Ubiquitin-activating Enzyme E1 Research Articles

Abstract LB427: UBA1 blockade induces homologous recombination deficiency and is synthetic lethal with PARP inhibition

Abstract Therapeutic strategies targeting DNA damage have revolutionized cancer treatment, yet their efficacy often comes at the cost of substantial adverse effects. The emergence of poly-ADP-ribose polymerase (PARP) inhibition as a targeted approach to disrupt DNA repair in tumors deficient in homologous recombination (HR) has shown promise. However, the limited response of most cancers to PARP inhibitors has posed a significant challenge. This study employs a genome-wide CRISPR knockout functional genomics screen, revealing that depletion of ubiquitin-activating enzyme E1 (UBA1) enhances sensitivity to PARP inhibition in HR-proficient ovarian cancer cells. Validation across different ovarian and breast cancer cell lineages, utilizing cell line and organoid models, establishes that pharmacological inhibition of UBA1, a pivotal player in ubiquitination, sensitizes cells to PARP inhibition and other DNA damaging therapies. In vivo experiments combining PARP and UBA1 inhibition demonstrate not only tolerability but also significant extension of survival in patient-derived xenografts compared to individual therapies. Mechanistic studies uncover that UBA1 inhibition not only impedes HR repair, sensitizing cells to PARP inhibition, but also induces widespread increases in PARylation, subsequently targeted by PARP inhibition. These findings underscore the multifaceted pathways through which UBA1 inhibition potentiates the efficacy of PARP inhibition. The demonstrated tolerability and substantial therapeutic gains establish the combined approach as a promising strategy to maximize the benefits of PARP inhibition in cancer therapy. Citation Format: Sharad Awasthi, Daniel J. McGrail, S. Stephen Yi, Nidhi Sahni. UBA1 blockade induces homologous recombination deficiency and is synthetic lethal with PARP inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB427.

F-box protein FBXB-65 regulates anterograde transport of the kinesin-3 motor UNC-104 through a PTM near its cargo-binding PH domain.

Axonal transport in neurons is essential for cargo movement between the cell body and synapses. Caenorhabditis elegans UNC-104 and its homolog KIF1A are kinesin-3 motors that anterogradely transport precursors of synaptic vesicles (pre-SVs) and are degraded at synapses. However, in C. elegans, touch neuron-specific knockdown of the E1 ubiquitin-activating enzyme, uba-1, leads to UNC-104 accumulation at neuronal ends and synapses. Here, we performed an RNAi screen and identified that depletion of fbxb-65, which encodes an F-box protein, leads to UNC-104 accumulation at neuronal distal ends, and alters UNC-104 net anterograde movement and levels of UNC-104 on cargo without changing synaptic UNC-104 levels. Split fluorescence reconstitution showed that UNC-104 and FBXB-65 interact throughout the neuron. Our theoretical model suggests that UNC-104 might exhibit cooperative cargo binding that is regulated by FBXB-65. FBXB-65 regulates an unidentified post-translational modification (PTM) of UNC-104 in a region beside the cargo-binding PH domain. Both fbxb-65 and UNC-104, independently of FBXB-65, regulate axonal pre-SV distribution, transport of pre-SVs at branch points and organismal lifespan. FBXB-65 regulates a PTM of UNC-104 and the number of motors on the cargo surface, which can fine-tune cargo transport to the synapse.

Reconstitution of the in vitro STUB1-mediated ubiquitination reaction system for α-1 antitrypsin mutant Z protein

The α-1 antitrypsin Z-mutant protein (ATZ) is the primary cause of α-1 antitrypsin deficiency (AATD). Studying the ubiquitination modification and degradation of ATZ protein is importance for developing treatments for AATD. STUB1 is an important E3 ubiquitin ligase that regulates ubiquitination modification of various proteins. However, whether STUB1 in involved in the ubiquitination modification of ATZ has not been fully elucidated. In this study, the ATZ and STUB1 coding genes were first cloned into the pET28a plasmid, constructing 2 protein expression plasmids. The recombinant plasmids were then transferred into the Escherichia coli for expression. With the optimization of induction temperature and IPTG dosage, the recombinant proteins were successfully expressed. The target proteins were then efficiently purified from cell lysates using metal-chelating affinity chromatography, and the accuracy of the amino acid sequence was verified through protein mass spectrometry analysis. Using the purified ATZ and STUB1, we established an in vitro ubiquitination reaction system. Experimental results showed that, in the presence of ATP, E1 ubiquitin-activating enzyme, and E2 ubiquitin-conjugating enzyme, STUB1 catalyzed the ubiquitination modification of ATZ. This study provides a method for obtaining the ATZ protein in vitro, elucidates the mechanism of STUB1 mediating ATZ ubiquitination, thereby advancing our understanding of the intracellular degradation mechanism of the α-1 antitrypsin Z-mutant.

A Survey on the Expression of the Ubiquitin Proteasome System Components HECT- and RBR-E3 Ubiquitin Ligases and E2 Ubiquitin-Conjugating and E1 Ubiquitin-Activating Enzymes during Human Brain Development.

Human brain development involves a tightly regulated sequence of events that starts shortly after conception and continues up to adolescence. Before birth, neurogenesis occurs, implying an extensive differentiation process, sustained by changes in the gene expression profile alongside proteome remodeling, regulated by the ubiquitin proteasome system (UPS) and autophagy. The latter processes rely on the selective tagging with ubiquitin of the proteins that must be disposed of. E3 ubiquitin ligases accomplish the selective recognition of the target proteins. At the late stage of neurogenesis, the brain starts to take shape, and neurons migrate to their designated locations. After birth, neuronal myelination occurs, and, in parallel, neurons form connections among each other throughout the synaptogenesis process. Due to the malfunctioning of UPS components, aberrant brain development at the very early stages leads to neurodevelopmental disorders. Through deep data mining and analysis and by taking advantage of machine learning-based models, we mapped the transcriptomic profile of the genes encoding HECT- and ring-between-ring (RBR)-E3 ubiquitin ligases as well as E2 ubiquitin-conjugating and E1 ubiquitin-activating enzymes during human brain development, from early post-conception to adulthood. The inquiry outcomes unveiled some implications for neurodevelopment-related disorders.

Disease-associated polyalanine expansion mutations impair UBA6-dependent ubiquitination

Expansion mutations in polyalanine stretches are associated with a growing number of diseases sharing a high degree of genotypic and phenotypic commonality. These similarities prompted us to query the normal function of physiological polyalanine stretches and to investigate whether a common molecular mechanism is involved in these diseases. Here, we show that UBA6, an E1 ubiquitin-activating enzyme, recognizes a polyalanine stretch within its cognate E2 ubiquitin-conjugating enzyme USE1. Aberrations in this polyalanine stretch reduce ubiquitin transfer to USE1 and, subsequently, polyubiquitination and degradation of its target, the ubiquitin ligase E6AP. Furthermore, we identify competition for the UBA6-USE1 interaction by various proteins with polyalanine expansion mutations in the disease state. The deleterious interactions of expanded polyalanine tract proteins with UBA6 in mouse primary neurons alter the levels and ubiquitination-dependent degradation of E6AP, which in turn affects the levels of the synaptic protein Arc. These effects are also observed in induced pluripotent stem cell-derived autonomic neurons from patients with polyalanine expansion mutations, where UBA6 overexpression increases neuronal resilience to cell death. Our results suggest a shared mechanism for such mutations that may contribute to the congenital malformations seen in polyalanine tract diseases.

Clinical and genetic features of MDS associated with VEXAS syndrome

VEXAS syndrome is a new disease entity characterized by the presence of cytoplasmic vacuoles in blood cells, X-linked autoinflammatory symptoms, and somatic variants in UBA1, which encodes an E1 ubiquitin-activating enzyme. Around 30-50% of VEXAS syndrome patients have concurrent MDS. We and others have recently analyzed clinical and genetic features of MDS associated with VEXAS syndrome and found that most of these cases are categorized in the low-risk subgroup with low bone marrow blast percentages. MDS associated with VEXAS syndrome tended to involve a smaller number of genes and lower-risk genetic alterations than classical MDS. In addition, anemia in MDS associated with VEXAS syndrome with active inflammation before treatment tended to respond well to steroids. In this review, we will present our recent findings together with others, focusing on the new disease entity and pathophysiology of VEXAS syndrome and clinical/genetic features of associated MDS.

Role of the Mitochondrial E3 Ubiquitin Ligases as Possible Therapeutic Targets in Cancer Therapy.

Ubiquitination is a post-translational modification that targets specific proteins on their lysine residues. Depending on the type of ubiquitination, this modification ultimately regulates the stability or degradation of the targeted proteins. Ubiquitination is mediated by three different classes of enzymes: the E1 ubiquitin-activating enzymes, the E2 ubiquitin-conjugating enzymes and, most importantly, the E3 ubiquitin ligases. E3 ligases are responsible for the final step of the ubiquitin cascade, interacting directly with the target proteins. E3 ligases can also be involved in DNA repair, cell cycle regulation and response to stress; alteration in their levels can be involved in oncogenic transformation and cancer progression. Of all the six hundred E3 ligases of the human genome, only three of them are specific to the mitochondrion: MARCH5, RNF185 and MUL1. Their alterations (that reflect on the alteration of the mitochondria functions) can be related to cancer progression, as underlined by the increasing research performed in recent years on these three mitochondrial enzymes. This review will focus on the function and mechanisms of the mitochondrial E3 ubiquitin ligases, as well as their important targets, in cancer development and progression, also highlighting their potential use for cancer therapy.

The ubiquitin-proteasome pathway inhibitor TAK-243 has major effects on calcium handling in mammalian cells

The ubiquitin-proteasome pathway (UPP) is a major route for protein degradation and a key regulatory mechanism in mammalian cells. UPP inhibitors, including TAK-243, a first-in-class inhibitor of the E1 ubiquitin-activating enzyme, are currently being used and tested for treatment of a range of diseases, particularly cancer. Here, we reveal that TAK-243 has major effects on Ca2+ handling in a range of cultured mammalian cells (αT3, HeLa and SH-SY5Y). Effects were seen on agonist-induced Ca2+ mobilization, basal cytosolic Ca2+ levels, Ca2+ leak from the endoplasmic reticulum (ER), store-operated Ca2+ entry and mitochondrial Ca2+ uptake. These effects correlated with induction of ER stress, as measured by PERK activation / eIF2α phosphorylation, and most seemed to be underpinned by enhanced Ca2+ leak from the ER. Overall, these data indicate that TAK-243 reprograms the Ca2+-handling properties of mammalian cells and that these effects should be considered when UPP inhibitors are employed as therapeutic agents.

Total Synthesis of the 2,5-Disubstituted γ-Pyrone E1 UAE Inhibitor Himeic Acid A.

The first total synthesis of the E1 ubiquitin-activating enzyme inhibitor, himeic acid A, is reported. A McCombie reaction was used to form the core γ-pyrone via a 6π-electrocyclization. A dioxenone ring-opening/acyl ketene trapping reaction with a primary amide provided the unusual unsymmetrical imide functionality. Other key steps include the use of an Evans auxiliary alkylation (d.r. ≥ 95:5) to install the (S)-2-methyl succinic acid fragment and a cross-metathesis to install the unsaturated side-chain.

The Ubiquitin-Proteasome System Facilitates Membrane Fusion and Uncoating during Coronavirus Entry.

Although the involvement of the ubiquitin-proteasome system (UPS) in several coronavirus-productive infections has been reported, whether the UPS is required for infectious bronchitis virus (IBV) and porcine epidemic diarrhea virus (PEDV) infections is unclear. In this study, the role of UPS in the IBV and PEDV life cycles was investigated. When the UPS was suppressed by pharmacological inhibition at the early infection stage, IBV and PEDV infectivity were severely impaired. Further study showed that inhibition of UPS did not change the internalization of virus particles; however, by using R18 and DiOC-labeled virus particles, we found that inhibition of UPS prevented the IBV and PEDV membrane fusion with late endosomes or lysosomes. In addition, proteasome inhibitors blocked the degradation of the incoming viral protein N, suggesting the uncoating process and genomic RNA release were suppressed. Subsequently, the initial translation of genomic RNA was blocked. Thus, UPS may target the virus-cellular membrane fusion to facilitate the release of incoming viruses from late endosomes or lysosomes, subsequently blocking the following virus uncoating, initial translation, and replication events. Similar to the observation of proteasome inhibitors, ubiquitin-activating enzyme E1 inhibitor PYR-41 also impaired the entry of IBV, enhanced the accumulation of ubiquitinated proteins, and depleted mono-ubiquitin. In all, this study reveals an important role of UPS in coronavirus entry by preventing membrane fusion and identifies UPS as a potential target for developing antiviral therapies for coronavirus.

PYR-41, an inhibitor of ubiquitin-activating enzyme E1, attenuates 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin lesions in mice.

PYR-41 is an irreversible and cell permeable inhibitor of ubiquitin-activating enzyme E1, and has been reported to inhibit the degradation of IκB protein. Previous studies have shown that PYR-41 has effects on anti-inflammatory, but whether it has therapeutic effects on allergic dermatitis is unclear. The aim of this research was to explore the therapeutic effects of PYR-41 on atopic dermatitis. The effects of PYR-41 on the activation of NF-κB signaling pathway and the expression of inflammatory genes in HaCat cells were tested by western blot and qPCR. A mouse model was built, and the AD-like skin lesions were induced by 2,4-dinitrochlorobenzene (DNCB). Then, the treatment effects of PYR-41 were examined by skin severity score, ear swelling, ELISA, and qPCR. The results showed that PYR-41 can significantly reduce the K63-linked ubiquitination level of nuclear factor-κB essential modulator (NEMO) and tumor necrosis factor receptor associated factor 6 (TRAF6), inhibit the proteasomal degradation of IκBα, thereby activate TNF-α-induced NF-κB signaling pathway in HaCat cells. In addition, DNCB-treated mice have significant reduction in symptoms after treated by PYR-41, including reduced ear thickening and reduced skin damage. Serum tests showed that PYR-41 significantly reduced the expression of IgE, IFN-γ, and TNF-α. In conclusion, the current results suggest that PYR-41 has potential to reduce the symptoms of atopic dermatitis.

Auranofin targets UBA1 and enhances UBA1 activity by facilitating ubiquitin trans-thioesterification to E2 ubiquitin-conjugating enzymes

UBA1 is the primary E1 ubiquitin-activating enzyme responsible for generation of activated ubiquitin required for ubiquitination, a process that regulates stability and function of numerous proteins. Decreased or insufficient ubiquitination can cause or drive aging and many diseases. Therefore, a small-molecule enhancing UBA1 activity could have broad therapeutic potential. Here we report that auranofin, a drug approved for the treatment of rheumatoid arthritis, is a potent UBA1 activity enhancer. Auranofin binds to the UBA1’s ubiquitin fold domain and conjugates to Cys1039 residue. The binding enhances UBA1 interactions with at least 20 different E2 ubiquitin-conjugating enzymes, facilitating ubiquitin charging to E2 and increasing the activities of seven representative E3s in vitro. Auranofin promotes ubiquitination and degradation of misfolded ER proteins during ER-associated degradation in cells at low nanomolar concentrations. It also facilitates outer mitochondrial membrane-associated degradation. These findings suggest that auranofin can serve as a much-needed tool for UBA1 research and therapeutic exploration.

E3 ubiquitin ligase components in GtoPdb v.2023.2

Ubiquitination (a.k.a. ubiquitylation) is a protein post-translational modification that typically requires the sequential action of three enzymes: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes), and E3 (ubiquitin ligases) [30]. Ubiquitination of proteins can target them for proteasomal degradation, or modulate cellular processes including cell cycle progression, transcriptional regulation, DNA repair and signal transduction. E3 ubiquitin ligases, of which there are >600 in humans, are a family of highly heterogeneous proteins and protein complexes that recruit ubiquitin-loaded E2 enzymes to mediate transfer of the ubiquitin molecule from the E2 to protein substrates. Target substrate specificity is determined by a substrate recognition subunit within the E3 complex. E3 ligases are being exploited as pharmacological targets to facilitate targeted protein degradation (TPD), as an alternative to small molecule inhibitors [3], through the development of proteolysis targeting chimeras (PROTACs) and molecular glues.

AXR1 modulates trichome morphogenesis through mediating ROP2 stability in Arabidopsis.

Cell differentiation and morphogenesis are crucial for the establishment of diverse cell types and organs in multicellular organisms. Trichome cells offer an excellent paradigm for dissecting the regulatory mechanisms of plant cell differentiation and morphogenesis due to their unique growth characteristics. Here, we report the isolation of an Arabidopsis mutant, aberrantly branched trichome 3-1 (abt3-1), with a reduced trichome branching phenotype. Positional cloning and molecular complementation experiments confirmed that abt3-1 is a new mutant allele of Auxin resistant 1 (AXR1), which encodes the N-terminal half of ubiquitin-activating enzyme E1 and functions in auxin signaling pathway. Meanwhile, we found that transgenic plants expressing constitutively active version of ROP2 (CA-ROP2) caused a reduction of trichome branches, resembling that of abt3-1. ROP2 is a member of Rho GTPase of plants (ROP) family, serving as versatile signaling switches involved in a range of cellular and developmental processes. Our genetic and biochemical analyses showed AXR1 genetically interacted with ROP2 and mediated ROP2 protein stability. The loss of AXR1 aggravated the trichome defects of CA-ROP2 and induced the accumulation of steady-state ROP2. Consistently, elevated AXR1 expression levels suppressed ROP2 expression and partially rescued trichome branching defects in CA-ROP2 plants. Together, our results presented a new mutant allele of AXR1, uncovered the effects of AXR1 and ROP2 during trichome development, and revealed a pathway of ROP2-mediated regulation of plant cell morphogenesis in Arabidopsis.

Resonance assignments of the PUB domain of the RNF31 protein.

E3 ubiquitin protein ligase RNF31 is present in human proteins and is involved in linear ubiquitin chain assembly complex (LUBAC) activity and cell growth. RNF31 is involved in ubiquitination, which is the post-translational modification of proteins. Ubiquitin molecules connect with amino acid residues of target proteins under the action of ubiquitin-activating enzyme E1, ubiquitin binding enzyme E2 and ubiquitin ligase E3, so as to achieve certain physiological functions. The abnormal expression of ubiquitination promotes the formation of cancer. In studies of breast cancer, RNF31 mRNA levels were found to be higher in cancer cells than in other tissues. The PUB domain of RNF31 is the binding site of the ubiquitin thioesterase otulin. Here, we report the backbone and side-chain resonance assignments of the PUB domain of RNF31 and study the backbone relaxation of the domain. These studies will contribute to further understanding of the structural and functional relationship of RNF31 protein, which may also be a target for drug research.

The role of E3 ubiquitin ligases and deubiquitinases in bladder cancer development and immunotherapy.

Bladder cancer is one of the common malignant urothelial tumors. Post-translational modification (PTMs), including ubiquitination, acetylation, methylation, and phosphorylation, have been revealed to participate in bladder cancer initiation and progression. Ubiquitination is the common PTM, which is conducted by E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin-protein ligase. E3 ubiquitin ligases play a key role in bladder oncogenesis and progression and drug resistance in bladder cancer. Therefore, in this review, we summarize current knowledge regarding the functions of E3 ubiquitin ligases in bladder cancer development. Moreover, we provide the evidence of E3 ubiquitin ligases in regulation of immunotherapy in bladder cancer. Furthermore, we mention the multiple compounds that target E3 ubiquitin ligases to improve the therapy efficacy of bladder cancer. We hope our review can stimulate researchers and clinicians to investigate whether and how targeting E3 ubiquitin ligases acts a novel strategy for bladder cancer therapy.

Abstract LB043: Pharmacological inhibition of ubiquitination sensitizes to DNA damaging agents by preventing homologous recombination repair

Abstract Therapies inducing DNA damage have been the backbone of cancer treatment for decades, but they often cause significant adverse effects reducing quality of life. Recently, inhibition of poly-ADP-ribose polymerase (PARP) has been shown to target tumor cells with deficient DNA homologous recombination (HR) double-strand break repair by converting single-strand DNA breaks into toxic double-strand breaks that cannot be resolved. This approach has been advantageous as healthy cells with functional HR repair are less effective, resulting in a more tolerable treatment. However, most cancers will not respond to PARP inhibition. Here, using a genome-wide CRISPR knockout functional genomics screen we identified that depletion of ubiquitination enzymes induced sensitivity to PARP inhibition in HR proficient ovarian cancer cells, as well as KRAS-mutant colorectal cancer cells which are generally resistant to PARP and other DNA damaging agents. Validation studies across multiple cancer lineages using cell line and organoid models confirmed that either silencing or pharmacological inhibition of ubiquitin-activating enzyme E1 (UBA1), which is required for ubiquitination, sensitized to PARP inhibition and other DNA damaging therapies. In vivo testing of combination PARP and UBA1 inhibition was well tolerated, and significantly extended survival of patient-derived xenografts compared to either therapy alone. Mechanistically, we found that UBA1 inhibition not only blocked HR repair to sensitize to PARP inhibition, but it also induced global increases in PARylation, which was subsequently inhibited by PARP inhibition. Taken together, these results demonstrate that inhibition of UBA1 sensitizes to PARP via multiple pathways, and the combination is well tolerated making it an ideal strategy to expand the therapeutic benefit of PARP inhibition. Citation Format: Sharad Awasthi, S. Stephen Yi, Daniel J. McGrail, Nidhi Sahni. Pharmacological inhibition of ubiquitination sensitizes to DNA damaging agents by preventing homologous recombination repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB043.

Biosynthesis of long polyubiquitin chains in high yield and purity

As one of the most prevalent protein post-translational modifications, ubiquitin modification plays a momentous role in regulating varied cellular functions. Different polyubiquitin linkage types have diverse effects on cell signaling. However, compared with short ubiquitin chains, the preparation of long ubiquitin chains remains difficult and expensive to purchase commercially. In this study, we constructed an enzyme library of ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin-ligase E3, which are specific for synthesizing K63, K48, and M1 linked polyubiquitin chains. We demonstrate that these distinctly linked polyubiquitin chains could be synthesized and purified with high yield and purity. More importantly, this method can synthesize longer ubiquitin chains, the longest can reach more than fifteen ubiquitin molecules, which provides great convenience for ubiquitin-related structural and functional studies.

AZGP1P2/UBA1/RBM15 Cascade Mediates the Fate Determinations of Prostate Cancer Stem Cells and Promotes Therapeutic Effect of Docetaxel in Castration-Resistant Prostate Cancer via TPM1 m6A Modification.

Prostate cancer (PCa) is a common malignant tumor with high morbidity and mortality worldwide. The prostate cancer stem cell (PCSC) model provides novel insights into the pathogenesis of PCa and its therapeutic response. However, the roles and molecular mechanisms of specific genes in mediating fate decisions of PCSCs and carcinogenesis of PCa remain to be elusive. In this study, we have explored the expression, function, and mechanism of AZGP1P2, a pseudogene of AZGP1, in regulating the stemness and apoptosis of PCSCs and treatment resistance of docetaxel in castration-resistant prostate cancer (CRPC). We revealed that AZGP1P2 was downregulated in CRPC cell lines and PCSCs, while it was positively associated with progression-free interval. Upregulation of the AZGP1P2 enhanced the sensitivity of docetaxel treatment in CRPCs via inhibiting their stemness. RNA pull-down associated with mass spectrometry analysis, co-immunoprecipitation assay, and RNA immunoprecipitation assay demonstrated that AZGP1P2 could bind to UBA1 and RBM15 as a "writer" of methyltransferase to form a compound. UBA1, an E1 ubiquitin-activating enzyme, contributed to RBM15 protein degradation via ubiquitination modification. Methylated RNA immunoprecipitation assay displayed that RBM15 controlled the mRNA decay of TPM1 in m6A methylation. Furthermore, a xenograft mouse model and patient-derived organoids showed that the therapeutic effect of docetaxel in CRPC was increased by AZGP1P2 invivo. Collectively, these results imply that AZGP1P2 mediates the stemness and apoptosis of PCSCs and promotes docetaxel therapeutic effect by suppressing tumor growth and metastasis via UBA1/RBM15-mediated TPM1 mRNA decay in CRPC.

Identification and Characterization of Physiological Pairing of E2 Ubiquitin-Conjugating Enzymes and E3 Ubiquitin Ligases.

The posttranslational attachment of the small protein modifier ubiquitin (Ub) is best known for its function in targeting proteins for degradation by the proteasome. However, ubiquitination also serves as a signal determining protein localization, activity, and interaction. Ubiquitination requires the sequential activity of E1 ubiquitin-activating enzyme (UBA), E2 ubiquitin-conjugating enzyme (UBC), and E3 ubiquitin ligase. Recognition of a target protein by an Ub-E2-E3 complex can result in its mono-ubiquitination (attachment of a single Ub moiety) or poly-ubiquitination, i.e., attachment of Ub chains. While the E3 ligase is important for the reaction specificity, the E2s catalyze the attachment of Ub to the target and to Ub itself to generate chains. In Arabidopsis thaliana, there are two E1s, 37 UBCs (and two ubiquitin-like conjugating enzymes) and more than 1400 E3 ligases, working in a combinatorial way. Therefore, in order to understand E3 ligase function, it is important to frame it within its possible E2s interactors. In this chapter, we propose a two-step identification and characterization of physiological E2-E3 pairs. In a first step, in vivo interacting E2s are identified through bimolecular fluorescence complementation (BiFC) using transient expression in Arabidopsis protoplast. In the second step, the activity of E2-E3 pairs is analyzed by a synthetic biology approach in which autoubiquitination is reconstituted in bacteria.