▎ WuXi AppTec content team editor
Today, the journal Nature published online the latest paper from a research team from Peking University. The scientific research team led by Professor Mao Youdong used international cutting-edge innovative technology to greatly improve the time resolution analysis accuracy of cryo-EM and reveal the kinetic regulation mechanism of human proteasome.
The research team pointed out that as a living molecular machine that affects the degradation of proteins in cells, proteasomes are drug targets for the treatment of a series of major diseases, so accurate observation of its kinetic regulation and conformational changes is expected to promote a new round of major innovation in drug research and development.
Within eukaryotic cells, including human cells, the ubiquitin-protease system is a major way of directed degradation of proteins. In 2004, the Nobel Prize in Chemistry was awarded to three scientists for their historic discovery of the degradation mechanism of the ubiquitinated protein. In this process, the protein to be degraded is marked with "ubiquitin" as a substrate, which is then recognized by the proteasome; then, the proteasome is like a protein crusher, cutting the substrate into fragments to achieve precise degradation. Proteasome dysfunction is associated with a series of human diseases such as cancer, neurodegenerative diseases, and immune diseases.
Around the proteasome, Professor Mao Youdong's laboratory has gradually revealed its atomic structure, assembly principle and kinetic basic law of degradation of ubiquitinated substrates based on cryo-EM technology in a series of previous work. The proteasome whole enzyme, also known as 26S proteasome, consists of a cylindrical 20S core particle in the middle and one or two 19S regulatory particles covered at both ends. 19S contains a circular heterologous hexamer motor, AAA-ATPase, that regulates the degradation of ubiquitinated substrates by proteasomes through multiple synergistic ATP hydrolysis patterns.
▲Professor Mao Youdong, the corresponding author of the study (Source: Peking University official website)
In normal cells, the function of the proteasome is strictly regulated at multiple levels. The deubiquitinase USP14 is a major regulatory molecule of the proteasome, which removes the ubiquitin chain on the substrate by reversibly binding to the proteasome.
However, this process is extremely fast, and the time scale for proteasome degradation of substrates is between milliseconds and seconds, so it is always a world-class challenge to see how USP14 is activated by the proteasome and regulates proteasome function.
And that's where the breakthrough of this study lies: the conformational continuum of USP14 and proteasomes during protein degradation is presented at the atomic level.
▲One of the atomic structure models of the degradation of multiubiquitinated substrates by the proteasome complex under the regulation of USP14 (A), time-resolution cryo-EM to analyze the temporal evolution of the statistical distribution of 13 intermediate states with the process of protein degradation (B) (Source: Prof. Youdong Mao/CC BY 4.0)
In order to capture the intermediate structure of the process with cryo-EM techniques, the research team first managed to slow down the process. Through extensive conditional exploration, reconstruction of reaction kinetics systems and optimization of reaction conditions, the researchers obtained more than 45,000 cryo-EM transmission patterns containing time-containing USP14-degradation of ubiquitin substrates, and selected more than 3 million particle images of USP14-26S-ubiquitin substrate complexes.
Next, a more critical step is to classify such a large number of images, showing the dynamic process of protein responses. To this end, with the help of artificial intelligence, the research team used a new deep learning high-precision three-dimensional classification and four-dimensional reconstruction method independently developed over several years to capture the high-resolution (3.0-3.6 angstroms) non-equilibrium conformation of 13 different functional intermediate states of the degradation of multi-ubiquitination substrate process of the USP14-26S complex, and reconstructed the complete kinetic working cycle of the controlled proteasome.
"This is the first time in the world that artificial intelligence four-dimensional reconstruction technology has been used to greatly improve the accuracy of time-resolved cryo-EM analysis." The research team emphasized.
▲Parallel path model of USP14 regulating proteasome substrate degradation obtained by time-resolved cryo-EM analysis (Source: Prof. Youdong Mao/CC BY 4.0)
Combining molecular biological function and genetic mutation studies, this work ultimately elucidates the atomic structural basis and non-equilibrium kinetic mechanisms of USP14 and 26S proteasomes mutually regulating activity.
The study found that activation of USP14 relies on both ubiquitin recognition and binding of the proteasome RPT1 subunit. Surprisingly, USP14 induces conformational changes in the proteasome along two parallel state transition pathways simultaneously through allosteric effects. The study successfully captured the transient transition from the intermediate state of substrate degradation to the intermediate state of substrate inhibition, providing a new high-resolution insight into the complete functional cycle of USP14 to regulate the 26S proteasome.
In a review article published by Nature at the same time, the reviewers spoke highly of the study, stating that "this work is a major study that finally solves the problem of the mechanism by which USP14 activates and regulates the function of the proteasome at the atomic level."
"This breakthrough has multiple implications for the discovery of targeted drugs." Professor Mao Youdong pointed out, "In the past, our structure-based drug design was a static 'dead structure', and now we can design drug molecules in a targeted manner for the high-resolution conformations in the dynamic processes in which the target protein actually performs its functions.' Second, by accurately defining the dynamic process of the target protein, we can better predict the mechanism and kinetics of drug molecules, and there are more intermediate conformations that can be selected for targeting, thus greatly expanding the design space of drug molecules and improving the probability of success of molecular design. In addition, compared to the molecular dynamics calculation simulation method, our technology looks at the real dynamics, and there is no upper limit to the molecular weight of the target protein. More importantly, we are looking at 'functional dynamics', which is the target conformation of real drug molecules to be combined, which is difficult to achieve with molecular dynamics calculation simulations in the foreseeable future. ”
Resources:
[2] Control of human protein-degradation machinery revealed. Nature
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