HPPCn相互作用的膜蛋白筛选
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摘要
哺乳动物肝脏具有非常强大的再生能力。通常情况下,人的肝脏在受损后,3天内启动肝再生,2-3周后可以基本恢复肝脏功能,3-6个月后肝脏可恢复到与受损前一样大小。肝脏再生是一个复杂的过程,成千上万的物质参与了这个过程。
近两年,我们实验室的研究人员利用传统的生化分离与现代蛋白质组学相结合的分离方法从新生小牛肝脏中分离到一个能够特异的促进肝细胞增殖的蛋白因子,将其命名为HPPCn(hepatopoietin Cn, HPPCn)。随后,又从人胎肝cDNA文库中筛到了它的人源序列。
生物活性分析显示,重组人HPPCn蛋白能够特异地促进原代培养大鼠肝细胞、正常肝细胞系L02和肝癌细胞系SMMC7721的DNA合成和细胞分裂,而且能够促进70%肝脏切除小鼠的肝再生,初步的动物实验显示重组HPPCn蛋白对乙醇诱导的急性肝损伤有明显的保护作用。
序列分析显示,HPPCn与富含亮氨酸的酸性核蛋白(Leucine-rich acidic nuclear protein,LANP)家族其他成员序列同源性达到70%以上,且同已知的LANP蛋白一样,具有保守并且特殊的结构特征:1)其N端含有一个由25个亮氨酸组成的高度重复序列,称之为富含亮氨酸重复序列(leucine rich-repeats,LRRs),它具有特征性的βαβ的马靴型结构,有利于蛋白与蛋白之间的相互作用;2)C端第169-230位的氨基酸残基是一段由谷氨酸与天冬氨酸组成的重复序列,称之为酸性尾结构(acidic tail domain),这有利于它们在核内与染色体相结合,从而调控基因的转录。
蛋白质结构分析显示,HPPCn拥有核蛋白的结构特点,但是它又不具有典型的信号肽结构,那么它是如何作为一个生长因子来发挥作用的呢?在已有的报道中,相当数量的核蛋白(如HMGB1,HDGF等)能够同时在细胞内外发挥作用,属于一类可在核内外同时发挥作用的双功能细胞因子。HPPCn与其有极其相似的结构,这种结构的相似性,能否产生功能的相似性?即HPPCn是否具有能在细胞内外同时发挥作用的双功能性?前期的实验表明,FITC标记的重组HPPCn蛋白与肝细胞表面呈斑块样特异性结合,并能激活细胞中增殖相关的激酶活性,如SPK,MAPK,STAT3。流式分析显示,不论是结合实验还是竞争性抑制实验,均表明HPPCn能够与肝细胞特异结合。
本研究主要目的是寻找HPPCn相互作用的的膜蛋白。我们首先制备了携带His标签的HPPCn蛋白,并利用改进的His Pull-down的方法寻找HPPCn相互作用的膜蛋白,通过免疫共沉淀,转染细胞等方式对他们的相互作用做了进一步验证。具体的实验方法如下:
首先,表达纯化带His标签的HPPCn蛋白并分离肝癌细胞系SMMC7721的膜蛋白。构建带His标签的HPPCn的原核表达载体pET-24a(+)-HPPCn,并将其转入大肠杆菌BL21,构建表达带His标签的HPPCn重组蛋白的工程菌,利用工程菌发酵生产大量带His标签的HPPCn蛋白,并通过Q-XL阴离子交换层析和His亲和层析进行蛋白纯化。通过蔗糖密度梯度离心的方法获得肝癌细胞系SMMC7721的膜蛋白。
第二,通过改进的His Pull-down方法,寻找HPPCn相互作用的膜蛋白。将光敏感交联剂SANPAH、带His标签的HPPCn蛋白与提取的膜表面蛋白一起用紫外照射,由于化学键的断裂重新连接,带His标签的HPPCn蛋白与提取的膜表面蛋白进行交联,超速离心分离交联复合物。His亲和层析进一步纯化交联复合体,得到带His标签的HPPCn蛋白和交联复合体的混合物, SDS-PAGE电泳对其进行分离,并进一步通过Western blot用抗His的抗体和抗HPPCn的抗体对分离的条带进行专一性鉴定,与相应的SDS-PAGE电泳结果比对找到目的条带,割胶回收,质谱鉴定。
最后,进一步验证HPPCn与角蛋白8/18之间的相互作用。我们首先用角蛋白8/18抗体通过细胞免疫组化的方式,对SMMC7721细胞表面的角蛋白8/18的分布进行分析。通过细胞外的结合实验和免疫共沉淀进一步确定两者之间的相互作用。为了在细胞内确定HPPCn与角蛋白8/18相互作用的存在,将角蛋白8/18的表达载体pCDNA3.1-K8-IRES-K18转入HPPCn作用阴性的小鼠成纤维细胞系NIH-3T3,筛选稳定表达株,观察HPPCn对其是否有促增殖抗凋亡的活性。
实验结果显示,通过不同浓度IPTG,不同诱导时间诱导,最后确定0.3mM IPTG,诱导4小时,工程菌可以大量表达带His标签的HPPCn,因此表达带His标签的HPPCn的工程菌构建成功。大量摇瓶培养,收集菌体超声破碎,得到的超声上清经过Q-XL阴离子交换层析和His亲和层析,去除大部分杂蛋白,获得较为纯净的带His标签的HPPCn,灰度扫描纯度达到90%以上,符合后续实验的要求。通过蔗糖密度梯度离心的方法逐级分离大量的肝癌细胞系SMMC7721的膜蛋白,为后续两者的相互作用奠定基础。通过改进的His Pull-down和质谱相结合,筛选到HPPCn相互作用的膜蛋白角蛋白8/18。免疫组化实验确定了角蛋白8/18在膜表面的分布,PVDF膜上的蛋白结合实验和免疫共沉淀结果验证了HPPCn与角蛋白8/18的相互作用。通过角蛋白8稳定转染株的初步实验发现HPPCn对转化了pCDNA3.1-K8表达载体的NIH-3T3细胞系促增殖作用不太明显。
角蛋白8/18是正常成年人肝细胞中唯一的中间纤维蛋白。越来越多的实验发现,角蛋白8/18不仅能够对抗机械压力,而且可以对抗各种毒素压力以及Fas介导的细胞凋亡。而临床数据显示,角蛋白8/18特定氨基酸残基的突变将会导致各种肝脏疾病。分子机制研究发现,角蛋白8/18一方面可以与各种骨架相互蛋白作用维持细胞形态,另一方面与各种激酶、接头蛋白和细胞凋亡蛋白相互作用构成复杂的信号通路系统。因此角蛋白8/18在研究肝脏细胞对抗毒素压力,细胞凋亡以及各种肝脏疾病方面有重要的意义。
本研究通过His Pull-down的方法获得与HPPCn相互作用的膜蛋白角蛋白8/18,并通过进一步的免疫共沉淀等体外验证实验初步证实了两者的相互作用。构建了角蛋白8/18的真核表达载体,转染HPPCn作用阴性的NIH-3T3细胞。角蛋白8的单独表达解除了HPPCn对NIH-3T3细胞生长的抑制作用。
The mammalian liver possesses a remarkable capacity of regeneration in response to a variety of stimuli. Under normal circumstances, the human liver initiates regeneration within 3 days and has reached its original size by 3-6 months. Liver regeneration has been confirmed to be a very complex process, and hundreds of substances participate in it. The molecular mechanism of liver regeneration is always one of hot areas, which was concerned by many researchers in last several decades.
Recently, a cytokine stimulating the hepatocyte proliferation specifically was isolated from the extract of weanling calf liver by our lab and was named HPPCn. And its humanized counterpart was cloned from the cDNA library of human fetal liver. The bioactivity analysis showed that recombinant human HPPCn protein presented the specific stimulation activity on DNA synthesis and cytodieresis of primary cultured rat hepatocyte, human normal liver cell line L02 and human liver cancer cell line SMMC-7721, and hepatectomized mice liver. Experiments in vivo proved the protective effects of rhHPPCn on the acute liver injury and liver fibrosis induced by ethanol and CCl4.
The sequence analysis showed that the homogenization of HPPCn with the members of leucine-rich acidic nuclear protein (LANP) family was beyond 70 % and the conserved and special domains were just the same as the other family members of LANP: 1. The N-terminal repeat sequence including 25 leucine residues which was named leucine rich-repeats (LRRs), conformed the characteristic boot-shaped construction withβ-αunits, facilitating the protein-protein interaction; 2. The C-terminal 169-230 residues constituted a highly repeat sequence with GLUs and ASPs which was named acidic tail domain. This domain enabled LANPs to bind chromosome in the nucleus and regulate transcription accordingly. The LANP family members as a kind of multifunctional nucleoprotein involved in various biologic processes including cell proliferation, differentiation and apoptosis and tumor-inhibiting.
The protein structure analysis showed that although HPPCn represented the character of nucleoprotein, the typical signal peptide was absent within its structure. The function by which HPPCn exhibits growth factor activity needed to be revealed. A considerable number of nucleoprotein such as HMGB1 and HDGF has been reported to be able to take effects both inside and outside cells. They represent a group of bifunctional cytokines which are active either inside or outside nucleus. The similarity in the structures of HPPCn and these cytokines may produce the similarity in the functions. We wondered that whether HPPCn was a bifunctional cytokine as well. The early study using FITC-tagged rhHPPCn indicated that the rhHPPCn bound to the hepatocyte surface in a plaque-like manner and activated proliferation-related kinases such as SPK, MAPK, STAT-3. Besides, flow cytometry analysis showed either in binding experiment or in competitive inhibition, HPPCn bound to hepatocyte specifically.
The present study aims to identify the membrane protein that interacts with HPPCn. His-tagged HPPCn protein was produced and an improved His Pull-down method was used to search the membrane protein which interacted with HPPCn. The interaction between them was confirmed by co-immunoprecipitation and cell transfection. The experimental process was detailed as follow:
Firstly, prepare His-tagged HPPCn protein and membrane proteins of SMMC-7721 cell line. The His-tagged HPPCn protein was produced using E. coli. engineering strain BL21 containing express vector pET-24a(+)-HPPCn and purified by Q-XL anion-exchange chromatography and His affinity chromatography. The membrane protein of SMMC-7721 was gained applying the sucrose density gradient centrifugation.
Secondly, search the membrane protein interacting with HPPCn by improved His Pull-down. SANPAH (a light-sensitive cross-linker), His-tagged HPPCn protein and membrane protein were exposed to ultraviolet. The cross linkage between His-tagged HPPCn protein and membrane protein occurred because of the cleavage and reset of chemical bonds. The cross-linked complex was isolated by ultracentrifugation and purified by His affinity chromatography. SDS-PAGE was applied to separate the complex. The specificity was determined by Western blot using anti-His and anti-HPPCn antibodies sequentially. The targeted band was identified by comparing the results from SDS-PAGE and Western blot. The mass spectra analysis was applied to determine the component in the recovered gel band.
Finally, validate the interaction betweent HPPCn and keratin 8/18. The distribution of keratin 8/18 on SMMC-7721 surface was examined by cellular immunohistochemistry. The interaction between them was further validated by outside-cell binding experiment and co-immunoprecipitation. To determine the presence of this interaction inside cells, the keratin 8/18 express vector pCNA3.1-K8-IRES-K18 was transferred into NIH-3T3 cell line which responds negatively to HPPCn and the proliferation-promotion and anti-apoptosis activity of HPPCn on the transferred cell line was assessed.
The results showed that 0.3 mM was the most appropriate IPTG concentration to induce the expression of HPPCn in the engineering strain. After induction for 4 hours, large amount of His-tagged HPPCn protein was yield, indicating that the construction of E.coli. engineering strain expressing His-tagged HPPCn was successful. The bacteria were lysed by ultra-sonication and the supernatant was purified by Q-XL anion-exchange chromatography and His affinity chromatography. The gray scale scan showed that the protein purity was above 90% which met the requirement of the subsequent experiments. A considerable amount of SMMC-7721 membrane protein was gained by adopting sucrose density gradient centrifugation, establishing the foundation for the evaluation of their interaction. The membrane protein interacting with HPPCn was proved to be keratin 8/18 by improved His Pull-down and mass spectrum. The distribution of keratin 8/18 on the surface of cellular membrane was determined by immunohistochemistry. The interaction between HPPCn and keratin 8/18 was validated by protein binding assay and co-immunoprecipitation. Experiments using keratin 8 stable transfected cell line indicated that HPPCn exhibited proliferation-promotion and anti-apoptosis activity in keratin 8 transfected NIH-3T3 cell line.
Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair. The data accumμlated in recent years provide that K8/K18 can provide resistance not only to a mechanical stress but toxic stress and Fas-mediated apoptosis.Clinical dates show that the specific amino acid residues of K8/18 lead to various liver diseases. On the one hand, K8/18 maintain cell shape together with other cytoskeletal elements, integral membrane proteins and motor proteins, on the other hand they form complex signaling platforms and interact with various kinases, forms of toxic stress , apoptosis and various liver disease. Therefore K8/18 has a significant importance in the research of hepatocyte resistance to toxic stress, apoptosis and various liver diseases.
In this study the membrane protein that interactive with HPPCn was found by His Pull-down and proved to be keratin 8/18. The interaction between them was validated in vitro by co-immunoprecipitation. The keratin 8/18 eukaryotic expression vector was constructed and transferred into NIH-3T3 cell line which responded negatively to HPPCn. The suppression of HPPCn on the proliferation of NIH-3T3 cells was compromised by the expression of keratin 8 alone.
近两年,我们实验室的研究人员利用传统的生化分离与现代蛋白质组学相结合的分离方法从新生小牛肝脏中分离到一个能够特异的促进肝细胞增殖的蛋白因子,将其命名为HPPCn(hepatopoietin Cn, HPPCn)。随后,又从人胎肝cDNA文库中筛到了它的人源序列。
生物活性分析显示,重组人HPPCn蛋白能够特异地促进原代培养大鼠肝细胞、正常肝细胞系L02和肝癌细胞系SMMC7721的DNA合成和细胞分裂,而且能够促进70%肝脏切除小鼠的肝再生,初步的动物实验显示重组HPPCn蛋白对乙醇诱导的急性肝损伤有明显的保护作用。
序列分析显示,HPPCn与富含亮氨酸的酸性核蛋白(Leucine-rich acidic nuclear protein,LANP)家族其他成员序列同源性达到70%以上,且同已知的LANP蛋白一样,具有保守并且特殊的结构特征:1)其N端含有一个由25个亮氨酸组成的高度重复序列,称之为富含亮氨酸重复序列(leucine rich-repeats,LRRs),它具有特征性的βαβ的马靴型结构,有利于蛋白与蛋白之间的相互作用;2)C端第169-230位的氨基酸残基是一段由谷氨酸与天冬氨酸组成的重复序列,称之为酸性尾结构(acidic tail domain),这有利于它们在核内与染色体相结合,从而调控基因的转录。
蛋白质结构分析显示,HPPCn拥有核蛋白的结构特点,但是它又不具有典型的信号肽结构,那么它是如何作为一个生长因子来发挥作用的呢?在已有的报道中,相当数量的核蛋白(如HMGB1,HDGF等)能够同时在细胞内外发挥作用,属于一类可在核内外同时发挥作用的双功能细胞因子。HPPCn与其有极其相似的结构,这种结构的相似性,能否产生功能的相似性?即HPPCn是否具有能在细胞内外同时发挥作用的双功能性?前期的实验表明,FITC标记的重组HPPCn蛋白与肝细胞表面呈斑块样特异性结合,并能激活细胞中增殖相关的激酶活性,如SPK,MAPK,STAT3。流式分析显示,不论是结合实验还是竞争性抑制实验,均表明HPPCn能够与肝细胞特异结合。
本研究主要目的是寻找HPPCn相互作用的的膜蛋白。我们首先制备了携带His标签的HPPCn蛋白,并利用改进的His Pull-down的方法寻找HPPCn相互作用的膜蛋白,通过免疫共沉淀,转染细胞等方式对他们的相互作用做了进一步验证。具体的实验方法如下:
首先,表达纯化带His标签的HPPCn蛋白并分离肝癌细胞系SMMC7721的膜蛋白。构建带His标签的HPPCn的原核表达载体pET-24a(+)-HPPCn,并将其转入大肠杆菌BL21,构建表达带His标签的HPPCn重组蛋白的工程菌,利用工程菌发酵生产大量带His标签的HPPCn蛋白,并通过Q-XL阴离子交换层析和His亲和层析进行蛋白纯化。通过蔗糖密度梯度离心的方法获得肝癌细胞系SMMC7721的膜蛋白。
第二,通过改进的His Pull-down方法,寻找HPPCn相互作用的膜蛋白。将光敏感交联剂SANPAH、带His标签的HPPCn蛋白与提取的膜表面蛋白一起用紫外照射,由于化学键的断裂重新连接,带His标签的HPPCn蛋白与提取的膜表面蛋白进行交联,超速离心分离交联复合物。His亲和层析进一步纯化交联复合体,得到带His标签的HPPCn蛋白和交联复合体的混合物, SDS-PAGE电泳对其进行分离,并进一步通过Western blot用抗His的抗体和抗HPPCn的抗体对分离的条带进行专一性鉴定,与相应的SDS-PAGE电泳结果比对找到目的条带,割胶回收,质谱鉴定。
最后,进一步验证HPPCn与角蛋白8/18之间的相互作用。我们首先用角蛋白8/18抗体通过细胞免疫组化的方式,对SMMC7721细胞表面的角蛋白8/18的分布进行分析。通过细胞外的结合实验和免疫共沉淀进一步确定两者之间的相互作用。为了在细胞内确定HPPCn与角蛋白8/18相互作用的存在,将角蛋白8/18的表达载体pCDNA3.1-K8-IRES-K18转入HPPCn作用阴性的小鼠成纤维细胞系NIH-3T3,筛选稳定表达株,观察HPPCn对其是否有促增殖抗凋亡的活性。
实验结果显示,通过不同浓度IPTG,不同诱导时间诱导,最后确定0.3mM IPTG,诱导4小时,工程菌可以大量表达带His标签的HPPCn,因此表达带His标签的HPPCn的工程菌构建成功。大量摇瓶培养,收集菌体超声破碎,得到的超声上清经过Q-XL阴离子交换层析和His亲和层析,去除大部分杂蛋白,获得较为纯净的带His标签的HPPCn,灰度扫描纯度达到90%以上,符合后续实验的要求。通过蔗糖密度梯度离心的方法逐级分离大量的肝癌细胞系SMMC7721的膜蛋白,为后续两者的相互作用奠定基础。通过改进的His Pull-down和质谱相结合,筛选到HPPCn相互作用的膜蛋白角蛋白8/18。免疫组化实验确定了角蛋白8/18在膜表面的分布,PVDF膜上的蛋白结合实验和免疫共沉淀结果验证了HPPCn与角蛋白8/18的相互作用。通过角蛋白8稳定转染株的初步实验发现HPPCn对转化了pCDNA3.1-K8表达载体的NIH-3T3细胞系促增殖作用不太明显。
角蛋白8/18是正常成年人肝细胞中唯一的中间纤维蛋白。越来越多的实验发现,角蛋白8/18不仅能够对抗机械压力,而且可以对抗各种毒素压力以及Fas介导的细胞凋亡。而临床数据显示,角蛋白8/18特定氨基酸残基的突变将会导致各种肝脏疾病。分子机制研究发现,角蛋白8/18一方面可以与各种骨架相互蛋白作用维持细胞形态,另一方面与各种激酶、接头蛋白和细胞凋亡蛋白相互作用构成复杂的信号通路系统。因此角蛋白8/18在研究肝脏细胞对抗毒素压力,细胞凋亡以及各种肝脏疾病方面有重要的意义。
本研究通过His Pull-down的方法获得与HPPCn相互作用的膜蛋白角蛋白8/18,并通过进一步的免疫共沉淀等体外验证实验初步证实了两者的相互作用。构建了角蛋白8/18的真核表达载体,转染HPPCn作用阴性的NIH-3T3细胞。角蛋白8的单独表达解除了HPPCn对NIH-3T3细胞生长的抑制作用。
The mammalian liver possesses a remarkable capacity of regeneration in response to a variety of stimuli. Under normal circumstances, the human liver initiates regeneration within 3 days and has reached its original size by 3-6 months. Liver regeneration has been confirmed to be a very complex process, and hundreds of substances participate in it. The molecular mechanism of liver regeneration is always one of hot areas, which was concerned by many researchers in last several decades.
Recently, a cytokine stimulating the hepatocyte proliferation specifically was isolated from the extract of weanling calf liver by our lab and was named HPPCn. And its humanized counterpart was cloned from the cDNA library of human fetal liver. The bioactivity analysis showed that recombinant human HPPCn protein presented the specific stimulation activity on DNA synthesis and cytodieresis of primary cultured rat hepatocyte, human normal liver cell line L02 and human liver cancer cell line SMMC-7721, and hepatectomized mice liver. Experiments in vivo proved the protective effects of rhHPPCn on the acute liver injury and liver fibrosis induced by ethanol and CCl4.
The sequence analysis showed that the homogenization of HPPCn with the members of leucine-rich acidic nuclear protein (LANP) family was beyond 70 % and the conserved and special domains were just the same as the other family members of LANP: 1. The N-terminal repeat sequence including 25 leucine residues which was named leucine rich-repeats (LRRs), conformed the characteristic boot-shaped construction withβ-αunits, facilitating the protein-protein interaction; 2. The C-terminal 169-230 residues constituted a highly repeat sequence with GLUs and ASPs which was named acidic tail domain. This domain enabled LANPs to bind chromosome in the nucleus and regulate transcription accordingly. The LANP family members as a kind of multifunctional nucleoprotein involved in various biologic processes including cell proliferation, differentiation and apoptosis and tumor-inhibiting.
The protein structure analysis showed that although HPPCn represented the character of nucleoprotein, the typical signal peptide was absent within its structure. The function by which HPPCn exhibits growth factor activity needed to be revealed. A considerable number of nucleoprotein such as HMGB1 and HDGF has been reported to be able to take effects both inside and outside cells. They represent a group of bifunctional cytokines which are active either inside or outside nucleus. The similarity in the structures of HPPCn and these cytokines may produce the similarity in the functions. We wondered that whether HPPCn was a bifunctional cytokine as well. The early study using FITC-tagged rhHPPCn indicated that the rhHPPCn bound to the hepatocyte surface in a plaque-like manner and activated proliferation-related kinases such as SPK, MAPK, STAT-3. Besides, flow cytometry analysis showed either in binding experiment or in competitive inhibition, HPPCn bound to hepatocyte specifically.
The present study aims to identify the membrane protein that interacts with HPPCn. His-tagged HPPCn protein was produced and an improved His Pull-down method was used to search the membrane protein which interacted with HPPCn. The interaction between them was confirmed by co-immunoprecipitation and cell transfection. The experimental process was detailed as follow:
Firstly, prepare His-tagged HPPCn protein and membrane proteins of SMMC-7721 cell line. The His-tagged HPPCn protein was produced using E. coli. engineering strain BL21 containing express vector pET-24a(+)-HPPCn and purified by Q-XL anion-exchange chromatography and His affinity chromatography. The membrane protein of SMMC-7721 was gained applying the sucrose density gradient centrifugation.
Secondly, search the membrane protein interacting with HPPCn by improved His Pull-down. SANPAH (a light-sensitive cross-linker), His-tagged HPPCn protein and membrane protein were exposed to ultraviolet. The cross linkage between His-tagged HPPCn protein and membrane protein occurred because of the cleavage and reset of chemical bonds. The cross-linked complex was isolated by ultracentrifugation and purified by His affinity chromatography. SDS-PAGE was applied to separate the complex. The specificity was determined by Western blot using anti-His and anti-HPPCn antibodies sequentially. The targeted band was identified by comparing the results from SDS-PAGE and Western blot. The mass spectra analysis was applied to determine the component in the recovered gel band.
Finally, validate the interaction betweent HPPCn and keratin 8/18. The distribution of keratin 8/18 on SMMC-7721 surface was examined by cellular immunohistochemistry. The interaction between them was further validated by outside-cell binding experiment and co-immunoprecipitation. To determine the presence of this interaction inside cells, the keratin 8/18 express vector pCNA3.1-K8-IRES-K18 was transferred into NIH-3T3 cell line which responds negatively to HPPCn and the proliferation-promotion and anti-apoptosis activity of HPPCn on the transferred cell line was assessed.
The results showed that 0.3 mM was the most appropriate IPTG concentration to induce the expression of HPPCn in the engineering strain. After induction for 4 hours, large amount of His-tagged HPPCn protein was yield, indicating that the construction of E.coli. engineering strain expressing His-tagged HPPCn was successful. The bacteria were lysed by ultra-sonication and the supernatant was purified by Q-XL anion-exchange chromatography and His affinity chromatography. The gray scale scan showed that the protein purity was above 90% which met the requirement of the subsequent experiments. A considerable amount of SMMC-7721 membrane protein was gained by adopting sucrose density gradient centrifugation, establishing the foundation for the evaluation of their interaction. The membrane protein interacting with HPPCn was proved to be keratin 8/18 by improved His Pull-down and mass spectrum. The distribution of keratin 8/18 on the surface of cellular membrane was determined by immunohistochemistry. The interaction between HPPCn and keratin 8/18 was validated by protein binding assay and co-immunoprecipitation. Experiments using keratin 8 stable transfected cell line indicated that HPPCn exhibited proliferation-promotion and anti-apoptosis activity in keratin 8 transfected NIH-3T3 cell line.
Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair. The data accumμlated in recent years provide that K8/K18 can provide resistance not only to a mechanical stress but toxic stress and Fas-mediated apoptosis.Clinical dates show that the specific amino acid residues of K8/18 lead to various liver diseases. On the one hand, K8/18 maintain cell shape together with other cytoskeletal elements, integral membrane proteins and motor proteins, on the other hand they form complex signaling platforms and interact with various kinases, forms of toxic stress , apoptosis and various liver disease. Therefore K8/18 has a significant importance in the research of hepatocyte resistance to toxic stress, apoptosis and various liver diseases.
In this study the membrane protein that interactive with HPPCn was found by His Pull-down and proved to be keratin 8/18. The interaction between them was validated in vitro by co-immunoprecipitation. The keratin 8/18 eukaryotic expression vector was constructed and transferred into NIH-3T3 cell line which responded negatively to HPPCn. The suppression of HPPCn on the proliferation of NIH-3T3 cells was compromised by the expression of keratin 8 alone.
引文
1) Court FG, Wemyss-Holden SA, Dennison AR, et al. The mystery of liver regeneration. Bri J Surg, 2002, 89 (9): 1089-1095.
2)杜劭君,一种新的肝再生刺激因子HPPCn的克隆、表达、分离纯化及其生物活性的研究,军事医学科学院,2006,5。
3) Fausto N, Campbell JS, Riehle KJ. Liver regeneration. Hepatology, 2006, 43(2 Suppl 1): S45-53.
4)张达矜,新的肝细胞生长因子(HPPCn)生物活性及作用机制的研究,军事医学科学院,2007,5。
5) Fausto N, Laired AD, Webber EM. Role of growth factors and cytokines in hepatic regeneration. FASEB J, 1995, 9(15): 1527-1536.)
6) Blomquist K. Growth stimμlation in the liver following intraperitoneal injections of liver homogenates in the rat. Acta Pathol Microbiol Scand, 1957; 121(Suppl): 375
7) LaBrecque DR, Pesch LA. Partial characterization of hepatic regeneration stimμlator substance from rat liver. J Physiol, 1975; 248:273.
8) He FC, Wu CT, Tu Q, Xing G. Human hepatic stimμlator substance: a product of gene expression of human fetal liver tissue. Hepatology 1993; 17: 225.
9) Wu CT, Tu Q, He FC. Hepatokine and methods for its use. US Patent 1995; 5,440,022.
10) Francavilla A, Barone M, Vanthiel DH. Further steps of hepatic stimμlator substance purification. Dig Dis Sci, 1991;36:674.
11) Hagiya M, Francavilla A, Polimeno L, Ihara I, Sakai H, Seki T, Shimonishi M, Porter KA, Starzl TE. Cloning and sequence analysis of the rat augmentor of liver regeneration(ALR) gene: expression of biologically active recombinant ALR and demonstration of tissue distribution. PNAS, 1995;92: 3076.
12) Cui CP, Zhang DJ, Wu DL, Shi BX, Du SJ, Zhong GS, Wei P, Guo ZK, Liu Y, Wang LS, Chu-Tse Wu et al, Isolation and Functional Identification of a Novel Human Hepatic Growth Factor: Hepatopoietin Cn, Hepatology,, 2008; 47: 985.
13) Kobe B, Deisenhofer J. Crystal structure of procine ribonuclease inhibitor, a protein with leucine-rich repeats. Nature, 1993;366: 751.
14) Jiang XJ, Kim HE, Shu HJ. Distinctive roles of PHAPI proteins and prothymosin-αin a death regμlatory pathway. Science, 2003; 299: 223.
15) Brody JR, Kadkol SS, Hauer MC, Rajaii F, Lee J, Pasternack GR. Pp32 reduction induces differentiation of TSU-Pr1 cells. Am J Pathol, 2004;16:273.
16) Seo SB, Macfarlan T, McNamara P. The oncoprotein set/TAF-1-beta, an inhibitor of histone acetyltransferase, inhibits active demethylation of DNA, integrating DNA methylation and transcription signaling. J Biol Chem, 2002;277:14005.
17) Adegbola O, Pasternack GR. Phosphorylated Rb complexes with pp32 and inhibits pp32-mediated apoptosis. J Biol Chem, 2005; 304: 702.
18) El-Tahir HM, Dietz F, Dringen R, Schwabe K, Strenge K, Kelm S, Abouzied MM. Expression of Hepatoma-derived growth factor family members in the adμlt central nervous system. BMC Neurosci. 2006; 23:6.
19) Enomoto H, Yoshida K, Kishima Y, Kinoshita T, Everett, AD, Miyajima A., and Nakamura H. Hepatoma-derived growth factor is highly expressed in developing liver and promotes fetal hepatocyte proliferation. Hepatology, 2002; 36: 1519.
20) Everett AD., Lobe DR., Matsumura ME., Nakamura H. Hepatoma-derived growth factor stimμlates smooth muscle cell growth and is expressed in vascμlar development. J Clin Invest, 2000; 105: 567.
21) oshida K, Tomita Y, Okuda Y, Yamamoto S, Enomoto H, Uyama H, Ito H, Hoshida Y. Hepatoma-derived growth factor is a novel prognostic factor for hepatocellμlar carcinoma. Ann Surg Oncol, 2006;13:159.
22) Kishima Y, Yamamoto H, Izumoto Y, Yoshida K. Hepatoma-derived Growth Factor Stimμlates Cell Growth after Translocation to the Nucleus by Nuclear Localization Signals. J Biol Chem, 2002;277: 10315.
23) Mμller S, Scaffidi P, Degryse B, Bonaldi T. New EMBO members' review: the double life of HMGB1 chromatin protein: architectural factor and extracellμlar signal. EMBO J, 2001; 20: 4337.
24) Lotze MT, Tracey KJ. High-mobility group box1 protein(HMGB1): nuclear weapon in the immune arsenal. Nature Review Immun, 2005; 5: 330.
25) Evans A, Lennard TW, Davies BR. High-mobility group protein 1(Y): metastasis-associated or metastasis-inducing? J Surg Oncol. 2004; 88: 86.
26) Drewes G,Bouwmeester T. Curr Opin Cell Biol,2003,15(2):199~205.
27) Bauer A,Kuster B. Eur J Biochem,2003,270(4):570~578.
28) Monti M,Orru S,Pagnozzi D,et al. Clinica Chimica Acta,2005,357:140~150.
29) Swartz, J.R., Advances in Escherichia coli production of therapeutic proteins. Curr Opin Biotechnol, 2001. 12(2): p. 195-201.
30) Donovan, R.S., C.W. Robinson, and B.R. Glick, Review: optimizing inducer and cμlture conditions for expression of foreign proteins under the control of the lac promoter. J Ind Microbiol, 1996. 16(3): p. 145-54.
31) Hisamatsu T, Watanabe M, Ogata H, et al. Interferon-inducible gene family 1-8U expression in colitis-associated colon cancer and severely inflamed mucosa inμlcerative colitis [J]. Cancer Res,1999, 59(23): 5927-31.
32) Link AJ, Yates JR III, Schieltz D. Direct analysis of proteins in mixtures. Application to protein complexes [J]. Nat Biotechnol, 1999, 17: 676-82.
[1] Franke WW, Denk H, Kalt R,et al, Schmid E. Biochemical and immunological identification of cytokeratin proteins present in hepatocytes of mammalian liver tissue. Exp Cell Res, 1981, 131:299–318. 2.
[2] Moll R, Franke WW, Schiller D, et al, The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cμltured cells. Cell, 1982, 31: 11–24.
[3] Ingber, D.E. Opposing views on tensegrity as a structural framework for understanding cell mechanics. J. Appl. Physiol , 2000, 1663–1670
[4] Lane EB, Hogan BL, Kurkinen M, et al, Coexpression of vimentin and cytokeratins in parietal endoderm cells of early mouse embryo. Nature (London), 1983, 303: 701–704.
[5] Omary MB, and Ku NO. Intermediate filament proteins of the liver: emerging disease association and functions. Hepatology, 1997, 25: 1043–1048。
[6] Ku NO, Liao J, and Omary MB, et al, Phosphorylation of human keratin 18 serine regμlates binding to 14-3-3 proteins. EMBO J, 1998, 17: 1892–1906.
[7] Omary MB, Ku NO, Liao J, et al, Keratin modifications and solubility properties in epithelial cells and in vitro . Subcell. Biochem, 1998, 31: 105–140
[8] Ku NO, Liao J, Chou CF, et al, Implications of intermediate filament protein phosphorylation. Cancer Metastasis Rev, 1996, 15: 429–444.
[9] Heidemann SR, Lamoureaux P, Buxbaum RE, et al, Opposing views on tensegrityas a structural framework for understanding cell mechanics . J. Appl. Physiol, 2000, 89: 1663–1670.
[10] Loranger A, Duclos S, Grenier A, et al, Simple epithelium keratins are required for maintenance of hepatocyte integrity. Am. J. Pathol, 1997, 151: 1673–1683
[11] Bauman PA, Dalton WS, Anderson JM, et al, Expression of cytokeratin confers mμltiple drug resistance. Proc. Natl. Acad. Sci. U.S.A, 1994, 91: 5311–5314。
[12] Kurt Z, Cornelia S, Manfred L et al, Cytokeratin 8 protects from hepatotoxicity, and its ratio to Cytokeratin 18 determines the ability of hepatocytes toform Mallory Bodies, 2000, 156:1263-1274.
[13] Cadrin M., Marceau N, Baribaμlt H, et al, Griseofμlvin hepatotoxicity-related effects in keratin 8 deficient FVB/N mice. Mol. Biol. Cell, 1996, 6S: 2171.
[14] Huot J, Hoμle F, Marceau F, et al, Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascμlar endothelial cells. Circ. Res, 1997, 80:383–392.
[15] Omary MB, Ku NO, Liao J, et al, Keratin modifications and solubility properties in epithelial cells and in vitro. Subcell Biochem, 1998, 31:105–140.
[16] Gilbert S, Loranger A, Daigle N, et al, Simple epithelium keratins 8/18 provide resistance to Fas-mediated apoptosis. The protection occurs through a receptor-targeting modμlation. J. Cell Biol. 2001, 154:763-773.
[17] McLean WH, Lane EB. Intermediate filament s in disease [J] .Curr Opin Cell Biol, 1995, 7 (1)∶118-152.
[18] Ku NO, Liao J, Omary MB, et al, Phosphorylation of human keratin 18 serine 33 regμlates binding to 142323 proteins. EMBO J , 1998, 17 (7)∶1892.
[19] Ku NO, Wright TL, Terraμlt NA, et al. Mutation of human keratin 18 in association wit h cryptogenic cirrhosis. J Clin Invest ; 1997, 99 (1)∶19.
[20] Kriho VK, Yang HY, Moskal J R, et al. Keratin expression in ast rocytomas : an immunofluorescent and biochemical reassess-ment. Virchows Arch, 1997, 431 (2)∶139.
[21] Getsios S, Huen AC, Green K J, et al, Working out the strength and flexibility of desmosomes. Nat. Rev. Mol. Cell. Biol, 2004, 5, 271-281.
[22] Green KJ, B?hringer M, Gocken T, et al, Intermediate filament associated proteins. Adv Protein Chem , 2005, 70:143–202
[23] Normand M, Anne L, Stéphane G,et al. Keratin-mediated resistance to stress and apoptosis in simple epithelial cells in relation to health and disease. NRC Research, 2001, 543-555
[24] Miwako N, Ichiro I, Akihito IN, et al. Identification of trichoplein, a novel keratin filament binding protein. Journal of Cell Science, 2005, 1081-1090.
[25] Ku NO, Strnad P, Zhang BH, et al, Keratins let liver live: mutations predispose to liver disease and cross linking generates Mallory-Denk bodies. Hepatology, 2007, 46:1639–1649
[26] Kim S, Coμlombe PA, et al, Intermediate filament scaffolds fμlfill mechanical, organizational, and signaling functions in the cytoplasm. Genes Dev, 2007, 21:1581–1597.
[27] Oshima R.G. Apoptosis and keratin intermediate filaments. Cell Death Differ, 2002, 9: 486–492.
[28] Inada H, Izawa I, Nishizawa M, et al. Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. J. Cell Biol, 2001, 155: 415–426.
2)杜劭君,一种新的肝再生刺激因子HPPCn的克隆、表达、分离纯化及其生物活性的研究,军事医学科学院,2006,5。
3) Fausto N, Campbell JS, Riehle KJ. Liver regeneration. Hepatology, 2006, 43(2 Suppl 1): S45-53.
4)张达矜,新的肝细胞生长因子(HPPCn)生物活性及作用机制的研究,军事医学科学院,2007,5。
5) Fausto N, Laired AD, Webber EM. Role of growth factors and cytokines in hepatic regeneration. FASEB J, 1995, 9(15): 1527-1536.)
6) Blomquist K. Growth stimμlation in the liver following intraperitoneal injections of liver homogenates in the rat. Acta Pathol Microbiol Scand, 1957; 121(Suppl): 375
7) LaBrecque DR, Pesch LA. Partial characterization of hepatic regeneration stimμlator substance from rat liver. J Physiol, 1975; 248:273.
8) He FC, Wu CT, Tu Q, Xing G. Human hepatic stimμlator substance: a product of gene expression of human fetal liver tissue. Hepatology 1993; 17: 225.
9) Wu CT, Tu Q, He FC. Hepatokine and methods for its use. US Patent 1995; 5,440,022.
10) Francavilla A, Barone M, Vanthiel DH. Further steps of hepatic stimμlator substance purification. Dig Dis Sci, 1991;36:674.
11) Hagiya M, Francavilla A, Polimeno L, Ihara I, Sakai H, Seki T, Shimonishi M, Porter KA, Starzl TE. Cloning and sequence analysis of the rat augmentor of liver regeneration(ALR) gene: expression of biologically active recombinant ALR and demonstration of tissue distribution. PNAS, 1995;92: 3076.
12) Cui CP, Zhang DJ, Wu DL, Shi BX, Du SJ, Zhong GS, Wei P, Guo ZK, Liu Y, Wang LS, Chu-Tse Wu et al, Isolation and Functional Identification of a Novel Human Hepatic Growth Factor: Hepatopoietin Cn, Hepatology,, 2008; 47: 985.
13) Kobe B, Deisenhofer J. Crystal structure of procine ribonuclease inhibitor, a protein with leucine-rich repeats. Nature, 1993;366: 751.
14) Jiang XJ, Kim HE, Shu HJ. Distinctive roles of PHAPI proteins and prothymosin-αin a death regμlatory pathway. Science, 2003; 299: 223.
15) Brody JR, Kadkol SS, Hauer MC, Rajaii F, Lee J, Pasternack GR. Pp32 reduction induces differentiation of TSU-Pr1 cells. Am J Pathol, 2004;16:273.
16) Seo SB, Macfarlan T, McNamara P. The oncoprotein set/TAF-1-beta, an inhibitor of histone acetyltransferase, inhibits active demethylation of DNA, integrating DNA methylation and transcription signaling. J Biol Chem, 2002;277:14005.
17) Adegbola O, Pasternack GR. Phosphorylated Rb complexes with pp32 and inhibits pp32-mediated apoptosis. J Biol Chem, 2005; 304: 702.
18) El-Tahir HM, Dietz F, Dringen R, Schwabe K, Strenge K, Kelm S, Abouzied MM. Expression of Hepatoma-derived growth factor family members in the adμlt central nervous system. BMC Neurosci. 2006; 23:6.
19) Enomoto H, Yoshida K, Kishima Y, Kinoshita T, Everett, AD, Miyajima A., and Nakamura H. Hepatoma-derived growth factor is highly expressed in developing liver and promotes fetal hepatocyte proliferation. Hepatology, 2002; 36: 1519.
20) Everett AD., Lobe DR., Matsumura ME., Nakamura H. Hepatoma-derived growth factor stimμlates smooth muscle cell growth and is expressed in vascμlar development. J Clin Invest, 2000; 105: 567.
21) oshida K, Tomita Y, Okuda Y, Yamamoto S, Enomoto H, Uyama H, Ito H, Hoshida Y. Hepatoma-derived growth factor is a novel prognostic factor for hepatocellμlar carcinoma. Ann Surg Oncol, 2006;13:159.
22) Kishima Y, Yamamoto H, Izumoto Y, Yoshida K. Hepatoma-derived Growth Factor Stimμlates Cell Growth after Translocation to the Nucleus by Nuclear Localization Signals. J Biol Chem, 2002;277: 10315.
23) Mμller S, Scaffidi P, Degryse B, Bonaldi T. New EMBO members' review: the double life of HMGB1 chromatin protein: architectural factor and extracellμlar signal. EMBO J, 2001; 20: 4337.
24) Lotze MT, Tracey KJ. High-mobility group box1 protein(HMGB1): nuclear weapon in the immune arsenal. Nature Review Immun, 2005; 5: 330.
25) Evans A, Lennard TW, Davies BR. High-mobility group protein 1(Y): metastasis-associated or metastasis-inducing? J Surg Oncol. 2004; 88: 86.
26) Drewes G,Bouwmeester T. Curr Opin Cell Biol,2003,15(2):199~205.
27) Bauer A,Kuster B. Eur J Biochem,2003,270(4):570~578.
28) Monti M,Orru S,Pagnozzi D,et al. Clinica Chimica Acta,2005,357:140~150.
29) Swartz, J.R., Advances in Escherichia coli production of therapeutic proteins. Curr Opin Biotechnol, 2001. 12(2): p. 195-201.
30) Donovan, R.S., C.W. Robinson, and B.R. Glick, Review: optimizing inducer and cμlture conditions for expression of foreign proteins under the control of the lac promoter. J Ind Microbiol, 1996. 16(3): p. 145-54.
31) Hisamatsu T, Watanabe M, Ogata H, et al. Interferon-inducible gene family 1-8U expression in colitis-associated colon cancer and severely inflamed mucosa inμlcerative colitis [J]. Cancer Res,1999, 59(23): 5927-31.
32) Link AJ, Yates JR III, Schieltz D. Direct analysis of proteins in mixtures. Application to protein complexes [J]. Nat Biotechnol, 1999, 17: 676-82.
[1] Franke WW, Denk H, Kalt R,et al, Schmid E. Biochemical and immunological identification of cytokeratin proteins present in hepatocytes of mammalian liver tissue. Exp Cell Res, 1981, 131:299–318. 2.
[2] Moll R, Franke WW, Schiller D, et al, The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cμltured cells. Cell, 1982, 31: 11–24.
[3] Ingber, D.E. Opposing views on tensegrity as a structural framework for understanding cell mechanics. J. Appl. Physiol , 2000, 1663–1670
[4] Lane EB, Hogan BL, Kurkinen M, et al, Coexpression of vimentin and cytokeratins in parietal endoderm cells of early mouse embryo. Nature (London), 1983, 303: 701–704.
[5] Omary MB, and Ku NO. Intermediate filament proteins of the liver: emerging disease association and functions. Hepatology, 1997, 25: 1043–1048。
[6] Ku NO, Liao J, and Omary MB, et al, Phosphorylation of human keratin 18 serine regμlates binding to 14-3-3 proteins. EMBO J, 1998, 17: 1892–1906.
[7] Omary MB, Ku NO, Liao J, et al, Keratin modifications and solubility properties in epithelial cells and in vitro . Subcell. Biochem, 1998, 31: 105–140
[8] Ku NO, Liao J, Chou CF, et al, Implications of intermediate filament protein phosphorylation. Cancer Metastasis Rev, 1996, 15: 429–444.
[9] Heidemann SR, Lamoureaux P, Buxbaum RE, et al, Opposing views on tensegrityas a structural framework for understanding cell mechanics . J. Appl. Physiol, 2000, 89: 1663–1670.
[10] Loranger A, Duclos S, Grenier A, et al, Simple epithelium keratins are required for maintenance of hepatocyte integrity. Am. J. Pathol, 1997, 151: 1673–1683
[11] Bauman PA, Dalton WS, Anderson JM, et al, Expression of cytokeratin confers mμltiple drug resistance. Proc. Natl. Acad. Sci. U.S.A, 1994, 91: 5311–5314。
[12] Kurt Z, Cornelia S, Manfred L et al, Cytokeratin 8 protects from hepatotoxicity, and its ratio to Cytokeratin 18 determines the ability of hepatocytes toform Mallory Bodies, 2000, 156:1263-1274.
[13] Cadrin M., Marceau N, Baribaμlt H, et al, Griseofμlvin hepatotoxicity-related effects in keratin 8 deficient FVB/N mice. Mol. Biol. Cell, 1996, 6S: 2171.
[14] Huot J, Hoμle F, Marceau F, et al, Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascμlar endothelial cells. Circ. Res, 1997, 80:383–392.
[15] Omary MB, Ku NO, Liao J, et al, Keratin modifications and solubility properties in epithelial cells and in vitro. Subcell Biochem, 1998, 31:105–140.
[16] Gilbert S, Loranger A, Daigle N, et al, Simple epithelium keratins 8/18 provide resistance to Fas-mediated apoptosis. The protection occurs through a receptor-targeting modμlation. J. Cell Biol. 2001, 154:763-773.
[17] McLean WH, Lane EB. Intermediate filament s in disease [J] .Curr Opin Cell Biol, 1995, 7 (1)∶118-152.
[18] Ku NO, Liao J, Omary MB, et al, Phosphorylation of human keratin 18 serine 33 regμlates binding to 142323 proteins. EMBO J , 1998, 17 (7)∶1892.
[19] Ku NO, Wright TL, Terraμlt NA, et al. Mutation of human keratin 18 in association wit h cryptogenic cirrhosis. J Clin Invest ; 1997, 99 (1)∶19.
[20] Kriho VK, Yang HY, Moskal J R, et al. Keratin expression in ast rocytomas : an immunofluorescent and biochemical reassess-ment. Virchows Arch, 1997, 431 (2)∶139.
[21] Getsios S, Huen AC, Green K J, et al, Working out the strength and flexibility of desmosomes. Nat. Rev. Mol. Cell. Biol, 2004, 5, 271-281.
[22] Green KJ, B?hringer M, Gocken T, et al, Intermediate filament associated proteins. Adv Protein Chem , 2005, 70:143–202
[23] Normand M, Anne L, Stéphane G,et al. Keratin-mediated resistance to stress and apoptosis in simple epithelial cells in relation to health and disease. NRC Research, 2001, 543-555
[24] Miwako N, Ichiro I, Akihito IN, et al. Identification of trichoplein, a novel keratin filament binding protein. Journal of Cell Science, 2005, 1081-1090.
[25] Ku NO, Strnad P, Zhang BH, et al, Keratins let liver live: mutations predispose to liver disease and cross linking generates Mallory-Denk bodies. Hepatology, 2007, 46:1639–1649
[26] Kim S, Coμlombe PA, et al, Intermediate filament scaffolds fμlfill mechanical, organizational, and signaling functions in the cytoplasm. Genes Dev, 2007, 21:1581–1597.
[27] Oshima R.G. Apoptosis and keratin intermediate filaments. Cell Death Differ, 2002, 9: 486–492.
[28] Inada H, Izawa I, Nishizawa M, et al. Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. J. Cell Biol, 2001, 155: 415–426.