MS/MS, and flexibility in the 20S gate observed by cryo-EM, reflect the ability of the 20S proteasome to proteolyze an isopeptide-linked ubiquitin-conjugate. Peptidomics identifies proteasome-trapped ubiquitin-derived peptides and peptides of potential 20S substrates in Hi20S cells, hypoxic cells, and human failing-heart. Moreover, elevated levels of 20S proteasomes appear to contribute to cell survival under stress associated with damaged proteins.

Inhibition of the ubiquitin-proteasome system by an NQO1-activatable compound

Malignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes. In vivo efficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.