Omentin-1 May Be One Treatment Factor for Intravenous Thrombolysis of Acute Cerebral Infarction Through the Inhibition of NLRP3 Ubiquitination by AMPK Function: Preliminary Findings.

BACKGROUND: Acute cerebral infarction (ACI) is a common neurological disease that is associated with high morbidity, disability and mortality rates. At present, antiplatelet therapy is a necessary treatment for ACI. The present study aimed to investigate the effects of omentin-1 on the intravenous thrombolysis of ACI. OBJECTIVE: The present study aimed to investigate the effects of omentin-1 on the intravenous thrombolysis of ACI. MATERIAL AND METHODS: The mouse model of ACI was induced using male C57BL/6 mice through middle cerebral artery occlusion (MCAO). Meanwhile, the murine BV2 microglial cells were pretreated with 0.1 mg/ml of lipopolysaccharide (LPS), and then induced with 2 mM of adenosine triphosphate (ATP). RESULTS: The omentin-1 mRNA expression in patients receiving intravenous thrombolysis for ACI was down-regulated compared with the normal group. Additionally, the serum level of omentin-1 was negatively correlated with National Institute of Health Stroke Scale (NIHSS) score or serum level of IL-1ß or MMP-2 in patients receiving intravenous thrombolysis for ACI. Meanwhile, the serum mRNA expression of omentin-1 was positively correlated with Barthel index or high-sensitivity C-reactive protein (hs-CRP) in patients undergoing intravenous thrombolysis for ACI. As observed from the in vitro model, Omentin-1 reduced inflammation, promoted cell growth, alleviated ROS-induced oxidative stress, and enhanced AMPK activity through activating NLRP3 ubiquitination. Omentin-1 presented ACI in the mouse model of ACI. Regulating AMPK activity contributed to controlling the effects of Omentin-1 on the in vitro model. CONCLUSIONS: Omentin-1 reduced neuroinflammation and ROS-induced oxidative stress in the mouse model of ACI, which was achieved by inhibiting NLRP3 ubiquitination through regulating AMPK activity. Therefore, omentin-1 may serve as a treatment factor for the intravenous thrombolysis of ACI in further clinical application.

Sinensetin protects against periodontitis through binding to Bach1 enhancing its ubiquitination degradation and improving oxidative stress.

Periodontitis is a chronic inflammatory and immune reactive disease induced by the subgingival biofilm. The therapeutic effect for susceptible patients is often unsatisfactory due to excessive inflammatory response and oxidative stress. Sinensetin (Sin) is a nature polymethoxylated flavonoid with anti-inflammatory and antioxidant activities. Our study aimed to explore the beneficial effect of Sin on periodontitis and the specific molecular mechanisms. We found that Sin attenuated oxidative stress and inflammatory levels of periodontal ligament cells (PDLCs) under inflammatory conditions. Administered Sin to rats with ligation-induced periodontitis models exhibited a protective effect against periodontitis in vivo. By molecular docking, we identified Bach1 as a strong binding target of Sin, and this binding was further verified by cellular thermal displacement assay and immunofluorescence assays. Chromatin immunoprecipitation-quantitative polymerase chain reaction results also revealed that Sin obstructed the binding of Bach1 to the HMOX1 promoter, subsequently upregulating the expression of the key antioxidant factor HO-1. Further functional experiments with Bach1 knocked down and overexpressed verified Bach1 as a key target for Sin to exert its antioxidant effects. Additionally, we demonstrated that Sin prompted the reduction of Bach1 by potentiating the ubiquitination degradation of Bach1, thereby inducing HO-1 expression and inhibiting oxidative stress. Overall, Sin could be a promising drug candidate for the treatment of periodontitis by targeting binding to Bach1.

USP19 regulates DNA methylation damage repair and confers temozolomide resistance through MGMT stabilization.

OBJECTIVE: To elucidate the relationship between USP19 and O(6)-methylguanine-DNA methyltransferase (MGMT) after temozolomide treatment in glioblastoma (GBM) patients with chemotherapy resistance. METHODS: Screening the deubiquitinase pannel and identifying the deubiquitinase directly interacts with and deubiquitination MGMT. Deubiquitination assay to confirm USP19 deubiquitinates MGMT. The colony formation and tumor growth study in xenograft assess USP19 affects the GBM sensitive to TMZ was performed by T98G, LN18, U251, and U87 cell lines. Immunohistochemistry staining and survival analysis were performed to explore how USP19 is correlated to MGMT in GBM clinical management. RESULTS: USP19 removes the ubiquitination of MGMT to facilitate the DNA methylation damage repair. Depletion of USP19 results in the glioblastoma cell sensitivity to temozolomide, which can be rescued by overexpressing MGMT. USP19 is overexpressed in glioblastoma patient samples, which positively correlates with the level of MGMT protein and poor prognosis in these patients. CONCLUSION: The regulation of MGMT ubiquitination by USP19 plays a critical role in DNA methylation damage repair and GBM patients' temozolomide chemotherapy response.

An estrogen receptor α-derived peptide improves glucose homeostasis during obesity.

Estrogen receptor α (ERα) plays a crucial role in regulating glucose and energy homeostasis during type 2 diabetes mellitus (T2DM). However, the underlying mechanisms remain incompletely understood. Here we find a ligand-independent effect of ERα on the regulation of glucose homeostasis. Deficiency of ERα in the liver impairs glucose homeostasis in male, female, and ovariectomized (OVX) female mice. Mechanistic studies reveal that ERα promotes hepatic insulin sensitivity by suppressing ubiquitination-induced IRS1 degradation. The ERα 1-280 domain mediates the ligand-independent effect of ERα on insulin sensitivity. Furthermore, we identify a peptide based on ERα 1-280 domain and find that ERα-derived peptide increases IRS1 stability and enhances insulin sensitivity. Importantly, administration of ERα-derived peptide into obese mice significantly improves glucose homeostasis and serum lipid profiles. These findings pave the way for the therapeutic intervention of T2DM by targeting the ligand-independent effect of ERα and indicate that ERα-derived peptide is a potential insulin sensitizer for the treatment of T2DM.

Identification of a novel histone H2A mono-ubiquitination-inhibiting cell-active small molecule.

Histone H2A mono-ubiquitination plays important roles in epigenetic gene expression and is also involved in tumorigenesis. Small molecules controlling H2A ubiquitination are of interest as potential chemical tools and anticancer drugs. To identify novel small molecule inhibitors of H2A ubiquitination, we synthesized and evaluated several compounds designed based on PRT4165 (1), which is a reported histone ubiquitin ligase RING1A inhibitor. We found that compound 11b strongly inhibited the viability and reduced histone H2A mono-ubiquitination in human osteosarcoma U2OS cells. Therefore, compound 11b is a promising lead compound for the development of H2A histone ubiquitination-inhibiting small molecules.

Inhibition of Immunoproteasome Attenuates NLRP3 Inflammasome Response by Regulating E3 Ubiquitin Ligase TRIM31.

Excessive secretion of pro-inflammatory cytokines leads to the disruption of intestinal barrier in inflammatory bowel disease (IBD). The inflammatory cytokine tumor necrosis factor alpha (TNFα) induces the assembly of the NLRP3 inflammasome, resulting in the augmented secretion of inflammatory cytokines implicated in the pathogenesis of inflammatory bowel disease (IBD). TNFα has also been known to induce the formation of immunoproteasome (IP), which incorporates immunosubunits LMP2, LMP7, and MECL-1. Inhibition of IP activity using the IP subunit LMP2-specific inhibitor YU102, a peptide epoxyketone, decreased the protein levels of NLRP3 and increased the K48-linked polyubiquitination levels of NLRP3 in TNFα-stimulated intestinal epithelial cells. We observed that inhibition of IP activity caused an increase in the protein level of the ubiquitin E3 ligase, tripartite motif-containing protein 31 (TRIM31). TRIM31 facilitated K48-linked polyubiquitination and proteasomal degradation of NLRP3 with an enhanced interaction between NLRP3 and TRIM31 in intestinal epithelial cells. In addition, IP inhibition using YU102 ameliorated the symptoms of colitis in the model mice inflicted with dextran sodium sulfate (DSS). Administration of YU102 in the DSS-treated colitis model mice caused suppression of the NLRP3 protein levels and accompanied inflammatory cytokine release in the intestinal epithelium. Taken together, we demonstrated that inhibiting IP under inflammatory conditions induces E3 ligase TRIM31-mediated NLRP3 degradation, leading to attenuation of the NLRP3 inflammatory response that triggers disruption of intestinal barrier.

MiR-2110 induced by chemically synthesized cinobufagin functions as a tumor-metastatic suppressor via targeting FGFR1 to reduce PTEN ubiquitination degradation in nasopharyngeal carcinoma.

Tumor cell metastasis is the key cause of death in patients with nasopharyngeal carcinoma (NPC). MiR-2110 was cloned and identified in Epstein-Barr virus (EBV)-positive NPC, but its role is unclear in NPC. In this study, we investigated the effect of miR-2110 on NPC metastasis and its related molecular basis. In addition, we also explored whether miR-2110 can be regulated by cinobufotalin (CB) and participate in the inhibition of CB on NPC metastasis. Bioinformatics, RT-PCR, and in situ hybridization were used to observe the expression of miR-2110 in NPC tissues and cells. Scratch, Boyden, and tail vein metastasis model of nude mouse were used to detect the effect of miR-2110 on NPC metastasis. Western blot, Co-IP, luciferase activity, colocalization of micro confocal and ubiquitination assays were used to identify the molecular mechanism of miR-2110 affecting NPC metastasis. Finally, miR-2110 induced by CB participates in CB-stimulated inhibition of NPC metastasis was explored. The data showed that increased miR-2110 significantly suppresses NPC cell migration, invasion, and metastasis. Suppressing miR-2110 markedly restored NPC cell migration and invasion. Mechanistically, miR-2110 directly targeted FGFR1 and reduced its protein expression. Decreased FGFR1 attenuated its recruitment of NEDD4, which downregulated NEDD4-induced phosphatase and tensin homolog (PTEN) ubiquitination and degradation and further increased PTEN protein stability, thereby inactivating PI3K/AKT-stimulated epithelial-mesenchymal transition signaling and ultimately suppressing NPC metastasis. Interestingly, CB, a potential new inhibitory drug for NPC metastasis, significantly induced miR-2110 expression by suppressing PI3K/AKT/c-Jun-mediated transcription inhibition. Suppression of miR-2110 significantly restored cell migration and invasion in CB-treated NPC cells. Finally, a clinical sample assay indicated that reduced miR-2110 was negatively correlated with NPC lymph node metastasis and positively related to NPC patient survival prognosis. In summary, miR-2110 is a metastatic suppressor involving in CB-induced suppression of NPC metastasis.

Inhibition the ubiquitination of ENaC and Na,K-ATPase with erythropoietin promotes alveolar fluid clearance in sepsis-induced acute respiratory distress syndrome.

Sepsis-induced acute respiratory distress syndrome (ARDS) causes significant fatalities worldwide and lacks pharmacological intervention. Alveolar fluid clearance (AFC) plays a pivotal role in the remission of ARDS and is markedly impaired in the pathogenesis of ARDS. Here, we demonstrated that erythropoietin could effectively ameliorate lung injury manifestations and lethality, restore lung function and promote AFC in a rat model of lipopolysaccharide (LPS)-induced ARDS. Moreover, it was proven that EPO-induced restoration of AFC occurs through triggering the total protein expression of ENaC and Na,K-ATPase channels, enhancing their protein abundance in the membrane, and suppressing their ubiquitination for degeneration. Mechanistically, the data indicated the possible involvement of EPOR/JAK2/STAT3/SGK1/Nedd4-2 signaling in this process, and the pharmacological inhibition of the pathway markedly eliminated the stimulating effects of EPO on ENaC and Na,K-ATPase, and subsequently reversed the augmentation of AFC by EPO. Consistently, in vitro studies of alveolar epithelial cells paralleled with that EPO upregulated the expression of ENaC and Na,K-ATPase, and patch-clamp studies further demonstrated that EPO substantially strengthened sodium ion currents. Collectively, EPO could effectively promote AFC by improving ENaC and Na,K-ATPase protein expression and abundance in the membrane, dependent on inhibition of ENaC and Na,K-ATPase ubiquitination, and resulting in diminishing LPS-associated lung injuries.

Saikosaponin F ameliorates depression-associated dry eye disease by inhibiting TRIM8-induced TAK1 ubiquitination.

AIMS: Saikosaponin F (SsF) is one of the major active ingredients of Radix Bupleuri, an herb widely used in the treatment of depression. Studies have shown that dry eye disease often occurs together with depression. The aim of this study is to investigate whether SsF can improve depression-associated dry eye disease and explore the underlying mechanism. METHODS: Behavioral test was used to verify the effect of SsF on CUMS-induced depression-like behaviors in mice. Corneal fluorescein staining, phenol red cotton thread test and periodic acid-Schiff (PAS) staining were used to observe the effect of SsF on depression-associated dry eye disease. Western blot (WB) was performed to observe the expression of TAK1 protein and key proteins of NF-κB and MAPK (P38) inflammatory pathways in the hippocampus and cornea. Immunohistochemical staining was used to observe the expression of microglia, and immunoprecipitation was used to observe K63-linked TAK1 ubiquitination. Subsequently, we constructed a viral vector sh-TAK1 to silence TAK1 protein to verify whether SsF exerted its therapeutic effect based on TAK1. The expression of inflammatory factors such as IL-1ß, TNF-α and IL-18 in hippocampus and cornea were detected by ELISA. Overexpression of TRIM8 (OE-TRIM8) by viral vector was used to verify whether SsF improved depression-associated dry eye disease based on TRIM8. RESULTS: SsF treatment significantly improved the depression-like behavior, increased tear production and restored corneal injury in depression-related dry eye model mice. SsF treatment downregulated TAK1 expression and TRIM8-induced K63-linked TAK1 polyubiquitination, while inhibiting the activation of NF-κB and MAPK (P38) inflammatory pathways and microglial expression. In addition, selective inhibition of TAK1 expression ameliorated depression-associated dry eye disease, while overexpression of TRIM8 attenuated the therapeutic effect of SsF on depression-associated dry eye disease. CONCLUSION: SsF inhibited the polyubiquitination of TAK1 by acting on TRIM8, resulting in the downregulation of TAK1 expression, inhibition of inflammatory response, and improvement of CUMS-induced depression-associated dry eye disease.

Regulation of the water channel aquaporin-2 by cullin E3 ubiquitin ligases.

Aquaporin 2 (AQP2) is a vasopressin (VP)-regulated water channel in the renal collecting duct. Phosphorylation and ubiquitylation of AQP2 play an essential role in controlling the cellular abundance of AQP2 and its accumulation on the plasma membrane in response to VP. Cullin-RING ubiquitin ligases (CRLs) are multisubunit E3 ligases involved in ubiquitylation and degradation of their target proteins, eight of which are expressed in the collecting duct. Here, we used an established cell model of the collecting duct (mpkCCD14 cells) to study the role of cullins in modulating AQP2. Western blotting identified Cul-1 to Cul-5 in mpkCCD14 cells. Treatment of cells for 4 h with a pan-cullin inhibitor (MLN4924) decreased AQP2 abundance, prevented a VP-induced reduction in AQP2 Ser261 phosphorylation, and attenuated VP-induced plasma membrane accumulation of AQP2 relative to the vehicle. AQP2 ubiquitylation levels were significantly higher after MLN4924 treatment compared with controls, and they remained higher despite VP treatment. Cullin inhibition increased ERK1/2 activity, a kinase that regulates AQP2 Ser261 phosphorylation, and VP-induced reductions in ERK1/2 phosphorylation were absent during MLN4924 treatment. Furthermore, the greater Ser261 phosphorylation and reduction in AQP2 abundance during MLN4924 treatment were attenuated during ERK1/2 inhibition. MLN4924 increased intracellular calcium levels via calcium release-activated calcium channels, inhibition of which abolished MLN4924 effects on Ser261 phosphorylation and AQP2 abundance. In conclusion, CRLs play a vital role in mediating some of the effects of VP to increase AQP2 plasma membrane accumulation and AQP2 abundance. Whether modulation of cullin activity can contribute to body water homeostasis requires further studies.NEW & NOTEWORTHY Aquaporin 2 (AQP2) is essential for body water homeostasis and is regulated by the antidiuretic hormone vasopressin. The posttranslational modification ubiquitylation is a key regulator of AQP2 abundance and plasma membrane localization. Here we demonstrate that cullin-RING E3 ligases play a vital role in mediating some of the effects of vasopressin to increase AQP2 abundance and plasma membrane accumulation. The results suggest that manipulating cullin activity could be a novel strategy to alter kidney water handling.

Modulation of Cullin-RING E3 ubiquitin ligase-dependent ubiquitination by small molecule compounds.

Cullin (CUL)-RING (Really Interesting New Gene) E3 ubiquitin (Ub) ligases (CRLs) are the largest E3 family. The E3 CRL core ligase is a subcomplex formed by the CUL C-terminal domain bound with the ROC1/RBX1 RING finger protein, which acts as a hub that mediates and organizes multiple interactions with E2, Ub, Nedd8, and the ARIH family protein, thereby resulting in Ub transfer to the E3-bound substrate. This report describes the modulation of CRL-dependent ubiquitination by small molecule compounds including KH-4-43, #33, and suramin, which target the CRL core ligases. We show that both KH-4-43 and #33 inhibit the ubiquitination of CK1α by CRL4CRBN. However, either compound's inhibitory effect on this reaction is significantly reduced when a neddylated form of CRL4CRBN is used. On the other hand, both #33 and KH-4-43 inhibit the ubiquitination of ß-catenin by CRL1ß-TrCP and Nedd8-CRL1ß-TrCP almost equally. Thus, neddylation of CRL1ß-TrCP does not negatively impact the sensitivity to inhibition by #33 and KH-4-43. These findings suggest that the effects of neddylation to alter the sensitivity of CRL inhibition by KH-4-43/#33 is dependent upon the specific CRL type. Suramin, a compound that targets CUL's basic canyon, can effectively inhibit CRL1/4-dependent ubiquitination regardless of neddylation status, in contrast to the results observed with KH-4-43/#33. This observed differential drug sensitivity of KH-4-43/#33 appears to echo CUL-specific Nedd8 effects on CRLs as revealed by recent high-resolution structural biology efforts. The highly diversified CRL core ligase structures may provide opportunities for specific targeting by small molecule modulators.

Blueberry anthocyanins improve liver fibrosis by regulating NCOA4 ubiquitination through TRIM7 to affect ferroptosis of hepatic stellate cells.

This study aims to investigate the role and molecular mechanism of anthocyanin in improving liver fibrosis through ferroptosis, providing a basis for drug development and targeted therapy. In this study, a mouse model of liver fibrosis was established using CCl4, and the anthocyanin treatment groups were administered 100 mg/kg anthocyanin daily via gavage. Furthermore, real-time fluorescent quantitative PCR (qRT-PCR), Western blotting (WB), and enzyme-linked immunosorbent assay were used to assess liver fibrosis indicators and liver injury markers. Histopathological methods were used to confirm the morphology of liver injury in different treatment groups. The effects of anthocyanins on ferroptosis markers, NCOA4 and FTH1 expression, were examined through qRT-PCR, WB, and Co-IP. Confocal microscopy was used to validate the colocalization of ferritin and lysosomes. A differential expression model of TRIM7 was constructed to verify its impact on the progression of liver fibrosis. The present study demonstrates the hepatoprotective effects of anthocyanins in liver fibrosis, highlighting their ability to enhance hepatic stellate cell (HSC) ferroptosis and regulate ferritin autophagy. Moreover, TRIM7 is identified as a key mediator of anthocyanin-induced regulation of hepatic stellate cells activation for liver fibrosis treatment through modulation of ferroautophagy. Mechanistic investigations further reveal that TRIM7 exerts its influence on the process of ferroautophagy by controlling NCOA4 ubiquitination. Our study discovered that anthocyanins could improve liver fibrosis by regulating NCOA4 ubiquitination through TRIM7, thereby affecting hepatic stellate cells' ferroptosis levels.NEW & NOTEWORTHY This was the first study to demonstrate that anthocyanins can improve the progression of liver fibrosis by promoting hepatic stellate cell (HSC) ferroptosis. Anthocyanins could affect the content of Fe2+ by promoting ferroautophagy in HSCs, thereby promoting the level of ferroptosis. This study demonstrates for the first time that anthocyanins can inhibit the expression of TRIM7 and then affect the ubiquitination of NCOA4 to regulate the level of ferritin autophagy and ferroptosis.

Stress response silencing by an E3 ligase mutated in neurodegeneration.

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health1-3. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.

Cellular Assays for Dynamic Quantification of Deubiquitinase Activity and Inhibition.

Deubiquitinases (DUBs) are proteolytic enzymes that catalyze the removal of ubiquitin from protein substrates. The critical role of DUBs in regulating protein ubiquitination makes them attractive drug targets in oncology, neurodegenerative disease, and antiviral development. Biochemical assays for quantifying DUB activity have enabled characterization of substrate preferences and discovery of small molecule inhibitors. However, assessing the efficacy of these inhibitors in cellular contexts to support clinical drug development has been limited by a lack of tractable cell-based assays. To address this gap, we developed a two-color flow cytometry-based assay that allows for sensitive quantification of DUB activity and inhibition in living cells. The utility of this system was demonstrated by quantifying the potency of GRL0617 against the viral DUB SARS-CoV-2 PLpro, identifying potential GRL0617 resistance mutations, and performing structure-function analysis of the vOTU domain from the recently emerged Yezo virus. In addition, the system was optimized for cellular DUBs by modifying a GFP-targeting nanobody to recruit USP7 and USP28 to benchmark a panel of reported inhibitors and assess inhibition kinetics. Together, these results demonstrate the utility of these assays for studying DUB biology in a cellular context with potential to aid in inhibitor discovery and development.

Inhibiting von Hippel‒Lindau protein-mediated Dishevelled ubiquitination protects against experimental parkinsonism.

Dopaminergic neuron degeneration is a hallmark of Parkinson's disease (PD). We previously reported that the inactivation of von Hippel‒Lindau (VHL) alleviated dopaminergic neuron degeneration in a C. elegans model. In this study, we investigated the specific effects of VHL loss and the underlying mechanisms in mammalian PD models. For in vivo genetic inhibition of VHL, AAV-Vhl-shRNA was injected into mouse lateral ventricles. Thirty days later, the mice received MPTP for 5 days to induce PD. Behavioral experiments were conducted on D1, D3, D7, D14 and D21 after the last injection, and the mice were sacrificed on D22. We showed that knockdown of VHL in mice significantly alleviated PD-like syndromes detected in behavioral and biochemical assays. Inhibiting VHL exerted similar protective effects in MPP+-treated differentiated SH-SY5Y cells and the MPP+-induced C. elegans PD model. We further demonstrated that VHL loss-induced protection against experimental parkinsonism was independent of hypoxia-inducible factor and identified the Dishevelled-2 (DVL-2)/ß-catenin axis as the target of VHL, which was evolutionarily conserved in both C. elegans and mammals. Inhibiting the function of VHL promoted the stability of ß-catenin by reducing the ubiquitination and degradation of DVL-2. Thus, in vivo overexpression of DVL-2, mimicking VHL inactivation, protected against PD. We designed a competing peptide, Tat-DDF-2, to inhibit the interaction between VHL and DVL-2, which exhibited pharmacological potential for protection against PD in vitro and in vivo. We propose the therapeutic potential of targeting the interaction between VHL and DVL-2, which may represent a strategy to alleviate neurodegeneration associated with PD.

The E3 ligase adapter cereblon targets the C-terminal cyclic imide degron.

The ubiquitin E3 ligase substrate adapter cereblon (CRBN) is a target of thalidomide and lenalidomide1, therapeutic agents used in the treatment of haematopoietic malignancies2-4 and as ligands for targeted protein degradation5-7. These agents are proposed to mimic a naturally occurring degron; however, the structural motif recognized by the thalidomide-binding domain of CRBN remains unknown. Here we report that C-terminal cyclic imides, post-translational modifications that arise from intramolecular cyclization of glutamine or asparagine residues, are physiological degrons on substrates for CRBN. Dipeptides bearing the C-terminal cyclic imide degron substitute for thalidomide when embedded within bifunctional chemical degraders. Addition of the degron to the C terminus of proteins induces CRBN-dependent ubiquitination and degradation in vitro and in cells. C-terminal cyclic imides form adventitiously on physiologically relevant timescales throughout the human proteome to afford a degron that is endogenously recognized and removed by CRBN. The discovery of the C-terminal cyclic imide degron defines a regulatory process that may affect the physiological function and therapeutic engagement of CRBN.

Triad3A-Dependent TLR4 Ubiquitination and Degradation Contributes to the Anti-Inflammatory Effects of Pterostilbene on Vascular Dementia.

Pterostilbene, a methylated stilbene derived from many plant foods, has significant anti-inflammatory activity. Meanwhile, vascular dementia (VaD) is the second most common subtype of dementia, in which inflammation is one of the major pathogenic contributors. However, the protective effect of pterostilbene on VaD is not well understood. In this work, we investigated the effect of pterostilbene on VaD and explored its underlying mechanisms using in vivo and in vitro models. Y-maze and Morris water maze tests showed pterostilbene-attenuated cognitive impairment in mice with bilateral common carotid artery occlusion (BCCAO). The hippocampal neuronal death and microglial activation in BCCAO mice were also reduced by pterostilbene treatment. Further, pterostilbene inhibited the expression of TLR4 and downstream inflammatory cytokines in these mice, with similar results observed in an oxygen-glucose deprivation and reperfusion (OGD/R) BV-2 cell model. In addition, its anti-inflammatory effect on OGD/R BV-2 cells was partially blocked by TLR4 overexpression. Moreover, Triad3A-TLR4 interactions were increased by pterostilbene following enhanced ubiquitination and degradation of TLR4, and the inhibitory effect of pterostilbene on inflammation was blocked by Triad3A knockdown in OGD/R-stimulated BV-2 cells. Together, these results reveal that pterostilbene could reduce vascular cognitive impairment and that Triad3A-mediated TLR4 degradation might be the key target.

Releasing the trap: How the segregase p97 extracts PARP1 from chromatin.

Krastev et al. (2022) identify a cellular mechanism that counteracts cytotoxic trapping of PARP1 induced by clinical PARP inhibitors. SUMO-targeted ubiquitylation of trapped PARP1 is shown to trigger the enzymes' extraction from chromatin by the p97 ATPase.

Mechanisms involved in suppression of osteoclast supportive activity by transforming growth factor-ß1 via the ubiquitin-proteasome system.

Orthodontic treatment requires the regulation of bone remodeling in both compression and tension sides. Transforming growth factor-ß1 (TGF-ß1) is an important coupling factor for bone remodeling. However, the mechanism underlying the TGF-ß1-mediated regulation of the osteoclast-supporting activity of osteoblasts and stromal cells remain unclear. The current study investigated the effect of TGF-ß1 on receptor activator of nuclear factor kappa-B ligand (RANKL) expression in stromal cells induced by 1α,25(OH)2D3 (D3) and dexamethasone (Dex). TGF-ß1 downregulated the expression of RANKL induced by D3 and Dex in mouse bone marrow stromal lineage, ST2 cells. Co-culture system revealed that TGF-ß1 suppressed osteoclast differentiation from bone marrow cell induced by D3 and Dex-activated ST2 cells. The inhibitory effect of TGF-ß1 on RANKL expression was recovered by inhibiting the interaction between TGF-ß1 and the TGF-ß type I/activin receptor or by downregulating of smad2/3 expression. Interestingly, TGF-ß1 degraded the retinoid X receptor (RXR)-α protein which forms a complex with vitamin D receptor (VDR) and regulates transcriptional activity of RANKL without affecting nuclear translocation of VDR and phosphorylation of signal transducer and activator of transcription3 (STAT3). The degradation of RXR-α protein by TGF-ß1 was recovered by a ubiquitin-proteasome inhibitor. We also observed that poly-ubiquitination of RXR-α protein was induced by TGF-ß1 treatment. These results indicated that TGF-ß1 downregulates RANKL expression and the osteoclast-supporting activity of osteoblasts/stromal cells induced by D3 and Dex through the degradation of the RXR-α protein mediated by ubiquitin-proteasome system.

Cullin neddylation inhibitor attenuates hyperglycemia by enhancing hepatic insulin signaling through insulin receptor substrate stabilization.

Hepatic insulin resistance is a hallmark feature of nonalcoholic fatty liver disease and type-2 diabetes and significantly contributes to systemic insulin resistance. Abnormal activation of nutrient and stress-sensing kinases leads to serine/threonine phosphorylation of insulin receptor substrate (IRS) and subsequent IRS proteasome degradation, which is a key underlying cause of hepatic insulin resistance. Recently, members of the cullin-RING E3 ligases (CRLs) have emerged as mediators of IRS protein turnover, but the pathophysiological roles and therapeutic implications of this cellular signaling regulation is largely unknown. CRLs are activated upon cullin neddylation, a process of covalent conjugation of a ubiquitin-like protein called Nedd8 to a cullin scaffold. Here, we report that pharmacological inhibition of cullin neddylation by MLN4924 (Pevonedistat) rapidly decreases hepatic glucose production and attenuates hyperglycemia in mice. Mechanistically, neddylation inhibition delays CRL-mediated IRS protein turnover to prolong insulin action in hepatocytes. In vitro knockdown of either cullin 1 or cullin 3, but not other cullin members, attenuates insulin-induced IRS protein degradation and enhances cellular insulin signaling activation. In contrast, in vivo knockdown of liver cullin 3, but not cullin 1, stabilizes hepatic IRS and decreases blood glucose, which recapitulates the effect of MLN4924 treatment. In summary, these findings suggest that pharmacological inhibition of cullin neddylation represents a therapeutic approach for improving hepatic insulin signaling and lowering blood glucose.