Role of E3 ubiquitin ligases in lung cancer

E3 ubiquitin ligases are a large family of proteins that catalyze the ubiquitination of many protein substrates for targeted degradation by the 26S proteasome.Therefore,E3 ubiquitin ligases play an essential role in a variety of biological processes including cell cycle regulation,proliferation and apoptosis.E3 ubiquitin ligases are often found overexpressed in human cancers,including lung cancer,and their deregulation has been shown to contribute to cancer development.However,the lack of specific inhibitors in clinical trials is a major issue in targeting E3 ubiquitin ligases with currently only one E3 ubiquitin ligase inhibitor being tested in the clinical setting.In this review,we focus on E3 ubiquitin ligases that have been found deregulated in lung cancer.Furthermore,we discuss the processes in which they are involved and evaluate them as potential anti-cancer targets.By better understanding the mechanisms by which E3 ubiquitin ligases regulate biological processes and their exact role in carcinogenesis,we can improve the development of specific E3 ubiquitin ligase inhibitors and pave the way for novel treatment strategies for cancer patients.

Role of E3 ubiquitin ligases in gastric cancer

E3 ubiquitin ligases have an important role in carcinogenesis and include a large family of proteins that catalyze the ubiquitination of many protein substrates for targeted degradation by the 26S proteasome.So far,E3 ubiquitin ligases have been reported to have a role in a variety of biological processes including cell cycle regulation,cell proliferation,and apoptosis.Recently,several kinds of E3 ubiquitin ligases were demonstrated to be generally highly expressed in gastric cancer(GC) tissues and to contribute to carcinogenesis.In this review,we summarize thecurrent knowledge and information about the clinical significance of E3 ubiquitin ligases in GC.Bortezomib,a proteasome inhibitor,encouraged the evaluation of other components of the ubiquitin proteasome system for pharmaceutical intervention.The clinical value of novel treatment strategies targeting aberrant E3 ubiquitin ligases for GC are discussed in the review.

The role of E3 ubiquitin ligases in bone homeostasis and related diseases

The ubiquitin-proteasome system(UPS)dedicates to degrade intracellular proteins to modulate demic homeostasis and functions of organisms.These enzymatic cascades mark and modifies target proteins diversly through covalently binding ubiquitin molecules.In the UPS,E3 ubiquitin ligases are the crucial constituents by the advantage of recognizing and presenting proteins to proteasomes for proteolysis.As the major regulators of protein homeostasis,E3 ligases are indispensable to proper cell manners in diverse systems,and they are well described in physiological bone growth and bone metabolism.Pathologically,classic bone-related diseases such as metabolic bone diseases,arthritis,bone neoplasms and bone metastasis of the tumor,etc.,were also depicted in a UPS-dependent manner.Therefore,skeletal system is versatilely regulated by UPS and it is worthy to summarize the underlying mechanism.Furthermore,based on the current status of treatment,normal or pathological osteogenesis and tumorigenesis elaborated in this review highlight the clinical significance of UPS research.As a strategy possibly remedies the limitations of UPS treatment,emerging PROTAC was described comprehensively to illustrate its potential in clinical application.Altogether,the purpose of this review aims to provide more evidence for exploiting novel therapeutic strategies based on UPS for bone associated diseases.

Role of SKP1-CUL1-F-Box-Protein (SCF) E3 Ubiquitin Ligases in Skin Cancer

Many biological processes such as cell proliferation, differentiation, and cell death depend precisely on the timely synthesis and degradation of key regulatory proteins. While protein synthesis can be regulated at multiple levels, protein degradation is mainly controlled by the ubiquitin-proteasome system （UPS）, which consists of two distinct steps： （1） ubiquitylation of targeted protein by E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase, and （2） subsequent degradation by the 26S proteasome. Among all E3 ubiquitin ligases, the SCF （SKP1-CUL1-F-box protein） E3 ligases are the largest family and are responsible for the turnover of many key regulatory proteins. Aberrant regulation of SCF E3 ligases is associated with various human diseases, such as cancers, including skin cancer. In this review, we provide a comprehensive overview of all currently published data to define a promoting role of SCF E3 ligases in the development of skin cancer. The future directions in this area of research are also discussed with an ultimate goal to develop small molecule inhibitors of SCF E3 ligases as a novel approach for the treatment of human skin cancer. Furthermore, altered components or substrates of SCF E3 ligases may also be developed as the biomarkers for early diagnosis or predicting prognosis.

Plant E3 Ligases： Flexible Enzymes in a Sessile World

Since its discovery in the late 1970s, the ubiquitin proteasome pathway appears to be omnipresent in many research fields. Although originally discovered in animals, the pathway has a very central role in plants, which may be correlated to their sessile lifestyle. E3 ligases function as flexible and highly diverse key regulators within the path- way by targeting substrate proteins for ubiquitylation, and often proteolytic degradation via the 26S proteasome. This review provides a concise overview on the most common classes of E3 ligases so far described in plants, and emphasizes recent findings regarding these interesting and flexible enzymes and their diverse functions in plant biology.

PUB22 and PUB23 U-box E3 ubiquitin ligases negatively regulate 26S proteasome activity under proteotoxic stress conditions

The mechanism regulating proteasomal activity under proteotoxic stress conditions remains unclear.Here,we showed that arsenite-induced proteotoxic stress resulted in upregulation of Arabidopsis homologous PUB22 and PUB23 U-boxE3 ubiquitin ligases and that pub22 pub23 double mutants displayed arsenite-insensitive seed germination and root growth phenotypes.PUB22/PUB23 downregulated 26 S proteasome activity by promoting the dissociation of the 19 S regulatory particle from the holo-proteasome complex,resulting in intracellular accumulation of UbG76 VGFP,an artificial substrate of the proteasome complex,and insoluble poly-ubiquitinated proteins.These results suggest that PUB22/PUB23 play a critical role in arsenite-induced proteotoxic stress response via negative regulation of 26 S proteasome integrity.

Genetic investigation of the ubiquitin-protein ligase E3A gene as putative target in Angelman syndrome

BACKGROUND Angelman syndrome(AS)is caused by maternal chromosomal deletions,imprinting defects,paternal uniparental disomy involving chromosome 15 and the ubiquitin-protein ligase UBE3A gene mutations.However the genetic basis remains unclear for several patients.AIM To investigate the involvement of UBE3A gene in AS and identifying new potential genes using exome sequencing.METHODS We established a cohort study in 50 patients referred to Farhat Hached University Hospital between 2006 and 2021,with a strong suspicion of AS and absence of chromosomal aberrations.The UBE3A gene was screened for mutation detection.Two unrelated patients issued from consanguineous families were subjected to exome analysis.RESULTS We describe seven UBE3A variants among them 3 none previously described including intronic variants c.2220+14T>C(intron14),c.2507+43T>A(Exon15)and insertion in Exon7:c.30-47_30-46.The exome sequencing revealed 22 potential genes that could be involved in AS-like syndromes that should be investigated further.CONCLUSION Screening for UBE3A mutations in AS patients has been proven to be useful to confirm the diagnosis.Our exome findings could rise to new potential alternative target genes for genetic counseling.

The emerging roles of E3 ubiquitin ligases in ovarian cancer chemoresistance

Epithelial cancer of the ovary exhibits the highest mortality rate of all gynecological malignancies in women today,since the disease is often diagnosed in advanced stages.While the treatment of cancer with specific chemical agents or drugs is the favored treatment regimen,chemotherapy resistance greatly impedes successful ovarian cancer chemotherapy.Thus,chemoresistance becomes one of the most critical clinical issues confronted when treating patients with ovarian cancer.Convincing evidence hints that dysregulation of E3 ubiquitin ligases is a key factor in the development and maintenance of ovarian cancer chemoresistance.This review outlines recent advancement in our understanding of the emerging roles of E3 ubiquitin ligases in ovarian cancer chemoresistance.We also highlight currently available inhibitors targeting E3 ligase activities and discuss their potential for clinical applications in treating chemoresistant ovarian cancer patients.

Four Closely-related RING-type E3 Ligases,APD1-4,are Involved in Pollen Mitosis II Regulation in Arabidopsis

Ubiquitination of proteins is one of the critical regulatory mechanisms in eukaryotes. In higher plants, protein ubiquitination plays an essential role in many biological processes, including hormone signaling, photomorphogenesis, and pathogen defense. However, the roles of protein ubiquitination in the repro- ductive process are not clear. In this study, we identified four plant-specific RING-finger genes designated Aberrant_Pollen Development 1_ （APD1） to APD4, as regulators of pollen mitosis II （PMII） in Arabidopsis thaliana （L.）. The apdl apd2 double mutant showed a significantly increased percentage of bicellular-like pollen at the mature pollen stage. Further downregulation of the APD3 and APD4 transcripts in apdl apd2 by RNA interference （RNAi） resulted in more severe abnormal bicellular-like pollen phenotypes than in apdl apd2, suggesting that cell division was defective in male gametogenesis. All of the four genes were expressed in multiple stages at different levels during male gametophyte development. Confocal analysis using green florescence fusion proteins （GFP） GFP-APD1 and GFP-APD2 showed that APDs are associated with intracellular membranes. Furthermore, APD2 had E2-dependent E3 ligase activity in vitro, and five APD2-interacting proteins were identified. Our results suggest that these four genes may be involved, redundantly, in regulating the PMII process during male gametogenesis.

Identification of Arabidopsis MYB56 as a Nove Substrate for CRL3BPM E3 Ligases

Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work, MYB56 is identified as a novel target of a CULLIN3 （CUL3）-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ （BPM） proteins, which function as substrate adaptors to the E3 ligase. Genetic studies have indicated that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T （FT）, a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling flowering time in plants.

Immune regulation by protein ubiquitination: roles of the E3 ligases VHL and Itch

Protein ubiquitination is an important means of posttranslational modification which plays an essential role in the regulation of various aspects of leukocyte development and function. The specificity of ubiquitin tagging to a protein substrate is determined by E3 ubiquitin ligases via defined E3-substrate interactions. In this review, we will focus on two E3 ligases, VHL and Itch, to discuss the latest progress in understanding their roles in the differentiation and function of CD4+ T helper cell subsets, the stability of regulatory T cells, effector function of CD8+ T cells, as well as the development and maturation of innate lymphoid cells. The biological implications of these E3 ubiquitin ligases will be highlighted in the context of normal and dysregulated immune responses including the control of homeostasis, inflammation, auto-immune responses and anti-tumor immunity. Further elucidation of the ubiquitin system in immune cells will help in the design of new therapeutic interventions for human immunological diseases and cancer.

Endoplasmic reticulum-related E3 ubiquitin ligases: Key regulators of plant growth and stress responses

Accumulating evidence has revealed that the ubiquitin proteasome system plays fundamental roles in the regulation of diverse cellular activities in eukaryotes.The ubiquitin protein ligases(E3s)are central to the proteasome system because of their ability to determine its substrate specificity.Several studies have demonstrated the essential role of a group of ER(endoplasmic reticulum)-localized E3s in the positive or negative regulation of cell homeostasis.Most ER-related E3s are conserved between plants and mammals,and a few plant-specific components have been reported.In this review,we summarize the functions of ERrelated E3s in plant growth,ER-associated protein degradation and ER-phagy,abiotic and biotic stress responses,and hormone signaling.Furthermore,we highlight several questions that remain to be addressed and suggest directions for further research on ER-related E3 ubiquitin ligases.

Novel insights into the mechanism of reactive oxygen species-mediated neurodegeneration

Neurite degeneration,a major component of many neurodegenerative diseases,such as Parkinson’s disease,Alzheimer’s disease,and amyotrophic lateral sclerosis,is not part of the typical apoptosis signaling mechanism,but rather it appears that a self-destructive process is in action.Oxidative stress is a well-known inducer of neurodegenerative pathways:neuronal cell death and neurite degeneration.Although oxidative stress exerts cytotoxic effects leading to neuronal loss,the pathogenic mechanisms and precise signaling pathways by which oxidative stress causes neurite degeneration have remained entirely unknown.We previously reported that reactive oxygen species generated by NADPH oxidases induce activation of the E3 ubiquitin ligase ZNRF1 in neurons,which promotes neurite degeneration.In this process,the phosphorylation of an NADPH oxidase subunit p47-phox at the 345serine residue serves as an important checkpoint to initiate the ZNRF1-dependent neurite degeneration.Evidence provides new insights into the mechanism of reactive oxygen species-mediated neurodegeneration.In this review,we focus specifically on reactive oxygen species-induced neurite degeneration by highlighting a phosphorylation-dependent regulation of the molecular interaction between ZNRF1 and the NADPH oxidase complex.

Fine mapping of a major QTL qHYF_B06 for peanut yield

High yield is a major objective for peanut(Arachis hypogaea L.) breeding worldwide. However, fewer yield-related quantitative trait loci(QTL) have been reported in peanut than in other staple food crops such as rice(Oryza sativa), wheat(Triticum aestivum), and maize(Zea mays). This study aimed to identify stable major-effect QTL associated with pod yield per plant, hundred-pod weight for double-seeded pods,hundred-seed weight, shelling percentage, and pod number per plant, allowing us to predict candidate genes by means of transcriptome and genome sequencing. To this end, we used a population of recombinant inbred lines comprising 192 F9:11families derived from a JH6 × KX01-6 cross to construct a highresolution genetic map(1705.7 c M) consisting of 2273 polymorphic SNPs, with 0.75 c M(on average)between adjacent SNPs. We identified two high-confidence, yield-related QTL, qHYF_A08 and qHYF_B06, explaining 5.78%–31.40% of phenotypic variation and with LOD values of 5.10–24.48, in six environments. qHYF_A08 mainly explained the variation in shelling percentage, whereas qHYF_B06explained variation in hundred-pod weight and hundred-seed weight and accounted for 8.77%–31.40%of the variation in effective pod number per plant, pod number per plant, and shelling percentage. We narrowed down qHYF_B06 to an 890-kb interval using an advanced mapping population.Transcriptome and genome analyses revealed that only Arahy.129FS0 and Arahy.3R9A5K in the candidate mapping interval were differentially expressed between JH6 and KX01-6, with substantial structural variations in their promoter and coding regions. Genotypes of 208 peanut accessions determined using a diagnostic CAPS marker suggested that the two haplotypes of Arahy.3R9A5K were highly associated with hundred-seed weight and hundred-pod weight;this diagnostic CAPs marker could therefore be useful for selecting high-yielding lines during peanut breeding. Overall, our results provide valuable information for cloning alleles with favorable effects on peanut yield.

Research Progress in Function and Regulation of E3 Ubiquitin Ligase SMURF1

Smad ubiquitylation regulatory factor 1(Smurf1)is an important homologous member of E6-AP C-terminus type E3 ubiquitin ligase.Initially,Smurf1 was reportedly involved in the negative regulation of the bone morphogenesis protein(BMP)pathway.After further research,several studies have confirmed that Smurf1 is widely involved in various biological processes,such as bone homeostasis regulation,cell migration,apoptosis,and planar cell polarity.At the same time,recent studies have provided a deeper understanding of the regulatory mechanisms of Smurf1’s expression,activity,and substrate selectivity.In our review,a brief summary of recent important biological functions and regulatory mechanisms of E3 ubiquitin ligase Smurf1 is proposed.

抑制Ligase4或Xrcc6活性增强斑马鱼原始生殖细胞中DNA同源重组的效率(英文)

利用斑马鱼作为体内模型,研究旨在提高斑马鱼原始生殖细胞(Primordial germ cells,PGCs)中同源重组(Homologous recombination,HR)的效率。首先,将UAS:m RFP-nos1载体显微注射到Tg(kop:Kal TA4)转基因胚胎中标记转基因PGCs,结果表明筛选PGCs特异表达m RFP的胚胎能够相对提高转基因的生殖系传递效率。随后建立了PGCs中HR效率的评估体系,并且证明抑制DNA ligase IV(Lig4)和Xrcc6(曾用名Ku70)的活性不但在全胚胎水平,而且在PGCs水平都能够显著提高HR的效率。研究表明Tg(kop:Kal TA4)转基因品系是开展HR介导的基因打靶的一个有效平台。

Targeting the COP9 signalosome for cancer therapy

The COP9 signalosome(CSN)is a highly conserved protein complex composed of 8 subunits(CSN1 to CSN8).The individual subunits of the CSN play essential roles in cell proliferation,tumorigenesis,cell cycle regulation,DNA damage repair,angiogenesis,and microenvironmental homeostasis.The CSN complex has an intrinsic metalloprotease that removes the ubiquitin-like activator NEDD8 from cullin-RING ligases(CRLs).Binding of neddylated CRLs to CSN is sensed by CSN4 and communicated to CSN5 with the assistance of CSN6,thus leading to the activation of deneddylase.Therefore,CSN is a crucial regulator at the intersection between neddylation and ubiquitination in cancer progression.Here,we summarize current understanding of the roles of individual CSN subunits in cancer progression.Furthermore,we explain how the CSN affects tumorigenesis through regulating transcription factors and the cell cycle.Finally,we discuss individual CSN subunits as potential therapeutic targets to provide new directions and strategies for cancer therapy.

Overexpressed NEDD8 as a potential therapeutic target in esophageal squamous cell carcinoma

Objective:The hyperactivated neddylation pathway plays an important role in tumorigenesis and is emerging as a promising anticancer target.We aimed to study whether NEDD8(neural precursor cell expressed,developmentally down-regulated 8)might serve as a therapeutic target in esophageal squamous cell carcinoma(ESCC).Methods:The clinical relevance of NEDD8 expression was evaluated by using The Cancer Genome Atlas(TCGA)database and tissue arrays.NEDD8-knockdown ESCC cells generated with the CRISPR/Cas9 system were used to explore the anticancer effects and mechanisms.Quantitative proteomic analysis was used to examine the variations in NEDD8 knockdown-induced biological pathways.The cell cycle and apoptosis were assessed with fluorescence activated cell sorting.A subcutaneous-transplantation mouse tumor model was established to investigate the anticancer potential of NEDD8 silencing in vivo.Results:NEDD8 was upregulated at both the mRNA and protein expression levels in ESCC,and NEDD8 overexpression was associated with poorer overall patient survival(mRNA level:P=0.028,protein level:P=0.026,log-rank test).Downregulation of NEDD8 significantly suppressed tumor growth both in vitro and in vivo.Quantitative proteomic analysis revealed that downregulation of NEDD8 induced cell cycle arrest,DNA damage,and apoptosis in ESCC cells.Mechanistic studies demonstrated that NEDD8 knockdown led to the accumulation of cullin-RING E3 ubiquitin ligases(CRLs)substrates through inactivation of CRLs,thus suppressing the malignant phenotype by inducing cell cycle arrest and apoptosis in ESCC.Rescue experiments demonstrated that the induction of apoptosis after NEDD8 silencing was attenuated by DR5 knockdown.Conclusions:Our study elucidated the anti-ESCC effects and underlying mechanisms of NEDD8 knockdown,and validated NEDD8 as a potential target for ESCC therapy.

RNF31表达下调抑制TNF-α刺激的NF-κB通路的激活

目的利用慢病毒干涉下调内源性RNF31表达,研究NF-κB通路的活化及对细胞凋亡的影响。方法将人RNF31的shRNA片段克隆到慢病毒表达载体p Green Puro中,瞬时转染HEK293T细胞,筛选出有效的干涉片段。将重组表达质粒与包装质粒PMD、SPA共转染293T细胞,在24 h、48 h分2次收集慢病毒上清,用流式细胞术检测病毒滴度。将获得的病毒感染HEK293细胞,提取细胞蛋白,Real-time PCR以及Western Blot检测RNF31干涉效果;报告基因实验检测敲低RNF31对NF-κB转录活性的影响;Real-time PCR检测干涉RNF31对TNF-α诱导的NF-κB下游靶基因的影响;Western Blot检测下调RNF31对IκBα活化的影响;Hochest染色检测下调RNF31对细胞凋亡的影响。结果成功构建RNF31干涉慢病毒p Green Puro-RNF31载体并获得慢病毒颗粒,病毒滴度可达3×107pfu/ml。在HEK293细胞中下调RNF31,抑制TNF-α刺激的NF-κB的转录活性,并抑制NF-κB下游靶基因的表达;下调RNF31抑制TNF-α刺激的IκBα的活化;此外,在TNF-α刺激细胞24 h时,RNF31表达下调使细胞凋亡增多。结论 RNF31表达下调抑制TNF-α刺激的NF-κB通路的激活。

沉默Ku70促进PARP1、ligase3和XRCC1在DNA双链断裂染色质聚集

目的研究碱基切除修复途径(base excision repair,BER)关键因子PARP1、ligase3和XRCC1在Ku70缺失的条件下与DNA双链断裂(double-strand break,DSB)所在染色质的结合及抑制PARP1所产生的影响。方法采用可经药物诱导Ku70 shRNA的细胞株,以高效的DNA双链断裂诱导剂刺孢霉素calicheamicin诱导DSB。分离不同组分蛋白联合稳定同位素标记定量差异蛋白组分析法鉴定Ku70下调后在损伤染色质聚集增多的蛋白,Western blot进行验证并检测PARP1抑制剂DIQ对其染色质聚集的影响。结果在药物Doxy作用下,Ku70明显下调,其余蛋白的表达不受影响。稳定同位素标记定量蛋白组学方法鉴定出PARP1、ligase3和XRCC1在Ku70下调后在损伤染色质聚集增多。Western blot进行了验证并显示PARP1、ligase3和XRCC1在损伤染色质的聚集随DSB程度增加而增加。施加PARP1抑制剂DIQ,随着DIQ浓度的上升,ligase3在损伤染色质的聚集逐渐减少。结论沉默Ku70后,PARP1、ligase3和XRCC1在DSB染色质聚集增加,这种聚集与DSB程度变化一致,并且ligase3的染色质聚集具有一定程度的PARP1依赖性。