Research opens new possibilities for overcoming protein-induced neurodegenerative disorders and cancer
Professor Hyun Kyu Song’s research team discovers the mechanisms behind cell degradation via a p62/SQSTM1 autophagy adapter
Results published in Nature Communications
▲ Professor Hyun Kyu Song, Division of Life Sciences, College of Life Sciences
A KU research team has uncovered the mechanisms behind the degradation of cells in the human body and homeostasis.
The team, led by Professor Hyun Kyu Song from the Division of Life Sciences at Korea University, discovered the mechanism behind autophagy-based protein degradation at the molecular level.
In addition to the ubiquitin-proteasome system, autophagy is an essential degradation mechanism that removes harmful proteins from cells for homeostasis. This process is closely associated with aging, genetic variation, and cellular stress. Various stressors create harmful protein aggregates, and these cellular proteins are degraded via autophagy and the ubiquitin-proteasome system. The autophagic removal of protein aggregates had previously been studied only at the cellular level, with specific mechanisms at the molecular level yet to be examined.
Professor Song’s team succeeded in revealing the 3D structure of p62/SQSTM1, which creates and transports protein aggregates, and the Arg-BiP/GRP78 complex, a key product of cellular stress. The functions of the protein complex were also identified for the first time.
The team prepared mass samples of the p62:Arg-BiP/GRP78 complex, passed them through filtration columns, and analyzed their structure using electron microscopy and X-ray scattering. They obtained the structures of the ZZ-domain, the only p62 domain with previously unknown functions, and its substrate Arg-BiP, confirming that complex formation is affected by changes in pH.
Professor Song first had to employ X-ray scattering to purify recombinant proteins and produce crystals. However, in the case of the ZZ-domain of p62, crystals were hard to obtain because the highly concentrated proteins disappear quickly when purified due to their large size and the presence of small zinc atoms. The team enhanced the stability of the proteins by purifying the ZZ-domain after fusing it with LC3B, which is a protein with outstanding physical properties. The LC3B tag was removed with ATG4B protease to produce specific residues at the amino-terminal; the resulting chimeric protein helped to reveal the complex structure.
Professor Song said, “This study is the first to examine the structure of the p62 ZZ-domain and Arg-BiP/GRP78 in high resolution. It related changes in the pH of the p62 protein to protein activity. The results have enhanced our understanding of the degradation of proteins through autophagy, and will present new directions for research on protein-induced diseases such as neurodegenerative disorders and cancer.”
The study was funded by the Mid-Career Strategic Research Project at the National Research Foundation, and the results were published in the international journal Nature Communications.
* Title of Paper
Insights into degradation mechanism of N-end rule substrates by p62/SQSTM1 autophagy adaptor
Professor Hyun Kyu Song (corresponding author, Korea University), Do Hoon Kwon (first author, Korea University), Ok Hyun Park (Korea University), Leehyeon Kim (Korea University), Dr. Yang Ouk Jung (Korea University), Yeonkyoung Park (Korea University), Hyeongseop Jeong (KBSI), Dr. Jaekyung Hyun (KBSI), and Professor Yoon Ki Kim (Korea University)
(Figure 1) Structure of the ZZ-domain of p62 and the R-BiP complex
The figure on the left shows the entire structure of the ZZ-domain of p62. The areas colored red represent negatively charged surfaces, which play an important role in recognizing N-degrons. The figure on the right shows the structure of the ZZ-domain in a complex with Arg-BiP. Ionic interactions are observed with the negatively charged surfaced, colored in red on the left.
(Figure 2) Key residues in the binding of p62 and Arg-BiP
Five key amino acid residues (D129, N132, R139, D147, and D149) that play an important role in the binding of the p62 ZZ-domain and Arg-BiP were identified. If these residues are present in a mutated form, Arg-BiP is unable to be broken down properly in the cell.