RNF167调节蛋白质的泛素化与相关抑制剂调节自噬的研究
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摘要
ERAD (endoplasmic reticulum- associated degradation)与自噬系统曾被认为是几乎毫无关系的两种蛋白降解途径,越来越多的证据表明二者存在着紧密的联系。至少这两种途径都是以泛素来作为降解信号。ERAD是细胞清除可溶性蛋白的方式,在此途径中泛素链被作为降解信号,依次通过泛素活化酶E1,泛素交联酶E2和泛素连接酶E3的催化被连接到等待降解的蛋白上,并通过蛋白酶体降解为短肽;而在自噬途径降解的是蛋白聚集体或细胞器,同样需要泛素连接酶将泛素连接到蛋白聚集体或细胞器上,而后一系列Atg蛋白和p62或(和)NBR1等蛋白参与进来,把蛋白聚集体或细胞器带到溶酶体降解。虽然两种途径都是以泛素来作为降解信号,但是二者信号的具体形式是不同的。ERAD途径的信号是K48泛素链,而自噬途径的信号部分是K63泛素链,也有K48泛素链。显然,不同种类的泛素链具有不同的功能。然而,目前尚不存在一种能够观察活细胞中特定种类泛素链的方法。本文分为三部分,涉及了1)参与ERAD途径的泛素连接酶RNF167的研究;2)与RNF167有相互作用的另一种泛素连接酶gp78的天然抑制剂SVIP参与并调节自噬;3)研究活细胞中K48泛素链动态的强大工具UCS。
蛋白的错误折叠与许多疾病密切相关。细胞清除可溶性的错误折叠蛋白主要通过内质网相关的降解途径(ERAD),而泛素连接酶作为ERAD途径上的一种重要酶类,其活力表现为将泛素链这一降解信号连接到底物蛋白上。RNF167是一种未被人们了解的泛素连接酶,前期的实验表明它可能与CFTRΔF508的降解有关。在此研究中发现RNF167在细胞中主要定位于溶酶体,也有少量分布在内质网。RNF167在肝脏和脾脏中表达最丰富,中枢神经系统中次之。RNF167经过翻译后修饰剪切了24肽的信号序列,并存在N-糖基化修饰。实验中通过点突变的方式得到几种RNF167的突变体,并对它们的性质进行了分析,并与野生型做出比较。结果显示,泛素连接酶活性结构域RING domain的突变体RNF167rm丧失了泛素连接酶活力,并且比野生型蛋白更加稳定,而分拣信号的突变体RNF1673A改变了细胞定位,从溶酶体来到细胞表面。实验中也对RNF167的底物范围进行了分析。可以观察到RNF167与CFTRΔF508的共同定位,而且RNF167能特异性的降低CFTRΔF508的表达水平,通过加快CFTRΔF508的降解速率,但是对另两种典型ERAD底物NHK和CD3Δ没有作用。通过免疫沉淀的方法可以检测到RNF167与CFTRΔF508的相互作用,另外有证据表明RNF167参与了其他泛素连接酶gp78/Hrd1与VCP、derlin等组成的ERAD蛋白复合物。
SVIP作为ERAD途径的一种抑制剂被人们所认识。在HeLa细胞中大量表达SVIP会引起VCP,同时还有LC3和Lamp1的亚细胞定位的变化。这预示着SVIP可能与自噬系统有关。SVIP在细胞中水平过低时,会通过下调mRNA水平来使p62减少,也可以观察到自噬的标志物LC3的减少。过量表达SVIP增加了p62蛋白水平并且可以增强饥饿诱导的自噬,另外可以观察到更多的位于自噬性溶酶体中的p62和泛素化蛋白形成的p62体,而且细胞中泛素水平受到很大影响。这些结果表明SVIP在自噬系统起到了调节作用。有证据证明SVIP是与VCP一起调节自噬系统的。综合之前的报道,SVIP在ERAD途径和自噬途径都起了重要的作用。这些结果使我们重新认识这两条重要的蛋白降解途径之间的联系。8种泛素链在细胞中有着不同的功能。目前没有一种可以在活细胞中分析这8种泛素链之一的有效方法。本研究中开发的基于泛素相互作用基序和双分子报告蛋白互补的带有荧光的泛素链检测元件(UCS)可以达到要求。目前应用UCS可以成像并量化各种药物处理后和依赖于Parkin的线粒体特异性自噬过程中活细胞中K48泛素链的动态变化。
本文创新点:(1)对一种新的泛素连接酶RNF167性质进行了研究,明确了其底物特异性,发现其与其他ERAD组分的相互作用。(2)揭示了一种ERAD通路抑制剂SVIP对另一蛋白降解途径的激活调节作用,为研究蛋白酶体降解(ERAD)与溶酶体降解(自噬)之间的关系提供重要证据和了崭新思路。(3)开发了一种全新的K48泛素链特异性的活细胞实时观察和定量方法。对泛素及蛋白降解领域的研究会有深远影响。
ERAD (endoplasmic reticulum- associated degradation) and autophagy were belived had hardly any relationship between the two major protein degradation pathways. More and more evidence has been shown that ERAD and autophagy have something to do with each other. At least, uiquitin or poly-ubiquitin chain is validated as protein degradation signal in both pathways. ERAD, in which K48 ubiquitin chains work as degradation signal, degrades soluble proteins in the cell. The ubiquination approach is catalysed by three enzymes sequencially: ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3, thus, poly-ubiquitinated proteins are subjected to proteasome for degradation. In contrast, protein aggragates or insoluble proteins and organelles are degraded by autophagy. But ubiquitin chains which linked to the sustrates are still degradation signal. Atg proteins and p62 and/or NBR1 take part in autophagy and send the substrates to lysosome for degradation. Although ubquitin chains are used as degradation signal in these two pathways, the type of ubquitin chains are different between ERAD and autophagy. K48 poly-ubiquitin chains are the signal of ERAD, whereas K63 type is specific for autophagy. Obviously, different type of poly-ubiquitin chains has different function, but now no technology can be used to observe specific ubiquitin chain’s type in live cell. This thesis contains three sections: 1) ubiquitin ligase RNF167 takes part in ERAD, 2) the nature inhibitor of gp78, another ubiquitin ligase interacting with RNF167, named SVIP regulates autophagy, and 3) K48 ubiquitin chain imaging with UCS in live cell.
Misfolded proteins are related to some diseases closely. The clearance of misfolded soluble proteins is relied on ERAD. Ubiquitin ligase is one kind of most important enzymes in ERAD pathway, which links ubiquitin or ubiquin chain to the substrate proteins. RNF167 is a ubiquitin ligase not well-known. Previous result showed that RNF167 may regulate the degradation of CFTRΔF508. In this research, RNF167 was found localized to lysosome mainly, and a small part of which localized to ER. In liver and spleen of mice, RNF167 expressed stongly, and a little lower level was observed in central nervous system. After post-translational modification, 24-peptide signal sequence was cleaved for RNF167 precursor, and N-linked glycosilation was achivied. RNF167 mutants were constructed by site-direct mutagenesis. RNF167rm was a RING domain mutant, which had non-active ubiquitin ligase activity sites, lost the E3 activity. Sorting signal mutant, RNF1673A, localized to cytoplasm rather than lysosome. The substrates’range was analysed in the research. RNF167 colocalized well with CFTRΔF508 in lysosome, furthermore, RNF167 regulated the degradation of CFTRΔF508, but had no effect on other two typical ERAD substrates NHK and CD3Δ. RNF167 and CFTRΔF508’s interaction was confirmed with immuno-precipitation method. Extra evidence showed RNF167 may form a protein complex together with other ERAD componants, such as gp78/Hrd1, VCP and derlin.
The small p97/VCP-interacting protein (SVIP) functions as an inhibitor of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. Here we show that overexpression of SVIP in HeLa cells leads to localization of p97/VCP at the plasma membrane, intracellular foci and juxtanuclear vacuoles. The p97/VCP-positive vacuolar structures colocalized or associated with LC3 and lamp1, suggesting that SVIP may regulate autophagy. In support of this possibility, knockdown of SVIP diminished, whereas overexpression of SVIP enhanced LC3 lipidation. Surprisingly, knockdown of SVIP reduced the levels of p62 protein at least partially through downregulation of its mRNA, which was accompanied by a decrease in starvation-induced formation of p62 bodies. Overexpression of SVIP, on the other hand, increased the levels of p62 protein and enhanced starvation-activated autophagy as well as promoted sequestration of polyubiquitinated proteins polyubiquitin and p62 in autophagosomes. These results suggest that SVIP plays regulatory roles of p97 subcellular localization and is able to regulate autophagy.
Eight topologically distinct polyubiquitin chains signal for a remarkably diverse functions in cells, yet no technology is currently available for analysis of any of these chains in living cells. Here we developed a fluorescent ubiquitin chain sensor (UCS) based on ubiquitin-interacting motif-mediated bimolecular fluorescence complementation (BiFC). Application of the UCS enables imaging and quantification of the dynamic increases of K48-specific polyubiquitin chains in living cells under various stress conditions and during Parkin-dependent mitophagy.
蛋白的错误折叠与许多疾病密切相关。细胞清除可溶性的错误折叠蛋白主要通过内质网相关的降解途径(ERAD),而泛素连接酶作为ERAD途径上的一种重要酶类,其活力表现为将泛素链这一降解信号连接到底物蛋白上。RNF167是一种未被人们了解的泛素连接酶,前期的实验表明它可能与CFTRΔF508的降解有关。在此研究中发现RNF167在细胞中主要定位于溶酶体,也有少量分布在内质网。RNF167在肝脏和脾脏中表达最丰富,中枢神经系统中次之。RNF167经过翻译后修饰剪切了24肽的信号序列,并存在N-糖基化修饰。实验中通过点突变的方式得到几种RNF167的突变体,并对它们的性质进行了分析,并与野生型做出比较。结果显示,泛素连接酶活性结构域RING domain的突变体RNF167rm丧失了泛素连接酶活力,并且比野生型蛋白更加稳定,而分拣信号的突变体RNF1673A改变了细胞定位,从溶酶体来到细胞表面。实验中也对RNF167的底物范围进行了分析。可以观察到RNF167与CFTRΔF508的共同定位,而且RNF167能特异性的降低CFTRΔF508的表达水平,通过加快CFTRΔF508的降解速率,但是对另两种典型ERAD底物NHK和CD3Δ没有作用。通过免疫沉淀的方法可以检测到RNF167与CFTRΔF508的相互作用,另外有证据表明RNF167参与了其他泛素连接酶gp78/Hrd1与VCP、derlin等组成的ERAD蛋白复合物。
SVIP作为ERAD途径的一种抑制剂被人们所认识。在HeLa细胞中大量表达SVIP会引起VCP,同时还有LC3和Lamp1的亚细胞定位的变化。这预示着SVIP可能与自噬系统有关。SVIP在细胞中水平过低时,会通过下调mRNA水平来使p62减少,也可以观察到自噬的标志物LC3的减少。过量表达SVIP增加了p62蛋白水平并且可以增强饥饿诱导的自噬,另外可以观察到更多的位于自噬性溶酶体中的p62和泛素化蛋白形成的p62体,而且细胞中泛素水平受到很大影响。这些结果表明SVIP在自噬系统起到了调节作用。有证据证明SVIP是与VCP一起调节自噬系统的。综合之前的报道,SVIP在ERAD途径和自噬途径都起了重要的作用。这些结果使我们重新认识这两条重要的蛋白降解途径之间的联系。8种泛素链在细胞中有着不同的功能。目前没有一种可以在活细胞中分析这8种泛素链之一的有效方法。本研究中开发的基于泛素相互作用基序和双分子报告蛋白互补的带有荧光的泛素链检测元件(UCS)可以达到要求。目前应用UCS可以成像并量化各种药物处理后和依赖于Parkin的线粒体特异性自噬过程中活细胞中K48泛素链的动态变化。
本文创新点:(1)对一种新的泛素连接酶RNF167性质进行了研究,明确了其底物特异性,发现其与其他ERAD组分的相互作用。(2)揭示了一种ERAD通路抑制剂SVIP对另一蛋白降解途径的激活调节作用,为研究蛋白酶体降解(ERAD)与溶酶体降解(自噬)之间的关系提供重要证据和了崭新思路。(3)开发了一种全新的K48泛素链特异性的活细胞实时观察和定量方法。对泛素及蛋白降解领域的研究会有深远影响。
ERAD (endoplasmic reticulum- associated degradation) and autophagy were belived had hardly any relationship between the two major protein degradation pathways. More and more evidence has been shown that ERAD and autophagy have something to do with each other. At least, uiquitin or poly-ubiquitin chain is validated as protein degradation signal in both pathways. ERAD, in which K48 ubiquitin chains work as degradation signal, degrades soluble proteins in the cell. The ubiquination approach is catalysed by three enzymes sequencially: ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3, thus, poly-ubiquitinated proteins are subjected to proteasome for degradation. In contrast, protein aggragates or insoluble proteins and organelles are degraded by autophagy. But ubiquitin chains which linked to the sustrates are still degradation signal. Atg proteins and p62 and/or NBR1 take part in autophagy and send the substrates to lysosome for degradation. Although ubquitin chains are used as degradation signal in these two pathways, the type of ubquitin chains are different between ERAD and autophagy. K48 poly-ubiquitin chains are the signal of ERAD, whereas K63 type is specific for autophagy. Obviously, different type of poly-ubiquitin chains has different function, but now no technology can be used to observe specific ubiquitin chain’s type in live cell. This thesis contains three sections: 1) ubiquitin ligase RNF167 takes part in ERAD, 2) the nature inhibitor of gp78, another ubiquitin ligase interacting with RNF167, named SVIP regulates autophagy, and 3) K48 ubiquitin chain imaging with UCS in live cell.
Misfolded proteins are related to some diseases closely. The clearance of misfolded soluble proteins is relied on ERAD. Ubiquitin ligase is one kind of most important enzymes in ERAD pathway, which links ubiquitin or ubiquin chain to the substrate proteins. RNF167 is a ubiquitin ligase not well-known. Previous result showed that RNF167 may regulate the degradation of CFTRΔF508. In this research, RNF167 was found localized to lysosome mainly, and a small part of which localized to ER. In liver and spleen of mice, RNF167 expressed stongly, and a little lower level was observed in central nervous system. After post-translational modification, 24-peptide signal sequence was cleaved for RNF167 precursor, and N-linked glycosilation was achivied. RNF167 mutants were constructed by site-direct mutagenesis. RNF167rm was a RING domain mutant, which had non-active ubiquitin ligase activity sites, lost the E3 activity. Sorting signal mutant, RNF1673A, localized to cytoplasm rather than lysosome. The substrates’range was analysed in the research. RNF167 colocalized well with CFTRΔF508 in lysosome, furthermore, RNF167 regulated the degradation of CFTRΔF508, but had no effect on other two typical ERAD substrates NHK and CD3Δ. RNF167 and CFTRΔF508’s interaction was confirmed with immuno-precipitation method. Extra evidence showed RNF167 may form a protein complex together with other ERAD componants, such as gp78/Hrd1, VCP and derlin.
The small p97/VCP-interacting protein (SVIP) functions as an inhibitor of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. Here we show that overexpression of SVIP in HeLa cells leads to localization of p97/VCP at the plasma membrane, intracellular foci and juxtanuclear vacuoles. The p97/VCP-positive vacuolar structures colocalized or associated with LC3 and lamp1, suggesting that SVIP may regulate autophagy. In support of this possibility, knockdown of SVIP diminished, whereas overexpression of SVIP enhanced LC3 lipidation. Surprisingly, knockdown of SVIP reduced the levels of p62 protein at least partially through downregulation of its mRNA, which was accompanied by a decrease in starvation-induced formation of p62 bodies. Overexpression of SVIP, on the other hand, increased the levels of p62 protein and enhanced starvation-activated autophagy as well as promoted sequestration of polyubiquitinated proteins polyubiquitin and p62 in autophagosomes. These results suggest that SVIP plays regulatory roles of p97 subcellular localization and is able to regulate autophagy.
Eight topologically distinct polyubiquitin chains signal for a remarkably diverse functions in cells, yet no technology is currently available for analysis of any of these chains in living cells. Here we developed a fluorescent ubiquitin chain sensor (UCS) based on ubiquitin-interacting motif-mediated bimolecular fluorescence complementation (BiFC). Application of the UCS enables imaging and quantification of the dynamic increases of K48-specific polyubiquitin chains in living cells under various stress conditions and during Parkin-dependent mitophagy.
引文
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