Ubiquitin-dependent Proteasomal Degradation Research Articles

Fbxo11 maintains mitochondrial function and prevents podocyte injury in adriamycin-induced nephropathy by mediating the ubiquitin degradation of Fosl2

Mitochondrial dysfunction is a pivotal factor in the onset of podocyte damage, which is a central component in the pathogenesis of nephrotic syndrome (NS). However, the precise mechanisms underlying the changes in podocyte mitochondria remain elusive. Our study aims to clarify the potential mechanisms involved in the role of F-box protein 11 (Fbxo11) in NS, specifically concentrating on its impact on mitochondrial function. A mouse model was established by tail vein injection of adriamycin (ADR, 10 mg/kg) and was infected with lentivirus overexpressing Fbxo11. Mouse podocytes (MPC-5) were infected with lenti-Fbxo11-OE, followed by treatment with 0.4 μg/mL of ADR. We identified the decreased expression of Fbxo11 in mouse kidney tissues and MPC-5 cells induced by ADR. Lenti-Fbxo11-OE intervention relieved ADR-induced glomerular lesion, podocyte injury, and mitochondrial dysfunction. In vitro, overexpression of Fbxo11 in mouse podocytes improved mitochondrial function and reduced podocyte damage, thereby inhibiting podocyte apoptosis. Mechanistically, Fbxo11 decreased the protein expression of Fosl2 through ubiquitin-dependent proteasomal degradation. Rescue experiments revealed that overexpression of Fosl2 abolished the protective effects of Fbxo11 overexpression on mitochondrial damage and podocyte injury. Importantly, the regulatory effects of the Fbxo11/Fosl2 axis were reversed when treated with the mitochondrial fission inhibitor mdivi-1. Taken together, our results demonstrated that Fbxo11-mediated ubiquitin degradation of Fosl2 is critical for maintaining mitochondrial function and preventing podocyte injury during NS.

Activation of the 26S Proteasome to Reduce Proteotoxic Stress and Improve the Efficacy of PROTACs.

The 26S proteasome degrades the majority of cellular proteins and affects all aspects of cellular life. Therefore, the 26S proteasome abundance, proper assembly, and activity in different life contexts need to be precisely controlled. Impaired proteasome activity is considered a causative factor in several serious disorders. Recent advances in proteasome biology have revealed that the proteasome can be activated by different factors or small molecules. Thus, activated ubiquitin-dependent proteasome degradation has effects such as extending the lifespan in different models, preventing the accumulation of protein aggregates, and reducing their negative impact on cells. Increased 26S proteasome-mediated degradation reduces proteotoxic stress and can potentially improve the efficacy of engineered degraders, such as PROTACs, particularly in situations characterized by proteasome malfunction. Here, emerging ideas and recent insights into the pharmacological activation of the proteasome at the transcriptional and posttranslational levels are summarized.

The transcription factor BBX regulates phosphate homeostasis through the modulation of FGF23

Fibroblast growth factor 23 (FGF23) plays an important role in phosphate homeostasis, and increased FGF23 levels result in hypophosphatemia; however, the molecular mechanism underlying increased FGF23 expression has not been fully elucidated. In this study, we found that mice lacking the bobby sox homolog (Bbx−/−) presented increased FGF23 expression and low phosphate levels in the serum and skeletal abnormalities such as a low bone mineral density (BMD) and bone volume (BV), as well as short and weak bones associated with low bone formation. Osteocyte-specific deletion of Bbx using Dmp-1-Cre resulted in similar skeletal abnormalities, elevated serum FGF23 levels, and reduced serum phosphate levels. In Bbx−/− mice, the expression of sodium phosphate cotransporter 2a (Npt2a) and Npt2c in the kidney and Npt2b in the small intestine, which are negatively regulated by FGF23, was downregulated, leading to phosphate excretion/wasting and malabsorption. An in vitro Fgf23 promoter analysis revealed that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced transactivation of the Fgf23 promoter was significantly inhibited by BBX overexpression, whereas it was increased following Bbx knockdown. Interestingly, 1,25(OH)2D3 induced an interaction of the 1,25(OH)2D3 receptor (VDR) with BBX and downregulated BBX protein levels. Cycloheximide (CHX) only partially downregulated BBX protein levels, indicating that 1,25(OH)2D3 regulates BBX protein stability. Furthermore, the ubiquitination of BBX followed by proteasomal degradation was required for the increase in Fgf23 expression induced by 1,25(OH)2D3. Collectively, our data demonstrate that BBX negatively regulates Fgf23 expression, and consequently, the ubiquitin-dependent proteasomal degradation of BBX is required for FGF23 expression, thereby regulating phosphate homeostasis and bone development in mice.

N-butanol extract of Broussonetia papyrifera (L.) L′Hér. ex Vent root bark alleviates atopic dermatitis by targeting E3 ubiquitin ligase WWP1 to promote NLRP3 degradation

BackgroundBroussonetia papyrifera (L.) L′Hér. ex Vent (B. papyrifera) is a deciduous tree widely distributed in Asia. Previous studies have revealed that leaves of B. papyrifera ameliorated atopic dermatitis (AD)-like symptoms and inflammatory response. However, the impact and underlying mechanism of other parts of B. papyrifera on AD remain elusive. MethodsThe AD mice induced by 1-Chloro-2,4-dinitrochlorobenzene were used to observe the histopathological alterations in the skin tissues using hematoxylin-eosin staining and toluidine blue staining techniques. Serum levels of inflammatory factors were quantified utilizing ELISA. Pyroptosis was analyzed by lactate dehydrogenase release and flow cytometry in human keratinocytes. The activation of Nod-like receptor protein 3 (NLRP3) inflammasome was analyzed by western blots. Furthermore, the mechanism underlying the inhibition of NLRP3 inflammasome by N-butanol extracts of B. papyrifera root bark (NE-BPRB) was investigated using cellular thermal shift assay and surface plasmon resonance techniques. ResultsTreatment with NE-BPRB significantly ameliorated symptoms of AD mice and reduced serum levels of pro-inflammatory factors. NE-BPRB intervention exhibited inhibitory effects on NLRP3 inflammasome activation and pyroptosis in vitro and in vivo. NE-BPRB and its primary bioactive constituent chlorogenic acid (CA) promote the K48-linked ubiquitination of NLRP3, leading to its proteasomal degradation by binding WW domain containing E3 ubiquitin protein ligase 1 (WWP1). ConclusionsThe NE-BPRB and its primary bioactive constituent, CA, effectively inhibit the formation of the NLRP3 inflammasome and impede cell pyroptosis by promoting K48-linked ubiquitin-dependent proteasomal degradation of NLRP3 through binding to the E3 ubiquitin ligase WWP1, thereby resulting in improved AD.

Pepper RING-Type E3 Ligase CaFIRF1 Negatively Regulates the Protein Stability of Pepper Stress-Associated Protein, CaSAP14, in the Dehydration Stress Response.

As part of the cellular stress response in plants, the ubiquitin-proteasome system (UPS) plays a crucial role in regulating the protein stability of stress-related transcription factors. Previous study has indicated that CaSAP14 is functionally involved in enhancing pepper plant tolerance to dehydration stress by modulating the expression of downstream genes. However, the comprehensive regulatory mechanism underlying CaSAP14 remains incompletely understood. Here, we identified a RING-type E3 ligase, CaFIRF1, which interacts with and ubiquitinates CaSAP14. Pepper plants with silenced CaFIRF1 exhibited a dehydration-tolerant phenotype when subjected to dehydration stress, while overexpression of CaFIRF1 in pepper and Arabidopsis resulted in reduced dehydration tolerance. Co-silencing of CaFIRF1 and CaSAP14 in pepper increased sensitivity to dehydration, suggesting that CaFIRF1 acts upstream of CaSAP14. A cell-free degradation analysis demonstrated that silencing of CaFIRF1 led to decreased CaSAP14 protein degradation, implicating CaFIRF1 in the regulation of CaSAP14 protein via the 26S proteasomal degradation pathway. Our findings suggest a mechanism by which CaFIRF1 mediates the ubiquitin-dependent proteasomal degradation of CaSAP14, thereby influencing the response of pepper plants to dehydration stress.

DNA damage-induced proteasome phosphorylation controls substrate recognition and facilitates DNA repair

Upon DNA damage, numerous proteins are targeted for ubiquitin-dependent proteasomal degradation, which is an integral part of the DNA repair program. Although details of the ubiquitination processes have been intensively studied, little is known about whether and how the 26S proteasome is regulated in the DNA damage response (DDR). Here, we show that human Rpn10/PSMD4, one of the three ubiquitin receptors of the 26S proteasome, is rapidly phosphorylated in response to different types of DNA damage. The phosphorylation occurs at Rpn10-Ser266 within a conserved SQ motif recognized by ATM/ATR/DNA-PK. Blockade of S266 phosphorylation attenuates homologous recombination-mediated DNA repair and sensitizes cells to genotoxic insults. In vitro and in cellulo experiments indicate that phosphorylation of S266, located in the flexible linker between the two ubiquitin-interacting motifs (UIMs) of Rpn10, alters the configuration of UIMs, and actually reduces ubiquitin chain (substrate) binding. As a result, essential DDR proteins such as BRCA1 are spared from premature degradation and allowed sufficient time to engage in DNA repair, a scenario supported by proximity labeling and quantitative proteomic studies. These findings reveal an inherent self-limiting mechanism of the proteasome that, by controlling substrate recognition through Rpn10 phosphorylation, fine-tunes protein degradation for optimal responses under stress.

Identification of tomato F-box proteins functioning in phenylpropanoid metabolism.

Phenylpropanoids, a class of specialized metabolites, play crucial roles in plant growth and stress adaptation and include diverse phenolic compounds such as flavonoids. Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) are essential enzymes functioning at the entry points of general phenylpropanoid biosynthesis and flavonoid biosynthesis, respectively. In Arabidopsis, PAL and CHS are turned over through ubiquitination-dependent proteasomal degradation. Specific kelch domain-containing F-Box (KFB) proteins as components of ubiquitin E3 ligase directly interact with PAL or CHS, leading to polyubiquitinated PAL and CHS, which in turn influences phenylpropanoid and flavonoid production. Although phenylpropanoids are vital for tomato nutritional value and stress responses, the post-translational regulation of PAL and CHS in tomato remains unknown. We identified 31 putative KFB-encoding genes in the tomato genome. Our homology analysis and phylogenetic study predicted four PAL-interacting SlKFBs, while SlKFB18 was identified as the sole candidate for the CHS-interacting KFB. Consistent with their homolog function, the predicted four PAL-interacting SlKFBs function in PAL degradation. Surprisingly, SlKFB18 did not interact with tomato CHS and the overexpression or knocking out of SlKFB18 did not affect phenylpropanoid contents in tomato transgenic lines, suggesting its irreverence with flavonoid metabolism. Our study successfully discovered the post-translational regulatory machinery of PALs in tomato while highlighting the limitation of relying solely on a homology-based approach to predict interacting partners of F-box proteins.

SETBP1 activation upon MDM4-enhanced ubiquitination of NR3C1 triggers dissemination of colorectal cancer cells.

Colorectal cancer (CRC) presents a growing concern globally, marked by its escalating incidence and mortality rates, thus imposing a substantial health burden. This investigation delves into the role of nuclear receptor subfamily 3 group C member 1 (NR3C1) in CRC metastasis and explores the associated mechanism. Through a comprehensive bioinformatics analysis, NR3C1 emerged as a gene with diminished expression levels in CRC. This finding was corroborated by observations of a low-expression pattern of NR3C1 in both CRC tissues and cells. Furthermore, experiments involving NR3C1 knockdown revealed an exacerbation of proliferation, migration, and invasion of CRC cells in vitro. Subsequent assessments in mouse xenograft tumor models, established by injecting human HCT116 cells either through the tail vein or at the cecum termini, demonstrated a reduction in tumor metastasis to the lung and liver, respectively, upon NR3C1 knockdown. Functionally, NR3C1 (glucocorticoid receptor) suppressed SET binding protein 1 (SETBP1) transcription by binding to its promoter region. Notably, mouse double minute 4 (MDM4) was identified as an upstream regulator of NR3C1, orchestrating its downregulation via ubiquitination-dependent proteasomal degradation. Further investigations unveiled that SETBP1 knockdown suppressed migration and invasion, and epithelial to mesenchymal transition of CRC cells, consequently impeding in vivo metastasis in murine models. Conversely, upregulation of MDM4 exacerbated the metastatic phenotype of CRC cells, a propensity mitigated upon additional upregulation of NR3C1. In summary, this study elucidates a cascade wherein MDM4-mediated ubiquitination of NR3C1 enables the transcriptional activation of SETBP1, thereby propelling the dissemination of CRC cells.

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.

Abstract 4503: Preclinical characterization of PRT7732: A highly potent, selective, and orally bioavailable targeted protein degrader of SMARCA2

Abstract SWI/SNF (BAF) complexes play an important role in controlling gene expression by remodeling chromatin. SMARCA2 (BRM) and SMARCA4 (BRG1) are the core catalytic subunits of the SWI/SNF complexes, containing an ATPase domain and a bromodomain. SMARCA4 protein expression is lost in some cancers due to loss-of-function (LOF) mutations and homozygous deletions, and SMARCA4-deleted cancer cells are highly dependent on its paralog gene SMARCA2 for their survival. Therefore, targeting SMARCA2 in SMARCA4-deficient cancers using selective SMARCA2 degraders induces synthetic lethality while sparing SMARCA4 wild type (WT) normal cells. We have recently identified a series of orally bioavailable SMARCA2 selective degraders that demonstrate robust efficacy in pre-clinical animal models with favorable pharmacokinetic properties and safety profiles. Our nomination candidate PRT7732 exhibits >1000x selectivity for SMARCA2 over SMARCA4 in cell-based assays, with DC50 values in cancer cell lines in the low nanomolar range. The PRT7732-induced SMARCA2 degradation was rescued by a proteasome inhibitor and neddylation inhibitor, indicating ubiquitin-proteasome dependent degradation. Furthermore, PRT7732 does not change levels of known CRBN ligand neo-substrates such as IKZF2/3, GSPT1 and SALL4. PRT7732 inhibits only SMARCA4-deficient cancer cell proliferation with IC50 values ranging from 5.0-50 nM, but not SMARCA4 WT cells in vitro. Oral administration of PRT7732 resulted in near-total degradation of SMARCA2 protein with complete selectivity over SMARCA4 protein in a SMARCA4 WT lung cancer model in mice, consistent with the SMARCA2 degradation kinetics-based pharmacodynamic prediction model. PRT7732 oral daily administration showed significant tumor growth inhibition of SMARCA4-deficient lung cancer xenograft models at well tolerated doses. The treated tumor tissues show robust SMARCA2 protein reduction for 24h post dosing. In summary, our orally bioavailable SMARCA2 degraders induce synthetic lethality in SMARCA4-deficient cancers in vitro and in vivo. Efforts to further evaluate these compounds in additional models and in combination with other agents are ongoing. Citation Format: Artem Shvartsbart, Koichi Ito, Joseph Rager, Michael Hulse, Anjana Agarwal, Komali Vykuntam, Jessica Burtell, Min Wang, Justin Kurian, Miles Cowart, Joy Cote, Nick Stahl, Monisha Sivakumar, Anthony Reichelderfer, Jack Carter, Alexander Grego, Andrew Moore, Neha Bhagwat, Ross Kuskovsky, Shanthi Ganesan, Stefan Ruepp, Tom Emm, Philip Pitis, Corey Basch, Klare Bersch, Yongchun Pan, Song Mei, Raul Leal, John Rose, Dani Roth, Chaoyi Xu, Ganfeng Cao, Kris Vaddi, Sang Hyun Lee, Sandy Geeganage, Andrew Combs, Peggy Scherle. Preclinical characterization of PRT7732: A highly potent, selective, and orally bioavailable targeted protein degrader of SMARCA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4503.

Abstract 3170: Epigenetic identification and functional characterization of a novel RING finger protein tumor suppressor in nasopharyngeal carcinoma and esophageal carcinoma

Abstract RING finger proteins play crucial roles in normal physiology and pathophysiology as important regulators of ubiquitin-dependent proteasomal protein degradation. Many RING finger proteins have also been implicated in tumorigenesis either as oncogenes or tumor suppressors. Through cancer CpG methylome studies, we identified RING finger protein 150 (RNF150) as a methylated target gene in nasopharyngeal carcinoma (NPC) and esophageal carcinoma (ESCC). RNF150 was found to be ubiquitously expressed in human normal tissues but frequently downregulated in NPC and ESCC cells due to its promoter methylation. Its downregulation is associated with poor survival in patients with head and neck cancer and esophageal cancer. We also found that RNF150 is a functional tumor suppressor by inhibiting tumor cell growth and inducing cell cycle arrest in NPC and ESCC cells. Furthermore, ectopic expression of RNF150 inhibited NPC and ESCC cell migration/invasion by suppressing epithelial-mesenchymal transition (EMT) through upregulation of epithelial marker E-cadherin and downregulation of mesenchymal markers (vimentin, N-cadherin, Snail, and Slug). RNF150 is located in the cytoplasm and represses several oncogenic signaling pathways including the Wnt/β-catenin and NF-kB signaling pathways. Liquid chromatography-mass spectrometry (LC-MS) was performed to elucidate RNF150 binding proteins. KEGG enrichment analysis showed that glycolysis/gluconeogenesis, pyruvate metabolism, central carbon metabolism in cancer, and AMPK signaling were the top 4 pathways with significant enrichment, suggesting potential targeting of metabolic molecules by RNF150. Thus, our results provide insights into a new ring finger protein member, RNF150, as a functional tumor suppressor for NPC and ESCC. Our study also suggests a potential epigenetic biomarker for the early dection of NPC and ESCC. Citation Format: Xiaoxue Chai, Feifei Luo, Shujie Bao, Liuzhen Meng, Anthony T. Chan, Brigette B. MA, Qian Tao, li LiLi. Epigenetic identification and functional characterization of a novel RING finger protein tumor suppressor in nasopharyngeal carcinoma and esophageal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3170.

Abstract 2089: A pro-oncogenic RAS-GTP:RAN-GAP complex facilitates nuclear protein export and has clinical implications

Abstract The majority of RAS oncogenic activity is thought to be mediated by its canonical signaling, mainly via RAS-GTP interactions with its effectors RAF and PI3K at the plasma membrane, which respectively activate MEK/ERK and AKT/mTOR pathways. However, in this study, we have identified a new, fundamental, pro-oncogenic, non-canonical activity of RAS-GTP that increases the Exportin 1 (XPO1)-dependent export of nuclear proteins into the cytoplasm and is independent of RAF/MEK and PI3K/AKT signaling. The nuclear protein export machinery is known to depend on a trimeric protein complex composed of RAN-GTP:XPO1:nuclear protein cargo. Unexpectedly, we have found that RAS-GTP forms a stable complex with RAN GTPase-Activating Protein 1 (RAN-GAP) at the nuclear envelope, which facilitates the release of nuclear protein cargo by increasing the hydrolysis of RAN-GTP to RAN-GDP. We have determined that RAS-GTP and RAN-GAP form this complex in primary human lung tumors, patient derived xenografts, and human lung cancer cell lines. Interestingly, the RAS-GTP:RAN-GAP complex is found at the nuclear envelope and in the cytoplasm, but not at the plasma membrane. The RAS-dependent step mediated by the RAS-GTP:RAN-GAP complex acts downstream of XPO1 by increasing RAN-GTP hydrolysis, which promotes protein cargo release. Our fundamental findings have important clinical implications. For example, in human lung cancer models that harbor a KRAS-G12C mutation, the combined inhibition of MEK and PI3K (MEKi + PI3Ki) does not inhibit tumor growth to the same degree as a KRAS-G12C inhibitor. Remarkably, adding an XPO1 inhibitor to MEKi + PI3Ki inhibits tumor growth to the same degree as the KRAS-G12C inhibitor. One critical pro-oncogenic target of the RAS-GTP:RAN-GAP complex is nuclear export of the EZH2 methyltransferase, whose increase in the cytoplasm leads to a direct methylation of the cytoplasmic DLC1 tumor suppressor protein, leading to ubiquitin-dependent proteasomal degradation of DLC1 protein. Analysis of a KRAS mutant cell line from which the DLC1 gene has been disrupted by CRISPR-Cas9 technology indicates that DLC1 makes an important contribution to the growth inhibition by inhibitors that reduce nuclear protein export. These findings establish nuclear protein export as a critical, pro-oncogenic, non-canonical RAS function that is mediated by the RAS-GTP:RAN-GAP complex and provide a mechanistic explanation for poor clinical responses to combined inhibition of MEK and PI3K, which do not regulate the nuclear protein activity identified in this study. Citation Format: Brajendra K. Tripathi, Nicole H. Hirsh, David Hansen, Xiaolan Qian, Marian E. Durkin, Dunrui Wang, Alex G. Papageorge, Ross Lake, Yvonne A. Evrard, Adam Marcus, Suresh S. Ramalingam, Mary Dasso, Karen Vousden, James H. Doroshow, Kylie J. Walters, Douglas R. Lowy. A pro-oncogenic RAS-GTP:RAN-GAP complex facilitates nuclear protein export and has clinical implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2089.

LncRNA HOST2 promotes NSUN2-mediated breast cancer progression via interaction with ELAVL1

Breast cancer (BC) is the most prevalent malignant tumor in women worldwide with high morbidity and mortality. NSUN2, a crucial RNA methyltransferase, plays a pivotal role in regulating the proliferation and metastasis of tumor cells. Our study demonstrated that NSUN2 is upregulated in BC tissues and cell lines, and its high expression is associated with a poor prognosis in BC patients. Knockout of NSUN2 exerted inhibitory effects on the proliferation and migration of BC cells in vitro and in vivo. Mechanistic investigations revealed that the RNA-binding protein ELAVL1 can bind to NSUN2 mRNA and increase its stability. Additionally, we identified HOST2, a long non-coding RNA, as a key player in blocking the ubiquitin-dependent proteasomal degradation of ELAVL1, thereby influencing the stability of NSUN2 mRNA. In conclusion, this study revealed for the first time that HOST2 maintains NSUN2 mRNA stability by blocking ubiquitin-dependent degradation of ELAVL1, which in turn affects BC progression. HOST2/ELAVL1/NSUN2 oncogenic cascade has the potential to be a novel therapeutic target for BC.

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.

Hydrogen Peroxide Inhibits Hepatitis C Virus Replication by Downregulating Hepatitis C Virus Core Levels through E6-Associated Protein-Mediated Proteasomal Degradation.

Hepatitis C virus (HCV) is constantly exposed to considerable oxidative stress, characterized by elevated levels of reactive oxygen species, including hydrogen peroxide (H2O2), during acute and chronic infection in the hepatocytes of patients. However, the effect of oxidative stress on HCV replication is largely unknown. In the present study, we demonstrated that H2O2 downregulated HCV Core levels to inhibit HCV replication. For this purpose, H2O2 upregulated p53 levels, resulting in the downregulation of both the protein and enzyme activity levels of DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b, and activated the expression of E6-associated protein (E6AP) through promoter hypomethylation in the presence of HCV Core. E6AP, an E3 ligase, induced the ubiquitin-dependent proteasomal degradation of HCV Core in a p53-dependent manner. The inhibitory effect of H2O2 on HCV replication was almost completely nullified either by treatment with a representative antioxidant, N-acetyl-L-cysteine, or by knockdown of p53 or E6AP using a specific short hairpin RNA, confirming the roles of p53 and E6AP in the inhibition of HCV replication by H2O2. This study provides insights into the mechanisms that regulate HCV replication under conditions of oxidative stress in patients.

The PIN1-YTHDF1 axis promotes breast tumorigenesis via the m6A-dependent stabilization of AURKA mRNA.

The post-transcriptional processing of N6-methyladenosine (m6A)-modified mRNA by YTH domain-containing family protein 1 (YTHDF1) plays a crucial role in the regulation of gene expression. Although YTHDF1 expression is frequently upregulated in breast cancer, the regulatory mechanisms for this remain unclear. In this study, we examined the role of peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) in regulating YTHDF1 stability in breast cancer cells. The WW domain of PIN1 interacted with YTHDF1 in a phosphorylation-dependent manner. Additionally, PIN1 overexpression increased YTHDF1 stability by preventing ubiquitin-dependent proteasomal degradation. Furthermore, using the MS2-tagged RNA pull-down assay, we identified Aurora kinase A (AURKA) mRNA as a bona fide substrate of YTHDF1. PIN1-mediated YTHDF1 stabilization increased the stability of AURKA mRNA in an m6A-dependent manner. Furthermore, YTHDF1 knockout reduced AURKA protein expression levels, resulting in anticancer effects in breast cancer cells, including decreased cell proliferation, cell cycle arrest at the G0/G1 phase, apoptotic cell death, and decreased spheroid formation. The anticancer effects induced by YTHDF1 knockout were reversed by AURKA overexpression. Similarly, the knockout of PIN1 produced comparable anticancer effects to those observed in YTHDF1-knockout cells, and these effects were reversed upon overexpression of YTHDF1. In conclusion, the findings of our study suggest that increased YTHDF1 stability induced by PIN1 promotes breast tumorigenesis via the stabilization of AURKA mRNA. Targeting the PIN1/YTHDF1 axis may represent a novel therapeutic strategy for breast cancer.

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.

HADHA promotes ovarian cancer outgrowth via up-regulating CDK1

BackgroundOvarian cancer, a prevalent cause of cancer-related mortality among gynecological cancers, still lacks a clear understanding of its pathogenesis. In this study, our objective was to investigate the functional roles and pathogenic mechanisms of HADHA in ovarian cancer.MethodsWe utilized an ovarian cancer tissue microarray and three ovarian cancer cell lines (HO-8910, A2780, and SK-OV-3) for our analysis. Lentiviral-mediated short hairpin RNA (shRNA) was employed to interfere with HADHA expression in ovarian cancer cells. Various cellular events associated with tumor development were assessed using techniques such as Celigo cell counting assay, wound healing assay, Transwell assay, and flow cytometry analysis. Additionally, xenograft tumor models were developed to visualize the impacts of HADHA/CDK1 on ovarian cancer progression.ResultsOur data revealed significant HADHA overexpression in both ovarian cancer tissues and cell lines. Patients with elevated HADHA levels tended to experience poor survival outcomes. Moreover, HADHA upregulation correlated with several pathological parameters, including pathological stage, tumor size, tumor infiltrate, metastasis, and recurrence. Loss-of-function experiments targeting HADHA demonstrated that its suppression in ovarian cancer cells hindered cell growth and migration, while promoting apoptosis. To elucidate the underlying mechanism by which HADHA regulates ovarian cancer, we identified CDK1 as a target of HADHA. HADHA upregulated CDK1 expression by inhibiting its ubiquitination-dependent proteasomal degradation. Significantly, the overexpression of CDK1 reversed the impaired cell development caused by HADHA depletion, both in vitro and in vivo.ConclusionOur study highlights the involvement of HADHA in ovarian cancer tumorigenesis and suggests its potential as a promising prognostic marker in ovarian cancer. Through its regulation of CDK1, HADHA influences critical cellular processes in ovarian cancer, providing insights into its pathogenic mechanism.

Engineered a dual-targeting HA-TPP/A nanoparticle for combination therapy against KRAS-TP53 co-mutation in gastrointestinal cancers

KRAS-TP53 co-mutation is strongly associated with poor prognosis and high malignancy in gastrointestinal cancers. Therefore, a novel approach to oncotherapy may lie in combination therapy targeting both KRAS and TP53. Herein, we present a novel self-assembled nanoparticle (HA-TPP/A) that are functionalized nano-carrier hyaluronic acid (HA)-TPP conjugate (HA-TPP) to degrade mutant p53 proteins (mutp53) and co-deliver AMG510 for treating KRAS-TP53 co-alteration of gastrointestinal cancers by inhibiting the mutant KRAS and mutp53 signaling pathways. The HA-TPP/A nanoparticles led to ubiquitination-dependent proteasomal degradation of mutp53 by targeting damage to mitochondria. Furthermore, these nanoparticles abrogated the gain-of-function (GOF) phenotypes of mutp53 and increased sensitivity to AMG510-induced cell killing, thereby reducing cell proliferation and migration in gastrointestinal cancer with KRAS-TP53 co-mutation. The co-loaded HA-TPP/A nanoparticles demonstrated remarkable therapeutic efficacy in a tumor-bearing mouse model, particularly in KRAS-TP53 double mutant expressing cancer cells, compared with single drug and combined free drug groups. Notably, HA-TPP/A is the first reported nanoparticle with an ability to co-target KRAS-TP53, providing a promising approach for therapy in highly malignant gastrointestinal tumors and potentially expanding clinical indications for AMG510 targeted therapies in gastrointestinal tumors.

USP51 facilitates colorectal cancer stemness and chemoresistance by forming a positive feed-forward loop with HIF1A.

In the current study, we have shown that USP51 promotes colorectal cancer stemness and chemoresistance, and high expression of USP51 predicts survival disadvantage in colorectal cancer patients. Mechanically, USP51 directly binds to Elongin C (ELOC) and forms a larger functional complex with VHL E3 ligase (USP51/VHL/CUL2/ELOB/ELOC/RBX1) to regulate the ubiquitin-dependent proteasomal degradation of HIF1A. USP51 efficiently deubiquitinates HIF1A and activates hypoxia-induced gene transcription. Conversely, the activation of HIF1A under hypoxia transcriptionally upregulates the expression of USP51. Thus, USP51 and HIF1A form a positive feedback loop. Further, we found that the SUMOylation of ELOC at K32 inhibits its binding to USP51. SUMO-specific protease 1 (SENP1) mediates the deSUMOylation of ELOC, promoting the binding of USP51 to ELOC and facilitating the deubiquitination and stabilization of HIF1A by USP51. Importantly, USP51 plays a crucial role in promoting the HIF1A and SENP1-dependent proliferation, migration, stemness, and chemoresistance under hypoxia in colorectal cancer. Together, our data revealed that USP51 is an oncogene stabilizing the pro-survival protein HIF1A, offering a potential therapeutic target for colorectal cancer.