Pulification and identification of substrate proteins for selective ubquitin-dependent proteolysis during NGF-induced neurite outgrowth of PC12h cells.

NGF 诱导 PC12h 细胞神经突生长期间选择性泛素依赖性蛋白水解的底物蛋白的纯化和鉴定。

• 批准号: 10680723
• 负责人: TAKADA Koji
• 金额: $ 2.18万
• 依托单位: JIKEI UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680723/
• 关键词: Ubiquitin; Ubiquitinated proteins; Uniquitin-conjugating enzyme; Proteolysis; PC12 cell; Neurite; Purification; Amino acd sequence; ユビキチン結合酵素; プロテアソーム; 蛋白精製; アミノ酸配列解析; 熱ショック

Optimization of purification protocols.Using K562 cells we established methods for purifying cellular ubiquitinated proteins. The first step is an immunoaffinity chromatography using a monoclonal antibody FK2 to ubiquitin. One ml of FK2-Sepharose gel was able to adsorb abount 0.2 mg of ubiquitin equivalent, and the adsorbed materials were completely eluted with 3..5 M MgClィイD22ィエD2. Then the materials were fractionated by gel filtration using Superdex 75, and two characteristic fractions having high molecular weights (HMW) (>30 kDa) and low molecular weights (LMW) (<30 kDa) were obtained. The former was mainly composed of multi-ubiquitinated proteins. In the latter we found ubiquitin thioesters with ubiquitin-conjugating enzymes (E2) as well as ubiquitinated proteins.Ubiquitinated protein variety during neurite elongation.By the combination of immunoprecipitation using FK2 antibody and SDS-PAGE, we visualized ubiquitinated proteins in PC12h cells cultivated in the presence of nerve gro … More wth factor (NGF). Three bands, 18, 19, and 36-kDa, newly appeared, and intensities of six bands, 20, 23, 26, 51, 68, and 93-kDa, increased.Purification of ubiquitinated proteins.Ubiquitinated protein mixtures (0.56 mg protein) were obtained from NGF-treated PC12h cells (2 x 10ィイD19ィエD1 cells) by the immunoaffinity chromatography. The mixtures were further separated by the gel filtration, and resulted in the two distinctive fractions of HMW and LMW. Several protein bands were isolated from the latter by SDS-PAGE, and amino acid sequencing of their peptide fragments obtained by lysyl-endopeptidase is ongoing in turns. The analysis of the 26-kDa band resulted in two amino acid sequences of ubiquitin digests and five unknown sequences.A monoclonal antibody against ubiquitin C-terminal tag peptide.This antibody is required for identification of the target proteins. Because of antigen insolubility, we had to change the peptide design, and finally established the desired antibody, which will be applied to the analysis of HMW ubiquitinated proteins. Less

纯化方案的优化：利用K562细胞建立了纯化细胞泛素化蛋白的方法。第一步是使用泛素的单抗FK2进行免疫亲和层析。每毫升FK2-琼脂糖凝胶可吸附约0.2mg泛素当量，吸附的物质用3.5M氯化镁ィイD22ィエD2完全洗脱。再用Superdex 75凝胶过滤，得到高分子量(HMW)(30 KDa)和低相对分子质量(LMW)(&lt；30 kDa)两个特征组分。前者主要由多种泛素化蛋白组成。在后者中，我们发现了带有泛素结合酶(E2)的泛素硫酸酯和泛素化蛋白。泛素化蛋白在轴突伸长过程中发生了变化。采用FK2抗体免疫沉淀和SDSPAGE相结合的方法，我们显示了在神经GRO-…存在下培养的PC12h细胞中泛素化蛋白更多的神经生长因子(NGF)。泛素化蛋白的纯化。经神经生长因子处理的PC12h细胞(2×10ィイD19ィエD1细胞)经免疫亲和层析得到泛素化蛋白混合物(0.56 mg蛋白)。通过凝胶过滤将混合物进一步分离，得到HMW和LMW两个不同的组分。通过SDS-PAGE从后者中分离出几条蛋白带，并依次对其赖氨酰内肽酶获得的多肽片段进行氨基酸序列测定。对26 kDa条带的分析得到两个氨基酸序列和5个未知序列。抗泛素C末端标签肽的单抗。该抗体是鉴定目的蛋白所必需的。由于抗原的不溶性，我们不得不改变了多肽的设计，最终建立了所需的抗体，将其应用于HMW泛素化蛋白的分析。较少

项目成果

Daino, H.: "Induction of apoptosis by extracellular ubiquitin in human hematopoietic cells ; possible involvement of STAT3 degradation by proteasome pathway in interleukin 6-dependent hematopoietic cells."Blood. (in press). (2000)

Daino, H.：“人类造血细胞中胞外泛素诱导细胞凋亡；白细胞介素 6 依赖性造血细胞中蛋白酶体途径可能参与 STAT3 降解。”血液。

高木優: "アルコール性肝障害における血清ユビキチンの動態について。"アルコールと医学生物学. 18. 47-51 (1998)

Yu Takagi：“酒精性肝损伤中血清泛素的动态。” 18. 47-51 (1998)

Takagi, M: "Serum ubiquitin levels in patients with alcoholic liver disease."Alcohol. Clin. Exp. Res.. 23. 76S-80S (1999)

Takagi, M：“酒精性肝病患者的血清泛素水平。”酒精。

Ohtani-Kaneko, R.: "Proteasome inhibitors which induce neurite outgrowth from PC12h cells cause different subcellular accumulations of multi-ubiquitin chains"Neurochem. Res.. 23. 1435-1443 (1998)

Ohtani-Kaneko, R.：“诱导 PC12h 细胞神经突生长的蛋白酶体抑制剂导致多泛素链不同的亚细胞积累”Neurochem。

Daino, H.: "Induction of apoptosis by extracellular ubiquitin in human hematopoietic cells : possible involvement of STAT3 degradation by proteasome pathway in interleukin 6-dependent hematopoietic cells."Blood. (in press.). (2000)

Daino, H.：“人类造血细胞中细胞外泛素诱导细胞凋亡：白细胞介素 6 依赖性造血细胞中蛋白酶体途径可能参与 STAT3 降解。”血液。