Linkage reprogramming by tailor-made E3s reveals polyubiquitin chain requirements in DNA-damage bypass.

A polyubiquitin chain can adopt a variety of shapes, depending on how the ubiquitin monomers are joined. However, the relevance of linkage for the signaling functions of polyubiquitin chains is often poorly understood because of our inability to control or manipulate this parameter in vivo. Here, we present a strategy for reprogramming polyubiquitin chain linkage by means of tailor-made, linkage- and substrate-selective ubiquitin ligases. Using the polyubiquitylation of the budding yeast replication factor PCNA in response to DNA damage as a model case, we show that altering the features of a polyubiquitin chain in vivo can change the fate of the modified substrate. We also provide evidence for redundancy between distinct but structurally similar linkages, and we demonstrate by proof-of-principle experiments that the method can be generalized to targets beyond PCNA. Our study illustrates a promising approach toward the in vivo analysis of polyubiquitin signaling.

Transcriptional regulators with broad expression in the zebrafish spinal cord.

BACKGROUND: The spinal cord is a crucial part of the vertebrate CNS, controlling movements and receiving and processing sensory information from the trunk and limbs. However, there is much we do not know about how this essential organ develops. Here, we describe expression of 21 transcription factors and one transcriptional regulator in zebrafish spinal cord. RESULTS: We analyzed the expression of aurkb, foxb1a, foxb1b, her8a, homeza, ivns1abpb, mybl2b, myt1a, nr2f1b, onecut1, sall1a, sall3a, sall3b, sall4, sox2, sox19b, sp8b, tsc22d1, wdhd1, zfhx3b, znf804a, and znf1032 in wild-type and MIB E3 ubiquitin protein ligase 1 zebrafish embryos. While all of these genes are broadly expressed in spinal cord, they have distinct expression patterns from one another. Some are predominantly expressed in progenitor domains, and others in subsets of post-mitotic cells. Given the conservation of spinal cord development, and the transcription factors and transcriptional regulators that orchestrate it, we expect that these genes will have similar spinal cord expression patterns in other vertebrates, including mammals and humans. CONCLUSIONS: Our data identify 22 different transcriptional regulators that are strong candidates for playing different roles in spinal cord development. For several of these genes, this is the first published description of their spinal cord expression.

GPS-Uber: a hybrid-learning framework for prediction of general and E3-specific lysine ubiquitination sites.

As an important post-translational modification, lysine ubiquitination participates in numerous biological processes and is involved in human diseases, whereas the site specificity of ubiquitination is mainly decided by ubiquitin-protein ligases (E3s). Although numerous ubiquitination predictors have been developed, computational prediction of E3-specific ubiquitination sites is still a great challenge. Here, we carefully reviewed the existing tools for the prediction of general ubiquitination sites. Also, we developed a tool named GPS-Uber for the prediction of general and E3-specific ubiquitination sites. From the literature, we manually collected 1311 experimentally identified site-specific E3-substrate relations, which were classified into different clusters based on corresponding E3s at different levels. To predict general ubiquitination sites, we integrated 10 types of sequence and structure features, as well as three types of algorithms including penalized logistic regression, deep neural network and convolutional neural network. Compared with other existing tools, the general model in GPS-Uber exhibited a highly competitive accuracy, with an area under curve values of 0.7649. Then, transfer learning was adopted for each E3 cluster to construct E3-specific models, and in total 112 individual E3-specific predictors were implemented. Using GPS-Uber, we conducted a systematic prediction of human cancer-associated ubiquitination events, which could be helpful for further experimental consideration. GPS-Uber will be regularly updated, and its online service is free for academic research at http://gpsuber.biocuckoo.cn/.

Gstp1 negatively regulates blood pressure in hypertensive rat via promoting APLNR ubiquitination degradation mediated by Nedd4.

Hypertension is a leading risk factor for disease burden worldwide. Vascular contraction and remodeling contribute to the development of hypertension. Glutathione S-transferase P1 (Gstp1) plays several critical roles in both normal and neoplastic cells. In this study, we investigated the effect of Gstp1 on hypertension as well as on vascular smooth muscle cell (VSMC) contraction and phenotypic switching. We identified the higher level of Gstp1 in arteries and VSMCs from hypertensive rats compared with normotensive rats for the first time. We then developed Adeno-associated virus 9 (AAV9) mediated Gstp1 down-regulation and overexpression in rats and measured rat blood pressure by using the tail-cuff and the carotid catheter method. We found that the blood pressure of spontaneously hypertensive rats (SHR) rose significantly with Gstp1 down-regulation and reduced apparently after Gstp1 overexpression. Similar results were obtained from the observations of 2-kidney-1-clip renovascular (2K1C) hypertensive rats. Gstp1 did not influence blood pressure of normotensive Wistar-Kyoto (WKY) rats and Sprague-Dawley (SD) rats. Further in vitro study indicated that Gstp1 knockdown in SHR-VSMCs promoted cell proliferation, migration, dedifferentiation and contraction, while Gstp1 overexpression showed opposite effects. Results from bioinformatic analysis showed that the Apelin/APLNR system was involved in the effect of Gstp1 on SHR-VSMCs. The rise in blood pressure of SHR induced by Gstp1 knockdown could be reversed by APLNR antagonist F13A. We further found that Gstp1 enhanced the association between APLNR and Nedd4 E3 ubiquitin ligases to induce APLNR ubiquitination degradation. Thus, in the present study, we discovered a novel anti-hypertensive role of Gstp1 in hypertensive rats and provided the experimental basis for designing an effective anti-hypertensive therapeutic strategy.

Long Non-Coding RNA Nuclear-Enriched Abundant Transcript 1 (NEAT1) Facilitates Foam Cell Formation and Atherosclerosis Progression Through the miR-17-5p/Itchy E3 Ubiquitin Protein Ligase (ITCH)/Liver Kinase B1 (LKB1) Axis.

BACKGROUND: Foam cell formation is an important step for atherosclerosis (AS) progression. We investigated the mechanism by which the long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) regulates foam cell formation during AS progression.Methods and Results: An in vivo AS model was created by feeding ApoE-/-mice a high-fat diet. Oxidized low-density lipoprotein (ox-LDL)-stimulated macrophages were used as a cellular AS model. Interactions between NEAT1, miR-17-5p, itchy E3 ubiquitin protein ligase (ITCH) and liver kinase B1 (LKB1) were analyzed. NEAT1 and ITCH were highly expressed in clinical samples collected from 10 AS patients and in ox-LDL-treated macrophages, whereas expression of both miR-17-5p and LKB1 was low. ITCH knockdown inhibited ox-LDL-induced lipid accumulation and LDL uptake in macrophages. Mechanistically speakingly, ITCH promoted LDL uptake and lipid accumulation in macrophages by mediating LKB1 ubiquitination degradation. NEAT1 knockdown reduced LDL uptake and lipid accumulation in macrophages and AS progression in vivo. NEAT1 promoted ITCH expression in macrophages by acting as a sponge for miR-17-5p. Inhibition of miR-17-5p facilitated ox-LDL-induced increase in LDL uptake and lipid accumulation in macrophages, which was reversed by NEAT1/ITCH knockdown. CONCLUSIONS: NEAT1 accelerated foam cell formation during AS progression through the miR-17-5p/ITCH/LKB1 axis.

Conditional loss of Ube3d in the retinal pigment epithelium accelerates age-associated alterations in the retina of mice.

Several studies have suggested a correlation between the ubiquitin-proteasome system (UPS) and age-related macular degeneration (AMD), with its phenotypic severity ranging from mild visual impairment to blindness, but the mechanism for UPS dysfunction contributing to disease progression is unclear. In this study, we investigated the role of ubiquitin protein ligase E3D (UBE3D) in aging and degeneration in mouse retina. Conditional knockout of Ube3d in the retinal pigment epithelium (RPE) of mice led to progressive and irregular fundus lesions, attenuation of the retinal vascular system, and age-associated deterioration of rod and cone responses. Simultaneously, RPE-specific Ube3d knockout mice also presented morphological changes similar to the histopathological characteristics of human AMD, in which a defective UPS led to RPE abnormalities such as phagocytosis or degradation of metabolites, the interaction with photoreceptor outer segment, and the transport of nutrients or waste products with choroidal capillaries via Bruch's membrane. Moreover, conditional loss of Ube3d resulted in aberrant molecular characterizations associated with the autophagy-lysosomal pathway, oxidative stress damage, and cell-cycle regulation, which are implicated in AMD pathology. Thus, our findings strengthen and expand the impact of UPS dysfunction on retinal pathophysiology during aging, indicating that genetic Ube3d deficiency in the RPE could lead to the abnormal formation of pigment deposits and secondary fundus alterations. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A WWP2-PTEN-KLF5 signaling axis regulates odontoblast differentiation and dentinogenesis in mice.

WW domain-containing E3 Ubiquitin-protein ligase 2 (WWP2) has been found to positively regulate odontoblastic differentiation by monoubiquitinating the transcription factor Kruppel-like factor 5 (KLF5) in a cell culture system. However, the in vivo role of WWP2 in mouse teeth remains unknown. To explore this, here we generated Wwp2 knockout (Wwp2 KO) mice. We found that molars in Wwp2 KO mice exhibited thinner dentin, widened predentin, and reduced numbers of dentinal tubules. In addition, expression of the odontoblast differentiation markers Dspp and Dmp1 was decreased in the odontoblast layers of Wwp2 KO mice. These findings demonstrate that WWP2 may facilitate odontoblast differentiation and dentinogenesis. Furthermore, we show for the first time that phosphatase and tensin homolog (PTEN), a tumor suppressor, is expressed in dental papilla cells and odontoblasts of mouse molars and acts as a negative regulator of odontoblastic differentiation. Further investigation indicated that PTEN is targeted by WWP2 for degradation during odontoblastic differentiation. We demonstrate PTEN physically interacts with and inhibits the transcriptional activity of KLF5 on Dspp and Dmp1. Finally, we found WWP2 was able to suppress the interaction between PTEN and KLF5, which diminished the inhibition effect of PTEN on KLF5. Taken together, this study confirms the essential role of WWP2 and the WWP2-PTEN-KLF5 signaling axis in odontoblast differentiation and dentinogenesis in vivo.

Erk5 in Bone Marrow Mesenchymal Stem Cells Regulates Bone Homeostasis by Preventing Osteogenesis in Adulthood.

Extracellular signal-regulated kinase 5 (Erk5) belongs to the mitogen-activated protein kinase (MAPK) family. Previously, we demonstrated that Erk5 directly phosphorylates Smad-specific E3 ubiquitin protein ligase 2 (Smurf2) at Thr249 (Smurf2Thr249) to activate its E3 ubiquitin ligase activity. Although we have clarified the importance of Erk5 in embryonic mesenchymal stem cells (MSCs) on skeletogenesis, its role in adult bone marrow (BM)-MSCs on bone homeostasis remains unknown. Leptin receptor-positive (LepR+) BM-MSCs represent a major source of bone in adult bone marrow and are critical regulators of postnatal bone homeostasis. Here, we identified Erk5 in BM-MSCs as an important regulator of bone homeostasis in adulthood. Bone marrow tissue was progressively osteosclerotic in mice lacking Erk5 in LepR+ BM-MSCs with age, accompanied by increased bone formation and normal bone resorption in vivo. Erk5 deficiency increased the osteogenic differentiation of BM-MSCs along with a higher expression of Runx2 and Osterix, essential transcription factors for osteogenic differentiation, without affecting their stemness in vitro. Erk5 deficiency decreased Smurf2Thr249 phosphorylation and subsequently increased Smad1/5/8-dependent signaling in BM-MSCs. The genetic introduction of the Smurf2T249E mutant (a phosphomimetic mutant) suppressed the osteosclerotic phenotype in Erk5-deficient mice. These findings suggest that the Erk5-Smurf2Thr249 axis in BM-MSCs plays a critical role in the maintenance of proper bone homeostasis by preventing excessive osteogenesis in adult bone marrow.

AREL1 resists the apoptosis induced by TGF-ß by inhibiting SMAC in vascular endothelial cells.

Abnormal apoptosis of vascular endothelial cells is an important feature of arteriosclerosis (AS). Here, we induced apoptosis in human umbilical vein endothelial cells (HUVECs) using transforming growth factor-ß (TGF-ß), and investigated the role of antiapoptotic E3 ubiquitin ligase (AREL1) in the apoptosis of vascular endothelial cells. We proved that AREL1 is downregulated in TGF-ß treated HUVECs. The overexpression of AREL1 inhibits the activation of Caspase-3 and Caspase-9 and attenuates cell apoptosis induced by TGF-ß. According to the result of coimmunoprecipitation, AREL1 interacts with the proapoptotic proteins the second mitochondria-derived activator of caspases (SMAC) in TGF-ß treated HUVECs. In addition, miR-320b inhibits the expression of AREL1, and the overexpression of AREL1 attenuates the apoptosis induced by miR-320b mimics in HUVECs. In conclusion, AREL1 is downregulated by miR-320b. AREL1 overexpression inhibits TGF-ß induced apoptosis through downregulating SMAC in vascular endothelial cells. Our study explores pathogenesis regulation mechanism and new biological therapeutic targets for vascular disease.

Effects of UBE3A on the insulin resistance in polycystic ovary syndrome through the ubiquitination of AMPK.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a reproductive hormonal abnormality and a metabolic disorder, which is frequently associated with insulin resistance (IR). We aim to investigate the potential therapeutic effects of Ubiquitin-protein ligase E3A (UBE3A) on IR in the PCOS rats via Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activation. METHODS: The PCOS and IR rats model was established by dehydroepiandrosterone (DHEA) and high fat diet (HFD) treatment, and the fat rate, glucose tolerance and insulin tolerance were measured. The IR rats numbers were calculated. Besides, the mRNA levels of glucose transporter 4 (GLUT4) and UBE3A were detected by RT-qPCR. Furthermore, the relationship between was demonstrated by co-IP assay. The phosphorylation and ubiquitination of AMPK were analyzed by western blot. RESULTS: UBE3A was up-regulated in the PCOS rats. UBE3A knockdown significantly decreased the fat rate, glucose tolerance and insulin tolerance in the PCOS and IR rats. Additionally, the GLUT4 levels were significantly increased in PCOS + IR rats. Besides, after UBE3A knockdown, the IR rats were decreased, the p-IRS1 and p-AKT levels were significantly up-regulated. Furthermore, UBE3A knockdown enhanced phosphorylation of AMPK through decreasing the ubiquitination of AMPK. AMPK knockdown reversed the role of UBE3A knockdown in the PCOS + IR rats. CONCLUSIONS: UBE3A knockdown inhibited the IR in PCOS rats through targeting AMPK. Our study indicated that UBE3A might become a potential biological target for the clinical treatment of PCOS.

Ubc13-Mms2 cooperates with a family of RING E3 proteins in budding yeast membrane protein sorting.

Polyubiquitin chains linked via lysine (K) 63 play an important role in endocytosis and membrane trafficking. Their primary source is the ubiquitin protein ligase (E3) Rsp5/NEDD4, which acts as a key regulator of membrane protein sorting. The heterodimeric ubiquitin-conjugating enzyme (E2), Ubc13-Mms2, catalyses K63-specific polyubiquitylation in genome maintenance and inflammatory signalling. In budding yeast, the only E3 proteins known to cooperate with Ubc13-Mms2 so far is a nuclear RING finger protein, Rad5, involved in the replication of damaged DNA. Here, we report a contribution of Ubc13-Mms2 to the sorting of membrane proteins to the yeast vacuole via the multivesicular body (MVB) pathway. In this context, Ubc13-Mms2 cooperates with Pib1, a FYVE-RING finger protein associated with internal membranes. Moreover, we identified a family of membrane-associated FYVE-(type)-RING finger proteins as cognate E3 proteins for Ubc13-Mms2 in several species, and genetic analysis indicates that the contribution of Ubc13-Mms2 to membrane trafficking in budding yeast goes beyond its cooperation with Pib1. Thus, our results widely implicate Ubc13-Mms2 as an Rsp5-independent source of K63-linked polyubiquitin chains in the regulation of membrane protein sorting.This article has an associated First Person interview with the first author of the paper.

Circular RNA ITCH attenuates the progression of nasopharyngeal carcinoma by inducing PTEN upregulation via miR-214.

BACKGROUND: Circular RNA itchy E3 ubiquitin protein ligase (circ-ITCH) has previously been reported to play a key role in carcinogenesis. Nevertheless, the role of circ-ITCH in nasopharyngeal carcinoma (NPC) remains to be explored. METHODS: Gene expression analysis was performed using a quantitative real-time polymerase chain reaction, western blotting and immunohistochemistry. The role of circ-ITCH in NPC was explored using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, colony formation, transwell invasion, scratch healing and xenograft tumor assays. Furthermore, luciferase reporter assay was carried out to assess the interactions among circ-ITCH, microRNA-214 (miR-214) and phosphatase and tensin homolog (PTEN). RESULTS: The levels of circ-ITCH and PTEN were decreased, whereas the level of miR-214 was increased in NPC tissues collected from 28 subjects compared to normal nasopharynx tissues collected from 15 subjects. Moreover, a negative correlation between circ-ITCH and miR-214 expression and a positive correlation between circ-ITCH and PTEN expression were observed in NPC tissues. Downregulation of circ-ITCH expression was also observed in NPC cell lines. In addition, upregulation of circ-ITCH markedly inhibited NPC cell proliferation, migration and invasion. Furthermore, circ-ITCH was confirmed to exert its function by sponging miR-214. PTEN was found to be a direct target gene of miR-214 and its expression was negatively correlated with miR-214 expression in NPC tissues. Moreover, our results showed that the circ-ITCH/miR-214 axis regulated NPC proliferation, migration and invasion through regulating the expression of PTEN. Upregulation of circ-ITCH or PTEN blocked miR-214-mediated promotion of NPC tumorigenesis in vitro. Additionally, upregulation of circ-ITCH also suppressed NPC tumorigenesis in vivo. CONCLUSIONS: The present study demonstrated that circ-ITCH suppressed NPC tumorigenesis by upregulating PTEN expression through interacting with miR-214, thus proposing a novel mechanism for NPC inhibition.

The NEDD4 ubiquitin E3 ligase: a snapshot view of its functional activity and regulation.

Due to its fundamental role in all eukaryotic cells, a deeper understanding of the molecular mechanisms underlying ubiquitination is of central importance. Being responsible for chain specificity and substrate recognition, E3 ligases are the selective elements of the ubiquitination process. In this review, we discuss different cellular pathways regulated by one of the first identified E3 ligase, NEDD4, focusing on its pathophysiological role, its known targets and modulators. In addition, we highlight small molecule inhibitors that act on NEDD4 and discuss new strategies to effectively target this E3 enzyme.

The Hippocampal Response to Acute Corticosterone Elevation Is Altered in a Mouse Model for Angelman Syndrome.

Angelman Syndrome (AS) is a severe neurodevelopmental disorder, caused by the neuronal absence of the ubiquitin protein ligase E3A (UBE3A). UBE3A promotes ubiquitin-mediated protein degradation and functions as a transcriptional coregulator of nuclear hormone receptors, including the glucocorticoid receptor (GR). Previous studies showed anxiety-like behavior and hippocampal-dependent memory disturbances in AS mouse models. Hippocampal GR is an important regulator of the stress response and memory formation, and we therefore investigated whether the absence of UBE3A in AS mice disrupted GR signaling in the hippocampus. We first established a strong cortisol-dependent interaction between the GR ligand binding domain and a UBE3A nuclear receptor box in a high-throughput interaction screen. In vivo, we found that UBE3A-deficient AS mice displayed significantly more variation in circulating corticosterone levels throughout the day compared to wildtypes (WT), with low to undetectable levels of corticosterone at the trough of the circadian cycle. Additionally, we observed an enhanced transcriptomic response in the AS hippocampus following acute corticosterone treatment. Surprisingly, chronic corticosterone treatment showed less contrast between AS and WT mice in the hippocampus and liver transcriptomic responses. This suggests that UBE3A limits the acute stimulation of GR signaling, likely as a member of the GR transcriptional complex. Altogether, these data indicate that AS mice are more sensitive to acute glucocorticoid exposure in the brain compared to WT mice. This suggests that stress responsiveness is altered in AS which could lead to anxiety symptoms.

[Puerarin Inhibits the Proliferation,Invasion,and Migration of Non-small Cell Lung Cancer Cells through Regulating miR-490/Denticleless E3 Ubiquitin Protein Ligase].

Objective To explore the mechanism of puerarin inhibiting the proliferation,invasion,and migration of non-small cell lung cancer cells. Methods A549 cells were cultured and treated with different concentrations of puerarin.The inhibition rate (IR) on cell proliferation was detected by CCK-8,and qRT-PCR was performed to detect the mRNA levels of miR-490 and denticleless E3 ubiquitin protein ligase(DTL).Double luciferase reporter assay was employed to identify the targets of miR-490 and DTL based on the establishment of NC mimic group,miR-490 mimic group,NC inhibitor group,and miR-490 inhibitor group.The cells treated by 20 µmol/L puerarin were classified into six groups:DMSO,puerarin,puerarin+NC inhibitor,puerarin+miR-490 inhibitor,puerarin+miR-490 inhibitor+Si-NC,and puerarin+miR-490 inhibitor+Si-DTL.Transwell was used to detect cell migration and invasion.Western blotting was performed to detect the protein levels of epithelial-mesenchymal transition-related markers E-cadherin,N-cadherin,and Vimentin. Results With the increase in puerarin concentration,the IR gradually elevated (F=105.375,P<0.001),miR-490 expression gradually increased (F=32.919,P<0.001),and DTL expression gradually decreased (F=116.120,P<0.001).Compared with NC mimic group,miR-490 mimic group had decreased luciferase activity (t=7.762,P=0.016),raised miR-490 mRNA level (t=13.319,P<0.001),and declined DTL mRNA level (t=7.415,P=0.002).Compared with those in NC inhibitor group,miR-490 demonstrated decreased mRNA level (t=9.523,P=0.001) and DTL presented increased mRNA level (t=11.305,P<0.001) in miR-490 inhibitor group.Western blotting showed that the protein level of DTL was higher in NC mimic group (t=7.953,P=0.001) than in miR-490 mimic group and higher in miR-490 inhibitor group than in NC inhibitor group (t=10.552,P<0.001).Compared with DMSO group,puerarin group showed up-regulated mRNA level of miR-490 (t=10.255,P=0.001) while down-regulated mRNA level of DTL (t=6.682,P=0.003).Compared with those in puerarin+NC inhibitor group,the mRNA level of miR-490 declined (t=10.995,P<0.001) while that of DTL raised (t=12.478,P<0.001) in puerarin+miR-490 inhibitor group.The mRNA level of miR-490 had no significant difference between puerarin+miR-490 inhibitor+Si-NC group and puerarin+miR-490 inhibitor+Si-DTL group (t=1.081,P=0.341),and that of DTL was lower in the latter group (t=14.321,P<0.001).The protein level of DTL was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=11.423,P<0.001),and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=12.080,P<0.001).Compared with DMSO group,puerarin group showed inhibited cell proliferation (F=129.27,P<0.001).The activity of cell proliferation was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (F=75.12,P<0.001),and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (F=52.59,P<0.001).Compared with DMSO group,puerarin group had suppressed cell migration (t=8.963,P=0.001).The cell migration ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=12.117,P<0.001) and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (t=12.934,P<0.001).Puerarin group showed weakened cell invasion ability compared with DMSO group (t=4.710,P=0.009).The cell invasion ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=13.264,P<0.001) and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=13.476,P<0.001).Compared with DMSO group,puerarin group showed up-regulated protein level of E-cadherin (t=7.137,P=0.002) while down-regulated protein levels of N-cadherin (t=8.828,P=0.001) and vimentin (t=6.594,P=0.003).Compared with those in puerarin+NC inhibitor group,the protein level of E-cadherin (t=12.376,P<0.001) decreased while those of N-cadherin (t=13.436,P<0.001) and vimentin (t=11.467,P<0.001) increased in puerarin+miR-490 inhibitor group.Compared with puerarin+miR-490 inhibitor+Si-NC group,puerarin+miR-490 inhibitor+Si-DTL group up-regulated the protein level of E-cadherin (t=13.081,P<0.001) while down-regulated the protein levels of N-cadherin (t=10.835,P<0.001) and vimentin (t=11.862,P<0.001). Conclusion Puerarin could inhibit the proliferation,invasion,and migration of non-small cell lung cancer cells by up-regulating miR-490 and down-regulating DTL.

Ubiquitination of interleukin-1α is associated with increased pro-inflammatory polarization of murine macrophages deficient in the E3 ligase ITCH.

Macrophages play critical roles in homeostasis and inflammation. Macrophage polarization to either a pro-inflammatory or anti-inflammatory status is controlled by activating inflammatory signaling pathways. Ubiquitination is a posttranslational modification that regulates these inflammatory signaling pathways. However, the influence of protein ubiquitination on macrophage polarization has not been well studied. We hypothesized that the ubiquitination status of key proteins in inflammatory pathways contributes to macrophage polarization, which is regulated by itchy E3 ubiquitin ligase (ITCH), a negative regulator of inflammation. Using ubiquitin proteomics, we found that ubiquitination profiles are different among polarized murine macrophage subsets. Interestingly, interleukin-1α (IL-1α), an important pro-inflammatory mediator, was specifically ubiquitinated in lipopolysaccharide-induced pro-inflammatory macrophages, which was enhanced in ITCH-deficient macrophages. The ITCH-deficient macrophages had increased levels of the mature form of IL-1α and exhibited pro-inflammatory polarization, and reduced deubiquitination of IL-1α protein. Finally, IL-1α neutralization attenuated pro-inflammatory polarization of the ITCH-deficient macrophages. In conclusion, ubiquitination of IL-1α is associated with increased pro-inflammatory polarization of macrophages deficient in the E3 ligase ITCH.

The N-terminal domain of the non-receptor tyrosine kinase ABL confers protein instability and suppresses tumorigenesis.

Chromosome translocation can lead to chimeric proteins that may become oncogenic drivers. A classic example is the fusion of the BCR activator of RhoGEF and GTPase and the ABL proto-oncogene nonreceptor tyrosine kinase, a result of a chromosome abnormality (Philadelphia chromosome) that causes leukemia. To unravel the mechanism underlying BCR-ABL-mediated tumorigenesis, here we compared the stability of ABL and the BCR-ABL fusion. Using protein degradation, cell proliferation, 5-ethynyl-2-deoxyuridine, and apoptosis assays, along with xenograft tumor analysis, we found that the N-terminal segment of ABL, which is lost in the BCR-ABL fusion, confers degradation capacity that is promoted by SMAD-specific E3 ubiquitin protein ligase 1. We further demonstrate that the N-terminal deletion renders ABL more stable and stimulates cell growth and tumorigenesis. The findings of our study suggest that altered protein stability may contribute to chromosome translocation-induced cancer development.

E3 ubiquitin ligase HECW1 promotes the metastasis of non-small cell lung cancer cells through mediating the ubiquitination of Smad4.

Lung cancer is the leading cause of cancer-related death globally. Ubiquitin modification plays a crucial role in the regulation of gene expression, and is closely associated with cancer pathogenesis. The aim of our study was to clarify the role and mechanisms of action for HECT, C2 and WW domain containing E3 ubiquitin protein ligase 1 (HECW1) in non-small cell lung cancer (NSCLC). Herein, we demonstrate that the expression of HECW1 was significantly increased in NSCLC cell lines and tissues. Upregulation of HECW1 markedly enhanced the proliferation of NSCLC cells, whereas downregulation of HECW1 significantly inhibited proliferation. Moreover, the expression levels of HECW1 positively correlated with the migration and invasiveness of NSCLC cells. Upregulation or downregulation of HECW1 only affected the protein expression levels of SMAD family member 4 (Smad4), but had no effect on the mRNA expression levels. Furthermore, after treatment with MG-132, the relative protein level of Smad4 significantly increased in NSCLC cells. HECW1 promoted the proliferation, migration, and invasiveness of NSCLC cells by inducing the ubiquitination and degradation of Smad4, thus our data provide a novel target for NSCLC treatment.

Identification of CDC20 as an immune infiltration-correlated prognostic biomarker in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a malignancy with a poor prognosis. E3 ubiquitin-protein ligases play essential roles in HCC, such as regulating progression, migration, and metastasis. We aimed to explore a hub E3 ubiquitin-protein ligase gene and verify its association with prognosis and immune cell infiltration in HCC. Cell division cycle 20 (CDC20) was identified as a hub E3 ubiquitin-protein ligase in HCC by determining the intersecting genes in a protein-protein interaction (PPI) network of differentially expressed genes (DEGs) using HCC data from the International Cancer Genome Consortium (ICGC) and the gene list of 919 E3 ubiquitin-protein ligases. DEGs and their correlations with clinicopathological features were explored in The Cancer Genome Atlas (TCGA), ICGC, and Gene Expression Omnibus (GEO) databases via the Wilcoxon signed-rank test. The prognostic value of CDC20 was illustrated by Kaplan-Meier (K-M) curves and Cox regression analyses. Subsequently, the correlation between CDC20 and immune infiltration was demonstrated via the Tumor Immune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA). CDC20 expression was significantly higher in HCC than in normal tissues (all P < 0.05). High CDC20 expression predicted a poor prognosis and might be an independent risk factor in HCC (P < 0.05). Additionally, CDC20 was correlated with the immune infiltration of CD8 + T cells, T cells (general), monocytes, and exhausted T cells. This study reveals the potential prognostic value of CDC20 in HCC and demonstrates that CDC20 may be an immune-associated therapeutic target in HCC because of its correlation with immune infiltration.

ATG101 Degradation by HUWE1-Mediated Ubiquitination Impairs Autophagy and Reduces Survival in Cancer Cells.

Autophagy is a critical cytoprotective mechanism against stress, which is initiated by the protein kinase Unc-51-like kinase 1 (ULK1) complex. Autophagy plays a role in both inhibiting the progression of diseases and facilitating pathogenesis, so it is critical to elucidate the mechanisms regulating individual components of the autophagy machinery under various conditions. Here, we examined whether ULK1 complex component autophagy-related protein 101 (ATG101) is downregulated via ubiquitination, and whether this in turn suppresses autophagy activity in cancer cells. Knockout of ATG101 in cancer cells using CRISPR resulted in severe growth retardation and lower survival under nutrient starvation. Transfection of mutant ATG101 revealed that the C-terminal region is a key domain of ubiquitination, while co-immunoprecipitation and knockdown experiments revealed that HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1(HUWE1) is a major E3 ubiquitin ligase targeting ATG101. Protein levels of ATG101 was more stable and the related-autophagy activity was higher in HUWE1-depleted cancer cells compared to wild type (WT) controls, indicating that HUWE1-mediated ubiquitination promotes ATG101 degradation. Moreover, enhanced autophagy in HUWE1-depleted cancer cells was reversed by siRNA-mediated ATG101 knockdown. Stable ATG101 level in HUWE1-depleted cells was a strong driver of autophagosome formation similar to upregulation of the known HUWE1 substrate WD repeat domain, phosphoinositide interacting 2 (WIPI2). Cellular survival rates were higher in HUWE1-knockdown cancer cells compared to controls, while concomitant siRNA-mediated ATG101 knockdown tends to increase apoptosis rate. Collectively, these results suggest that HUWE1 normally serves to suppress autophagy by ubiquitinating and triggering degradation of ATG101 and WIPI2, which in turn represses the survival of cancer cells. Accordingly, ATG101-mediated autophagy may play a critical role in overcoming metabolic stress, thereby contributing to the growth, survival, and treatment resistance of certain cancers.