Background: The SAG/RBX2/RNF7, a dual E3 ligase promoting CUL5 neddylation and CRL5-dependent ubiquitylation, is essential for the growth and survival of cancer cells, and tumorigenesis in multiple genetically engineered mouse models. Despite SAG being an attractive anticancer target, no specific and selective small-molecule inhibitors have been reported, leaving a critical gap in SAG-dependent cancer therapy. Methods: A high-throughput screening of a diversified library was conducted, followed by lead compound optimization via structure-activity relationship assay. The selectivity was validated by biochemical pre-charged assay, and cell-based Western blotting. The target binding was confirmed by in vitro SPR assay, and in vivo CETSA assay, and molecular docking. Polyubiquitylation and protein half-life assays were conducted. Anticancer activity was monitored by growth, survival and apoptosis assays as well as in vivo xenograft tumor model. Pharmacokinetics profile was also conducted. Findings: HA-50-257 specifically binds the SAG-CUL5 complex, selectively inhibits CUL5 neddylation, inactivates CRL5 ligase activity, causes pro-apoptotic substrate NOXA accumulation, and suppresses tumor growth in vitro and in vivo in a NOXA-dependent manner, without observable toxicity and with a sound pharmacokinetic profile. Interpretation: HA-50-257 is the first SAG-CUL5 inhibitor with potent anti-tumor activity. The study validated SAG as a promising anticancer target, fills in the gap in the lack of small-molecule inhibitors targeting SAG, and sets the stage for further optimization of HA-50-257 as the first class of SAG-CRL5-targeting anticancer drugs, thus having potential for clinical translation.

The diagnosis of pulmonary hypertension (PH) relies primarily on invasive right heart catheterization (RHC). Although imaging modalities, including echocardiography, computed tomography angiography (CTA) and computed tomography (CT), offer clinical utility, their effectiveness in early detection remains limited. To clarify the relationship between group 1/2 PH and radiomic-morphological features of the pulmonary vasculature derived from non-contrast CT. We retrospectively enrolled control patients and patients with group 1 PH (PAH) and group 2 PH (PH-LHD) from two centers. All participants underwent RHC and chest CT. We extracted the radiomic features from pulmonary arteries and veins, identifying key radiomics markers and further exploring their association with early pathological changes through animal experiments. Subsequently, we analyzed morphological characteristics from 95,197 vascular centerlines. Multivariable models were employed to evaluate their efficacy in identifying PH and discriminating PH-LHD in validation cohort. The training cohort included 49 patients (20 controls, 13 with PAH, 16 with PH-LHD), and validation cohort included 30 patients. Among 212 radiomics features analysis, the pulmonary arterial surface volume ratio (SVR) correlated strongly with mean pulmonary artery pressure (mPAP) (r = -0.602, P < 0.001), while the venous SVR showed a strong correlation with pulmonary artery wedge pressure (PAWP) (r = -0.651, P < 0.001). In animal models, venous SVR demonstrated the ability to reflect early pulmonary venous remodeling in PH-LHD prior to hemodynamic changes. Morphologically, PH-LHD patients showed increased distal vessel tortuosity with reduced torsion and curvature (all P < 0.001). SVR and models combining morphological features demonstrated promising performance for identifying PH (AUC 0.895 (95

SAG (Sensitive to Apoptosis Gene), also known as RBX2/ROC2/RNF7, was originally cloned as a redox-inducible gene encoding a cysteine-enriched antioxidant protein. SAG was subsequently characterized as the second family member of the RBX with RING domain, essential for E3 ligase activity in both ubiquitylation and neddylation. Data accumulated over the past 26 years have shown that SAG is overexpressed in many types of human cancer tissues with positive correlation of poor patient survival. Functional studies have revealed that SAG is essential for cancer cell growth, and for tumorigenesis induced by oncogene activation and tumor suppressor inactivation in several genetically modified mouse models. Mechanistically, SAG acts as a catalytic subunit of CRL5 as well as CRL1 to ubiquitylate and degrade mainly tumor suppressor substrates, whereas SAG knockdown or knockout causes their accumulation to inhibit the growth and survival of cancer cells, and tumor progression. Thus, SAG E3 is emerging as an attractive anti-cancer target with drug discovery of small molecule inhibitors and PROTAC degraders being currently pursued. Here, we provide a comprehensive literature review on SAG, from its molecular cloning, biochemical activities, and biological function, to SAG validation as an anti-cancer target, and finally to the drug discovery efforts of SAG targeting agents. The perspectives are also proposed for current challenges and future directions on the study of SAG-associated neddylation-CRLs.

Coronary microvascular dysfunction (CMD) is prevalent in diabetes. Ten-eleven translocation-2 (TET2) as the major demethylase in endothelial cells (ECs) is decreased in diabetic CMD, and the role warrants further exploration. In this study, a multi-modality imaging, consist of transthoracic Doppler echocardiography and artery spin labeling cardiac magnetic resonance, is assessed for coronary microvascular function. The expression of TET2 is down-regulated in the heart of diabetic CMD mice. ECs TET2 conditional knockout increases the severity of CMD in diabetic mice. Mechanistically, TET2 deficiency declined the expression of CMPK2, a mtDNA synthetase. Additionally, the mitochondrial electron transport chain complexes encoded by mitochondrial DNA (mtDNA) are down-regulated, which contributes to the excessive production of reactive oxygen species. This, in turn, exacerbated mitochondrial dysfunction, manifesting as mitochondrial membrane potential depolarization, aberrant opening of the mitochondrial permeability transition pore, and structural abnormalities in mitochondria. Therapeutic research demonstrates that vitamin C improves ECs mitochondrial function in diabetic CMD through the TET2-CMPK2 pathway, revealing its potential clinical therapeutic value. In conclusion, we show that loss of endothelial TET2 impairs endothelial mitochondrial function and exacerbated diabetic CMD by regulating the expression of mitochondria enzyme CMPK2.

This study identifies and characterizes compound 24 (HA-218-6-31-80), a novel small-molecule NEDD8-activating enzyme (NAE) inhibitor. Discovered from a high-throughput screening, followed by structure-guided optimization, compound 24 possesses a distinct spirocyclic amide chemical core entirely different from the clinical-stage NAE inhibitor MLN4924. Biochemical assays confirm that compound 24 directly binds NAE and suppresses its catalytic activity, blocks the formation of E2-NEDD8 thioesters, and reduces the neddylation of CUL1 and CUL5 in purified protein systems. In human lung cancer cells, it potently suppresses neddylation of multiple CUL family members, leading to the accumulation of CRL substrates including p27 and NOXA. Functionally, it triggers G1-phase arrest and NOXA-dependent apoptosis, inhibiting lung cancer cell proliferation and survival. In A549 xenograft models, it significantly suppresses tumor growth without obvious systemic toxicity. Collectively, 24 (HA-218-6-31-80) represents a promising NAE inhibitor, offering a potential therapeutic candidate for lung cancer and a novel scaffold for neddylation targeted drug discovery.

BackgroundPatients with pulmonary embolism (PE) at high altitude face an increased risk of developing chronic thromboembolic pulmonary hypertension (CTEPH). This study aims to establish a diagnosis model of CTEPH patients at high altitude to optimize early screening.MethodsA retrospective cohort of CTEPH and PE patients was rigorously selected through inclusion/exclusion criteria. Clinical data encompassing biochemical profiles, echocardiography, and CT angiography (CTA) were collected, yielding 103 candidate variables. Feature parameters were screened using the Boruta algorithm, followed by predictive model development with seven machine learning architectures. The optimal model was identified based on area under the curve (AUC). The optimal Random Forest model was subsequently interpreted through Shapley Additive Explanations (SHAP) to quantify feature contributions.ResultsAmong 57 PE patients, 44% met echocardiographic criteria for pulmonary hypertension following PE. Diameter of right atrium, diameter of right ventricle, Vessel-Grade (of embolization) and Sup-inferior (superior or inferior of embolization) were key identified predictors. Random Forests model had the highest AUC of 0.842. Enlarged right heart, embolization of small vessels and superior pulmonary artery embolism increased the risk of CTEPH, while normal right heart structure and isolated inferior pulmonary embolism reduced it.ConclusionThe Random Forests model demonstrated potential for detecting CTEPH in PE patients, enabling early and rapid pulmonary hypertension assessment.

Broadening the application of covalent inhibitors requires the exploration of nucleophilic residues beyond cysteine. The covalent DNA-encoded chemical library (CoDEL) represents an advanced technology for covalent drug discovery. However, its application in lysine-targeting inhibitors remains uncharted territory. Here, we report the utilization of CoDEL selection guided by proteome-wide data to identify lysine-targeting covalent inhibitors. A comprehensive assessment of activity-based protein profiling (ABPP) data on lysine distribution and ligandability reveals potential targets for selective covalent inhibition, including phosphoglycerate mutase 1 (PGAM1), bromodomain (BRD) family proteins, and ubiquitin-conjugating enzyme E2 N (UBE2N). The 10.7-million-member CoDELs, featuring diverse lysine-reactive warheads, enable the discovery of a series of covalent inhibitors, covering photo-covalent, reversible covalent, and irreversible covalent reaction mechanisms. In-depth characterization of binding sites and modes of action provides structural and functional insights. Notably, irreversible covalent inhibitors unveil a novel mechanism for regulating UBE2N-mediated ubiquitination by modulating the conformation of the protein complex. Our work adopts the ABPP-CoDEL strategy, offering an efficient and versatile selection method for the development of covalent inhibitors targeting functional lysines.

BACKGROUND:Pulmonary venous remodeling is a key pathological feature of pulmonary hypertension associated with left heart disease (PH-LHD). This study aims to investigate the role of regulatory T (Treg) cells in this process. METHODS:We used mouse models with transverse aortic constriction and cell depletion of Foxp3-DTR/tdTomato mice to examine Treg cells' function around pulmonary veins in PH-LHD in vivo. To confirm the effect of Nlrc3-/- Treg cells on PH-LHD, we utilized 3 mouse models: Nlrc3 knockout mice, athymic mice, and endothelial cell lineage tracing Cdh5CreERT2+/--mT/mG+/- mice. The interaction proteins and signaling pathways of Treg cells during endothelial-to-mesenchymal transition were elucidated by protein docking prediction, coimmunoprecipitation and cocultivation of Treg cells with venous endothelial cells. RESULTS:Treg cells were abundant around pulmonary veins of transverse aortic constriction-induced PH-LHD and were essential for promoting inflammation resolution and inhibiting pulmonary venous remodeling. Nlrc3 expression was reduced in mice and patients with PH-LHD. NLRC3 (nucleotide-oligomerization domain-like receptor family CARD domain containing 3) deficiency inhibited Treg cell proliferation and impaired their immunosuppressive and endothelial-to-mesenchymal transition-protective effects. Mechanistically, NLRC3 interacted with TRAM (TRIF-related adaptor molecule) and regulated interferon regulatory factor 3 (IRF3)/NF-κB (nuclear factor-κB) p65 signaling in cluster differentiation 4+ (CD4+) T cells. NLRC3-deficient Treg cells promoted interleukin (IL)-18 expression through IRF3/NF-κB p65 signaling, and thus IL-18 secretion activated endothelial receptor tyrosine kinase (RTK) signaling, favoring endothelial-to-mesenchymal transition progression in pulmonary veins and PH-LHD progress. This process was reversible with IL-18 binding protein in vivo. CONCLUSIONS:NLRC3 is crucial for Treg cells to prevent pulmonary venous remodeling in PH-LHD, primarily by modulating IL-18 secretion, which inhibits endothelial-to-mesenchymal transition and thereby improves disease progression and prognosis.

NEDD8 (Neural precursor cell expressed developmentally downregulated protein 8) is an ubiquitin-like protein that is covalently attached to a lysine residue of a protein substrate through a process known as neddylation, catalyzed by the enzyme cascade, namely NEDD8 activating enzyme (E1), NEDD8 conjugating enzyme (E2), and NEDD8 ligase (E3). The substrates of neddylation are categorized into cullins and non-cullin proteins. Neddylation of cullins activates CRLs (cullin RING ligases), the largest family of E3 ligases, whereas neddylation of non-cullin substrates alters their stability and activity, as well as subcellular localization. Significantly, the neddylation pathway and/or many neddylation substrates are abnormally activated or over-expressed in various human diseases, such as metabolic disorders, liver dysfunction, neurodegenerative disorders, and cancers, among others. Thus, targeting neddylation becomes an attractive strategy for the treatment of these diseases. In this review, we first provide a general introduction on the neddylation cascade, its biochemical process and regulation, and the crystal structures of neddylation enzymes in complex with cullin substrates; then discuss how neddylation governs various key biological processes via the modification of cullins and non-cullin substrates. We further review the literature data on dysregulated neddylation in several human diseases, particularly cancer, followed by an outline of current efforts in the discovery of small molecule inhibitors of neddylation as a promising therapeutic approach. Finally, few perspectives were proposed for extensive future investigations.

Efferocytosis and metabolic reprogramming of macrophages play crucial roles in myocardial infarction (MI) repair. TREM2 has been proven to participate in phagocytosis and metabolism, but how it modulates myocardial infarction remains unclear. In this study, we showed that macrophage-specific TREM2 deficiency worsened cardiac function and impaired post-MI repair. Using RNA-seq, protein and molecular docking, and Targeted Metabolomics (LC–MS), our data demonstrated that macrophages expressing TREM2 exhibited decreased SLC25A53 transcription through the SYK-SMAD4 signaling pathway after efferocytosis, which impaired NAD + transport into mitochondria, downregulated SLC25A53 thereby causing the breakpoint in the TCA cycle and subsequently increased itaconate production. In vitro experiments confirmed that itaconate secreted by TREM2 + macrophages inhibited cardiomyocyte apoptosis and promoted fibroblast proliferation. Conversely, overexpression of TREM2 in macrophages could improve cardiac function. In summary, our study reveals a novel role for macrophage-specific TREM2 in MI, connecting efferocytosis to immune metabolism during cardiac repair.

Spinal cord injury (SCI) is a severe injury to the central nervous system, and its treatment is always a major medical challenge. Proinflammatory cell death is considered an important factor affecting neuroinflammation and the prognosis after injury. PANoptosis, a newly discovered type of proinflammatory cell death, regulates the activation of executioner molecules of apoptosis, pyroptosis and necroptosis through the PANoptosome, providing a new target for therapeutic intervention after SCI. However, its role and regulatory mechanism in SCI are not yet elucidated. Here, based on proteomic data, YBX1 expression is significantly increased in neurons after SCI. Guided by RIP-seq, subsequent experiments reveal that YBX1 promotes ZBP1 expression by stabilizing the Zbp1 mRNA, thereby aggravating ZBP1-mediated PANoptosis. Furthermore, the E3 ubiquitin ligase TRIM56 is identified as an endogenous inhibitor of YBX1 via molecular docking and IP/MS analysis. Mechanistically, TRIM56 bound to YBX1 and promoted its ubiquitination, thereby accelerating its degradation. Taken together, these findings reveal a novel function of YBX1 in regulating ZBP1-mediated PANoptosis in the pathogenesis of SCI and verified that TRIM56 functions as an endogenous inhibitor to promote the ubiquitin-proteasomal degradation of YBX1, providing new insights into SCI treatment strategies.

AbstractAcute lung injury/acute respiratory distress syndrome (ALI/ARDS) is characterised by an uncontrolled inflammatory response, and current treatment strategies have limited efficacy. Although the protective effect of M2‐like macrophages (M2φ) and their extracellular vesicles (EVs) has been well‐documented in other inflammatory diseases, the role of M2φ‐derived EVs (M2φ‐EVs) in the pathogenesis of ALI/ARDS remains poorly understood. The present study utilised a mouse model of lipopolysaccharide‐induced ALI to first demonstrate a decrease in endogenous M2‐like alveolar macrophage‐derived EVs. And then, intratracheal instillation of exogenous M2φ‐EVs from the mouse alveolar macrophage cell line (MH‐S) primarily led to a take up by alveolar macrophages, resulting in reduced lung inflammation and injury. Mechanistically, the M2φ‐EVs effectively suppressed the pyroptosis of alveolar macrophages and inhibited the release of excessive cytokines such as IL‐6, TNF‐α and IL‐1β both in vivo and in vitro, which were closely related to NF‐κB/NLRP3 signalling pathway inhibition. Of note, the protective effect of M2φ‐EVs was partly mediated by miR‐709, as evidenced by the inhibition of miR‐709 expression in M2φ‐EVs mitigated their protective effect against lipopolysaccharide‐induced ALI in mice. In addition, we found that the expression of miR‐709 in EVs derived from bronchoalveolar lavage fluid was correlated negatively with disease severity in ARDS patients, indicating its potential as a marker for ARDS severity. Altogether, our study revealed that M2φ‐EVs played a protective role in the pathogenesis of ALI/ARDS, partly mediated by miR‐709, offering a potential strategy for assessing disease severity and treating ALI/ARDS.

凋亡敏感基因蛋白(Sensitive to Apoptosis Gene,SAG)是一种结构上进化保守的锌环指蛋白(zinc RING finger protein),1997年由该文笔者的实验室首次克隆,并于1999年发表.大量研究先后证实SAG不仅是一个具有抗氧化能力、可抑制金属离子或ROS诱导的细胞凋亡的蛋白,同时还是具有促癌作用的重要E3泛素连接酶,也是一个极富潜力的新型抗肿瘤靶点.SAG是泛素化和拟素化修饰的双重E3连接酶,通过介导Cullin-5蛋白的拟素化修饰参与形成CRL5或CRL1 E3泛素连接酶复合体,介导多种抑癌底物蛋白的泛素化降解,促进肿瘤细胞增生、存活、血管生成和肿瘤形成.此外,SAG还参与病毒的复制与合成,并与多种人类疾病相关.目前,靶向SAG的抗肿瘤小分子抑制剂正在研发中.该文回顾多年来在SAG的结构和功能方面的研究进展,综述SAG的生物学功能,重点阐述SAG促进肿瘤发生发展的功能和作用机理,并探讨和展望SAG的基础研究方向和以SAG为靶点的新型抗肿瘤药物的研发策略.

Protein neddylation is catalyzed by a neddylation activating enzyme (NAE, E1), an E2 conjugating enzyme, and an E3 ligase. In various types of human cancers, the neddylation pathway is abnormally activated. Our previous study validated that the neddylation E2 UBE2F is a promising therapeutic target in lung cancer. Although the NAE inhibitor MLN4924/pevonedistat is currently under clinical investigation as an anti-cancer agent, there are no small molecules available that selectively target UBE2F. Here, we report, for the first time, the discovery, via structure-based virtual screen and chemical optimization, of such a small molecule, designated as HA-9104. HA-9104 binds to UBE2F, reduces its protein levels, and consequently inhibits cullin-5 neddylation. Blockage of cullin-5 neddylation inactivates cullin-RING ligase-5 (CRL5) activity, leading to accumulation of the CRL5 substrate, NOXA, to induce apoptosis. Moreover, HA-9104 appears to form the DNA adduct via its 7-azaindole group to induce DNA damage and G2/M arrest. Biologically, HA-9104 effectively suppresses the growth and survival of lung cancer cells and confers radiosensitization in both in vitro cell culture and in vivo xenograft tumor models. In summary, we discovered a small molecule, designated HA-9104, that targets the UBE2F-CRL5 axis with anti-cancer activity alone or in combination with radiation.

Cullin-RING E3 ligases (CRLs) are the largest family of E3 ubiquitin ligases, responsible for about 20% of the protein degradation by the ubiquitin-proteasome system (UPS). Given their vital roles in multiple cellular processes, and over-activation in many human cancers, CRLs are validated as promising targets for anti-cancer therapies. Activation of CRLs requires cullin neddylation, a process catalysed by three neddylation enzymes. Recently, our group established an AlphaScreen-based in vitro cullin neddylation assay and employed it for high-throughput screening to search for small-molecule inhibitors targeting cullin neddylation. During our pilot screen, gossypol, a natural product extracted from cottonseeds, was identified as one of the most potent neddylation inhibitors of cullin-1 and cullin-5. We further demonstrated that gossypol blocks cullin neddylation by binding to cullin-1/-5 to inactivate CRL1/5 ligase activity, leading to accumulation of MCL-1 and NOXA, the substrates of CRL1 and CRL5, respectively. The combination of gossypol and an MCL-1 inhibitor synergistically enhanced the anti-proliferative effect in multiple human cancer cell lines. Our study unveiled a rational combination of two previously known inhibitors of the Bcl-2 family for enhanced anti-cancer efficacy and identified a novel activity of gossypol as an inhibitor of CRL1 and CRL5 E3s, thus providing a new possibility in the development of novel CRL inhibitors for anti-cancer therapy.

Protein neddylation is catalyzed by a three-enzyme cascade, namely an E1 NEDD8-activating enzyme (NAE), one of two E2 NEDD8 conjugation enzymes and one of several E3 NEDD8 ligases. The physiological substrates of neddylation are the family members of cullin, the scaffold component of cullin RING ligases (CRLs). Currently, a potent E1 inhibitor, MLN4924, also known as pevonedistat, is in several clinical trials for anti-cancer therapy. Here we report the discovery, through virtual screening and structural modifications, of a small molecule compound HA-1141 that directly binds to NAE in both in vitro and in vivo assays and effectively inhibits neddylation of cullins 1-5. Surprisingly, unlike MLN4924, HA-1141 also triggers non-canonical endoplasmic reticulum (ER) stress and PKR-mediated terminal integrated stress response (ISR) to activate ATF4 at an early stage, and to inhibit protein synthesis and mTORC1 activity at a later stage, eventually leading to autophagy induction. Biologically, HA-1141 suppresses growth and survival of cultured lung cancer cells and tumor growth in in vivo xenograft lung cancer models at a well-tolerated dose. Taken together, our study has identified a small molecule compound with the dual activities of blocking neddylation and triggering ER stress, leading to growth suppression of cancer cells.

Cullin-RING E3 ligases (CRLs) are the major components of ubiquitin-proteasome system, responsible for ubiquitylation and subsequent degradation of thousands of cellular proteins. CRLs play vital roles in the regulation of multiple cellular processes, including cell cycle, cell apoptosis, DNA replication, signalling transduction among the others, and are frequently dysregulated in many human cancers. The discovery of specific neddylation inhibitors, represented by MLN4924, has validated CRLs as promising targets for anti-cancer therapies with a growing market. Recent studies have focused on the discovery of the CRLs inhibitors by a variety of approaches, including high through-put screen, virtual screen or structure-based drug design. The field is, however, still facing the major challenging, since CRLs are a large multi-unit protein family without typical active pockets to facilitate the drug design, and enzymatic activity is mainly dependent on undruggable protein-protein interactions and dynamic conformation changes. Up to now, most reported CRLs inhibitors are aiming at targeting the F-box family proteins (e.g., SKP2, β-TrCP and FBXW7), the substrate recognition subunit of SCF E3 ligases. Other studies reported few small molecule inhibitors targeting the UBE2M-DCN1 interaction, which specifically inhibits CRL3/CRL1 by blocking the cullin neddylation. On the other hand, several CRL activators have been reported, such as plant auxin and immunomodulatory imide drugs, thalidomide. Finally, proteolysis-targeting chimeras (PROTACs) has emerged as a new technology in the field of drug discovery, specifically targeting the undruggable protein-protein interaction. The technique connects the small molecule that selectively binds to a target protein to a CRL E3 via a chemical linker to trigger the degradation of target protein. The PROTAC has become a hotspot in the field of E3-ligase-based anti-cancer drug discovery.

Cullin-RING E3 ligase (CRL) is the largest family of E3 ubiquitin ligase, responsible for ubiquitylation of ∼20% of cellular proteins. CRL plays an important role in many biological processes, particularly in cancers due to abnormal activation. CRL activation requires neddylation, an enzymatic cascade transferring small ubiquitin-like protein NEDD8 to a conserved lysine residue on cullin proteins. Recent studies have validated that neddylation is an attractive anticancer target. In this study, we report the establishment of an Alpha-Screen-based high throughput screen (HTS) assay for in vitro CUL5 neddylation, and screened a library of 17,000 compounds including FDA approved drugs, natural products and synthetic drug-like small-molecule compounds. Gossypol, a natural compound derived from cotton seed, was identified as an inhibitor of cullin neddylation. Biochemical studies showed that gossypol blocked neddylation of both CUL5 and CUL1 through direct binding to SAG-CUL5 or RBX1-CUL1 complex, and CUL5-H572 plays a key role for gossypol binding. On cellular level, gossypol inhibited cullin neddylation in a variety of cancer cell lines and selectively caused accumulation of NOXA and MCL1, the substrates of CUL5 and CUL1, respectively, in multiple cancer cell lines. Combination of gossypol with specific MCL1 inhibitor synergistically suppress growth of human cancer cells. Our study revealed a previously unknown anti-cancer mechanism of gossypol with potential to develop a new class of neddylation inhibitors.

Nucleotide-binding domain, leucine-rich repeat family with a caspase activation and recruitment domain 3 (NLRC3) participates in both immunity and cancer. The aim of this study was to determine the role of NLRC3 in human hepatocellular carcinoma (HCC) and the underlying mechanisms. We collected human liver tissues from nonalcoholic steatohepatitis (NASH), HCC, and adjacent normal tissues to characterize the pattern of NLRC3 expression by real-time quantitative polymerase chain reaction and immunohistochemistry. Then, we used the HCC cell line, HuH-7, transfected with small interfering RNA to silence the NLRC3 expression. 5-Ethynyl-2'-deoxyuridine assay, scratch assay, and transwell invasion assay were used for assessing proliferation, migration, and invasion, respectively. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were conducted to assess cell apoptosis. The expression of NLRC3 was reduced in human HCC tissues, compared with normal liver and nonalcoholic steatohepatitis tissues. After knocking down of NLRC3, the proliferation, migration, and invasion were increased in HuH-7 cells. And flow cytometry and TUNEL assay showed that HuH-7 cell apoptosis was suppressed after NLRC3 knockdown. As for the underlying mechanisms, knockdown of NLRC3 in HuH-7 cells was associated with the activation of Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) pathway under interleukin-6 (IL-6) stimulation. NLRC3 expression was downregulated in human HCC tissues. NLRC3 silencing in HuH-7 cells can promote the proliferation, migration, and invasion of hepatocellular carcinoma cells. JAK2/STAT3 pathway activation induced by IL-6 may be the underlying mechanism for HCC when NLRC3 expression is silenced. And the invasion of HuH-7 cells was partially suppressed by the STAT3 specific inhibitor (cryptotanshinone). Therefore, NLRC3 may play a significant role in HCC and might be a therapeutic target for the treatment of HCC.

Protein neddylation is a post-translational modification which transfers the ubiquitin-like protein NEDD8 to a lysine residue of the target substrate through a three-step enzymatic cascade. The best-known substrates of neddylation are cullin family proteins, which are the core component of Cullin–RING E3 ubiquitin ligases (CRLs). Given that cullin neddylation is required for CRL activity, and CRLs control the turn-over of a variety of key signal proteins and are often abnormally activated in cancers, targeting neddylation becomes a promising approach for discovery of novel anti-cancer therapeutics. In the past decade, we have witnessed significant progress in the field of protein neddylation from preclinical target validation, to drug screening, then to the clinical trials of neddylation inhibitors. In this review, we first briefly introduced the nature of protein neddylation and the regulation of neddylation cascade, followed by a summary of all reported chemical inhibitors of neddylation enzymes. We then discussed the structure-based targeting of protein–protein interaction in neddylation cascade, and finally the available approaches for the discovery of new neddylation inhibitors. This review will provide a focused, up-to-date and yet comprehensive overview on the discovery effort of neddylation inhibitors.