Ubiquitin-proteasome Degradation Research Articles

EMC2 suppresses ferroptosis via regulating TFRC in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy with poorly understood underlying molecular mechanisms. Ferroptosis, a form of programmed cell death, is not fully elucidated in NPC. We conducted quantitative proteomics to detect dysregulated proteins in NPC tissues. The levels of endoplasmic reticulum membrane protein complex 2 (EMC2) in NPC tissue microarrays were evaluated by immunohistochemistry, and the prognostic value of EMC2 was analyzed in NPC patients. The role of EMC2 in ferroptosis and carcinogenesis was determined through in vitro and in vivo experiments. Quantitative proteomics, protease inhibition, ubiquitin detection, and rescue experiments were performed to explore the mechanism of EMC2-regulated ferroptosis. Significantly upregulated EMC2 was detected in NPC, and it was closely related to the characteristics of tumor progression. Elevated EMC2 was obviously correlated with poor survival in patients with NPC. EMC2 knockdown promoted ferroptosis, inhibiting cell viability, migration, and invasion, and enhancing the efficacy of cisplatin in NPC cells. Conversely, EMC2 overexpression contributed to ferroptosis repression, malignant progression, and reduced the efficacy of cisplatin. In addition, EMC2 knockdown suppressed xenograft tumor growth and enhanced ferroptosis in nude mice. Mechanistically, we identified transferrin receptor (TFRC) as a critical downstream protein. EMC2 interacted with TFRC and promoted its ubiquitin-proteasomal degradation. EMC2 regulated ferroptosis by mediating the level of TFRC. EMC2 suppresses ferroptosis and promotes tumor progression, and the EMC2-TFRC axis is a novel ferroptosis regulatory pathway. EMC2 is a potentially biomarker and therapeutic target for NPC.

SENP2 negatively regulates RIG-I/MDA5 mediated innate immunity in black carp.

Mammalian SUMO specific peptidase 2 (SENP2) plays vital roles in a variety of biological procedures including the immune response. However, the effects of teleost SENP2 are still mostly unexplored. In this study, the SENP2 of black carp (Mylopharyngodon piceus) was cloned and characterized. The open reading frame of black carp SENP2 (bcSENP2) consists of 1800 nucleotides, which encode 600 amino acids. The reporter assay results showed that over-expression of bcSENP2 alone had a weak effect on interferon (IFN) promoter transcription activity, whereas it significantly reduced bcMDA5/bcRIG-I mediated IFN promoter transcription activity. The interaction between bcSENP2 and bcMDA5 or bcRIG-I was detected by immunoprecipitation experiments. The plaque assay and qPCR results indicated that bcMDA5 or bcRIG-I mediated antiviral capacity was attenuated by bcSENP2, while knockdown of bcSENP2 led to enhanced antiviral resistance to SVCV in host cells. In addition, the expression level of bcMDA5/bcRIG-I protein was attenuated by co-expressed bcSENP2 and MG132 treatment rescued this attenuating effect. All of these data support the conclusion that bcSENP2 inhibits bcMDA5/bcRIG-I mediated antiviral signaling by enhancing ubiquitin-proteasome mediated degradation of bcMDA5/bcRIG-I in black carp.

SKP1-CUL1-F-box: Key molecular targets affecting disease progression.

The correct synthesis and degradation of proteins are vital for numerous biological processes in the human body, with protein degradation primarily facilitated by the ubiquitin-proteasome system. The SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase, a member of the Cullin-RING E3 ubiquitin ligase (CRL) family, plays a crucial role in mediating protein ubiquitination and subsequent 26S proteasome degradation during normal cellular metabolism. Notably, SCF is intricately linked to the pathogenesis of various diseases, including malignant tumors. This paper provides a comprehensive overview of the functional characteristics of SCF complexes, encompassing their assembly, disassembly, and regulatory factors. Furthermore, we discuss the diverse effects of SCF on crucial cellular processes such as cell cycle progression, DNA replication, oxidative stress response, cell proliferation, apoptosis, cell differentiation, maintenance of stem cell characteristics, tissue development, circadian rhythm regulation, and immune response modulation. Additionally, we summarize the associations between SCF and the onset, progression, and prognosis of malignant tumors. By synthesizing current knowledge, this review aims to offer a novel perspective for a holistic and systematic understanding of SCF complexes and their multifaceted functions in cellular physiology and disease pathogenesis.

Limiting cap-dependent translation increases 20S proteasomal degradation and protects the proteomic integrity in autophagy-deficient skeletal muscle

ABSTRACT Postmitotic skeletal muscle critically depends on tightly regulated protein degradation to maintain proteomic stability. Impaired macroautophagy/autophagy-lysosomal or ubiquitin-proteasomal protein degradation causes the accumulation of damaged proteins, ultimately accelerating muscle dysfunction with age. While in vitro studies have demonstrated the complementary nature of these systems, their interplay at the organism levels remains poorly understood. Here, our study reveals novel insights into this complex relationship in autophagy-deficient skeletal muscle. We demonstrated that despite a compensatory increase in proteasome level in response to autophagy impairment, 26S proteasome activity was not proportionally enhanced in autophagy-deficient skeletal muscle. This functional deficit was partly attributed to reduced ATP levels to fuel the 26S proteasome. Remarkably, we found that activation of EIF4EBP1, a crucial inhibitor of cap-dependent translation, restored and even augmented proteasomal function through dual mechanisms. First, genetically activating EIF4EBP1 enhanced both ATP-dependent 26S proteasome and ATP-independent 20S proteasome activities, thereby expanding overall protein degradation capacity. Second, EIF4EBP1 activation caused muscle fiber transformation and increased mitochondrial biogenesis, thus replenishing ATP levels for 26S proteasome activation. Notably, the improved performance of the 20S proteasome in EIF4EBP1-activated skeletal muscle was attributed to an increased abundance of the immunoproteasome, a subtype specially adapted to function under oxidative stress conditions. This dual action of EIF4EBP1 activation preserved proteomic integrity in autophagy-deficient skeletal muscle. Our findings uncover a novel role of EIF4EBP1 in improving protein quality control, presenting a promising therapeutic strategy for autophagy-related muscular disorders and potentially other conditions characterized by proteostatic imbalance.

KEAP1-NRF2 Interaction in Cancer: Competitive Interactors and Their Role in Carcinogenesis

The KEAP1−NRF2 signaling pathway is one of the main regulators of the cellular response to oxidative and electrophilic stress. NRF2 is a transcription factor that controls more than 200 cytoprotective genes encoding proteins involved in detoxification, antioxidant protection, carbohydrate metabolism, NADPH regeneration, lipid metabolism, heme and iron metabolism, transcription factors, and the regulation of the proteasome and autophagy. KEAP1 is an inhibitory protein that maintains low levels of NRF2 under basal conditions by targeting it for degradation via the CUL3-RBX1 E3 ubiquitin ligase complex, leading to NRF2 ubiquitination and subsequent proteasomal degradation. Dysregulation of the KEAP1-NRF2 pathway has been observed in various non-communicable diseases, including cancer. NRF2 activity has a dual role in cancer: it prevents cancer initiation by protecting the cells from oxidative and electrophilic damage that can lead to genomic instability and DNA damage. However, in later stages of cancer, overactivation of NRF2 promotes cancer progression by protecting cancer cells from the reactive oxygen species (ROS) induced cell death, enabling the detoxification of chemotherapeutics, promoting metabolic reprogramming, and suppressing the immune response by reducing inflammation. One mechanism of NRF2 activation in cancer involves the disruption of the KEAP1-NRF2 interaction through the binding of competitive disruptor proteins to KEAP1. This prevents NRF2 ubiquitination and degradation. This review provides an overview of the most prominent competitive interactors of KEAP1, including SQSTM1, MCM3, PALB2, IKKβ, DPP3, PGAM5, PTMA, FAM129B, and WTX, as well as several less well-characterized KEAP1 interactors, and their potential roles in carcinogenesis.

Ras Guanine Nucleotide-Releasing Protein-4 Inhibits Erythropoietin Production in Diabetic Mice with Kidney Disease by Degrading HIF2A.

In acute and chronic renal inflammatory diseases, the activation of inflammatory cells is involved in the defect of erythropoietin (EPO) production. Ras guanine nucleotide-releasing protein-4 (RasGRP4) promotes renal inflammatory injury in type 2 diabetes mellitus (T2DM). Our study aimed to investigate the role and mechanism of RasGRP4 in the production of renal EPO in diabetes. The degree of tissue injury was observed by pathological staining. Inflammatory cell infiltration was analyzed by immunohistochemical staining. Serum EPO levels were detected by enzyme-linked immunosorbent assay, and EPO production and renal interstitial fibrosis were analyzed by immunofluorescence. Quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression of key inflammatory factors and the activation of signaling pathways. In vitro, the interaction between peripheral blood mononuclear cells (PBMCs) and C3H10T1/2 cells was investigated via cell coculture experiments. RasGRP4 decreased the expression of hypoxia-inducible factor 2-alpha (HIF2A) via the ubiquitination-proteasome degradation pathway and promoted myofibroblastic transformation by activating critical inflammatory pathways, consequently reducing the production of EPO in T2DM mice. RasGRP4 participates in the production of renal EPO in diabetic mice by affecting the secretion of proinflammatory cytokines in PBMCs, degrading HIF2A, and promoting the myofibroblastic transformation of C3H10T1/2 cells.

The unique structure of the highly conserved PPLP region in HIV-1 Vif is critical for the formation of APOBEC3 recognition interfaces.

The human cellular cytidine deaminases APOBEC3s (A3s) inhibit virion infectivity factor (Vif)-deficient HIV-1 replication. However, virus-encoded Vifs abolish this defense system by specifically recruiting A3s to an E3 ubiquitin ligase complex to induce their degradation. The highly conserved Vif PPLP motif is critical for the Vif-mediated antagonism of A3s and is believed to be important for Vif multimerization. However, how the PPLP motif dictates the functions of Vif remains unclear. Here, we aimed to elucidate this mechanism using biochemical and structural biology approaches. First, we found that no stable Vif multimer complexes formed in our tandem coimmunoprecipitation assays. Next, a series of Vif truncation mutants were constructed, and the short α-helix α6 just downstream of PPLP was found to be the smallest fragment essential for efficient A3G degradation in cells. In silico structural analysis suggested that PPLP-α6 adopts a stable L-shaped conformation when complexed in Vif/CBF-β and contributes to the structural integrity of Vif. In vitro ubiquitination assays with recombinant proteins confirmed that PPLP-α6 is necessary to form the functional complex of the E3 ligase adaptor of Vif/CBF-β/elongin B/elongin C. Additionally, mutations of the highly conserved PPLP-α6 hydrophobic residues severely disrupted Vif function. In the Vif structure, PPLP-α6 is positioned behind α1-α2 that constitutes the A3-binding Vif interfaces. Therefore, both the PPLP motif and α6 play critical allosteric roles in maintaining the integrity of the A3 interaction interfaces. Our findings will also provide important data for the design of novel anti-HIV-1 compounds that disrupt the A3-binding Vif interfaces.IMPORTANCEThe APOBEC3 (A3) family enzymes potently block the replication of retroviruses, such as HIV-1. However, HIV-1 expresses Vif, a small multifaceted protein that binds and specifically eliminates A3s in infected cells via ubiquitination-proteasome degradation. Thus, A3-Vif interactions are attractive targets for anti-HIV-1 drug development. The Vif PPLP motif that is distal from these interfaces is necessary for A3 degradation; however, the mechanism by which PPLP participates in A3 degradation is unknown. In this study, we performed biochemical and structural biology analyses to elucidate the underlying mechanisms involved. We found that the PPLP motif, in addition to the short downstream fragment α6, forms a stable L-shaped conformation and acts as a scaffold for the A3 recognition interfaces. Importantly, mutations in α6 abolished Vif function to antagonize multiple A3 family enzymes. These findings provide important data for the development of novel HIV-1 inhibitors that utilize A3s as cellular defense enzymes.

Age‑related changes in endoplasmic reticulum stress response‑associated protein expression in rat tibial nerves.

In age-related peripheral neurodegeneration, changes in the promotion or inhibition of endoplasmic reticulum (ER) stress response related to the ubiquitin-proteasome degradation system (UPS), autophagy and apoptosis signaling factors during aging remain unclear. In the present study, the expression of ER stress response signaling-related protein factors was examined in tibial nerves during aging in rats. Tibial nerves were extracted from continuously housed rats at 20, 50, 70, 90 and 105 weeks of age. Expression of factors associated with ER stress-related degradation, including X-box binding protein 1 (XBP1s), eukaryotic translation initiation factor 2 subunit 1 (eIF2α), Beclin-1 (Becn1), and Caspase-3 (Casp3); ER stress-related repair, including binding immunoglobulin protein [also known as 78 kDa glucose-regulated protein (BiP/GRP78)], protein disulfide isomerase (PDI), brain-derived neurotrophic factor (BDNF) and the inflammatory cytokine IL6, was assessed by western blotting of tibial nerves from rats in each age group. Expression of XBP1s and Becn1, which promote UPS and autophagy, decreased significantly after 50 weeks of age. However, expression of eIF2α and Casp3, which inhibit new protein biosynthesis and promote apoptosis, increased significantly after 50 weeks. Expression of BiP/GRP78 and PDI, which are refolding factors for denatured proteins, showed a significant decrease after 50 (or 70) weeks of age. The expression of BDNF, a ligand protein for the repair cascade, showed a significant increase after 70 weeks of age, while that of IL6 increased significantly after 50 weeks of age. These results indicate that ER stress-related degradation (UPS and autophagy) and refolding repair functions are reduced in rat tibial nerves after 50 weeks, followed by enhanced apoptosis and inflammation. These findings shed light on the progression of age-related peripheral neurodegeneration in rats.

TRIM8 inhibits porcine epidemic diarrhoea virus replication by targeting and ubiquitinately degrading the nucleocapsid protein

Porcine epidemic diarrhoea virus (PEDV) is an enteric pathogen that causes acute diarrhoea, dehydration and high mortality rates in suckling pigs. Tripartite motif 8 (TRIM8) has been shown to play multiple roles in the host’s defence against viral infections. However, the functions of TRIM8 in regulating PEDV infection are still not well understood. In our study, we found a significant upregulation of TRIM8 following PEDV infection. We created TRIM8 knockout and overexpression cell lines and discovered that TRIM8 can inhibit PEDV replication within host cells. Co-immunoprecipitation assays revealed that TRIM8 directly interacts with the nucleocapsid protein (N) of PEDV, specifically within the coiled-coil structural domain of TRIM8. Furthermore, TRIM8 was shown to reduce the expression of the PEDV N protein in a dose-dependent manner. Mechanistically, TRIM8 inhibits the expression of PEDV N through K48-linked ubiquitin proteasome degradation. Transcriptomics analysis revealed that TRIM8 facilitates the expression of genes associated with several pathways, including the IL-17 signalling pathway, chemokine signalling pathway, and cytokine-cytokine receptor interaction. This suggests that TRIM8 plays a crucial role in boosting antiviral immune responses against PEDV infection. Our findings provide new insights into the functions and mechanisms of TRIM8 in regulating PEDV infection and highlight its potential as a molecular target for the prevention and control of this virus.

Protein phosphatase EYA1 regulates the dephosphorylation and turnover of BCL2L12 to promote glioma development.

Glioma is the most prevalent and deadly type of intracranial tumor. Understanding the molecular drivers and their underlying mechanisms in glioma development is urgently needed. EYA1 is a unique protein phosphatase that drives gliomagenesis, yet its substrates remain largely uncharacterized. In this study, we identify BCL2L12 (BCL2-like 12), a critical oncoprotein in glioma, as a novel substrate of EYA1 phosphatase in glioma cells. Our findings demonstrate that EYA1 dephosphorylates BCL2L12 at threonine-33 (T33), which in turn protects BCL2L12 from ubiquitination and subsequent proteasomal degradation. Our results indicate that BCL2L12 partially mediates the oncogenic roles of EYA1 in promoting glioma cell proliferation, highlighting the significance of EYA1's dephosphorylation of BCL2L12 in tumor progression. Moreover, we validate a positive correlation between EYA1 and BCL2L12 protein levels in glioma patient samples. In summary, our study reveals how EYA1-BCL2L12 interaction functions in glioma development, implicating EYA1 as a potential therapeutic target for glioma treatment.

P4HA3 depletion induces ferroptosis and inhibits colorectal cancer growth by stabilizing ACSL4 mRNA.

Colorectal cancer (CRC) is a malignancy with high global incidence and mortality rates, posing a serious threat to human health. Despite favorable outcomes following early detection and surgical intervention, the asymptomatic nature of CRC often results in delayed diagnoses, limiting surgical treatment options. Furthermore, effective therapeutic drugs for CRC remain lacking in clinical practice, highlighting an urgent need to identify novel therapeutic targets. In this study, we identified that prolyl 4-hydroxylase subunit alpha 3 (P4HA3) is significantly upregulated in CRC and is associated with poor prognosis in patients. Both in vitro and in vivo experiments demonstrated that knockdown of P4HA3 induces ferroptosis, thereby inhibiting tumor growth. This ferroptosis induction is closely linked to increased lipid peroxidation, and P4HA3 knockdown promotes ferroptosis by upregulating acyl-CoA synthetase long-chain family member 4 (ACSL4), which regulates polyunsaturated fatty acid-containing phospholipids (PUFA-PLs) biosynthesis. Mechanistically, P4HA3 knockdown stabilizes ACSL4 mRNA by downregulating AUF1, an important RNA-binding protein (RBP) that binds to AU-rich elements (AREs) in the ACSL4 mRNA 3' untranslated region (UTR), thereby preventing its degradation. Additionally, given the lack of research on P4HA3 inhibitors, we employed virtual screening and identified Tubuloside A as a potential therapeutic agent. Tubuloside A promotes the ubiquitin-proteasome degradation of P4HA3, exerting anti-CRC effects. In summary, our findings demonstrate that P4HA3 protects CRC cells from ferroptosis by regulating ACSL4 mRNA stability via AUF1, and Tubuloside A serves as a potential P4HA3 degrader, offering a promising therapeutic strategy for CRC treatment.

IL-6 Promotes Muscle Atrophy by Increasing Ubiquitin–Proteasome Degradation of Muscle Regeneration Factors After Cerebral Infarction in Rats

IL-6 Promotes Muscle Atrophy by Increasing Ubiquitin–Proteasome Degradation of Muscle Regeneration Factors After Cerebral Infarction in Rats

Aspirin-based PROTACs as COX-2 degraders for anti-inflammation.

Cyclooxygenase-2 (COX-2) is a key enzyme in the biosynthesis of prostaglandins and plays a special role in the process of inflammatory response. COX-2 is a target of non-steroidal anti-inflammatory drugs (NSAIDs), which can effectively relieve inflammation, pain and fever responses by inhibiting COX-2. Despite the significant study progress of inhibitors targeting COX-2, the development of COX-2 degraders remains insufficient. Proteolysis targeting chimaeras (PROTACs) have recently emerged as a fascinating technology for targeted protein degradation and drug discovery. In this report, we present the design, synthesis and detection of aspirin-based PROTACs that demonstrate effective ubiquitin-proteasome pathway degradation of COX-2 in lipopolysaccharide-stimulated RAW264.7 cells, and the aspirin-based negative PROTACs does not promote the degradation of COX-2. Moreover, we show AspPROTACs could significantly affect proteasome degradation and inflammatory signaling pathways through quantitative proteomic data analysis. These COX-2 degraders offer valuable chemical tools and novel insights for research in anti-inflammatory drugs.

Disturbed shear stress promotes atherosclerosis through TRIM21-regulated MAPK6 degradation and consequent endothelial inflammation.

Coronary artery plaques often develop in regions subjected to disturbed shear stress (DSS), yet the mechanisms underlying this phenomenon remain poorly understood. Our study aimed to elucidate the unknown role of MAPK6 in shear stress and plaque formation. In vitro and in vivo experiments, RNA-seq, CO-IP and proteomic analysis, combined with single-cell RNA-seq datasets were used to reveal the upstream and downstream mechanisms involved. AAV-MAPK6, ApoE-/-MAPK6flox/floxTEKCre mice and the CXCL12 neutraligand were used to confirm the beneficial effects of MAPK6 against atherosclerosis. Our study revealed a substantial decrease in MAPK6 protein levels in endothelial cells in response to DSS, both in vivo and in vitro, which was contingent on the binding of the ubiquitin ligase TRIM21 to MAPK6. Endothelium-specific MAPK6 overexpression exerts antiatherosclerotic effects in ApoE-/- mice, elucidating the unexplored role of MAPK6 in atherosclerosis. Comprehensive RNA-seq, integrated single-cell mapping and further experiments unveiled the involvement of MAPK6 in inflammation through the EGR1/CXCL12 axis. ApoE-/-MAPK6flox/floxTEKCre mice finally confirmed that conditional MAPK6 knockout resulted in endothelial inflammation and significant increases in plaque areas. Notably, these effects could be reversed through the neutralization of CXCL12. Our study illuminates the advantages of MAPK6 in decelerating plaque progression, highlighting the potential of safeguarding MAPK6 as a novel therapeutic strategy against atherosclerosis. Disturbed flow activates the ubiquitin‒proteasome degradation pathway of MAPK6 in endothelial cells, which is contingent on the binding of the ubiquitin ligase TRIM21 to MAPK6. Endothelial MAPK6 has an advantageous impact on decelerating plaque progression. MAPK6 regulates endothelial inflammation via the EGR1/CXCL12 axis.

Non-canonical Function of Prolyl Hydroxylase Domain 2 in Breast Cancer Cell Growth and Progression: Role of Peptidyl-prolyl Cis-trans Isomerase NIMA-interacting 1.

Prolyl hydroxylase domain 2 (PHD2) is the primary oxygen sensing enzyme involved in hydroxylation of hypoxia-inducible factor (HIF). Under normoxic conditions, PHD2 hydroxylates specific proline residues in HIF-1α and HIF-2α, promoting their ubiquitination and subsequent proteasomal degradation. Although PHD2 activity decreases in hypoxia, notable residual activity persists, but its function in these conditions remains unclear. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) targets proteins with phosphorylated serine/threonine-proline (pSer/Thr-Pro) motifs. As PHD2 contains several pSer/Thr-Pro motifs, it may be a potential substrate of Pin1. In the present study, we found Pin1 and PHD2 interactions in human breast cancer MDA-MB-231 cells. The breast cancer tissue array revealed higher levels of PHD2 and Pin1 in tumors compared to adjacent normal tissues. Through liquid chromatography-tandem mass spectrometry spectrometry, three phosphorylation sites (S125, T168, and S174) on PHD2 were identified, with serine 125 as the main site for Pin1 binding. As a new Pin1 binding partner, oncogenic PHD2 could be a potential therapeutic target for breast cancer treatment.

VCP controls KCC2 degradation through FAF1 recruitment and accelerates emergence from anesthesia

Ubiquitin-proteasomal degradation of K+/Cl- cotransporter 2 (KCC2) in the ventral posteromedial nucleus (VPM) has been demonstrated to serve as a common mechanism by which the brain emerges from anesthesia and regains consciousness. Ubiquitin-proteasomal degradation of KCC2 during anesthesia is driven by E3 ligase Fbxl4. However, the mechanism by which ubiquitinated KCC2 is targeted to the proteasome has not been elucidated. We report in cultured neuro-2a cells that the valosin-containing protein (VCP) transported ubiquitinated KCC2 to the proteasome and in mice in vivo experiments that inhibition of VCP restored KCC2 expression in the VPM and enhanced the effects of anesthesia. In cultured neuro-2a cells, propofol-induced degradation of KCC2 was inhibited by VCP inhibitor DBeQ and VCP knockout plasmid sgRNA(VCP). Propofol-induced enhanced interaction between VCP and KCC2 was inhibited by knockout of Fbxl4 or Fas-associated factor 1 (FAF1). In in vivo studies, pharmacological or genetic inhibition of VCP in the VPM significantly prevented KCC2 degradation and enhanced propofol anesthesia; these effects were abrogated by a KCC2 antagonist VU0463271. These results demonstrate that the VCP controls ubiquitin-proteasomal degradation of KCC2 dependent on FAF1 recruitment and serves as a mechanism for the ubiquitin-proteasomal degradation of KCC2, which is responsible for the subsequent emergence from anesthesia.

Targeting CDK2 Confers Vulnerability to Lenvatinib Via Driving Senescence in Anaplastic Thyroid Cancer

AbstractAnaplastic thyroid cancer (ATC) is the most lethal tumor arising from thyroid follicular epithelium. Lenvatinib is an off‐label use option for ATC patients in many countries but an approved prescription in Japan. However, lenvatinib resistance is a substantial clinical challenge. Clinical ATC samples including lenvatinib‐resistant tumors are used to build patient‐derived cells and patient‐derived xenografts. High‐throughput drug screening and synergy analyses are performed to identify an effective combination partner for lenvatinib. Cellular functions are detected by cell senescence, apoptosis, cell cycle, cell viability and colony formation assays. CDK2 inhibition showed the significant synthetic lethality with lenvatinib via inhibiting G1/S transition and inducing cell senescence in ATC. High expression of CDK2 is associated with lenvatinib resistance and poor clinical outcomes of ATC patients. Lenvatinib increased protein expression of CDK2 in lenvatinib‐resistant ATC cells. Mechanistically, lenvatinib inhibited protein degradation of CDK2 via reducing CDK2's interaction with the RACK1‐FBW7 complex, which is involved in ubiquitination and subsequent proteasomal degradation of CDK2. Combination of CDK2 inhibitors in clinical trials (Dinaciclib or PF‐07104091) and lenvatinib markedly suppressed growth of xenograft tumors from the lenvatinib‐resistant patient. The findings support the combination therapy strategy of lenvatinib and CDK2 inhibitor for lenvatinib‐resistant ATC patients with high CDK2 expression.

Adverse Effects of Nrf2 in Different Organs and the Related Diseases.

Significance: Under normal physiological conditions, Nrf2 undergoes ubiquitination and subsequent proteasome degradation to maintain its basal activity. Oxidative stress can trigger Nrf2 activation, prompting its translocation to the nucleus where it functions as a transcription factor, activating various antioxidant pathways, and conferring antioxidant properties. Recent Advances: While extensive research has shown Nrf2's protective role in various diseases, emerging evidence suggests that Nrf2 activation can also produce harmful effects. Critical Issues: This review examines the pathological contexts in which Nrf2 assumes different roles, emphasizing the mechanisms and conditions that result in adverse outcomes. Future Directions: Persistent Nrf2 activation may have deleterious consequences, necessitating further investigation into the specific conditions and mechanisms through which Nrf2 exerts its harmful effects. Antioxid. Redox Signal. 00, 000-000.

Diacylglycerol Kinase ζ Attenuates Doxorubicin-Induced Cardiotoxicity Through p53 Degradation.

Doxorubicin-induced cardiotoxicity is still an important medical problem associated with a high mortality rate in cancer survivors. p53 plays a key role in doxorubicin-induced cardiotoxicity. Diacylglycerol kinase ζ (Dgkζ), a 130-kDa enzyme abundant in cardiomyocytes, regulates the p53 protein expression level in neurons. To elucidate the mechanism of doxorubicin-induced cardiotoxicity, we focused on the functional role of Dgkζ and its interaction with heat shock protein 70 (Hsp70)-related ubiquitin E3 ligases such as E6-associated protein (E6ap) and C-terminus of Hsp70-interacting protein. Protein interactions of Dgkζ with Hsp70 and E6ap were confirmed by immunoprecipitation, but not C-terminus of Hsp70-interacting protein. We administered doxorubicin in cardiac-specific overexpression of Dgkζ transgenic (Dgkζ-Tg) mice and wild-type littermates. Dgkζ-Tg mice showed lower p53 protein expression levels, preserved cardiac function, and improved survival rates compared with wild-type littermates after doxorubicin administration. RNA sequence analysis of myocardial tissues from Dgkζ-Tg after doxorubicin stimulation identified Hspa1b encoding Hsp70 as the differentially expressed gene. Dgkζ overexpression increased proteasomal p53 degradation and attenuated cardiomyocyte apoptosis after doxorubicin stimulation in cardiomyocytes, which was reversed by knockdown of E6ap. Dgkζ interacted with E6ap through ankyrin-like repeats. The overexpression of mutant Dgkζ, lacking ankyrin-like repeats, failed to inhibit p53 protein expression after doxorubicin stimulation. In Dgkζ-overexpressing cardiomyocytes, expression levels of p53 and caspase-3 were increased by knockdown of the C-terminus of Hsp70-interacting protein. We demonstrated for the first time that Dgkζ augments p53 ubiquitin-proteasome degradation and ameliorates doxorubicin-induced cardiotoxicity by interacting with Hsp70 and E3 ligases such as E6ap and C-terminus of Hsp70-interacting protein.

Targeting PPARγ via SIAH1/2-mediated ubiquitin-proteasomal degradation as a new therapeutic approach in luminal-type bladder cancer

Bladder cancer (BC) is the second most prevalent genitourinary malignancy worldwide. Despite recent approvals of immune checkpoint inhibitors and targeted therapy for muscle invasive or recurrent BC, options remain limited for patients with non-muscle invasive BC (NMIBC) refractory to Bacillus Calmette-Guérin (BCG) and chemotherapy. NMIBC is more frequently classified as a luminal subtype, in which increased PPARγ activity is a key feature in promoting tumor growth and evasion of immunosurveillance. Cinobufotalin is one of the major compound of bufadienolides, the primary active components of toad venom that has been utilized in the clinical treatment of cancer. We herein focused on cinobufotalin, examining its anticancer activity and molecular mechanisms in luminal-type NMIBC. Our results newly reveal that cinobufotalin strongly suppresses the viability and proliferation of luminal BC cells with minimal cytotoxic effects on normal uroepithelial cells, and exhibits significant antitumor activity in a RT112 xenograft BC model. Mechanistically, our sub-G1-phase cell accumulation, Annexin V staining, caspase-3/8/9 activation, and PARP activation analyses show that cinobufotalin induces apoptosis in luminal-type BC cells. Cinobufotalin significantly inhibited the levels of PPARγ and its downstream targets, as well as lipid droplet formation and free fatty acid levels in RT112 cells. PPARγ overexpression rescued RT112 cells from cinobufotalin-induced apoptosis and mitigated the downregulation of FASN and PLIN4. Finally, we show seemingly for the first time that cinobufotalin promotes SIAH1/2-mediated proteasomal degradation of PPARγ in luminal BC cells. Together, these findings compellingly support the idea that cinobufotalin could be developed as a promising therapeutic agent for treating luminal-type NMIBC.