泛素E3连接酶TRIM10在心肌细胞肥大中的作用

目的:探讨泛素E3连接酶TRIM10在心肌细胞肥大中的作用及分子机制。方法:培养原代的大鼠乳鼠心肌细胞,siRNA-TRIM10与siRNA对照(siRNA-control)或过表达腺病毒Ad-TRIM10与空载对照Ad-GFP转染细胞24 h,然后用苯肾上腺素(phenylephrine,PE)处理细胞24 h。Western blot检测TRIM10、AKT和ERK1/2的蛋白水平;免疫荧光染色观察心肌细胞的大小;实时荧光定量PCR检测心房钠尿肽(ANP)和脑钠尿肽(BNP)的mRNA的表达水平。结果:与对照组相比,PE处理明显上调心肌细胞中TRIM10蛋白的表达水平。siRNA-TRIM10敲低内源性TRIM10表达后明显减小PE诱导的心肌细胞体积,抑制ANP和BNP的mRNA的表达以及降低AKT和ERK1/2的磷酸化水平;而过表达TRIM10则呈现出与siRNA-TRIM10完全相反的结果。结论:TRIM10可调节心肌细胞肥大,其作用可能与AKT和ERK信号相关。     

人睾丸组织TRIM69蛋白具有催化多聚泛素链形成的活性

目的 验证人源TRIM69蛋白是否具有催化多聚泛素链形成的活性.方法 把人源TRIM69基因构建到原核细胞表达载体中,经过转化、诱导、表达和纯化获得TRIM69纯化蛋白,通过细胞外泛素化实验分析其是否具有催化多聚泛素链形成的活性;另外构建了TRIM69基因的全长、RING结构域的点突变、以及RING结构域截短序列的真核细胞表达载体,瞬时转染293T细胞或稳定表达泛素的HeLa细胞株,通过免疫沉淀结合免疫印迹方法检测其是否具有催化多聚泛素链形成的活性.结果 泛素化分析实验表明TRIM69具有催化多聚泛素链形成的活性并且其活性依赖于它的RING结构域.结论 成功鉴定了人源TRIM69蛋白具有催化多聚泛素链形成的活性,为进一步证明其是一个新的E3泛素连接酶提供依据.     

TRIM在病毒免疫防御机制中的作用

TRIM(tripartite motif)蛋白是一类含有RING结构域的蛋白质,在人类中发现超过70种,还有许多新成员正在研究中.这类蛋白除了参与细胞增殖、分化、发育、形态发生和细胞凋亡.也在免疫相关信号通路和抗病毒免疫反应中发挥重要作用.TRIM25能够泛素化病毒RNA受体视黄酸诱导基因-Ⅰ(RIG-Ⅰ)的N末端,这种改变能够激活RIG-Ⅰ与其下游配体结合参与线粒体抗病毒信号通路(MAVS).这个过程直接引起I型干扰素(IFN)的产生从而抑制病毒复制.近来的研究发现甲型流感病毒能够靶向结合TRIM25,使其抗病毒的功能失效从而抑制宿主的干扰素活性.本文对TRIM蛋白在抗病毒防御机制中的功能以及一种流感病毒通过靶向结合某种TRIM蛋白而产生的免疫逃逸机制的研究进展进行总结.     

TRIM家族在宫颈癌中的研究进展

宫颈癌是女性中常见恶性肿瘤之一,病死率较高,全球死亡女性超过88％发生在发展中国家,其中以宫颈鳞癌最为常见.三方基序蛋白(tripartite motif-containing protein,TRIM)家族大多数具有E3泛素连接酶活性,且与多种生理过程有关,包括细胞增殖、DNA修复、信号转导和转录.近年大量研究发现TRIM家族通过多种通路参与宫颈癌的发生和发展.     

TRIM69可抑制紫外线和依托泊苷刺激时HeLa和HEK293T细胞系内活化型caspase7和剪切型PARP的表达

目的验证TRIM69蛋白是否能够抑制He La和HEK293T细胞系内活化型caspase7(cleaved caspase7)和剪切型PARP(cleaved PARP)的蛋白水平。方法构建含人源TRIM69的真核表达质粒,转染He La细胞和HEK293T细胞,筛选稳定表达TRIM69的细胞系,通过紫外线照射和凋亡诱导剂依托泊苷(etoposide)刺激处理,Western blot检测细胞内TRIM69蛋白、活化型caspase7和剪切型PARP蛋白水平的变化。结果紫外线照射和依托泊苷刺激后,与对照组相比,TRIM69过表达的细胞内活化型caspase7和剪切型PARP的蛋白水平均下降(P0.05)。结论TRIM69蛋白的表达可抑制紫外线或依托泊苷刺激时He La和HEK293T细胞内凋亡相关蛋白活化型caspase7和剪切型PARP的蛋白水平。     

TRIM21胞内免疫功能与治疗应用

三基序蛋白21(tripartite -motif protein 21,TRIM21)是一种存在于细胞胞质内的泛素连接酶,也是一类胞内抗体受体或者感应器,成为抵御病毒入侵的固有免疫防线。对于在细胞外已经逃逸了中和抗体作用,并被非中和抗体吸附而通过细胞膜侵入细胞内的病毒,TRIM21可以通过与病毒-抗体复合物中的抗体F...     

天然免疫分子TRIM5α限制HIV-1感染作用机制的研究进展

逆转录病毒的侵染能造成严重的人和动物的疾病，包括获得性免疫缺陷综合征（AIDS，艾滋病）、白血病、淋巴瘤、癌症和肉瘤。哺乳动物在长期的进化过程中，已经产生了各种机制来抑制逆转录病毒的感染和复制。细胞内抗病毒因子介导的抗病毒反应是宿主机体阻止逆转录病毒感染的第一道防线。早在多年以前就发现多个细胞内病毒限制因子可以在逆转录病毒的感染中起抑制作用，例如，Ref1、Lv1及Fv1等。近年来，又有多个胞内抗病毒分子包括APO-BEC3G、ZAP、TRIM5α等陆续被发现。     

固有免疫因子APOBEC3蛋白及其抗病毒作用的研究进展

载脂蛋白B mRNA编辑酶催化多肽样蛋白3(APOBEC3)是APOBEC蛋白家族成员,是新发现的固有免疫抗病毒因子,具有脱氨酶活性,可使负链胞嘧啶脱氨变为尿嘧啶(C→U),新合成的正链DNA降解或产生致死性G→A突变,从而达到抑制病毒复制的作用.APOBEC3在抗HIV的研究较多,对HBV的抗病毒作用也得到了证实.因...     

TRIM19通过调控p53信号促进卵巢癌进展的作用及机制研究

目的:探讨TRIM19在卵巢癌进展中的作用及其分子机制。方法:qPCR检测人卵巢表皮细胞IOSE80、卵巢癌细胞SKOV-3、A2780、OVCAR3中TRIM19表达水平;以siRNA敲减SKOV-3细胞中TRIM19基因表达;以CCK-8方法、Transwell实验、流式细胞术分别检测细胞增殖能力、迁移行为及凋亡率;以RT-qPCR技术检测信号通路分子表达。结果:在卵巢癌细胞SKOV-3、A2780、OVCAR3中,TRIM19表达量较正常表皮细胞IOSE80中明显增强。当TRIM19基因在卵巢癌SKOV-3细胞中被成功敲减后,细胞增殖能力降低74.2%,侵袭能力降低70.0%,凋亡率增加约30倍。在分子水平,敲减TRIM19后,p53、p21、CyclinD、CDK2、Bax基因表达显著增加;同时敲减TRIM19与p53后,p53、p21、CyclinD、CDK2、Bax基因表达略有提高。结论:沉默TRIM19基因抑制卵巢癌细胞增殖、侵袭,诱导凋亡,并且激活p53依赖的凋亡信号,TRIM19促进卵巢癌进程。TRIM19具有成为卵巢癌治疗的新靶点潜力。     

平顶猴TRIM5α基因变异影响其抗HIV-1功能

目的：解释平顶猴成为唯一能够被HIV-1感染的旧大陆猴以及在被SIV感染时出现较其他旧大陆猴严重临床症状的原因。方法：参考人TRIM5α基因的结构和序列合成引物,以平顶猴DNA为模板,分别扩增其TRIM5α基因编码序列的所有外显子。利用生物学软件分析获得的编码序列,分析在已经明确与TRIM5α抗病毒功能密切相关的几个位点上是否发生可能影响其功能的突变。结果：在平顶猴基因组上CypA基因融合到TRIM5α基因第八外显子的下游,融合方式与在鹰猴中发现的CypA与TRIM5α的融合方式完全不同。恒河猴TRIM5α基因具有限制HIV-1复制的功能,第332位脯氨酸对其抗病毒功能非常重要,但在平顶猴的TRIM5α上,该氨基酸突变成谷氨酰胺;第335位以后8个氨基酸对恒河猴TRIM5α抗病毒功能也有重要影响,但在平顶猴的TRIM5α上,与其对应的氨基酸片段不仅缺失了两个氨基酸,还有两个氨基酸发生突变,附近区域还有另外两个发生突变。在平顶猴TRIM5α基因的Coiled-Coil结构域插入了13个氨基酸,但没有影响到编码框。结论：在平顶猴的基因组上CypA与TRIM5α的融合以及平顶猴TRIM5α基因抗病毒功能关键位点上的氨基酸突变可能影响其抗病毒功能,是其成为唯一能够被HIV-1感染的旧大陆猴以及在被SIV感染时出现较其他旧大陆猴严重临床症状的重要原因。     

TRIM21: a cytosolic Fc receptor with broad antibody isotype specificity

Antibodies are key molecules in the fight against infections. Although previously thought to mediate protection solely in the extracellular environment, recent research has revealed that antibody-mediated protection extends to the cytosolic compartment of cells. This postentry viral defense mechanism requires binding of the antibody to a cytosolic Fc receptor named tripartite motif containing 21 (TRIM21). In contrast to other Fc receptors, TRIM21 shows remarkably broad isotype specificity as it does not only bind IgG but also IgM and IgA. When viral pathogens coated with these antibody isotypes enter the cytosol, TRIM21 is rapidly recruited and efficient neutralization occurs before the virus has had the time to replicate. In addition, inflammatory signaling is induced. As such, TRIM21 acts as a cytosolic sensor that engages antibodies that have failed to protect against infection in the extracellular environment. Here, we summarize our current understanding of how TRIM21 orchestrates humoral immunity in the cytosolic environment.     

TRIM21 controls Toll-like receptor 2 responses in bone-marrow-derived macrophages

TRIM21 is an interferon-stimulated E3 ligase that controls the activity of pattern-recognition signaling via ubiquitination of interferon regulatory factors and DDX41. Previous studies on the role of TRIM21 in innate immune responses have yielded contradictory results, suggesting that the role of TRIM21 is cell specific. Here, we report that bone-marrow-derived macrophages (BMDMs) generated from Trim21^−/− mice have reduced expression of mature macrophage markers. Reflecting their reduced differentiation in response to macrophage colony-stimulating factor (M-CSF), Trim21^−/− BMDMs had decreased expression of M-CSF signature genes. Although Trim21^−/− BMDMs responded normally to Toll-like receptor 9 (TLR9) activation, they produced lower levels of pro-inflammatory cytokines in response to the TLR2 agonist PAM3CSK4. In line with this, the response to infection with the Bacillus Calmette–Guérin strain of Mycobacterium bovis was also diminished in Trim21^−/− BMDMs. Our results indicate that TRIM21 controls responses to TLR2 agonists.     

Alterations of TRIM21-mRNA expression during monocyte maturation

Tripartite motif-containing protein 21 (TRIM21) play a dual role in the cytoplasm of the cells where it facilitates destruction of some antibody-coated viruses and some bacteria, and initiates synthesis of proinflammatory cytokines. Macrophages and CD16⁺ monocyte subset can particularly participate in a proinflammatory response caused by viral infection, however, the molecular mechanisms underlying these processes are not fully understood. The aim of this study was to determine the level of TRIM21-mRNA expression in monocyte subsets including: classical (CD14⁺⁺CD16⁻), intermediate (CD14⁺⁺CD16⁺) and non-classical (CD14⁺CD16⁺⁺) monocytes, as well as during in vitro differentiation of the isolated monocytes towards dendritic cells or macrophages.Our results revealed that the level of TRIM21 mRNA expression was significantly lower in CD16- monocytes, when compared to CD16⁺ cells and the whole monocyte population, yet no significant differences were observed when CD16⁺ population was divided into intermediate and non-classical subsets. More pronounced differences were observed in the case of monocyte-derived macrophages (MDM) and dendritic cells (DCs). TRIM21-mRNA expression level was app. 6-fold higher in DCs, and app. 16-fold higher in MDM (p < 0,01), when compared to freshly isolated monocytes.Our results may suggest the new mechanism of increased proinflammatory cytokine production by CD16⁺ (intermediate and non-classical) monocytes and macrophages, at least in patients with acute or chronic infections, caused by enveloped viruses. We suggest that TRIM21 may be one of the factors associated with the “switching on” the proinflammatory programme in CD16⁺ monocytes or monocyte-derived macrophages.     

干扰素刺激基因15(ISG15)在天然免疫中的抗病毒作用研究进展

干扰素刺激基因15(ISG15)是干扰素刺激下产生的最丰富的产物,也是宿主对病毒感染应答环节中的重要环节。距离ISG15第一次被报道已有42年,越来越多研究揭示了其蛋白复杂和多变的性质,除可对细胞内多种蛋白进行修饰外,也可作为细胞因子分泌至细胞外。ISG15存在物种间作用差异。本文介绍ISG15在天然免疫中的抗病毒功能及其发挥这一功能的不同机制,并阐述其研究进展。     

Reprint of: Alterations of TRIM21-mRNA expression during monocyte maturation

Tripartite motif-containing protein 21 (TRIM21) play a dual role in the cytoplasm of the cells where it facilitates destruction of some antibody-coated viruses and some bacteria, and initiates synthesis of proinflammatory cytokines. Macrophages and CD16⁺ monocyte subset can particularly participate in a proinflammatory response caused by viral infection, however, the molecular mechanisms underlying these processes are not fully understood. The aim of this study was to determine the level of TRIM21-mRNA expression in monocyte subsets including: classical (CD14⁺⁺CD16⁻), intermediate (CD14⁺⁺CD16⁺) and non-classical (CD14⁺CD16⁺⁺) monocytes, as well as during in vitro differentiation of the isolated monocytes towards dendritic cells or macrophages.Our results revealed that the level of TRIM21 mRNA expression was significantly lower in CD16- monocytes, when compared to CD16⁺ cells and the whole monocyte population, yet no significant differences were observed when CD16⁺ population was divided into intermediate and non-classical subsets. More pronounced differences were observed in the case of monocyte-derived macrophages (MDM) and dendritic cells (DCs). TRIM21-mRNA expression level was app. 6-fold higher in DCs, and app. 16-fold higher in MDM (p < 0,01), when compared to freshly isolated monocytes.Our results may suggest the new mechanism of increased proinflammatory cytokine production by CD16⁺ (intermediate and non-classical) monocytes and macrophages, at least in patients with acute or chronic infections, caused by enveloped viruses. We suggest that TRIM21 may be one of the factors associated with the “switching on” the proinflammatory programme in CD16⁺ monocytes or monocyte-derived macrophages.     

TRIM21 Polymorphisms are associated with Susceptibility and Clinical Status of Oral Squamous Cell Carcinoma patients

Squamous cell cancer of head and neck (HNSCC) is the sixth most common malignancy worldwide. One of the most common HNSCC types is oral squamous cell carcinoma (OSCC), which is the fifth leading cause of cancer death in Taiwan. Tripartite motif 21 (TRIM21) has been reported to play an important role in different cancer types. We found a correlation between TRIM21 and survival of HNSCC patients, but little information exists about how altered TRIM21 expression contributes to tumorigenesis. Thus, we investigated the combined effect of TRIM21 polymorphisms and exposure to environmental carcinogens on the susceptibility and clinicopathological characteristics of OSCC. Two single-nucleotide polymorphisms (SNPs) of TRIM21 (rs4144331, rs915956) from 1194 healthy controls and 1192 OSCC patients were analyzed by real-time PCR. Among 1632 smokers, TRIM21 polymorphism carriers with the betel-nut chewing habit had a ~4.8-fold greater risk of OSCC than TRIM21 wild-type carriers without the betel-nut chewing habit. After adjusting for other covariants, OSCC patients with G/T at TRIM21 rs4144331 had a high risk for distant metastasis compared with G/G homozygotes. This study is the first to examine the risk factors associated with TRIM21 SNPs in OSCC progression and development. Thus, our findings suggest that this study is the first to examine the risk factors associated with TRIM21 SNPs in OSCC progression and development and suggest that interactions between mutant genes may alter the susceptibility to OSCC.     

沉默TRIM23基因抑制骨肉瘤细胞的增殖

目的 探讨TRIM23基因对骨肉瘤细胞增殖能力的影响.方法 应用Western blot实验检测TRIM23基因在骨肉瘤细胞中的表达;应用shRNA质粒转染骨肉瘤细胞系U2OS,通过MTT与平板克隆形成实验评估细胞增殖能力;应用Westernblot实验检测U2OS细胞Akt/P53/P21通路的表达改变.结果 TRI...