Trim 蛋白家族与肿瘤

研究发现部分 Trim 蛋白家族成员与肿瘤发生发展过程相关。不同的 Trim 家族成员在肺癌、肝癌、结直肠癌、胃癌、乳腺癌等肿瘤中呈差异性表达,并通过调控 p53活性、核转录因子-κB 信号通路等影响肿瘤相关基因的表达,参与肿瘤进程。Trim 家族成员可以作为抑癌基因或癌基因影响肿瘤细胞的增殖、分化、凋亡、侵袭迁移等生物学过程。     

Inhibition of NEDD8-conjugation pathway by novel molecules: potential approaches to anticancer therapy

Cancer cells can survive through the upregulation of cell cycle and the escape from apoptosis induced by numerous cellular stresses. In the normal cells, these biological cascades depend on scheduled proteolytic degradation of regulatory proteins via the ubiquitin-proteasome pathway. Therefore, interruption of regulated proteolytic pathways leads to abnormal cell-proliferation. Ubiquitin ligases called SCF complex (consisting of Skp-1, cullin, and F-box protein) or CRL (cullin-RING ubiquitin ligase) are predominant in a family of E3 ubiquitin ligases that control a final step in ubiquitination of diverse substrates. To a great extent, the ubiquitin ligase activity of the SCF complex requires the conjugation of NEDD8 to cullins, i.e. scaffold proteins. This review is anticipated to review the downregulation system of NEDD8 conjugation by several factors including a chemical compound such as MLN4924 and protein molecules (e.g. COP9 signalosome, inactive mutant of Ubc12, and NUB1/NUB1L). Since the downregulation of NEDD8 conjugation affects cell-cycle progression by inhibiting the ligase activity of SCF complexes, such knowledge in the NEDD8-conjugation pathway will contribute to the more magnificent therapies that selectively suppress tumorigenesis.     

Nedd4L对前列腺癌患者TrkA抑癌性泛素化的影响研究

目的 探讨Nedd4L对前列腺癌患者TrkA抑癌性泛素化的影响.方法 采用荧光定量PCR法,检测60例前列腺癌组织(PC组)及24例前列腺良性增生组织(BPH组)中Nedd4L及TrkA的水平,分析Nedd4L和TrkA的相关性,同时分析NEDD4L和TrkA的水平分别与前列腺癌临床特征(年龄、Gleason评分、TNM分期)的关系.采用免疫印迹法进一步检测TrkA蛋白水平和TrkAde泛素化水平.结果 ①PC组Nedd4L的mRNA水平显著低于BPH组Nedd4L的mRNA水平(t=13.01,P<0.0001);PC组TrkA mRNA水平明显高于BPH组TrkA mRNA水平(t=8.097,P<0.0001),且Nedd4L mRNA水平与TrkA mRNA水平呈显著负相关(γ=-0.8029,P=0.0156).②Nedd4L水平与患者年龄、Glea-son评分、TNM分期呈明显负相关(P<0.05),TrkA mRNA水平与患者年龄、Gleason评分、TNM分期呈显著正相关(P<0.05).③PC组TrkA蛋白水平显著高于BPH组TrkA蛋白水平(t=11.06,P<0.0001),而PC组TrkA泛素化水平显著低于BPH组TrkA泛素化水平,差异具有统计学意义(t=5.538,P<0.0001).结论 前列腺癌患者中Nedd4L表达水平下调,TrkA表达水平上调,TrkA泛素化水平的降低与前列腺癌发生、发展及转移密切相关.     

TJ-5抑制ubch5c介导NEMO泛素化调控NF-κB通路对MHD患者体内微炎症的影响

目的观察倍半萜内酯化合物TJ-5调控泛素结合酶(ubch5c)介导NF-κB必须调节蛋白(NEMO)泛素化对维持性血液透析(MHD)患者体内微炎症的影响。方法选取2018年2月-2019年2月浙江中医药大学附属湖州中医院收治的MHD患者60例,同期选取体检中心健康体检者20名。抽取MHD患者和健康者外周血,分离单一核细胞(PBMC)及CD4+T细胞。分离纯化得到的外周血CD4+T细胞使用不同浓度TJ-5处理,MHD患者血样本分离纯化得到的CD4+T细胞分为空白组(0μM/mL TJ-5处理)和高、低浓度组(10、5μM/mL TJ-5处理),每组20份,健康对照组(健康者不予处理)20份。ELISA法检测MHD患者外周血CD4+T细胞经TJ-5处理后肿瘤坏死因子-α(TNF-α)、白细胞介素1β(IL-1β)、白介素-6(IL-6)及白介素-10(IL-10)表达情况;Western Blot法检测MHD患者和健康志愿者外周血CD4+T细胞核转录因子(P65)、I-κB(NF-κB抑制蛋白)蛋白及其磷酸化;采用镍柱亲和纯化获得ubch5c,通过体外泛素化实验检测泛素连接酶ubch5c活性变化情况;免疫共沉淀法检测NEMO线性泛素化情况。结果 MHD患者外周血Th17/Treg比例显著高于健康志愿者[(1.57±0.25)%比(0.93±0.11)%,P<0.05];与空白组比较,TJ-5处理后,低、高浓度组CD4+T细胞促炎性因子TNF-α、IL-1β和IL-6明显下调[TNF-α:(5.53±0.37)ng/L、(2.27±0.26)ng/L比(8.11±1.05)ng/L,P均<0.05;IL-1β:(1.59±0.30)pg/L、(0.84±0.22)pg/L比(2.75±0.46)pg/L,P均<0.05;IL-6:(30.94±6.47)pg/L、(12.81±5.51)pg/L比(41.26±7.25)pg/L,P均<0.05];抗炎性因子IL-10明显上调[(15.82±1.01)pg/L、(20.47±1.16)pg/L比(7.34±1.71),P均<0.05];与空白组比较,核转录因子(P65)、磷酸化p65(p-P65)水平显著下调[P65:(0.72±0.12)、(0.40±0.10)比(1.19±0.14),P均<0.05;p-P65:(0.27±0.15)、(0.18±0.11)比(0.32±0.11),P均<0.05];核因子κB(NF-κB)抑制蛋白(I-κB)显著上调[(0.40±0.14)、(0.56±0.12)比(0.27±0.08),P均<0.05;磷酸化I-κB(p-I-κB)显著下调[(0.53±0.17)、(0.33±0.08)比(0.76±0.14),P均<0.05];ubch5c活性抑制率增高[(11.18±2.11)、(26.80±2.68)比(0.00±0.00),P均<0.05];NEMO线性泛素化减弱[(0.573±0.036)、(0.234±0.037)比(0.833±0.079),P均<0.05]。结论倍半萜内酯化合物TJ-5能拮抗CD4+T细胞ubch5c活性,抑制NEMO线性泛素化调控NF-κB通路及炎症因子表达,改善MHD患者微炎症。     

指环蛋白31对表皮生长因子受体表达的调控作用及其机制

目的：探讨指环蛋白31 (RNF31)对表皮生长因子受体(EGFR)表达的影响,并分析RNF31对EGFR的调控机制。方法：采用免疫共沉淀(Co-IP)法验证RNF31蛋白与EGFR蛋白的相互作用。HEK293T细胞分为空载体组(转染pc DNA3.1空载体)和RNF31组(转染RNF31-Flag质粒),采用实时荧光定量PCR (RT-q PCR)法和Western blotting法检测细胞中EGFR m RNA和蛋白表达水平。RNF31的泛素连接酶功能位点(RNF31^C699/702S)和去泛素化酶结合位点(RNF31^{N84A Y93A})突变后,采用Western blotting法检测各组细胞中EGFR蛋白表达水平。HEK293细胞分为空载体组、RNF31组(转染RNF31-Flag质粒)、RNF31+EGFR抑制剂吉非替尼(Gefitinib)组和RNF31+EGFR抑制剂厄洛替尼(Erlotinib)组,采用Western blotting法检测各组细胞中EGFR及其下游信号通路蛋白表达水平。结果：Co-IP法检测,RNF31蛋白...     

单纯疱疹病毒2型泛素化新型DNA疫苗构建

目的构建单纯疱疹病毒2型(HSV-2)早期表达蛋白感染细胞蛋白(ICP)27强制泛素化的DNA疫苗表达质粒。方法利用聚合酶链反应(PCR)技术从人基因组DNA和HSV-2基因组中分别扩增出泛素全长基因和HSV-2、ICP27 377-459,PCR鉴定后酶切连接获得融合基因片段,插入真核表达质粒pcDNA3．1载体中,构建出重组真核表达质粒Ub-ICP-pcDNA3．1,并对其进行测序鉴定。结果构建的重组质粒Ub-ICP-pcDNA3．1克隆基因插入方向正确,与预期序列完全一致。结论成功构建了HSV-2早期表达蛋白ICP27强制泛素化DNA疫苗。     

RIP1 modulates death receptor mediated apoptosis and autophagy in macrophages

Macrophages are responsible for defending against diverse pathogens and play a crucial role in the innate immune system. Macrophage''s lifespan is determined by homeostatic balance between survival and apoptosis. Here we report that tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) triggers both apoptosis and autophagy in human U937 cells. Inhibition of autophagy facilitates TRAIL‐induced apoptosis, suggesting that autophagy of macrophages protects against TRAIL‐induced apoptosis. TRAIL treatment influences the expression of death receptors, indicating that TRAIL‐induced apoptosis and autophagy are mediated by death receptors. RIP1 ubiquitination and expression regulate apoptosis and autophagy. Furthermore, expression and bioactivity of the p43/41‐caspase‐8 variant are critical to TRAIL‐induced autophagy and apoptosis. Knockdown of RIP1 suppresses autophagy in macrophage. These data demonstrate that RIP1 is essential for the regulation of death receptor mediated autophagy and apoptosis. The results in this study contribute to understanding the regulation of autophagy and apoptosis in macrophages, and shed lights on death receptor‐targeted therapy for cancer, inflammation and autoimmune diseases.     

A bacterial protein promotes the recognition of the Legionella pneumophila vacuole by autophagy

Legionella pneumophila (L. pneumophila) is an intracellular bacterium of human alveolar macrophages that causes Legionnaires’ disease. In contrast to humans, most inbred mouse strains are restrictive to L. pneumophila replication. We demonstrate that autophagy targets L. pneumophila vacuoles to lysosomes and that this process requires ubiquitination of L. pneumophila vacuoles and the subsequent binding of the autophagic adaptor p62/SQSTM1 to ubiquitinated vacuoles. The L. pneumophila legA9 encodes for an ankyrin‐containing protein with unknown role. We show that the legA9 mutant replicate in WT mice and their bone marrow‐derived macrophages. This is the first L. pneumophila mutant to be found to replicate in WT bone marrow‐derived macrophages other than the Fla mutant. Less legA9 mutant‐containing vacuoles acquired ubiquitin labeling and p62/SQSTM1 staining, evading autophagy uptake and avoiding lysosomal fusion. Thus, we describe a bacterial protein that targets the L. pneumophila‐containing vacuole for autophagy uptake.     

视神经蛋白结构域的功能性作用

视神经蛋白 （OPTN） 是一种多功能蛋白,与一系列蛋白质相互作用,参与多种细胞功能,与青光眼和肌萎缩侧索硬化症等神经退行性疾病有关。OPTN 含有多个结构域使其能够参与多种细胞功能调节,包括 NF-κB 调节、膜运输、 胞吐、囊泡运输、维持高尔基体形态、细胞周期控制、泛素化调节和自噬。OPTN结构和功能的多样性,为探讨其参与疾病的具体作用机制带来了挑战。本文梳理了近年来有关OPTN的研究文献,总结了OPTN各个结构域参与的功能性作用,为进一步理解OPTN在神经退行性疾病中所发挥的作用提供了思路和参考。