Signalling functions of polyubiquitin chains and ubiquitin-binding domains

摘要

The ubiquitin signalling system has been shown to regulate many important biologicaludevents, ranging from DNA repair to the immune response. Different polyubiquitinudchains linked by various linkages have been identified in vivo, and can be recognised byudproteins containing ubiquitin-binding domains that act as downstream effectors.udHowever, functions for many of them are not well understood. I have studied theudfunction of K63-linked and linear polyubiquitin chains on a common substrate. Theudother branch of my study was to investigate the role of ubiquitin binding for a noveludubiquitin-interacting protein, SPC25.udK63-linked and linear polyubiquitin chains have a similar topology, but whether theyudconvey a similar signal in vivo remains unclear. I have used the eukaryotic replicationudclamp PCNA, a natural substrate of K63-linked polyubiquitylation, as a model substrateudto directly compare the consequences of modification by different types ofudpolyubiquitin chains. I have shown that K63-polyubiquitylated PCNA is not subject toudproteasomal degradation. In contrast, linear, non-cleavable ubiquitin chains do notudpromote DNA damage tolerance, but function as general degradation signals. I foundudthat a linear tetraubiquitin chain is sufficient to afford proteasomal targeting through theudCdc48-Npl4-Ufd1 complex without further modification.udIn the second part of my thesis, I describe the identification of SPC25, a subunit of theudNdc80 complex, as a novel ubiquitin-binding protein, using tetra-ubiquitin chains asudbaits in a genome-wide two-hybrid screen. I have shown that the C-terminal region ofudSPC25 interacts with ubiquitin in vivo and in vitro. This region does not exhibitudsignificant similarity with any known ubiquitin-binding domains. Further geneticudevidence suggests that this ubiquitin-binding domain contributes to the stability of theudkinetochore complex.