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.