Ubiquitin-dependent Proteasome Research Articles

Impaired cyclin D3 protein degradation contributes to trastuzumab resistance in HER2 positive breast cancer

As the first anti-HER2 targeted agent approved by FDA in 1998, Trastuzumab has significantly improved the outcome of patients with HER2 positive metastatic breast cancer. Unfortunately, resistance to trastuzumab is a severe obstacle to its therapeutic efficacy in clinical application, and its mechanism has not yet been fully elucidated. In our study, we found that stabilization of cyclin D3 could be one reason for trastuzumab resistance. Trastuzumab could induce G1/G0 phase arrest by downregulating cyclin D3 protein expression. However, the protein expression of cyclin D3 was not affected in trastuzumab-resistant cells, which might be related to aberrant activation of ERK signaling pathway. Furthermore, degradation of cyclin D3 protein by trastuzumab was mainly resulted from ubiquitin-dependent proteasome mechanism instead of transcriptional regulation. In trastuzumab-resistant breast cancer cells, trastuzumab-induced degradation of cyclin D3 protein was abrogated. When the ubiquitin pathway was inhibited, cells would show a predisposition to resistance to trastuzumab. Further, CDK4/6 inhibitor can inhibit the proliferation of trastuzumab-resistant HER-2 positive breast cancer cells. Therefore, combination of CDK4/6 inhibitors and anti-HER2 targeted therapy may be an alternative and promising strategy to overcome trastuzumab resistance in the future.

Ring finger protein 138 inhibits transcription factor C/EBPα protein turnover leading to differentiation arrest in acute myeloid leukemia.

E3 ubiquitin ligase, ring finger protein 138 (RNF138) is involved in several biological processes; however, its role in myeloid differentiation or tumorigenesis remains unclear. RNAseq data from TNMplot showed that RNF138 mRNA levels are highly elevated in acute myeloid leukemia (AML) bone marrow samples as compared with bone marrow of normal volunteers. Here, we show that RNF138 serves as an E3 ligase for the tumor suppressor CCAAT/enhancer binding protein (C/EBPα) and promotes its degradation leading to myeloid differentiation arrest in AML. Wild-type RNF138 physically interacts with C/EBPα and promotes its ubiquitin-dependent proteasome degradation while a mutant RNF-138 deficient in ligase activity though interacts with C/EBPα, fails to down-regulate it. We show that RNF138 depletion enhances endogenous C/EBPα levels in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers. Our data further shows that RNF138-mediated degradation of C/EBPα negatively affects its transactivation potential on its target genes. Furthermore, RNF138 overexpression inhibits all-trans-retinoic acid-induced differentiation of HL-60 cells whereas RNF138 RNAi enhances. In line with RNF138 inhibiting C/EBPα protein turnover, we also observed that RNF138 overexpression inhibited β-estradiol (E2)-induced C/EBPα driven granulocytic differentiation in C/EBPα inducible K562-p42C/EBPα-estrogen receptor cells. Furthermore, we also recapitulated these findings in PBMCs isolated from AML patients where depletion of RNF138 increased the expression of myeloid differentiation marker CD11b. These results suggest that RNF138 inhibits myeloid differentiation by targeting C/EBPα for proteasomal degradation and may provide a plausible mechanism for loss of C/EBPα expression often observed in myeloid leukemia. Also, targeting RNF138 may resolve differentiation arrest by restoring C/EBPα expression in AML.

CRIP1 involves the pathogenesis of multiple myeloma via dual-regulation of proteasome and autophagy

Multiple myeloma (MM) is an incurable hematological malignancy of the plasma cells. The maintenance of protein homeostasis is critical for MM cell survival. Elevated levels of paraproteins in MM cells are cleared by proteasomes or lysosomes, which are independent but inter-connected with each other. Proteasome inhibitors (PIs) work as a backbone agent and successfully improved the outcome of patients; however, the increasing activity of autophagy suppresses the sensitivity to PIs treatment. The transcription levels of CRIP1 were explored in plasma cells obtained from healthy donors, patients with newly diagnosed multiple myeloma (NDMM), and relapsed/refractory multiple myeloma (RRMM) using Gene expression omnibus datasets. Doxycycline-inducible CRIP1-shRNA and CRIP1 overexpressed MM cell lines were constructed to explore the role of CRIP1 in MM pathogenesis. Proliferation, invasion, migration, proteasome activity and autophagy were examined in MM cells with different CRIP1 levels. Co-immunoprecipitation (Co-IP) with Tandem affinity purification/Mass spectrum (TAP/MS) was performed to identify the binding proteins of CRIP1. The mouse xenograft model was used to determine the role of CRIP1 in the proliferation and drug-resistance of MM cells. High CRIP1 expression was associated with unfavorable clinical outcomes in patients with MM and served as a biomarker for RRMM with shorter overall survival. Invitro and invivo studies showed that CRIP1 plays a critical role in protein homeostasis via the dual regulation of the activities of proteasome and autophagy in MM cells. A combined analysis of RNA-seq, Co-IP and TAP/MS demonstrated that CRIP1 promotes proteasome inhibitors resistance in MM cells by simultaneously binding to de-ubiquitinase USP7 and proteasome coactivator PA200. CRIP1 promoted proteasome activity and autophagosome maturation by facilitating the dequbiquitination and stabilization of PA200. Our findings clarified the pivotal roles of the CRIP1/USP7/PA200 complex in ubiquitin-dependent proteasome degradation and autophagy maturation involved in the pathogenesis of MM. A full list of funding sources can be found in the acknowledgements section.

CRIP1 Involves the Pathogenesis of Multiple Myeloma Via Dual-Regulation of Proteasome and Autophagy

Multiple myeloma (MM) is an incurable hematological malignancy of plasma cells. The maintenance of protein homeostasis is critical for the survival of MM cells. Elevated paraprotein in MM cells would be cleared by proteasomes or lysosomes, which are independent but inter-connected with each other. Proteasome inhibitors (PIs) work as the backbone agent and successfully improved the outcome of patients, but the increasing activity of autophagy suppresses the sensitivity to PIs treatment. In the present study, we elucidated that CRIP1 was a critical player in protein homeostasis via the dual regulation of proteasome and autophagy in MM. A combination analysis of RNA-seq, Co-IP and Tandem affinity purification/mass spectrometry identified that high-level CRIP1 represents a biomarker for relapsed/refractory MM with shorter overall-survival of patients. CRIP1 plays a critical role in MM progression and PIs resistance by binding to deubiquitinase USP7 and proteasome coactivator PA200. CRIP1 promotes proteasome activity and autophagosome maturation in MM by stabilizing PA200. Therefore, our findings clarified the pivotal roles of CRIP1/USP7/PA200 complex formation in ubiquitin-dependent proteasome degradation and autophagy maturation involved in the progression of MM.

ODF2 Negatively Regulates CP110 Levels at the Centrioles/Basal Bodies to Control the Biogenesis of Primary Cilia.

Primary cilia are essential sensory organelles that develop when an inhibitory cap consisting of CP110 and other proteins is eliminated. The degradation of CP110 by the ubiquitin-dependent proteasome pathway mediated by NEURL4 and HYLS1 removes the inhibitory cap. Here, we investigated the suitability of rapamycin-mediated dimerization for centriolar recruitment and asked whether the induced recruitment of NEURL4 or HYLS1 to the centriole promotes primary cilia development and CP110 degradation. We used rapamycin-mediated dimerization with ODF2 to induce their targeted recruitment to the centriole. We found decreased CP110 levels in the transfected cells, but independent of rapamycin-mediated dimerization. By knocking down ODF2, we showed that ODF2 controls CP110 levels. The overexpression of ODF2 is not sufficient to promote the formation of primary cilia, but the overexpression of NEURL4 or HYLS1 is. The co-expression of ODF2 and HYLS1 resulted in the formation of tube-like structures, indicating an interaction. Thus, ODF2 controls primary cilia formation by negatively regulating the concentration of CP110 levels. Our data suggest that ODF2 most likely acts as a scaffold for the binding of proteins such as NEURL4 or HYLS1 to mediate CP110 degradation.

A novel antimycin analogue antimycin A2c, derived from marine Streptomyces sp., suppresses HeLa cells via disrupting mitochondrial function and depleting HPV oncoproteins E6/E7

AimsNovel antimycin alkaloid antimycin A2c (AE) was isolated from the culture of a marine derived Streptomyces sp. THS-55. We elucidated its chemical structure by extensive spectra and clarified the specific mechanism in HPV infected-cervical cancer. Materials and methodsColony formation assay, cell cycle analysis, hoechst 33342 staining assay, et.al were used to detect the inhibitory effect of AE on cervical cancer cells. Meanwhile, flow cytometry, western blotting, immunoprecipitation, RNA interference and molecular docking were used to analyze the mechanism of AE. Key findingsAE exhibited potent cytotoxicity in vitro against HPV-transformed cervical cancer HeLa cell line. AE inhibited the proliferation, arrested cell cycle distribution, and triggered caspase dependent apoptosis in HeLa cells. Further studies revealed AE-induced apoptosis is mediated by the degradation of E6/E7 oncoproteins. Molecular mechanic investigation showed that AE degraded the levels of E6/E7 oncoproteins through reactive oxygen (ROS)-mediated ubiquitin-dependent proteasome system activation, and the increased ROS generation was due to the disruption of the mitochondrial function. SignificanceThis present work revealed that this novel marine derived antimycin alkaloid could target the mitochondria and subsequently degrade HPV E6/E7 oncoproteins, and have potential application in the design and development of lead compound for cervical cancer cells, as well as the development for tool compounds to dissect E6/E7 functions.

The H protein of attenuated canine distemper virus is degraded via endoplasmic reticulum-associated protein degradation.

Canine distemper (CD) caused by canine distemper virus (CDV) is considered a highly contagious and acutely febrile disease in various animals around the world. Endoplasmic reticulum-associated protein degradation (ERAD) is an important biological effect induced by endoplasmic reticulum (ER) stress (ERS) for the degradation of unfolded/misfolded proteins in the ER of cells. CDV H glycoprotein is translocated into the ER for post-translational modifications. The effects of CDV H and ER on each other are unclear. In this study, we found that CDV H protein induced ERS through the PERK-mediated signaling pathway. The inhibition of ERS by 4-Phenylbutyric acid (4-PBA) increased the H protein amounts of an attenuated CDV, which was reduced by dithiothreitol (DTT)-induced ERS. Further, the H protein levels were increased when ERAD was inhibited by using Eeyarestatin I or interfering E3 ligase Hrd1 in ERAD, suggesting that the attenuated CDV H protein is degraded via ERAD. ERAD involved ubiquitin-dependent proteasome degradation (UPD) and/or autophagic-lysosome degradation (ALD). The attenuated CDV H protein was ubiquitinated and significantly increased after treatment with UPD inhibitor MG132 but not ALD inhibitor chloroquine (CQ), suggesting that ERAD degrading the attenuated CDV H protein selectively depends on UPD. Moreover, the inhibition of the degradation of CDV H protein with 4-PBA or MG132 treatment increased viral replication, whereas treatment with DTT promoting degradation of H protein was found to reduce viral replication. These findings suggest that the degradation of CDV H protein via ERAD negatively affects viral replication and provide a new idea for developing CDV prevention and control strategies.

Abstract CT128: Phase 1 study of HSK29116, a Bruton tyrosine kinase (BTK) proteolysis-targeting chimera (PROTAC) agent, in patients with relapsed or refractory B-cell malignancies

Abstract BACKGROUND: BTK, a non-receptor kinase in the B-cell receptor signaling pathway, plays an essential role in B-cell development and differentiation. BTK is critical for malignant B-cell survival. Oral BTK inhibitors (BTKis), standard treatments for patients (pts) with B-cell malignancies, prevent proliferation and induce apoptosis of B cells. Currently approved BTKis covalently bind to BTK residue C481, irreversibly inhibiting phosphorylation of downstream kinases and blocking B-cell activation. Because these agents are often given continuously, their use may lead to clinical resistance and unacceptable toxicities. Indeed, acquired resistance to covalent BTKis is common and caused by the expansion of clones with a cysteine-to-serine mutation at residue 481. A novel strategy being developed to overcome BTKi resistance, is PROTAC-induced degradation of BTK. PROTACs are small molecules with one domain that binds the target protein linked to another domain that binds to ubiquitin E3 ligase, resulting in ubiquitin-dependent proteasome degradation of the target protein. HSK29116 is a small BTK PROTAC molecule that is currently undergoing clinical investigation for the treatment of B-cell malignancies. In preclinical studies, HSK29116 overcame drug resistance caused by the C481S BTK mutation. Also, HSK29116 has demonstrated inhibition of B-cell proliferation without measurable loss of other BTK-family kinase activities. This kinase selectivity may limit some of the off-target toxicities seen with currently available BTKis. METHODS: This first-in-human, multicenter, open-label, phase 1 study (NCT04861779) began recruiting in 2021 to evaluate the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics of oral HSK29116. Eligible pts are ≥18 years old with diagnosis of a B-cell malignancy that has relapsed or become refractory to standard therapy (≥2 prior systemic therapies, which may have included a BTKi) and for which no other treatments known to provide clinical benefit exist. Pts must have an Eastern Cooperative Oncology Group performance status ≤2 and life expectancy >3 months. Phase 1a includes dose escalation to identify dose-limiting toxicities and establish the maximum tolerated dose and/or recommended dose of HSK29116 for a phase 1b dose expansion, initiating in the US. Approximately 36 pts will be enrolled in phase 1a and 90 pts in phase 1b (≥40 pts must have previously received covalent BTKis). The primary endpoint is safety. Secondary endpoints include PK and investigator-assessed overall response rate, duration of response, time to response, and progression-free survival. Exploratory analyses will evaluate the relationship between HSK29116 anti-tumor activity and BTK gene mutations, as well as BTK protein degradation effects of HSK29116. Descriptive statistics will be used to summarize all data. Citation Format: Jianyong Li, Wei Xu, Pangke Yan, Yong Cao, Mengyue Hu, William Daley. Phase 1 study of HSK29116, a Bruton tyrosine kinase (BTK) proteolysis-targeting chimera (PROTAC) agent, in patients with relapsed or refractory B-cell malignancies [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 CT128.

The E3 ligase RBCK1 reduces the sensitivity of ccRCC to sunitinib through the ANKRD35-MITD1-ANXA1 axis.

Despite the promise of targeted tyrosine kinase inhibitors (TKIs), such as sunitinib, in the extension of survival time in patients with clear cell renal cell carcinoma (ccRCC) progression or metastasis, the patients eventually succumb to inevitable drug resistance. Protein degradation executed by the ubiquitin-dependent proteasome system played an important role in determining the sensitivity of ccRCC to sunitinib. Here, we applied the bioinformatic analysis to identify that E3 ligase RBCK1 was elevated in the sunitinib-resistant renal cancer cell lines or patient specimens. The subsequent in vitro or in vivo studies demonstrated that RBCK1 contributed to decreasing the sensitivity of ccRCC to sunitinib. Then, we showed that inhibition of RBCK1 inactivated the AKT and MAPK signaling pathways, which might be one of the main reasons why RBCK1 induces sunitinib resistance in ccRCC cells. Mechanistically, our results indicated that RBCK1 promotes the degradation of ANKRD35 and that ANKRD35 destabilizes MITD1 by binding with SUMO2 in ccRCC cells. In addition, we showed that the RBCK1-ANKRD35-MITD1-ANXA1 axis regulates the phosphorylation of AKT and ERK and contributes to the dysregulation of sunitinib in ccRCC cells. Therefore, we identified a novel mechanism for regulating the sensitivity of sunitinib in ccRCC. Therefore, we elucidated a novel mechanism by which RBCK1 regulates sunitinib sensitivity in ccRCC.

2-Deoxy-D-glucose Alleviates Cancer Cachexia-Induced Muscle Wasting by Enhancing Ketone Metabolism and Inhibiting the Cori Cycle.

Cachexia is characterized by progressive weight loss accompanied by the loss of specific skeletal muscle and adipose tissue. Increased lactate production, either due to the Warburg effect from tumors or accelerated glycolysis effects from cachectic muscle, is the most dangerous factor for cancer cachexia. This study aimed to explore the efficiency of 2-deoxy-D-glucose (2-DG) in blocking Cori cycle activity and its therapeutic effect on cachexia-associated muscle wasting. A C26 adenocarcinoma xenograft model was used to study cancer cachectic metabolic derangements. Tumor-free lean mass, hindlimb muscle morphology, and fiber-type composition were measured after in vivo 2-DG administration. Activation of the ubiquitin-dependent proteasome pathway (UPS) and autophagic–lysosomal pathway (ALP) was further assessed. The cachectic skeletal muscles of tumor-bearing mice exhibited altered glucose and lipid metabolism, decreased carbohydrate utilization, and increased lipid β-oxidation. Significantly increased gluconeogenesis and decreased ketogenesis were observed in cachectic mouse livers. 2-DG significantly ameliorated cancer cachexia-associated muscle wasting and decreased cachectic-associated lean mass levels and fiber cross-sectional areas. 2-DG inhibited protein degradation-associated UPS and ALP, increased ketogenesis in the liver, and promoted ketone metabolism in skeletal muscle, thus enhancing mitochondrial bioenergetic capacity. 2-DG effectively prevents muscle wasting by increasing ATP synthesis efficiency via the ketone metabolic pathway and blocking the abnormal Cori cycle.

PAD4-dependent citrullination of nuclear translocation of GSK3β promotes colorectal cancer progression via the degradation of nuclear CDKN1A

Peptidylarginine deiminase 4 (PAD4), a Ca2+-dependent enzyme, catalyzes the conversion of arginine to citrulline and has been strongly associated with many malignant tumors. However, the molecular mechanisms of PAD4 in the development and progression of colorectal cancer (CRC) remain unclearly defined. In our study, PAD4 expression was increased in CRC tissues and cells, and was closely related to tumor size, lymph node metastasis. Moreover, the transcription factor KLF9 directly bound to PADI4 gene promoter, leading to overexpression of PAD4 in CRC cells, which augmented cell growth and migration. We revealed that PAD4 interacted with and citrullinated glycogen synthase kinase-3β (GSK3β) in CRC cells, and GSK3β Arg-344 was the dominating PAD4-citrullination site. Furthermore, IgL2 and catalytic domains of PAD4 directly bound to the kinase domain of GSK3β in CRC cells. Mechanistically, PAD4 promoted the transport of GSK3β from the cytoplasm to the nucleus, thereby increasing the ubiquitin-dependent proteasome degradation of nuclear cyclin-dependent kinase inhibitor 1 (CDKN1A). Our study is the first to reveal the details of a critical PAD4/GSK3β/CDKN1A signaling axis for CRC progression, and provides evidence that PAD4 is a potential diagnosis biomarker and therapeutic target in CRC.

A novel long noncoding RNA SP100-AS1 induces radioresistance of colorectal cancer via sponging miR-622 and stabilizing ATG3

Although radiotherapy is an essential modality in the treatment of colorectal cancer (CRC), the incidence of radioresistance remains high clinically. Long noncoding RNAs (lncRNAs) reportedly play critical roles in CRC radioresistance by regulating genes or proteins at the transcriptional or post-translational levels. This study aimed to identify novel lncRNAs involved in radioresistance. We found that SP100-AS1 (lncRNA targeting antisense sequence of SP100 gene) was upregulated in radioresistant CRC patient tissues using RNA-seq analysis. Importantly, knockdown of SP100-AS1 significantly reduced radioresistance, cell proliferation, and tumor formation in vitro and in vivo. Mechanistically, mass spectrometry and bioinformatics analyses were used to identify the interacting proteins and microRNAs of SP100-AS1, respectively. Moreover, SP100-AS1 was found to interact with and stabilize ATG3 protein through the ubiquitination-dependent proteasome pathway. In addition, it could serve as a sponge for miR-622, which targeted ATG3 mRNA and affected autophagic activity. Thus, lncRNA SP100-AS1 could act as a radioresistance factor in CRC patients via RNA sponging and protein stabilizing mechanisms. In conclusion, the present study indicates that SP100-AS1/miR-622/ATG3 axis contributes to radioresistance and autophagic activity in CRC patients, suggesting it has huge prospects as a therapeutic target for improving CRC response to radiation therapy.

TPI1 activates the PI3K/AKT/mTOR signaling pathway to induce breast cancer progression by stabilizing CDCA5

BackgroundTriosephosphate isomerase 1 (TPI1), as a key glycolytic enzyme, is upregulated in multiple cancers. However, expression profile and regulatory mechanism of TPI1 in breast cancer (BRCA) remain mysterious.MethodsWestern blotting and immunohistochemistry (IHC) assays were used to investigate the expression of TPI1 in BRCA specimens and cell lines. TPI1 correlation with the clinicopathological characteristics and prognosis of 362 BRCA patients was analyzed using a tissue microarray. Overexpression and knockdown function experiments in cells and mice models were performed to elucidate the function and mechanisms of TPI1-induced BRCA progression. Related molecular mechanisms were clarified using co-IP, IF, mass spectrometric analysis, and ubiquitination assay.ResultsWe have found TPI1 is highly expressed in BRCA tissue and cell lines, acting as an independent indicator for prognosis in BRCA patients. TPI1 promotes BRCA cell glycolysis, proliferation and metastasis in vitro and in vivo. Mechanistically, TPI1 activates phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway to regulate epithelial–mesenchymal transformation (EMT) and aerobic glycolysis, which is positively mediated by cell division cycle associated 5 (CDCA5). Moreover, TPI1 interacts with sequestosome-1 (SQSTM1)/P62, and P62 decreases the protein expression of TPI1 by promoting its ubiquitination in MDA-MB-231 cells.ConclusionsTPI1 promotes BRCA progression by stabilizing CDCA5, which then activates the PI3K/AKT/mTOR pathway. P62 promotes ubiquitin-dependent proteasome degradation of TPI1. Collectively, TPI1 promotes tumor development and progression, which may serve as a therapeutic target for BRCA.

Smurf2 suppresses the metastasis of hepatocellular carcinoma via ubiquitin degradation of Smad2.

PurposeSmurf2, one of C2-WW-HECT domain E3 ubiquitin ligases, is closely related to the development and progression in different cancer types, including hepatocellular carcinoma (HCC). This study aims to illustrate the expression and molecular mechanism of Smurf2 in regulating the progression of HCC.MethodsThe expression of Smurf2 in human HCC and adjacent non-tumor liver specimens was detected using tissue microarray studies from 220 HCC patients who underwent curative resection. The relationships of Smurf2 and HCC progression and survival were analyzed using the chi-square test, Kaplan–Meier analysis, and Cox proportional hazards model. For Smurf2 was low expression in HCC cell lines, Smurf2 overexpression cell lines were established. The effect of Smurf2 on cell proliferation and migration was detected by Cell Counting Kit-8 and colony formation assay, and the epithelial–mesenchymal transition (EMT) markers and its transcription factors were tested by immunoblotting. The interaction and ubiquitination of Smad2 by Smurf2 were detected by co-immunoprecipitation and immunoprecipitation assay. Finally, the effect of Smurf2 on HCC was verified using the mouse lung metastasis model.ResultsSmurf2 was downregulated in HCC tissues compared to that of corresponding non-tumor liver specimens. The low expression of Smurf2 in HCC was significantly associated with macrovascular or microvascular tumor thrombus and the impairment of overall survival and disease-free survival. In vitro and in vivo analysis showed that Smurf2 overexpression decreased the EMT potential of HCC cells by promoting the ubiquitination of Smad2 via the proteasome-dependent degradation pathway.ConclusionThe expression of Smurf2 was downregulated in HCC specimens and affected the survival of patients. Smurf2 inhibited the EMT of HCC by enhancing Smad2 ubiquitin-dependent proteasome degradation.

BRD7 Stabilizes P53 via Dephosphorylation of MDM2 to Inhibit Tumor Growth in Breast Cancer Harboring Wild-type P53.

Bromodomain-containing protein 7 (BRD7) was found to be down-expressed in nasopharyngeal carcinoma as well as breast cancer and to function as a potential tumor suppressor. BRD7 interacts with p53 and is required for p53-dependent oncogene-induced senescence. However, the mechanism how BRD7 functions as tumor suppressor roles in breast cancer remains unclear. MTT, colony formation assay, cell cycle, cell apoptosis, and tumorigenicity assays were performed to evaluate the biological functions of BRD7 in breast cancer cells in vitro and in vivo. Real-time PCR, western blot, luciferase reporter gene assays, and co-immunoprecipitation were used to examine the gene expression, transcription activation and protein-protein interaction. We reported that BRD7 effectively suppressed cell proliferation and tumor growth in vitro and in vivo. In addition, BRD7 increased p53 protein stability through ubiquitin-dependent proteasome pathway and regulated the expression of p53 downstream target genes by activating its transcriptional activity in breast cancers harboring wild-type p53. Mechanistically, BRD7 decreased phosphorylation and activation of MDM2 via inactivating its upstream kinase AKT depending on the bromodomain of BRD7, therefore BRD7 significantly reduced the amounts of phosphorylated MDM2 binding with p53 eventually decreasing ubiquitination level of p53. Furthermore, silencing the expression of p53 at least partly reversed the inhibition effect of BRD7 on cell proliferation and tumor growth in vitro and in vivo. Our studies identify that BRD7 stabilizes p53 by inhibiting the phosphorylation of MDM2 via AKT pathway dependent on its bromodomain to function as a tumor suppressor in breast cancer harboring wild-type p53.

Selective autophagy controls the stability of TBK1 via NEDD4 to balance host defense.

As a core kinase of antiviral immunity, the activity and stability of TANK-binding kinase 1 (TBK1) is tightly controlled by multiple post-translational modifications. Although it has been demonstrated that TBK1 stability can be regulated by ubiquitin-dependent proteasome pathway, it is unclear whether another important protein degradation pathway, autophagosome pathway, can specifically affect TBK1 degradation by cargo receptors. Here we report that E3 ubiquitin ligase NEDD4 functions as a negative regulator of type I interferon (IFN) signaling by targeting TBK1 for degradation at the late stage of viral infection, to prevent the host from excessive immune response. Mechanically NEDD4 catalyzes the K27-linked poly-ubiquitination of TBK1 at K344, which serves as a recognition signal for cargo receptor NDP52-mediated selective autophagic degradation. Taken together, our study reveals the regulatory role of NEDD4 in balancing TBK1-centered type I IFN activation and provides insights into the crosstalk between selective autophagy and antiviral signaling.

Cysteine Modification by Ebselen Reduces the Stability and Cellular Levels of 14-3-3 Proteins.

The 14-3-3 proteins constitute a family of adaptor proteins with many binding partners and biological functions, and they are considered promising drug targets in cancer and neuropsychiatry. By screening 1280 small-molecule drugs using differential scanning fluorimetry (DSF), we found 15 compounds that decreased the thermal stability of 14-3-3ζ Among these compounds, ebselen was identified as a covalent, destabilizing ligand of 14-3-3 isoforms ζ, ε, γ, and η Ebselen bonding decreased 14-3-3ζ binding to its partner Ser19-phosphorylated tyrosine hydroxylase. Characterization of site-directed mutants at cysteine residues in 14-3-3ζ (C25, C94, and C189) by DSF and mass spectroscopy revealed covalent modification by ebselen of all cysteines through a selenylsulfide bond. C25 appeared to be the preferential site of ebselen interaction in vitro, whereas modification of C94 was the main determinant for protein destabilization. At therapeutically relevant concentrations, ebselen and ebselen oxide caused decreased 14-3-3 levels in SH-SY5Y cells, accompanied with an increased degradation, most probably by the ubiquitin-dependent proteasome pathway. Moreover, ebselen-treated zebrafish displayed decreased brain 14-3-3 content, a freezing phenotype, and reduced mobility, resembling the effects of lithium, consistent with its proposed action as a safer lithium-mimetic drug. Ebselen has recently emerged as a promising drug candidate in several medical areas, such as cancer, neuropsychiatric disorders, and infectious diseases, including coronavirus disease 2019. Its pleiotropic actions are attributed to antioxidant effects and formation of selenosulfides with critical cysteine residues in proteins. Our work indicates that a destabilization of 14-3-3 may affect the protein interaction networks of this protein family, contributing to the therapeutic potential of ebselen. SIGNIFICANCE STATEMENT: There is currently great interest in the repurposing of established drugs for new indications and therapeutic targets. This study shows that ebselen, which is a promising drug candidate against cancer, bipolar disorder, and the viral infection coronavirus disease 2019, covalently bonds to cysteine residues in 14-3-3 adaptor proteins, triggering destabilization and increased degradation in cells and intact brain tissue when used in therapeutic concentrations, potentially explaining the behavioral, anti-inflammatory, and antineoplastic effects of this drug.

CDK2-instigates C/EBPα degradation through SKP2 in Acute myeloid leukemia.

Transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPα) regulates myelopoiesis by coupling growth arrest with differentiation of myeloid progenitors. Mutations in one or both alleles are observed in 10-14% AML cases that render C/EBPα functionally inactive. Besides, antagonistic protein-protein interactions also impair C/EBPα expression and function. In recent independent studies, we showed that CDK2 and SKP2 downregulated C/EBPα expression in an ubiquitin-dependent proteasome degradation manner leading to differentiation block in AML. Here, we demonstrate that CDK2-instigated C/EBPα downregulation is actually mediated by SKP2. Mechanistically, we show that CDK2 stabilizes SKP2 by phosphorylating it at Ser64 and thereby potentiates C/EBPα ubiquitination and subsequent degradation in AML cells. Immunoblot experiments showed that CDK2 inhibition downregulated SKP2 levels and concomitantly enhanced C/EBPα levels in myeloid cells. We further show that while CDK2 promoted C/EBPα ubiquitination and inhibited its protein levels, negatively affected its transactivation potential and DNA binding ability, simultaneous SKP2 depletion abrogated CDK2-promoted ubiquitination and restored C/EBPα expression and function. Taken together, these findings consolidate that CDK2 potentiates SKP2-mediated C/EBPα degradation in AML and targeting CDK2-SKP2 axis can be harnessed for therapeutic benefit in AML. Hypothetical model depicts that SKP2-mediated C/EBPα proteasomal degradation is reinforced by CDK2. CDK2 phopshorylates SKP2 leading to its enhanced stabilization which in turn exaggerates C/EBPα degradation leading to differentiation arrest in AML.

A novel c.-652C>T mutation in UCHL1 gene is associated with the growth performance in Yangzhou goose

As a member of the ubiquitin-dependent proteasome degradation pathway, Ubiquitin carboxyl-terminal hydrolase-L1 (UCHL1) plays a key role in post-translational modification and protein degradation, and it is extensive and important for the regulation of various biological functions of the organism. However, its function remains unclear in goose growth performance. In this study, the full-length genomic DNA and coding region of UCHL1 gene was firstly cloned and characterized in Yangzhou goose. Tissue expression profile revealed that UCHL1 was exclusively expressed in brain and gonads. A novel single nucleotide polymorphisms c.-652C>T which is significantly related to 64-d body weight of Yangzhou goose was found in UCHL1 promoter region by comparative sequencing. Correlation analysis in a population of 405 geese showed that TT genotype individuals had higher body weight than CC individuals in male, but not in female geese. Dual-luciferase reporter assay indicated that the single nucleotide polymorphisms c.-652C>T is located at the core promoter region of UCHL1, and the promoter transcription activity was significantly increased (P < 0.01) when allele C changed to T. Geese with TT genotype had higher mRNA level of UCHL1 in brain tissue than those of CC genotype (P < 0.01). Compared with CC individuals, neuropeptide Y and AdipoR1 mRNA level was significantly higher in TT individuals (P < 0.05), while FAS mRNA level was lower in the TT individuals (P < 0.05). In summary, we identify a novel mutation in the promoter of UCHL1 gene, which can alter transcriptional activity of UCHL1 gene, and affect the growth performance of male goose.

A novel de novo heterozygous DYRK1A mutation causes complete loss of DYRK1A function and developmental delay

Dual-specificity tyrosine phosphorylation-regulated kinase 1 A (DYRK1A) is essential for human development, and DYRK1A haploinsufficiency is associated with a recognizable developmental syndrome and variable clinical features. Here, we present a patient with DYRK1A haploinsufficiency syndrome, including facial dysmorphism, delayed motor development, cardiovascular system defects, and brain atrophy. Exome sequencing identified a novel de novo heterozygous mutation of the human DYRK1A gene (c.1185dup), which generated a translational termination codon and resulted in a C-terminally truncated protein (DYRK1A-E396ter). To study the molecular effect of this truncation, we generated mammalian cell and Drosophila models that recapitulated the DYRK1A protein truncation. Analysis of the structure and deformation energy of the mutant protein predicted a reduction in protein stability. Experimentally, the mutant protein was efficiently degraded by the ubiquitin-dependent proteasome pathway and was barely detectable in mammalian cells. More importantly, the mutant kinase was intrinsically inactive and had little negative impact on the wild-type protein. Similarly, the mutant protein had a minimal effect on Drosophila phenotypes, confirming its loss-of-function in vivo. Together, our results suggest that the novel heterozygous mutation of DYRK1A resulted in loss-of-function of the kinase activity of DYRK1A and may contribute to the developmental delay observed in the patient.