Regulation of the CUL3 Ubiquitin Ligase by a Calcium-Dependent Co-adaptor.

The ubiquitin ligase CUL3 is an essential regulator of neural crest specification whose aberrant activation has been linked to autism, schizophrenia, and hypertension. CUL3 exerts its roles by pairing with ∼90 distinct substrate adaptors, yet how the different CUL3-complexes are activated is poorly understood. Here, we show that CUL3 and its adaptor KLHL12 require two calcium-binding proteins, PEF1 and ALG2, for recognition of their substrate SEC31. PEF1 and ALG2 form a target-specific co-adaptor that translates a transient rise in cytosolic calcium levels into more persistent SEC31 ubiquitylation, which in turn triggers formation of large COPII coats and promotes collagen secretion. As calcium also instructs chondrocyte differentiation and collagen synthesis, calcium-dependent control of CUL3KLHL12 integrates collagen secretion into broader programs of craniofacial bone formation. Our work, therefore, identifies both calcium and CUL3 co-adaptors as important regulators of ubiquitylation events that control human development.

Co-adaptor driven assembly of a CUL3 E3 ligase complex.

Cullin-RING E3 ligases (CRLs) are essential ubiquitylation enzymes that combine a catalytic core built around cullin scaffolds with ∼300 exchangeable substrate adaptors. To ensure robust signal transduction, cells must constantly form new CRLs by pairing substrate-bound adaptors with their cullins, but how this occurs at the right time and place is still poorly understood. Here, we show that formation of individual CRL complexes is a tightly regulated process. Using CUL3KLHL12 as a model, we found that its co-adaptor PEF1-ALG2 initiates CRL3 formation by releasing KLHL12 from an assembly inhibitor at the endoplasmic reticulum, before co-adaptor monoubiquitylation stabilizes the enzyme for substrate modification. As the co-adaptor also helps recruit substrates, its role in CRL assembly couples target recognition to ubiquitylation. We propose that regulators dedicated to specific CRLs, such as assembly inhibitors or co-adaptors, cooperate with target-agnostic adaptor exchange mechanisms to establish E3 ligase complexes that control metazoan development.

A synthetic KLHL20 ligand to validate CUL3KLHL20 as a potent E3 ligase for targeted protein degradation.

Targeted protein degradation (TPD) has risen as a promising therapeutic modality. Leveraging the catalytic nature of the ubiquitin-proteasome enzymatic machinery, TPD exhibits higher potency to eliminate disease-causing target proteins such as oncogenic transcription factors that may otherwise be difficult to abrogate by conventional inhibitors. However, there are challenges that remain. Currently, nearly all degraders engage CUL4CRBN or CUL2VHL as the E3 ligase for target ubiquitination. While their immediate efficacies are evident, the narrowed E3 ligase options make TPD vulnerable to potential drug resistance. In addition, E3 ligases show differential tissue expression and have intrinsic limitations in accessing varying types of disease-relevant targets. As the success of TPD is closely associated with the ability of E3 ligases to efficiently polyubiquitinate the target of interest, the long-term outlook of TPD drug development will depend on whether E3 ligases such as CUL4CRBN and CUL2VHL are accessible to the targets of interest. To overcome these potential caveats, a broad collection of actionable E3 ligases is required. Here, we designed a macrocyclic degrader engaging CUL3KLHL20 for targeting BET proteins and validated CUL3KLHL20 as an E3 ligase system suitable for TPD. This work thus contributes to the expansion of usable E3 ligases for potential drug development.

Cancer Mutations of the Tumor Suppressor SPOP Disrupt the Formation of Active, Phase-Separated Compartments.

Mutations in the tumor suppressor SPOP (speckle-type POZ protein) cause prostate, breast, and other solid tumors. SPOP is a substrate adaptor of the cullin3-RING ubiquitin ligase and localizes to nuclear speckles. Although cancer-associated mutations in SPOP interfere with substrate recruitment to the ligase, mechanisms underlying assembly of SPOP with its substrates in liquid nuclear bodies and effects of SPOP mutations on assembly are poorly understood. Here, we show that substrates trigger phase separation of SPOP in vitro and co-localization in membraneless organelles in cells. Enzymatic activity correlates with cellular co-localization and in vitro mesoscale assembly formation. Disease-associated SPOP mutations that lead to the accumulation of proto-oncogenic proteins interfere with phase separation and co-localization in membraneless organelles, suggesting that substrate-directed phase separation of this E3 ligase underlies the regulation of ubiquitin-dependent proteostasis.

Cullin3-KLHL25 ubiquitin ligase targets ACLY for degradation to inhibit lipid synthesis and tumor progression.

Increased lipid synthesis is a key characteristic of many cancers that is critical for cancer progression. ATP-citrate lyase (ACLY), a key enzyme for lipid synthesis, is frequently overexpressed or activated in cancer to promote lipid synthesis and tumor progression. Cullin3 (CUL3), a core protein for the CUL3-RING ubiquitin ligase complex, has been reported to be a tumor suppressor and frequently down-regulated in lung cancer. Here, we found that CUL3 interacts with ACLY through its adaptor protein, KLHL25 (Kelch-like family member 25), to ubiquitinate and degrade ACLY in cells. Through negative regulation of ACLY, CUL3 inhibits lipid synthesis, cell proliferation, and xenograft tumor growth of lung cancer cells. Furthermore, ACLY inhibitor SB-204990 greatly abolishes the promoting effect of CUL3 down-regulation on lipid synthesis, cell proliferation, and tumor growth. Importantly, low CUL3 expression is associated with high ACLY expression and poor prognosis in human lung cancer. In summary, our results identify CUL3-KLHL25 ubiquitin ligase as a novel negative regulator for ACLY and lipid synthesis and demonstrate that decreased CUL3 expression is an important mechanism for increased ACLY expression and lipid synthesis in lung cancer. These results also reveal that negative regulation of ACLY and lipid synthesis is a novel and critical mechanism for CUL3 in tumor suppression.

A Comprehensive Analysis of the Structural Recognition between KCTD Proteins and Cullin 3.

KCTD ((K)potassium Channel Tetramerization Domain-containing) proteins constitute an emerging class of proteins involved in fundamental physio-pathological processes. In these proteins, the BTB domain, which represents the defining element of the family, may have the dual role of promoting oligomerization and favoring functionally important partnerships with different interactors. Here, by exploiting the potential of recently developed methodologies for protein structure prediction, we report a comprehensive analysis of the interactions of all KCTD proteins with their most common partner Cullin 3 (Cul3). The data here presented demonstrate the impressive ability of this approach to discriminate between KCTDs that interact with Cul3 and those that do not. Indeed, reliable and stable models of the complexes were only obtained for the 15 members of the family that are known to interact with Cul3. The generation of three-dimensional models for all KCTD-Cul3 complexes provides interesting clues on the determinants of the structural basis of this partnership as clear structural differences emerged between KCTDs that bind or do not bind Cul3. Finally, the availability of accurate three-dimensional models for KCTD-Cul3 interactions may be valuable for the ad hoc design and development of compounds targeting specific KCTDs that are involved in several common diseases.

JA-induced FtBPM3 accumulation promotes FtERF-EAR3 degradation and rutin biosynthesis in Tartary buckwheat.

Buckwheat accumulates abundant flavonoids, which exhibit excellent health-promoting value. Flavonoids biosynthesis is mediated by a variety of phytohormones, among which jasmonates (JAs) induce numerous transcription factors, taking part in regulation of flavonoids biosynthesis genes. However, some transcriptional repressors appeared also induced by JAs. How these transcriptional repressors coordinately participate in JA signaling remains unclear. Here, we found that the disruption of the GCC-box in FtF3H promoter was associated with flavonoids accumulation in Tartary buckwheat. Further, our study illustrated that the nucleus-localized FtERF-EAR3 could inhibit FtF3H expression and flavonoids biosynthesis through binding the GCC-box in the promoter of FtF3H. The JA induced FtERF-EAR3 gene expression while facilitating FtERF-EAR3 protein degradation via the FtBPM3-dependent 26S proteasome pathway. Overall, these results illustrate a precise modulation mechanism of JA-responsive transcription suppressor participating in flavonoid biosynthesis, and will further help to improve the efficiency of flavonoids biosynthesis in Tartary buckwheat.

Are CUL3 variants an underreported cause of congenital heart disease?

Complex heart defects (CHD) are a common malformation associated with disruption of developmental pathways. The Cullin-RING ligases (CRLs) are multi-subunit E3 ubiquitin ligases in which Cullin 3 (CUL3) serves as a scaffolding subunit. Heterozygous CUL3 variants have been associated with neurodevelopmental disorders and pseudohypoaldosteronism type IIE. We report a fetus with CHD and a de novo CUL3 variant (NM_003590.4:c.[1549_1552del];[=], p.(Ser517Profs*23)) and review CUL3 variants reported with CHD. We postulate that CUL3 variants predispose to CHD and hypothesize mechanisms of pathogenesis.

Exo-miR-1290-induced by COX-2 overexpression promotes cancer-associated fibroblasts activation and tumor progression by CUL3-Nrf2 pathway in lung adenocarcinoma.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are critically involved in tumor progression by maintaining extracellular mesenchyma (ECM) production and improving tumor development. Cyclooxygenase-2 (COX-2) has been proved to promote ECM formation and tumor progression. However, the mechanisms of COX-2 mediated CAFs activation have not yet been elucidated. Therefore, we conducted this study to identify the effects and mechanisms of COX-2 underlying CAFs activation by tumor-derived exosomal miRNAs in lung adenocarcinoma (LUAD) progression. METHODS: As measures of CAFs activation, the expressions of fibroblasts activated protein-1 (FAP-1) and α-smooth muscle actin (α-SMA), the main CAFs markers, were detected by Western blotting and Immunohistochemistry. And the expression of Fibronectin (FN1) was used to analyze ECM production by CAFs. The exosomes were extracted by ultracentrifugation and exo-miRNAs were detected by qRT-PCR. Herein, we further elucidated the implicated mechanisms using online prediction software, luciferase reporter assays, co-immunoprecipitation, and experimental animal models. RESULTS: In vivo, a positive correlation was observed between the COX-2 expression levels in parenchyma and α-SMA/FN1 expression levels in mesenchyma in LUAD. However, PGE2, one of major product of COX-2, did not affect CAFs activation directly. COX-2 overexpression increased exo-miR-1290 expression, which promoted CAFs activation. Furthermore, Cullin3 (CUL3), a potential target of miR-1290, was found to suppress COX-2/exo-miR-1290-mediated CAFs activation and ECM production, consequently impeding tumor progression. CUL3 is identified to induce the Nuclear Factor Erythroid 2-Related Factor 2 (NFE2L2, Nrf2) ubiquitination and degradation, while exo-miR-1290 can prevent Nrf2 ubiquitination and increase its protein stability by targeting CUL3. Additionally, we identified that Nrf2 is direcctly bound with promoters of FAP-1 and FN1, which enhanced CAFs activation by promoting FAP-1 and FN1 transcription. CONCLUSIONS: Our data identify a new CAFs activation mechanism by exosomes derived from cancer cells that overexpress COX-2. Specifically, COX-2/exo-miR-1290/CUL3 is suggested as a novel signaling pathway for mediating CAFs activation and tumor progression in LUAD. Consequently, this finding suggests a novel strategy for cancer treatment that may tackle tumor progression in the future. Video Abstract.

Insights into the diverse mechanisms and effects of variant CUL3-induced familial hyperkalemic hypertension.

Familial hyperkalemic hypertension (FHHt), also known as Pseudohypoaldosteronism type II (PHAII) or Gordon syndrome is a rare Mendelian disease classically characterized by hyperkalemia, hyperchloremic metabolic acidosis, and high systolic blood pressure. The most severe form of the disease is caused by autosomal dominant variants in CUL3 (Cullin 3), a critical subunit of the multimeric CUL3-RING ubiquitin ligase complex. The recent identification of a novel FHHt disease variant of CUL3 revealed intricacies within the underlying disease mechanism. When combined with studies on canonical CUL3 variant-induced FHHt, these findings further support CUL3's role in regulating renal electrolyte transport and maintaining systemic vascular tone. However, the pathophysiological effects of CUL3 variants are often accompanied by diverse systemic disturbances in addition to classical FHHt symptoms. Recent global proteomic analyses provide a rationale for these systemic disturbances, paving the way for future mechanistic studies to reveal how CUL3 variants dysregulate processes outside of the renovascular axis. Video Abstract.

KLHL3-dependent WNK4 degradation affected by potassium through the neddylation and autophagy pathway.

BACKGROUND: Studies reported that kelch-like protein 3 (KLHL3)-Cullin3(CUL3) E3 ligase ubiquitinated with-no-lysine kinase 4 (WNK4). Impaired WNK4 ubiquitination plays a key role in Familial hyperkalemic hypertension (FHHt, also called pseudohypoaldosteronism type II) which results from overaction of thiazide-sensitive sodium chloride cotransport (NCC). In addition, researchers have also found that dietary potassium deficiency activates NCC along the renal distal convoluted tubule (DCT). However, the underlying mechanism remains unclear about the relationship between potassium and WNK4. METHODS: In the present study, we conducted in vitro and in vivo experiments to confirm that KLHL3-dependent WNK4 degradation is affected by potassium through the neddylation and autophagy pathway. In vitro, the WNK4 and KLHL3 plasmids were cotransfected into HEK293 cell lines by lipofectamine 2000, and then incubated with different potassium concentrations (1mmol/L and 10mmol/L) for 24 h, and further treated with MLN4924 or the autophagy inhibitor or both of MLN4924 and the autophagy inhibitor for another 24 h respectively. In vivo, we created mice that were fed with low or high potassium diets and then were injected MLN4924 in the experimental groups. The expression of WNK4, pWNK4, KLHL3, NEDD8, LC3 ,and P62 was detected by western blotting in vitro and vivo experiments. RESULTS: We found that the abundance and phosphorylation of WNK4 increase when neddylation is inhibited both in vitro and vivo. Furthermore, the abundance of pWNK4, WNK4, NEDD8, and KLHL3 was increased in the low potassium (LK) group. Inhibiting autophagy can ameliorate the effect of potassium on the abundance and activity of WNK4 to some extent. CONCLUSION: These findings suggest a complex regulation of potassium in the degradation of WNK4. Low potassium can activate WNK4, which may be related to neddylation and autophagy, but the mechanism needs to be further studied.

Kelch-like protein 3 in human disease and therapy.

Kelch-like protein 3 (KLHL3) is a substrate adaptor of Cullin3-RING ubiquitin ligase (CRL3), and KLHL3-CUL3 complex plays a vital role in the ubiquitination of specific substrates. Mutations and abnormal post-translational modifications of KLHL3-CUL3 affect substrate ubiquitination and may related to the pathogenesis of Gordon syndrome (GS), Primary Hyperparathyroidism (PHPT), Diabetes Mellitus (DM), Congenital Heart Disease (CHD), Pre-eclampsia (PE) and even cancers. Therefore, it is essential to understand the function and molecular mechanisms of KLHL3-CUL3 for the treatment of related diseases. In this review, we summary the structure and function of KLHL3-CUL3, the effect of KLHL3-CUL3 mutations and aberrant modifications in GS, PHPT, DM, CHD and PE. Moreover, we noted a possible role of KLHL3-CUL3 in carcinogenesis and provided ideas for targeting KLHL3-CUL3 for related disease treatment.

Restoration of Cullin3 gene expression enhances the improved effects of sonic hedgehog signaling activation for hypertension and attenuates the dysfunction of vascular smooth muscle cells.

BACKGROUND: Hypertension is known as a major factor for global mortality. We aimed to investigate the role of Cullin3 (CUL3) in the regulation of hypertension. MATERIAL AND METHODS: Human vascular smooth muscle cells (VSMCs) were treated with Angiotensin II (Ang II) to establish a hypertension in vitro model. Cell viability was detected by a cell counting kit-8 (CCK-8) assay. The content of reactive oxygen species (ROS) was evaluated by kit. Transwell assay and TUNEL staining were, respectively, used to assess cell migration and apoptosis. Additionally, the expression of sonic hedgehog (SHH) signaling-related proteins (SHH, smoothened homolog (Smo) and glioblastoma (Gli)) and CUL3 was tested with western blotting. Following treatment with Cyclopamine (Cycl), an inhibitor of SHH signaling, in Ang II-induced VSMCs, cell viability, migration, apoptosis and ROS content were determined again. Then, VSMCs were transfected with CUL3 plasmid or/and treated with sonic hedgehog signaling agonist (SAG) to explore the impacts on Ang II-induced VSMCs damage. In vivo, a hypertensive mouse model was established. Systolic blood pressure and diastolic blood pressure were determined. The histopathologic changes of abdominal aortic tissues were examined using H&E staining. The expression of SHH, Smo, Gli and CUL3 was tested with western blotting. RESULTS: Significantly increased proliferation, migration and apoptosis of VSMCs were observed after Ang II exposure. Moreover, Ang II induced upregulated SHH, Smo and Gli expression, whereas limited increase in CUL3 expression was observed. The content of ROS in Ang II-stimulated VSMCs presented the same results. Following Cycl treatment, the high levels of proliferation and migration in Ang II-treated VSMCs were notably remedied while the apoptosis and ROS concentration were further increased. Moreover, Cycl downregulated SHH, Smo, Gli and CUL3 expression. Above-mentioned changes caused by Ang II were reversed following SAG addition. Indeed, SAG treatment combined with restoration of CUL3 expression inhibited proliferation, migration, apoptosis and ROS level in Ang II-stimulated VSMCs. In vivo, SAG aggravated the histopathological changes of the aorta and with a worse tendency after both SAG intervention and CUL3 silencing. By contrast, SAG treatment and rebound in CUL3 expression alleviated the vascular damage. CONCLUSIONS: Collectively, restoration of CUL3 gene expression protected against hypertension through enhancing the effects of SHH activation in inhibition of apoptosis and oxidative stress for hypertension and alleviating the dysfunction of VSMCs.

Proteostasis in the Hedgehog signaling pathway.

The Hedgehog (Hh) signaling pathway is crucial for the development of vertebrate and invertebrate animals alike. Hh ligand binds its receptor Patched (Ptc), allowing the activation of the obligate signal transducer Smoothened (Smo). The levels and localizations of both Ptc and Smo are regulated by ubiquitination, and Smo is under additional regulation by phosphorylation and SUMOylation. Downstream of Smo, the Ci/Gli family of transcription factors regulates the transcriptional responses to Hh. Phosphorylation, ubiquitination and SUMOylation are important for the stability and localization of Ci/Gli proteins and Hh signaling output. Finally, Suppressor of Fused directly regulates Ci/Gli proteins and itself is under proteolytic regulation that is critical for normal Hh signaling.

Cul3 regulates cyclin E1 protein abundance via a degron located within the N-terminal region of cyclin E.

Cyclin E and its binding partner Cdk2 control the G1/S transition in mammalian cells. Increased levels of cyclin E are found in some cancers. Additionally, proteolytic removal of the cyclin E N-terminus occurs in some cancers and is associated with increased cyclin E-Cdk2 activity and poor clinical prognosis. Cyclin E levels are tightly regulated and controlled in part through ubiquitin-mediated degradation initiated by one of two E3 ligases, Cul1 and Cul3. Cul1 ubiquitylates phosphorylated cyclin E, but the mechanism through which Cul3 ubiquitylates cyclin E is poorly understood. In experiments to ascertain how Cul3 mediates cyclin E destruction, we identified a degron on cyclin E that Cul3 targets for ubiquitylation. Recognition of the degron and binding of Cul3 does not require a BTB domain-containing adaptor protein. Additionally, this degron is lacking in N-terminally truncated cyclin E. Our results describe a mechanism whereby N-terminally truncated cyclin E can avoid the Cul3-mediated degradation pathway. This mechanism helps to explain the increased activity that is associated with the truncated cyclin E variants that occurs in some cancers.

Cul3 is required for normal development of the mammary gland.

Cullin 3 (Cul3) has recently been implicated in a multitude of different processes, including the oxidative stress response, autophagy, tumorigenesis, and differentiation. To investigate the role of Cul3 in mammary gland development, we created a mouse model system using Cre-lox targeting where Cul3 is specifically deleted from the mammary gland. Such MMTV-Cre Cul3Flx/Flx mice examined at 2 and 3 months of age show delays and defects in mammary gland development. Mammary ductal trees from Cul3-deficient mammary glands exhibit delayed forward growth through the mammary fat pad, dilation of the ducts, and abnormal morphology of some of the epithelial structures within the gland. Additionally, terminal end buds are larger and less plentiful in MMTV-Cre Cul3Flx/Flx mammary glands, and there is significantly less primary and secondary branching compared to control animals. In contrast, by 6 months of age, the mammary ductal tree has grown to fill the entire mammary fat pad in glands lacking Cul3. However, distorted epithelial structures and dilated ducts persist. MMTV-Cre Cul3Flx/Flx mothers are able to nourish their litters, but the process of involution is slightly delayed in mammary glands lacking Cul3. Therefore, we conclude that while Cul3 is not essential for mammary gland function, Cul3 is required for the mammary gland to proceed normally through development.

The emerging role of the KCTD proteins in cancer.

The human family of Potassium (K+) Channel Tetramerization Domain (KCTD) proteins counts 25 members, and a significant number of them are still only partially characterized. While some of the KCTDs have been linked to neurological disorders or obesity, a growing tally of KCTDs are being associated with cancer hallmarks or involved in the modulation of specific oncogenic pathways. Indeed, the potential relevance of the variegate KCTD family in cancer warrants an updated picture of the current knowledge and highlights the need for further research on KCTD members as either putative therapeutic targets, or diagnostic/prognostic markers. Homology between family members, capability to participate in ubiquitination and degradation of different protein targets, ability to heterodimerize between members, role played in the main signalling pathways involved in development and cancer, are all factors that need to be considered in the search for new key players in tumorigenesis. In this review we summarize the recent published evidence on KCTD members' involvement in cancer. Furthermore, by integrating this information with data extrapolated from public databases that suggest new potential associations with cancers, we hypothesize that the number of KCTD family members involved in tumorigenesis (either as positive or negative modulator) may be bigger than so far demonstrated. Video abstract.

MYB106 is a negative regulator and a substrate for CRL3BPM E3 ligase in regulating flowering time in Arabidopsis thaliana.

Flowering time is crucial for successful reproduction in plants, the onset and progression of which are strictly controlled. However, flowering time is a complex and environmentally responsive history trait and the underlying mechanisms still need to be fully characterized. Post-translational regulation of the activities of transcription factors (TFs) is a dynamic and essential mechanism for plant growth and development. CRL3BPM E3 ligase is a CULLIN3-based E3 ligase involved in orchestrating protein stability via the ubiquitin proteasome pathway. Our study shows that the mutation of MYB106 induced early flowering phenotype while over-expression of MYB106 delayed Arabidopsis flowering. Transcriptome analysis of myb106 mutants reveals 257 differentially expressed genes between wild type and myb106-1 mutants, including Flowering Locus T (FT) which is related to flowering time. Moreover, in vitro electrophoretic mobility shift assays (EMSA), in vivo chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) assays and dual luciferase assays demonstrate that MYB106 directly binds to the promoter of FT to suppress its expression. Furthermore, we confirm that MYB106 interacts with BPM proteins which are further identified by CRL3BPM E3 ligases as the substrate. Taken together, we have identified MYB106 as a negative regulator in the control of flowering time and a new substrate for CRL3BPM E3 ligases in Arabidopsis.

Label-Free Interactome Analysis Revealed an Essential Role of CUL3-KEAP1 Complex in Mediating the Ubiquitination and Degradation of PHD2.

Prolyl hydroxylase domain-containing protein 2 (PHD2/EGLN1) is a key regulatory enzyme that plays a fundamental role in the cellular hypoxic response pathway, mediating proline hydroxylation-dependent protein degradation of selected target proteins. However, the regulation of PHD2 homeostasis at the protein level is not well understood. Here, we perform label-free quantitative interactome analysis through immunoprecipitation coupled with mass spectrometry analysis. To minimize the side effects caused by ectopic overexpression, in HeLa cells, we stably overexpressed Flag-tagged PHD2 while suppressing the endogenous PHD2 by using an shRNA targeting its 3' UTR region. We identified and validated Cullin 3 as a novel PHD2 interactor in vivo. Through candidate screening, we further identified CUL3-KEAP1 E3 ubiquitin ligase complex as the major enzyme that regulates PHD2 degradation. Overexpression of either CUL3, KEAP1, or both significantly increases PHD2 ubiquitination and reduces PHD2 protein abundance. The knockdown of CUL3 or KEAP1 decreased PHD2 ubiquitination and inhibited PHD2 degradation. Accordingly, loss of the CUL3-KEAP1 complex under hypoxia promoted PHD2 stabilization and led to significantly reduced abundance of the PHD2 target, hypoxia-inducible factor 1A (HIF1A). Thus, CUL3-KEAP1 is an essential pathway that regulates PHD2 ubiquitination and degradation in cells.

MBNL1 regulates resistance of HeLa cells to cisplatin via Nrf2.

Chemotherapy is an important method in the treatment of cervical cancer, but some patients will face drug resistance, which often indicates a poor prognosis. Moreover, there is no complete solution at present. Therefore, it is urgent to study the drug resistance mechanism of cervical cancer. Based on sequencing data mining, we predicted that MBNL1 might be involved in the occurrence and poor prognosis of cervical cancer, and verifed that MBNL1 could regulate the resistance of HeLa cells to cisplatin via Nrf2. In addition, we demonstrated that MBNL1 up regulated the degradation of Nrf2 protein by increasing the mRNA stability of Cul3. These results can provide theoretical basis for clinical development of new diagnosis and treatment targets for cisplatin resistance.