Final Report Summary - UBIQUITIN CHAINS (Mechanisms of poly-ubiquitin chain assembly by Ubc7, a ubiquitin conjugating enzyme of the protein quality control system)
The focus of the Marie Curie grant proposal was on the mechanism of the ubiquitylation of misfolded proteins by the ubiquitin-proteasome system. In particular, we wanted to follow up our previous observations that the yeast E2 conjugating enzyme Ubc7 undergoes self-ubiquitylation on a cysteine residue at the active site, and test if the cysteine-linked polyubiquitin chain is an intermediate step towards substrate ubiquitylation (preassembly model). Our objectives were designed to provide a comprehensive view of this subject and of related topics concerning Ubc7 degradation and ubiquitin-chain formation.
Initially, we wanted to examine if Ubc7 is polyubiquitylated on its active-site cysteine in vivo, at its natural site of activity- the endoplasmic reticulum (ER). The data presented demonstrate that indeed Ubc7 is ubiquitylated on a cysteine residue under physiological conditions, while still tethered to the ER membrane. We did not used proteasome inhibitors for the ubiquitylation assay, suggesting that Ubc7 that was polyubiquitylated was not a degradation substrate. Importantly, during this study, we observed that Ubc7 also undergoes self ubiquitylation on a lysine residue (Lys118). This observation led to the discovery of a new regulatory mode of Ubc7 that distinguishes the two protein quality control degradation pathways in the ER - the Hrd1 and the Doa10 pathways. During these studies, we developed a new tool for monitoring the kinetics of protein degradation in vivo by using a metabolic marker and calculating the cell growth rate. A degradation signal was attached to a metabolic marker leading to degradation. Stabilisation of the fusion protein led to enhanced growth in minimal growth media. This simple and convenient method is now widely used in our lab for studying protein degradation. We now continue studying the consequences of this novel modification and its regulatory mode at the molecular level.
To study the ubiquitylation of protein quality control substrates, we wanted to establish a model substrate in which the ubiquitin modification will be easily detected. We have chosen to focus on a nuclear quality control substrate of Ubc7, termed Ndc10. In a series of truncation / mutation studies we isolated the Ndc10 degradation signal and identified its mechanism of misfolding. Attaching the degradation signal (100 aa of Ndc10 C terminal region) to the otherwise stable ER-embedded protein Vma12 destabilised it, suggesting that the signal is autonomous. By using yeast strains in which the proteasome activity is partially impaired we were able to monitor polyubiquitin conjugates attached to the substrate. The focus of our current research is on the ubiquitin-transfer step. Our preliminary observations indicate that a complex enzymatic reaction that involved an additional E2 enzyme, termed Ubc6, is responsible for polyubiquitin attachment onto the Vma12-Ndc10 model substrate. How Ubc6 and Ubc7 work together to perform the ubiquitylation reaction is currently under investigation.
To provide a comprehensive view of the interface between Ubc7, and its co-factor Cue1, and to identify essential elements within the E2 enzyme that partake in substrate ubiquitylation, we isolated Ubc7 expressed in bacteria, together with Cue1, and purified them to homogeneity, using sizing and charge-based columns. As determined by in vitro ubiquitylation assays the purified enzyme was active, however, in spite of multiple efforts, we were not able to determine the crystal structure of the Ubc7-Cue1 complex. Yet, bacterially purified enzymes served as useful tools for mutagenesis studies that reveal once more the important role of lysine 118 for Ubc7 activity.
Overall, our findings provide the foundation for a comprehensive research of the enzymatic steps leading to protein quality control substrate ubiquitylation at the ER. These findings can provide new means for studying the enzymatic steps preceding protein quality control substrate degradation in higher eukaryotes, thus, to contribute to the ongoing effort to identify novel enzymatic steps for targeting the ubiquitin-proteasome system in the battle of multiple diseases such as neurodegeneration and cancer.
Initially, we wanted to examine if Ubc7 is polyubiquitylated on its active-site cysteine in vivo, at its natural site of activity- the endoplasmic reticulum (ER). The data presented demonstrate that indeed Ubc7 is ubiquitylated on a cysteine residue under physiological conditions, while still tethered to the ER membrane. We did not used proteasome inhibitors for the ubiquitylation assay, suggesting that Ubc7 that was polyubiquitylated was not a degradation substrate. Importantly, during this study, we observed that Ubc7 also undergoes self ubiquitylation on a lysine residue (Lys118). This observation led to the discovery of a new regulatory mode of Ubc7 that distinguishes the two protein quality control degradation pathways in the ER - the Hrd1 and the Doa10 pathways. During these studies, we developed a new tool for monitoring the kinetics of protein degradation in vivo by using a metabolic marker and calculating the cell growth rate. A degradation signal was attached to a metabolic marker leading to degradation. Stabilisation of the fusion protein led to enhanced growth in minimal growth media. This simple and convenient method is now widely used in our lab for studying protein degradation. We now continue studying the consequences of this novel modification and its regulatory mode at the molecular level.
To study the ubiquitylation of protein quality control substrates, we wanted to establish a model substrate in which the ubiquitin modification will be easily detected. We have chosen to focus on a nuclear quality control substrate of Ubc7, termed Ndc10. In a series of truncation / mutation studies we isolated the Ndc10 degradation signal and identified its mechanism of misfolding. Attaching the degradation signal (100 aa of Ndc10 C terminal region) to the otherwise stable ER-embedded protein Vma12 destabilised it, suggesting that the signal is autonomous. By using yeast strains in which the proteasome activity is partially impaired we were able to monitor polyubiquitin conjugates attached to the substrate. The focus of our current research is on the ubiquitin-transfer step. Our preliminary observations indicate that a complex enzymatic reaction that involved an additional E2 enzyme, termed Ubc6, is responsible for polyubiquitin attachment onto the Vma12-Ndc10 model substrate. How Ubc6 and Ubc7 work together to perform the ubiquitylation reaction is currently under investigation.
To provide a comprehensive view of the interface between Ubc7, and its co-factor Cue1, and to identify essential elements within the E2 enzyme that partake in substrate ubiquitylation, we isolated Ubc7 expressed in bacteria, together with Cue1, and purified them to homogeneity, using sizing and charge-based columns. As determined by in vitro ubiquitylation assays the purified enzyme was active, however, in spite of multiple efforts, we were not able to determine the crystal structure of the Ubc7-Cue1 complex. Yet, bacterially purified enzymes served as useful tools for mutagenesis studies that reveal once more the important role of lysine 118 for Ubc7 activity.
Overall, our findings provide the foundation for a comprehensive research of the enzymatic steps leading to protein quality control substrate ubiquitylation at the ER. These findings can provide new means for studying the enzymatic steps preceding protein quality control substrate degradation in higher eukaryotes, thus, to contribute to the ongoing effort to identify novel enzymatic steps for targeting the ubiquitin-proteasome system in the battle of multiple diseases such as neurodegeneration and cancer.
