The carefully orchestrated process of ubiquitination, or protein degradation, is involved in a wide range of cellular processes, including cell division, DNA repair, and immune responses. By employing cryo-electron microscopy (cryo-EM), researchers have now captured the process in cells. In doing so, a team from the University of Chicago describe the structure of a key enzyme that helps mediate ubiquitination in yeast—part of the N-degron pathway that may be responsible for determining the rate of degradation for up to 80% of equivalent proteins in humans. Malfunctions in this pathway can lead to accumulation of damaged or misfolded proteins, which underlies the aging process, neurodegeneration, and some rare autosomal recessive disorders.
This work is published in a new study in Nature titled, “Structural insights into Ubr1-mediated N-degron polyubiquitination.”
The group studied an E3 ligase called Ubr1. In yeast, Ubr1 helps initiate the ubiquitination process as it attaches ubiquitin to proteins and elongates it into a polymer. But how Ubr1 mediates these processes remains unknown.
“Until this study, we didn’t know that much about how ubiquitin polymers are structurally formed,” said Minglei Zhao, PhD, assistant professor of biochemistry and molecular biology at the University of Chicago. “Now we are starting to get an idea of how it’s first installed onto the protein substrate, and then how the polymers are formed in a linkage-specific manner. This is a milestone in terms of understanding polyubiquitination at a near atomic level.”
In this study, Zhao and his team used some chemical biology techniques to mimic the initial steps of the process for attaching ubiquitin to proteins.
They were able to describe the structure of several intermediate enzyme complexes involved in the pathway, which will help researchers looking for ways to target proteins with drugs or intervene in a malfunctioning protein degradation process. More specifically, they developed strategies to mimic the reaction intermediates of the first and second ubiquitin transfer steps. In doing so, they could determine the cryo-EM structures of Ubr1 in complex with Ubc2, ubiquitin, and two N-degron peptides, representing the initiation and elongation steps of ubiquitination.
“Key structural elements, including a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1, were identified and characterized,” the authors wrote.
“Cryo-EM is exciting because after the data processing is done, a new structure pops out that you’ve never seen before,” Zhao said. “Now we can use what we’ve learned and repurpose the enzymes by introducing small molecules or mixture of peptides to degrade the proteins we want.”