New dimensions of cryo-electron microscopy uncover ‘multiverse’ of cancer targets for enabling drug discovery

Many therapeutic targets escape druggability by mysterious molecular motions that cannot be precisely visualized. This ‘dark-world’ reality is particularly augmented in the ubiquitin-proteasome system. The fate of all cellular proteins is determined by a chemical process known as protein degradation. The ubiquitin-proteasome system is the central ‘executioner’ of proteins to this purpose and regulates nearly all cellular activity. Such a powerful system is built around a highly dynamic molecular machine called the 26S proteasome¹. The proteasome is essential for all living cells, without which the cells cannot work properly and die quickly. This mechanism has been used as a therapy to kill cancer cells by targeting the ubiquitin-proteasome system.

Several US Food and Drug Agency (FDA)-approved proteasome inhibitors, most notably Bortezomib and Carfilzomib, have been used to treat multiple myeloma and mantle-cell lymphoma. Upstream of the proteasome are ubiquitin ligases and deubiquitylating enzymes, which may also be targeted by small-molecule inhibitors or degraders that provide more specificity and precision against different cancers.

Molecular gymnastics prohibit drug discovery

The proteasome functions through extremely complicated gymnastics^1-4. The deubiquitylating enzyme USP14 is activated by the proteasome and controls the proteasome function by removing ubiquitin tags on proteins, but how it does this is a longstanding puzzle. Preliminary USP14 inhibitors have shown preclinical promise in treating neurodegenerative disease by reducing the tau proteins in neuron, as well as exhibited efficacy in treating cancers like multiple myeloma in clinical studies. However, the improvement of USP14 inhibitors to meet the requirement of FDA-approved drugs has been hindered by the lack of knowledge of USP14 regulation of the proteasome activity. Among the greatest complex dynamics, USP14-proteasome association has failed all previous efforts for high-resolution structural visualization and analysis, preventing medicinal chemists from advancing drug discovery and optimization against USP14 for cancer therapy.

Writing in the international scientific journal Nature, a research team led by Professor Youdong Mao at Peking University eventually cracked this highly sought-after, longstanding problem by using time-resolved cryo-electron microscopy (cryo-EM) in conjunction with machine learning-based 4D reconstruction⁵. The team visualized 13 gigantic structures of USP14-proteasome in intermediate states at atomic level and tracked their time-dependent changes over the entire course of protein degradation, an extremely challenging task that has not been similarly accomplished for any other holoenzyme system of this size.

Uncovering ‘dark multiverse’ of cancer targets

The fourth dimension in their cryo-EM reconstruction, which was uniquely enabled by a deep-learning method developed by the Mao laboratory, reveals a surprising picture of ‘multiverse’ or parallel reality of USP14-proteasome gymnastics. Intriguingly, USP14 activation creates two parallel pathways of proteasome activities that effect oppositely. One pathway inhibits substrate degradation, while the other allows it to happen. Whether a substrate targeted to the proteasome will be degraded turns out to hinge on ‘rolling dice’ on the timing of USP14 activation, instead of absolute certainty. This bestows on USP14 the power of rescuing certain proteins from being degraded, while allowing some other proteins to be normally terminated.

‘Revealing how USP14 is activated and how it regulates the proteasome at atomic level expose the vulnerability of USP14 and enable structure-based design of novel inhibitors for therapeutic purpose,’ says Mao. The many intermediate structures now allow drug designers to explore more molecules to intervene USP14 for therapeutic purpose.

‘This is a significant study that finally reveals atomic details of USP14 activation by ubiquitylated substrates and how this is translated into proteasomal activation’, says a peer reviewer of the study⁶. ‘The data offer a conformational continuum of USP14 and the proteasome in the act of protein degradation’, the editorial team of Nature further comments in a Research Briefing article⁶ published online at the same time.

This new study demonstrates a new paradigm in directly visualizing previously inaccessible functional dynamics of complex enzymatic processes in atomic detail that may help in the discovery of new therapeutics that have failed existing approaches. ‘This is a watershed moment’, Mao says. Looking forward, unlocking new dimensions in cryo-EM bodes well for future translational research and development toward a new ultra-resolution imaging modality critical for enabling drug discovery against conventionally undruggable targets of notable dynamics.

References:

1. Mao, Y. Structure, Dynamics and Function of the 26S Proteasome. Subcell Biochem 96, 1-151, https://doi.org/10.1007/978-3-030-58971-4_1 (2021).
2. Chen, S. et al. Structural basis for dynamic regulation of the human 26S proteasome. Proc Natl Acad Sci U S A113, 12991-12996, https://doi.org/10.1073/pnas.1614614113 (2016).
3. Zhu, Y. et al. Structural mechanism for nucleotide-driven remodeling of the AAA-ATPase unfoldase in the activated human 26S proteasome. Nat Commun 9, 1360, https://doi.org/10.1038/s41467-018-03785-w (2018).
4. Dong, Y. et al. Cryo-EM structures and dynamics of substrate-engaged human 26S proteasome. Nature 565, 49-55, https://doi.org/10.1038/s41586-018-0736-4 (2019).
5. Zhang, S. et al. USP14-regualted allostery of the human proteasome by time-resolved cryo-EM. Nature 605, 567-574, https://doi.org/10.1038/s41586-022-04671-8 (2022). 
6. Mao, Y. & Zhang, S. Control of human protein-degradation machinery revealed. Nature https://doi.org/10.1038/d41586-022-01144-w (2022).