Use advanced microscopy to understand how cells break down proteins

Proteins are an integral part of all living things. From enzymes that carry out chemical reactions to messengers that transmit signals between cells, much research has been conducted on how and what these proteins are produced and do. In 2004, Aaron Ciechanover, Avram Hershko, and Irwin Rose won the Nobel Prize in Chemistry for a different but equally important mechanical process of proteins: how organisms break down proteins when they complete their work.

Protein degradation is a well-orchestrated process. Proteins are processed with a molecular marker called ubiquitin and then fed into the proteasome, a cell shredder that cuts proteins into small pieces. The process of ubiquitination or labeling proteins with ubiquitin is involved in a wide range of cellular processes, including cell division, DNA repair, and immune responses.

In a new study published in the November 17, 2021 issue of Nature, researchers at the University of Chicago studied the process of protein degradation in depth using advanced electron microscopy. They describe the structure of a key enzyme that helps mediate ubiquitination in yeast, a part of a cellular process called the N-degron pathway, which may determine the degradation rate of 80% of equivalent proteins in humans. Malfunction of the pathway leads to the accumulation of damaged or misfolded proteins, the basis of the aging process, neurodegeneration, and some rare autosomal recessive disorders, so a better understanding of it provides an opportunity to develop treatments.

Assistant Professor Minglei Zhao of the University of Chicago and colleagues are studying an ubiquitin E3 ligase, Ubr1. In baker's yeast, Ubr1 helps initiate the ubiquitination process because it attaches ubiquitin to proteins and elongates ubiquitin into molecular chains known as polymers.

"Before this study, we did not know much about how the structure of ubiquitin polymers is formed," Professor Zhao said. "Now we are beginning to understand how it is first installed on the protein matrix, and then how polymers are formed in a specific connection way. This is a milestone in understanding polyubiquitination at the near-atomic level."

In this study, the team initially used some chemobiological techniques to simulate the steps that attach ubiquitin to proteins. They then used cryo-electron microscopy (cryo-EM), another Nobel Prize winning innovation, to capture the process. Cryo-electron microscopy techniques are the rapid freezing of proteins followed by the generation of images of individual molecules or subcellular structures using high-power electron microscopy. About 10 years ago, breakthroughs in hardware and software created microscopes and detectors that could capture molecular images with higher resolution. In 2017, Jacques Dubochet, Joachim Frank, and Richard Henderson won the Nobel Prize in Chemistry for developing cryo-electron microscopy, which allows researchers to create a snapshot that freezes the "in vivo" activity of biological processes.

Professor Zhao's team used $10 million in investment from the bioscience department of advanced electron microscopy equipment to study ubiquitination in more detail using technology. They were able to describe the structure of several intermediate enzyme complexes involved in the protein pathway, which will help researchers find ways to target proteins with drugs or intervene in the failed protein degradation process.

"Cryo-electron microscopy is exciting because after data processing is complete, a new structure that you have never seen will appear, and now, we can use the knowledge we have learned to reuse enzymes to degrade the proteins we want by introducing small molecules or peptide mixtures."

Randi Warren from Creative Biostructure.