Tokyo Metropolitan Institute of Medical Science

Hikaru Tsuchiya

Keiji Tanaka

Yasushi Saeki

Ai Kaiho

Yukiko Yoshida

Arisa Murakami

An object of the present invention is to provide a method for efficiently identifying a polyubiquitinated substrate which is generally not easily identified. The method for identifying a polyubiquitinated substrate includes (1) a step of expressing a trypsin-resistant polyubiquitin chain-binding protein and a ubiquitin ligase in a cell, (2) a step of isolating a complex that contains the trypsin-resistant polyubiquitin chain-binding protein from the cell having undergone the step (1), (3) a step of subjecting the complex isolated by the step (2) to trypsin digestion, and (4) a step of identifying a peptide that has a ubiquitination site from a digested material obtained by the step (3).

Method for identifying polyubiquitinated substrate

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.

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Tokyo, Japan

Hikaru Tsuchiya