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    Home > Biochemistry News > Biotechnology News > The team of Professor Mao Youdong from the School of Physics has made a breakthrough in the reconstruction of protein dynamics regulation by artificial intelligence-assisted time-resolved cryo-EM

    The team of Professor Mao Youdong from the School of Physics has made a breakthrough in the reconstruction of protein dynamics regulation by artificial intelligence-assisted time-resolved cryo-EM

    • Last Update: 2022-05-13
    • Source: Internet
    • Author: User
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    The team of Professor Mao Youdong, Institute of Condensed Matter Physics and Materials Physics, School of Physics, Peking University, State Key Laboratory of Artificial Microstructure and Mesoscopic Physics, National Center for Biomedical Imaging Science, Peking University-Tsinghua Life Science Joint Center, and Center for Quantitative Biology The self-developed deep learning high-precision four-dimensional reconstruction technology, and the development and application of time-resolved cryo-electron microscopy have clarified the kinetic regulation and conformational reprogramming mechanism of the human proteasome at the atomic level
    .
    On April 27, 2022, the related research results are titled "Time-resolved cryo-electron microscopy analysis of the allostery of the human proteasome under the regulation of USP14" ( "USP14-regulated allostery of the human proteasome by time-resolved cryo-EM" ), Published online in the top international academic journal Nature .

    At the same time, the researchers, reviewers and the Nature editorial team jointly published a presentation titled "Control of human protein-degradation machinery revealed" in the Research Briefing column.
    In the article, the reviewers commented that "this work is a major study that finally solves the problem of USP14 activation and the mechanism of its regulation of proteasome function at the atomic level"; the Nature editorial team pointed out that "this work uses time-resolved cryo-electron microscopy, combined with Functional analysis .
    .
    .
    presents a conformational continuum of USP14 and the proteasome during protein degradation"
    .
    This is the first time that artificial intelligence four-dimensional reconstruction technology has been applied to greatly improve the analysis accuracy of time-resolved cryo-EM, aiming at major disease target protein complexes to achieve the international leading original achievement of atomic-level functional dynamics observation, demonstrating a new type of protein complex dynamics Research paradigms
    .

    Regulation of protein degradation is an extremely important fundamental biochemical process that plays a key role in major cellular and molecular processes such as cell cycle, signal transduction, immune response, gene regulation, metabolism, neurodegeneration, cancer tumors, viral infection, and protein toxicity responses.
    regulation
    .
    In eukaryotic cells, most intracellular proteins are degraded by the proteasome holoenzyme through the ubiquitin-proteasome pathway
    .
    In 2004, Aaron Ciechanover, Irwin Rose and Avram Hershko were awarded the Nobel Prize in Chemistry for "historic discovery of the ubiquitination pathway mediating protein degradation"
    .
    The proteasome holoenzyme, also known as the 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 heterohexameric motor, AAA-ATPase, that regulates proteasomal degradation of ubiquitinated substrates through multiple coordinated ATP hydrolysis modes
    .
    Proteasome dysfunction is associated with a variety of human diseases, such as cancer, neurodegenerative diseases, and immune diseases
    .
    The proteasome is the direct target of several marketed small-molecule drugs approved by the U.
    S.
    Food and Drug Administration (FDA) for the treatment of cancer
    .
    In normal cells, the function of the proteasome is tightly regulated at multiple levels
    .
    The deubiquitinating enzyme USP14 is the most important proteasome regulatory molecule and is considered to be an important target for the treatment of cancer and neurodegenerative diseases with great potential.
    A series of unresolved key questions about the functional mechanism greatly limit the development and clinical application of their targeted drug molecules
    .
    USP14 is activated by binding to 26S, and then cleaves the ubiquitin chain on the substrate on a millisecond time scale; but how it is activated by the proteasome and regulates proteasome function has always been the key to global research institutions and the biopharmaceutical field.
    scientific question
    .

    Molecular machines of life achieve their special functions through highly complex non-equilibrium kinetic processes and structural changes, which are then precisely regulated by various complex intermolecular interactions
    .
    How to directly observe the functional state dynamics of natural supermolecular machines at the atomic level presents an unprecedented challenge to the existing dynamic analysis techniques of atomic structures
    .

    Professor Mao Youdong's team has long been committed to the development of kinetic reconstruction methods based on cryo-electron microscopy, focusing on the structure, function, kinetic mechanism and targeted regulatory molecule design of target systems with great clinical application prospects such as proteasome and inflammasome.
    Frontier cross-study: In 2016, the 3.
    6 ? cryo-EM structure of the ground state of the human proteasome and three other sub-nanometer-resolved conformations were reported in the Proceedings of the National Academy of Sciences ( PNAS ), and a metastable conformation was found for the first time The core particle transport channel is open ( PNAS 2016; 113: 12991-12996); in 2017, the three-dimensional structure of the high-resolution proteasome 19S regulatory complex in the free state of the binding assembly partner p28 was analyzed by cryo-electron microscopy, explaining the assembly partner Assembly mechanism of conformational selection of protein Gankyrin/p28 during proteasome assembly ( Molecular Cell 2017; 67: 322-333); In April 2018, 6 ATPγS binding states were reported in Nature Communications The dynamic structure of the 26S proteasome, including the metastable degenerate near-atomic-resolved (4~5 ?) structure corresponding to the open state of the three core particle complexes ( Nature Communications 2018, 9: 1360); November 2018, in "Nature" ( Nature ) reported for the first time the high-resolution (2.
    8~3.
    6?) structures of seven intermediate state conformations of human proteasome 26S in the process of degrading substrates, showing the interaction between proteasome and substrate at the atomic level For the first time, the atomic-level observation of the complete process of the whole cycle of ATP hydrolysis in the AAA-ATPase hexameric motor has been realized ( Nature 2019; 565: 49-55)
    .
    This series of work revealed the atomic architecture of the proteasome, the principle of assembly and the basic laws of the kinetics of degradation of ubiquitinated substrates
    .

    (A) One of the atomic structural models of the degradation of polyubiquitinated substrates by the proteasome complex under the regulation of USP14; (B) Time-resolution cryo-electron microscopy analysis of the temporal evolution of the statistical distribution of 13 intermediate states with the process of protein degradation (Youdong Mao , CC BY 4.
    0)

    The process of scientific research is always difficult and tortuous.
    The first difficulty to be overcome is "time resolution": the process of proteasome degradation of substrates is very fast, and the time scale is between milliseconds and seconds.
    Under normal conditions, it is desirable to capture by cryo-electron microscopy.
    The intermediate state structure of this process is very difficult, so the first thing to do is to slow the process down
    .
    Through a lot of exploration of conditions, the research team reconstructed the reaction kinetic system and optimized the reaction conditions, including optimizing the buffer system, reaction temperature and other conditions, and optimized a more feasible experimental plan, which made the application of time-resolved cryo-EM technology possible.
    Cryo-EM transmission images of 45,193 USP14-26S complexes during the degradation of ubiquitin substrates were collected, and 3,556,806 particle images of USP14-26S-ubiquitin substrate complexes were picked
    .

    The next extreme challenge is "three-dimensional classification.
    " The complex images captured by cryo-EM need to undergo a series of classifications and classify them into different conformational categories in order to show the dynamic process of protein reactions
    .
    After USP14 binds to the 26S proteasome, the kinetic process of substrate degradation is more complicated.
    In order to identify the high-resolution non-equilibrium conformation of each time period of the degradation process in so many heterogeneous complex particle images, traditional The three-dimensional classification method is not possible
    .
    Low-accuracy 3D classification will lead to low-resolution 3D reconstructions, so that atomic-level kinetic information cannot be obtained and self-consistent dynamic changes cannot be given physical meaning to time-dependent data
    .
    Combined with a novel deep learning high-precision 3D classification and 4D reconstruction method independently developed over the years, the research team captured high-resolution (3.
    0~ 3.
    6?) Non-equilibrium conformation, the complete kinetic duty cycle of the controlled proteasome was reconstructed by time-resolved cryo-EM analysis, and combined with molecular biological function and gene mutation studies, the atomic structural basis of the reciprocal regulatory activity of USP14 and 26S was elucidated and non-equilibrium kinetic mechanisms
    .

    The study found that the activation of USP14 is dependent on both ubiquitin recognition and the binding of the proteasome RPT1 subunit
    .
    Unexpectedly, USP14 induced a conformational change of the proteasome along two parallel state transition paths simultaneously through an allosteric effect; the research team successfully captured the transient transition from an intermediate state of substrate degradation to an intermediate state of substrate inhibition
    .
    In the substrate degradation pathway, USP14 activation allosterically reprograms the conformational landscape and statistical distribution of the AAA-ATPase motor, and stimulates the opening of the 20S substrate channel, thereby observing ATPase hexamerization during continuous substrate transport.
    Motor asymmetric ATP hydrolysis and a near-complete all-round cycle
    .
    The dynamic interaction of USP14-ATPase enables ATPase motor substrate recognition and 26S's own deubiquitinase RPN11 to catalyze a decoupling effect, and in 26S ubiquitin recognition, substrate initiation translocation and ubiquitin chain recycling Three regulatory checkpoints (dynamic bifurcation points) are introduced in the process
    .
    These findings provide new high-resolution insights into the complete functional cycle of 26S regulated by USP14 and lay an extremely important mechanistic basis for USP14-targeted drug therapy discovery
    .

    Parallel pathway model of USP14-regulated proteasomal substrate degradation obtained by time-resolved cryo-EM analysis (Youdong Mao, CC BY 4.
    0)

    Zhang Shuwen, a postdoctoral fellow of Peking University's "Boya", and Zou Shitao, a 2019 doctoral student at the School of Physics, Peking University, are the co-first authors of the paper, and Mao Youdong is the corresponding author
    .
    All cryo-EM data in the research work were collected on the Peking University Electron Microscopy Laboratory and cryo-EM platform, and most of the data analysis was done on the Peking University high-performance computing platform
    .

    The above research work was supported by the Beijing Natural Science Foundation, the National Natural Science Foundation of China, the National Science Foundation for Distinguished Young Scholars, and the Peking University-Tsinghua Life Science Joint Center
    .

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