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.

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.

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.

Molecular cloning and functional characterization of apple U-box E3 ubiquitin ligase gene MdPUB29 reveals its involvement in salt tolerance

An E3 ubiquitin ligase gene(Genbank accession no.:MD01 G1010900) was cloned from the Royal Gala apple genome(Malus×domestica Borkh.).Sequence analysis showed that the length of the MdPUB29 gene was 1 275 bp,encoding 424 amino acids.Phylogenetic tree analysis indicated that the apple E3 ubiquitin ligase exhibited the greatest sequence similarity to Pyrus×bretschneideri.The predicted protein structural domain of MdPUB29 showed that it contained a U-box domain.qRT-PCR analysis showed that Md PUB29 was expressed widely in different tissues of the Royal Gala apple species,and was highly expressed in the root,while the expression of MdPUB29 was significantly inhibited by exogenous NaCl.Immunoblotting assays revealed that MdPUB29 protein abundance in tissue cultures of the Royal Gala apple accumulated under NaC l stress conditions.Three-dimensional protein structure prediction indicated that MdPUB29 was highly homologous with AtPUB29.The growing potential of MdPUB29-expressing apple calli and Arabidopsis were much stronger than that of the control under salt stress conditions,suggesting that MdPUB29 may positively regulate salt tolerance.

Human RING finger protein ZNF645 is a novel testis-specific E3 ubiquitin ligase

A large number of testis-specific genes are involved in the complex process of mammalian spermatogenesis. Identification of these genes and their roles is important for understanding the mechanisms underlying spermatogenesis. Here we report on a novel human RING finger protein, ZNF645, which contains a C3HC4 RING finger domain, a C2H2 zinc-finger domain, and a proline-rich region, indicating that it has a structure similar to that of the c-Cbl-like protein Hakai. ZNF645 was exclusively expressed in normal human testicular tissue. Immunohistochemical analysis confirmed that ZNF645 protein was present in spermatocytes, round and elongated spermatids, and Leydig cells. Immunofluorescence staining of mature sperms further showed that the ZNF645 protein was localized over the postacrosomal perinuclear theca region and the entire length of sperm tail. An in vitro ubiquitination assay indicated that the RING finger domain of the ZNF645 protein had E3 ubiquitin ligase activity. Therefore, we suggest that ZNF645 might act as an E3 ubiquitin-protein ligase and play a role in human sperm production and quality control.

RNF13： a novel RING-type ubiquitin ligase over-expressed in pancreatic cancer

Protein ubiquitination by E3 ubiquitin ligases plays an important role in cancer development. In this study, we provide experimental evidence that a RING-finger-containing protein RNF13 is an ER/Golgi membrane-associated E3 ubiquitin ligase and its RING finger domain is required for the ubiquitin ligase activity. Immunohistochemical analysis of pancreatic ductal adenocarcinoma （PDAC） and paracancerous normal tissues from 72 patients documented RNF13 over-expression in 30 tumor samples （41.7%, 30/72）, and its expression was significantly associated with histological grading （P= 0.024）. In addition, RNF13 was detected in precancerous lesions： tubular complexes in chronic pancreatitis （CP） and pancreatic intraepithelial neoplasia （PanIN） （79.3%, 23/29 and 62.8%, 22/35, respectively）. Moreover, RNF13 staining was significantly correlated with Tenascin-C expression （P = 0.004） in PDAC samples, further supporting the role of RNF13 in cancer progression. Over-expression of wild type but not RING domain-mutant RNF13 in pancreatic MiaPaca-2 cancer cells increased invasive potential and gelatinolytic activity by matrix metalloproteinase-9. Taken together, these findings reveal that RNF13 is a novel E3 ubiquitin ligase involved in pancreatic carcinogenesis; ubiqui-tin-mediated modification of proteins by RNF13 may participate in pancreatic cancer development.

Characterization of a Novel RING-Type Ubiquitin E3 Ligase GhRING2 Differentially Expressed in Cotton Fiber

The ubiquitin-proteasome proteolysis pathway is responsible for the degradation of abnormal and short-lived proteins to regulate many important biochemical activities in eukaryotes. By employing affymetrix microarray analysis, we have identified a novel ubiquitin ligase E3 gene GhRING2 that is differentially expressed between two Gossypium hirsutum lines-Texas Marker-1 (TM-1) and Chromosome Substitution Line CS-B25. The CS-B25 line has chromosome 25 from G. barbadense substituted into TM-1. The complete nucleotide sequences of GhRING2 along with its 5’-flanking region were obtained by genomic walking. GhRING2 was highly expressed in elongating fiber, and GUS expression directed by the GhRING2 promoter was found in hypocotyls and young stems of transgenic Arabidopsis plants. Using a yeast two-hybrid assay GhRING2 was found to interact with a PROTODERMAL FACTOR1 (GhPDF1) protein. GhPDF1 was expressed preferentially in immature ovules and fiber initials, and the GhPDF1 gene had been suggested to play a role in cell fate determination and fiber development. Pull down and plasmid swap assays further confirmed the interaction between GhRING2 and GhPDF1. The expression and protein interaction data indicate that GhRING2 may be involved in the turnover of GhPDF1 and participation in the transition from initiation to elongation stages during fiber development. Our data strongly suggest that the ubiquitin-proteasome pathway may regulate cotton fiber growth and development. The nucleotide sequence data of GhRING2 in this article have been submitted to the Gen Bank Nucleotide Sequence Data Bases under the accession number BankIt 1,742,008 SeqKM 108,000.

The E3 ubiquitin ligase seven in absentia homolog 1 may be a potential new therapeutic target for Parkinson's disease

In this study, we investigated the effect of an antibody against E3 ubiquitin ligase seven in absentia homolog 1（SIAH-1） in PC12 cells. 1-Methyl-4-phenylpyridinium（MPP＋） treatment increased α-synuclein, E1 and SIAH-1 protein levels in PC12 cells, and it reduced cell viability; however, there was no significant change in light chain 3 expression. Treatment with an SIAH-1 antibody decreased m RNA expression levels of α-synuclein, light chain 3 and SIAH-1, but increased E1 m RNA expression. It also increased cell viability. Combined treatment with MPP＋ and rapamycin reduced SIAH-1 and α-synuclein levels. Treatment with SIAH-1 antibody alone diminished α-synuclein immunoreactivity in PC12 cells, and reduced the colocalization of α-synuclein and light chain 3. These findings suggest that the SIAH-1 antibody reduces the monoubiquitination and aggregation of α-synuclein, promoting its degradation by the ubiquitin-proteasome pathway. Consequently, SIAH-1 may be a potential new therapeutic target for Parkinson’s disease.

Research status of E3 ubiquitin ligase Smurf2 and related signaling pathways in glioma

Glioma is the tumor with the highest incidence in the brain,and it is eager to seek new and efiective treatment.The interaction of ubiquitination and deubiquitination regulates many cell activities in organisms,and participates in tumor occurrence,development,migration,invasion and other processes.This article summarized the progress of E3 ubiquitination ligase smad ubiquitination regulatory factor 2(Smurf2)and glioma-related signaling pathways to assist clinical diagnosis and treatment of glioma.

Antiserum Preparation and Specific Detection of Banana RING-E3 Ubiquitin-Protein Ligase

According to the data of banana transcriptome sequencing, an E3 ubiquitin-protein ligase gene was cloned by RT-PCR method using the cDNA sample of banana leaves. The complete ORF of E3 ubiquitin-protein ligase is 681 bp long and its encoded protein showed 100% sequence identity to homologue RING-H2 finger protein (XP_009407047.1) of Musa_acuminata. Bioinformatic analysis indicated that E3 ubiquitin-protein ligase contains the Ring finger domain in C terminus and eight cross-brace motifs are found in the domain. The target gene was digested by EcoR V and EcoR I, and was inserted into prokaryotic expression vector pET-32a of the same digestions to obtain the plasmid pET32a-E3 ubiquitin-protein ligase. The recombinant plasmid was introduced into Escherichia coli strain BL21 (DE3), and induced at 25°C with 0.4 mmol/L IPTG for 6 hours. The soluble fusion protein was expressed and high purity fusion protein was obtained by Ni2+-NTA agarose affinity chromatography purification. The fusion protein was injected into mice 3 times to prepare the antiserum. Western blot analysis showed a specific protein band was detected in total protein sample of banana leaves, but not for the samples of wild-type Nicotiana benthamiana (N.B.) and wild-type Arabidopsis thaliana (A.T.), implying the antiserum was specific to banana E3 ubiquitin-protein ligase.

泛素化修饰关键酶在植物抗逆反应中的功能研究进展

泛素化修饰是植物蛋白质翻译后修饰的重要组成部分,通过对蛋白质的选择性降解,参与调控植物生长发育和多种逆境胁迫反应。泛素化修饰反应由3种关键酶协同作用完成。泛素分子通过与泛素激活酶的巯基酯键连接从而被激活,被激活的泛素分子再与泛素结合酶形成复合体,最后在泛素连接酶的作用下完成与靶蛋白的结合。随着蛋白质组学测序技术的不断发展,人们对泛素化修饰的研究更加普遍和深入。大量被泛素化修饰的蛋白及其修饰位点被鉴定出来,有助于深入了解蛋白质的调控机制,进一步解析蛋白质的功能。本文介绍了泛素-蛋白酶体系统的反应过程,泛素化修饰关键酶的结构、数量及分类,并重点围绕泛素激活酶、泛素结合酶和泛素连接酶在植物应对非生物及生物胁迫中的功能研究开展论述,同时也对植物泛素化修饰关键酶的功能研究所面临的问题进行总结,并对泛素化修饰与其他修饰之间的串扰进行了讨论与展望,对于泛素化修饰在植物逆境胁迫领域的深入研究具有重要意义。

灵芝孢子干预糖尿病大鼠睾丸组织线粒体自噬及细胞的凋亡

背景:男性生殖障碍作为糖尿病的常见并发症近年来受到越来越多的关注。灵芝孢子具有降糖、抗氧化、抗炎等功效,但其对于糖尿病睾丸组织的调控机制尚未完全阐明。目的:探讨灵芝孢子对糖尿病大鼠睾丸组织PTEN诱导激酶1/E3泛素蛋白连接酶通路及细胞凋亡的影响。方法:采用随机数字表法将40只雄性SD大鼠分为正常组、高脂高糖组、糖尿病组、灵芝孢子干预组,每组10只:高脂高糖组、糖尿病组、灵芝孢子干预组给予高脂高糖饮食至实验结束;高脂高糖饮食1个月后,糖尿病组、灵芝孢子干预组腹腔注射链脲佐菌素30 mg/kg建立2型糖尿病模型;造模成功后,灵芝孢子干预组灌胃给予灵芝孢子300 mg/(kg·d),其余3组灌胃给予等量生理盐水,持续给药12周。给药结束后,检测大鼠精子数量和形态、睾丸组织形态结构、血清睾酮和睾丸组织氧化应激水平,免疫组化分析PTEN诱导激酶1、E3泛素蛋白连接酶、抗核孔蛋白表达,Western blotting检测睾丸组织中PTEN诱导激酶1、E3泛素蛋白连接酶、抗核孔蛋白、程序性死亡受体1、微管相关蛋白轻链3Ⅱ/Ⅰ、caspase3、剪切caspase3的蛋白表达。结果与结论:①与正常组和高脂高糖组相比,糖尿病组大鼠精子数量减少(P<0.01),精子畸形率增加(P<0.01),血清睾酮浓度降低(P<0.01);与糖尿病组相比,灵芝孢子干预组大鼠精子数量增加(P<0.05),畸形率降低(P<0.01),血清睾酮浓度升高(P<0.01);②与正常组和高脂高糖组相比,糖尿病组大鼠睾丸组织中丙二醛水平升高(P<0.01),谷胱甘肽还氧化物酶和超氧化物歧化酶水平降低(P<0.01);与糖尿病组相比,灵芝孢子干预组大鼠睾丸组织中丙二醛水平降低(P<0.01),谷胱甘肽还氧化物酶和超氧化物歧化酶水平升高(P<0.01);③免疫组化染色显示,与正常组和高脂高糖组对比,糖尿病组大鼠睾丸组织中PTEN诱导激酶1、E3泛素蛋白连接酶阳性表达减少,抗核孔蛋白阳性表达增加;与糖尿病组相比,灵芝孢子干预组大鼠睾丸组织中PTEN诱导激酶1、E3泛素蛋白连接酶阳性表达增加,抗核孔蛋白阳性表达减少;④Western blotting检测显示,与正常组和高脂高糖组相比,糖尿病组PTEN诱导激酶1、E3泛素蛋白连接酶、程序性死亡受体1蛋白表达及微管相关蛋白轻链3Ⅱ/Ⅰ蛋白比值降低(P<0.05或P<0.01),抗核孔蛋白、caspase3、剪切caspase3的蛋白表达升高(P<0.01);与糖尿病组相比,灵芝孢子干预组PTEN诱导激酶1、E3泛素蛋白连接酶、程序性死亡受体1蛋白表达及微管相关蛋白轻链3Ⅱ/Ⅰ蛋白比值升高(P<0.05或P<0.01),抗核孔蛋白、caspase3、剪切caspase3的蛋白表达降低(P<0.05或P<0.01);⑤结果显示,灵芝孢子可能通过激活PTEN诱导激酶1/E3泛素蛋白连接酶信号通路增强睾丸组织自噬水平,减少组织细胞的凋亡,以此保护睾丸组织。

三孢布拉霉CrgA泛素连接酶功能的初步探究

CrgA已经被证实是三孢布拉霉和一些其他丝状真菌中类胡萝卜素生物合成的一个负调控因子。环指结构域的存在表明,CrgA可能具有泛素连接酶的功能,是泛素化调节系统中的一个关键酶。为了验证这一假设,从三孢布拉霉中分离并鉴定了一种泛素激活酶(BtE1)和18种假定的泛素结合酶(UBC)。生物信息学分析表明,18种UBC蛋白质都包含一个UBC的保守结构域,表明它们都属于泛素结合酶。系统发育关系分析表明,18个UBC蛋白质属于7个蛋白质亚家族。根据系统进化分析结果筛选出6种候选UBC蛋白质,通过异源表达及亲和纯化得到BtE1、6种BtUBC蛋白质、BtCrgA和BtWC-1b,并在体外进行泛素化反应,BtCrgA能在BtE1和BtUBC D的协助下完成对自身的泛素修饰功能。

小麦E3泛素连接酶TaRING1互作靶标筛选与验证

【目的】为探究TaRING1在小麦与条锈菌互作过程中的作用,解析其作用机理,为小麦条锈病的绿色防控提供理论依据。【方法】利用酵母双杂交技术筛选TaRING1的互作靶标蛋白,并通过荧火素酶互补实验(LCA)及双分子荧光互补(BiFC)验证互作,通过泛素化实验对TaRING1的泛素功能进行验证,通过烟草瞬时表达及小麦原生质体转化分析靶标TaRIP92的亚细胞定位。【结果】以TaRING1作为诱饵,利用酵母双杂交技术筛选到互作靶标TaRIP92,并通过LCA及BiFC验证TaRING1与TaRIP92互作,通过体外泛素化实验证明TaRING1可以泛素化底物TaRIP92,烟草瞬时表达及小麦原生质体转化发现TaRIP92蛋白定位于线粒体。【结论】TaRING1通过与线粒体蛋白TaRIP92互作并对其进行泛素化。

泛素化修饰及其调控果实胁迫响应的研究进展

简要介绍泛素化修饰在果实抵御外界逆境胁迫中的重要调控作用,总结泛素化修饰体系,特别是能特异性识别靶蛋白的E3泛素连接酶和具有蛋白酶水解活性的26S蛋白酶体的组成及该体系对靶蛋白的修饰过程。在此基础上,重点阐述以E3泛素连接酶为核心的泛素化修饰在果实应对包括干旱、盐、寒冷、重金属和衰老在内的非生物胁迫,以及病原菌侵染等生物胁迫中的调控作用及其可能的机制。最后指出目前果实中泛素化修饰研究中仍然存在包括泛素化基因和酶功能的鉴定不足、泛素化修饰存在时空调控与去泛素化现象、泛素化修饰位点鉴定手段的缺乏等问题,并提出解决这些问题的技术与方法,以期为深入解析泛素化修饰在果实品质形成和保持中的作用及其机制提供参考。

E3泛素连接酶调控果蔬生长发育及逆境响应的研究进展

蛋白质泛素化作为一种重要的翻译后修饰,在真核细胞的生命活动中发挥着广泛的作用。E3泛素连接酶在泛素-蛋白酶体降解系统中能够特异性地识别目标蛋白,在泛素化途径中起决定性作用。近年来研究表明,E3泛素连接酶在果蔬作物的生长发育及逆境胁迫响应中发挥着广泛的调控作用。本文介绍了果蔬作物中的泛素系统及E3泛素连接酶的种类,并重点综述了近年来E3泛素连接酶在果蔬生长发育及逆境胁迫响应中的调控作用及机制,旨在为深入揭示E3泛素连接酶在果蔬生命活动中的调控作用提供参考。

泛素连接酶MuRF2通过抑制线粒体自噬减轻心肌细胞缺氧损伤

目的探讨泛素连接酶肌肉环指状蛋白2(muscle ring finger protein 2,MuRF2)对缺氧心肌细胞活力和自噬的调控作用。方法构建大鼠源MuRF2基因过表达和敲减慢病毒载体,分别感染大鼠心肌细胞H9C2。将H9C2细胞分为阴性对照组(NC)、阳性对照组[MuRF2过表达空载体组(LV-Vector)、MuRF2敲减空载体组(LV-RNAi-Vector)]、Mu RF2过表达组(LV-MuRF2)和MuRF2敲减组(LV-RNAi-MuRF2),缺氧培养(5%CO_(2)、1%O_(2)、94%N_(2))后,采用CCK-8法和ELISA法检测心肌细胞损伤水平,利用透射电子显微镜检测心肌细胞超微结构和自噬水平变化,采用Western blot测定自噬相关蛋白LC3-Ⅱ和P62的表达水平。结果与对照组相比,缺氧组心肌细胞间隙增大、细胞皱缩、多见漂浮的死亡细胞和细胞碎片,心肌细胞活力下降、肌钙蛋白Ⅰ(cTnⅠ)含量增加(P均<0.05);电子显微镜下可观察到部分线粒体发生肿胀,并存在嵴断裂、嵴溶解现象,自噬小体散在胞质内;缺氧组LC3-Ⅱ蛋白表达水平升高,P62蛋白表达水平降低(P均<0.05)。缺氧培养后,与NC组和LV-Vector组相比,LV-MuRF2组细胞活力增加,偶见自噬小体,LC3-Ⅱ蛋白表达水平降低、P62蛋白表达水平增高(P均<0.05);与NC组和LV-RNAi-Vector组相比,LV-RNAi-MuRF2组细胞活力降低,可见大量自噬小体,LC3-Ⅱ蛋白表达水平升高、P62蛋白表达水平降低(P均<0.05)。结论泛素连接酶MuRF2可减轻缺氧所致的心肌细胞损伤,其机制可能与抑制心肌细胞线粒体自噬有关。

水稻泛素连接酶D3与抗病相关蛋白VOZ2的互作分析

多蘖矮秆基因dwarf-3(D3)是水稻独脚金内酯信号转导过程中的重要节点基因,拟南芥中的MAX2基因与D3同源,且MAX2参与拟南芥的抗病防卫反应。本研究以水稻泛素连接酶D3为诱饵进行酵母双杂筛库,发现水稻抗病相关蛋白维管植物单锌指蛋白VOZ2与D3存在潜在的相互作用。通过酵母双杂交试验证实,D3与VOZ2存在互作。通过荧光定量PCR证实,接种水稻白叶枯病菌后,VOZ2基因在转录水平上的表达受到显著诱导。利用水稻原生质体开展的亚细胞共定位实验发现,D3与VOZ2共定位于细胞核。双分子荧光互补实验发现,D3与VOZ2在烟草叶肉细胞的细胞核和细胞质均产生较强的荧光,进一步证实了D3与VOZ2的相互作用。研究结果为进一步探究D3和VOZ2在水稻抗病防卫反应中的功能与分子机理奠定了基础。