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    Method for determining ubiquitin chain length
    1.
    发明授权
    Method for determining ubiquitin chain length 有权

    公开(公告)号：US09891228B2

    公开(公告)日：2018-02-13

    申请号：US14533182

    申请日：2014-11-05

    申请人： Tokyo Metropolitan Institute of Medical Science

    发明人： Yasushi Saeki ,  Hikaru Tsuchiya ,  Ai Kaiho ,  Keiji Tanaka

    IPC分类号： G01N33/68 ,  C07K14/47

    CPC分类号： G01N33/6842 ,  C07K14/4702 ,  G01N33/6803

    摘要： Protein ubiquitylation, an essential post-translational modification, regulates almost every cellular process including protein degradation, protein trafficking, signal transduction, and DNA damage response in eukaryotic cells. The diverse functions of ubiquitylation are thought to be mediated by distinct chain topologies resulting from eight different ubiquitin linkages, chain lengths, and complexities. Currently, ubiquitin linkages are generally thought to be a critical determinant of ubiquitin signaling. However, ubiquitin chain lengths, another key element of ubiquitin signaling, have not been well documented especially in vivo situation during past three decades from the discovery of ubiquitin. The reason of this was simply because no method has been available for determination of ubiquitin chain length in endogenous ubiquitylated substrates. In the present invention, a practical technique for determining the actual length of substrate-attached polyubiquitin chains from biological samples is established. Using the method, the mean length of substrate-attached polyubiquitin chains was determined and the robustness of ubiquitin chain length regulation in cells is investigated. The following is a summary of findings in this invention: 1. A method for determining ubiquitin chain length was developed and this method was named ‘ubiquitin protection from trypsinization’ (Ub-ProT). 2. Using Ub-ProT, it was determined that the mean length of substrate-attached ubiquitin chains is in the dimer to decamer range. 3. By quantitative proteomics, it was found that the mean lengths of five major types of ubiquitin chains can be divided into two groups. 4. Proteasome-inhibition did not alter the mean length of substrate-attached polyubiquitin chains, indicating that cells have a robust system for regulating ubiquitin chain length.

    Method for determining ubiquitin chain length
    3.
    发明授权
    Method for determining ubiquitin chain length 有权

    公开(公告)号：US09891229B2

    公开(公告)日：2018-02-13

    申请号：US15711432

    申请日：2017-09-21

    申请人： Tokyo Metropolitan Institute of Medical Science

    发明人： Yasushi Saeki ,  Hikaru Tsuchiya ,  Ai Kaiho ,  Keiji Tanaka

    IPC分类号： G01N33/68 ,  C07K14/47

    CPC分类号： G01N33/6842 ,  C07K14/4702 ,  G01N33/6803

    摘要： Protein ubiquitylation, an essential post-translational modification, regulates almost every cellular process including protein degradation, protein trafficking, signal transduction, and DNA damage response in eukaryotic cells. The diverse functions of ubiquitylation are thought to be mediated by distinct chain topologies resulting from eight different ubiquitin linkages, chain lengths, and complexities. Currently, ubiquitin linkages are generally thought to be a critical determinant of ubiquitin signaling. However, ubiquitin chain lengths, another key element of ubiquitin signaling, have not been well documented especially in vivo situation during past three decades from the discovery of ubiquitin. The reason of this was simply because no method has been available for determination of ubiquitin chain length in endogenous ubiquitylated substrates. In the present invention, a practical technique for determining the actual length of substrate-attached polyubiquitin chains from biological samples is established. Using the method, the mean length of substrate-attached polyubiquitin chains was determined and the robustness of ubiquitin chain length regulation in cells is investigated. The following is a summary of findings in this invention: 1. A method for determining ubiquitin chain length was developed and this method was named ‘ubiquitin protection from trypsinization’ (Ub-ProT). 2. Using Ub-ProT, it was determined that the mean length of substrate-attached ubiquitin chains is in the dimer to decamer range. 3. By quantitative proteomics, it was found that the mean lengths of five major types of ubiquitin chains can be divided into two groups. 4. Proteasome-inhibition did not alter the mean length of substrate-attached polyubiquitin chains, indicating that cells have a robust system for regulating ubiquitin chain length.

    METHOD FOR DETERMINING UBIQUITIN CHAIN LENGTH
    5.
    发明申请
    METHOD FOR DETERMINING UBIQUITIN CHAIN LENGTH 有权

    公开(公告)号：US20180017571A1

    公开(公告)日：2018-01-18

    申请号：US15711432

    申请日：2017-09-21

    申请人： Tokyo Metropolitan Institute of Medical Science

    发明人： Yasushi Saeki ,  Hikaru Tsuchiya ,  Ai Kaiho ,  Keiji Tanaka

    IPC分类号： G01N33/68 ,  C07K14/47

    CPC分类号： G01N33/6842 ,  C07K14/4702 ,  G01N33/6803

    摘要： Protein ubiquitylation, an essential post-translational modification, regulates almost every cellular process including protein degradation, protein trafficking, signal transduction, and DNA damage response in eukaryotic cells. The diverse functions of ubiquitylation are thought to be mediated by distinct chain topologies resulting from eight different ubiquitin linkages, chain lengths, and complexities. Currently, ubiquitin linkages are generally thought to be a critical determinant of ubiquitin signaling. However, ubiquitin chain lengths, another key element of ubiquitin signaling, have not been well documented especially in vivo situation during past three decades from the discovery of ubiquitin. The reason of this was simply because no method has been available for determination of ubiquitin chain length in endogenous ubiquitylated substrates. In the present invention, a practical technique for determining the actual length of substrate-attached polyubiquitin chains from biological samples is established. Using the method, the mean length of substrate-attached polyubiquitin chains was determined and the robustness of ubiquitin chain length regulation in cells is investigated. The following is a summary of findings in this invention: 1. A method for determining ubiquitin chain length was developed and this method was named ‘ubiquitin protection from trypsinization’ (Ub-ProT). 2. Using Ub-ProT, it was determined that the mean length of substrate-attached ubiquitin chains is in the dimer to decamer range. 3. By quantitative proteomics, it was found that the mean lengths of five major types of ubiquitin chains can be divided into two groups. 4. Proteasome-inhibition did not alter the mean length of substrate-attached polyubiquitin chains, indicating that cells have a robust system for regulating ubiquitin chain length.

    Method for Determining Ubiqutin Chain Length
    7.
    发明申请
    Method for Determining Ubiqutin Chain Length 有权
    确定Ubiqutin链长度的方法

    公开(公告)号：US20150132779A1

    公开(公告)日：2015-05-14

    申请号：US14533182

    申请日：2014-11-05

    申请人： Tokyo Metropolitan Institute of Medical Science

    发明人： Yasushi Saeki ,  Hikaru Tsuchiya ,  Ai Kaiho ,  Keiji Tanaka

    IPC分类号： G01N33/68 ,  C07K14/00

    CPC分类号： G01N33/6842 ,  C07K14/4702 ,  G01N33/6803

    摘要： Protein ubiquitylation, an essential post-translational modification, regulates almost every cellular process including protein degradation, protein trafficking, signal transduction, and DNA damage response in eukaryotic cells. The diverse functions of ubiquitylation are thought to be mediated by distinct chain topologies resulting from eight different ubiquitin linkages, chain lengths, and complexities. Currently, ubiquitin linkages are generally thought to be a critical determinant of ubiquitin signaling. However, ubiquitin chain lengths, another key element of ubiquitin signaling, have not been well documented especially in vivo situation during past three decades from the discovery of ubiquitin. The reason of this was simply because no method has been available for determination of ubiquitin chain length in endogenous ubiquitylated substrates. In the present invention, a practical technique for determining the actual length of substrate-attached polyubiquitin chains from biological samples is established. Using the method, the mean length of substrate-attached polyubiquitin chains was determined and the robustness of ubiquitin chain length regulation in cells is investigated. The following is a summary of findings in this invention: 1. A method for determining ubiquitin chain length was developed and this method was named ‘ubiquitin protection from trypsinization’ (Ub-ProT). 2. Using Ub-ProT, it was determined that the mean length of substrate-attached ubiquitin chains is in the dimer to decamer range. 3. By quantitative proteomics, it was found that the mean lengths of five major types of ubiquitin chains can be divided into two groups. 4. Proteasome-inhibition did not alter the mean length of substrate-attached polyubiquitin chains, indicating that cells have a robust system for regulating ubiquitin chain length.

    摘要翻译： 蛋白质泛素化是必需的翻译后修饰，几乎可以调节真核细胞中几乎每个细胞过程，包括蛋白质降解，蛋白质转运，信号转导和DNA损伤反应。 泛素化的不同功能被认为是由八种不同的泛素键，链长度和复杂性引起的不同链拓扑进行介导的。 目前，泛素连接通常被认为是泛素信号传导的关键决定因素。 然而，遍在蛋白链长度，泛素信号传导的另一个关键因素，没有得到很好的记录，特别是在发现泛素过去三十年的体内情况。 其原因仅仅是因为没有方法可用于测定内源泛素化底物中的泛素链长度。 在本发明中，建立了用于确定生物样品中底物连接的多聚遍在蛋白链的实际长度的实用技术。 使用该方法，确定了底物连接的多聚遍在蛋白链的平均长度，并研究了细胞中泛素链长度调节的稳健性。 以下是本发明的发现摘要：1.开发了一种测定泛素链长度的方法，该方法被命名为“胰蛋白酶消化的泛素保护”（Ub-ProT）。 2.使用Ub-ProT，确定底物附着的泛素链的平均长度在二聚体到十聚体范围内。 通过定量蛋白质组学，发现五种主要类型的泛素链的平均长度可以分为两组。 蛋白酶体抑制不改变底物连接的多聚遍在蛋白链的平均长度，表明细胞具有稳定的泛素链长度的系统。