Biology subjects

Wang, W. L.

Publications and source records attributed to Wang, W. L..

5 recordsLinked to original sources

bioRxiv2025

Rapaprotin is Activated by an Endopeptidase to Disassemble 26S Proteasome

The 19S regulatory particle (RP) associates with the 20S core particle (CP) to form the 26S proteasome, an evolutionarily conserved holoenzyme that plays key roles in both physiological and pathological processes. Proteasome inhibitors that target the catalytic subunits within the 20S have proven to be valuable research tools and therapeutics for various cancers. Herein we report the discovery of rapaprotin, a 26S proteasome assembly inhibitor from our natural product-inspired hybrid macrocycle rapafucin library. Rapaprotin induces apoptosis in both myeloma and leukemia cell lines. Genome-wide CRISPR-Cas9 screen identified a cytosolic enzyme, prolyl endopeptidase (PREP) that is required for the pro-apoptotic activity of rapaprotin. Further mechanistic studies revealed that rapaprotin acts as a molecular transformer, changing from an inactive cyclic form into an active linear form, rapaprotin-L, upon PREP cleavage, to block 26S proteasome activity. Time-resolved cryogenic electron microscopy (cryo-EM) revealed that rapaprotin-L induces dissociation of the 19S RP from the 26S holoenzyme, which was verified in cells. Furthermore, rapaprotin exhibits a marked synergistic effect with FDA-approved proteasome inhibitors and resensitizes drug-resistant multiple myeloma cells from patients to bortezomib. Taken together, these results suggest that rapaprotin is a new chemical tool to probe the dynamics of the 26S proteasome assembly and a promising anticancer drug lead.

pharmacology and toxicology↗

bioRxiv2020

Deep manifold learning reveals hidden dynamics of proteasome autoregulation

The 26S proteasome regulates proteostasis and myriad cellular processes. Here we reconstitute the hidden dynamics of human 26S proteasome during protein degradation using cryo-EM data-driven deep learning, which identified 64 proteasomal conformers. These conformers revealed substrate-dependent conformational entanglement of two 19S regulatory particles in the doubly capped holocomplexes. The rate-limiting step in single-nucleotide exchange dynamics of the AAA-ATPase motor is ATPase re-engagement with the substrate, while the rate-limiting step in proteasomal degradation is the initiation of substrate unfolding by the AAA-ATPase motor following deubiquitylation. Nine rare conformers delineated novel ubiquitin-binding sites on the RPN2 and 5 subunits, along with a ubiquitylation site on RPN10, which were verified using site-directed mutagenesis and liquid chromatography-tandem mass spectrometry. Opening of the 20S core particle gate was found to upregulate proteolytic activity by allosterically promoting nucleophilic attack on the scissile peptide bond. Our systematic analyses illuminate a grand hierarchical allostery underlying ubiquitin-mediated proteasome autoregulation.

biochemistry↗

bioRxiv2018

Visualizing ATP-dependent substrate-processing dynamics of the human 26S proteasome at near-atomic resolution

The proteasome is an ATP-dependent 2.5-megadalton machine responsible for ubiquitylated protein degradation in all eukaryotic cells. Here we present cryo-EM structures of the substrate-engaged human 26S proteasome in seven conformational states at 2.8-3.6 [A] resolution, captured during polyubiquitylated protein degradation. These structures visualize a continuum of dynamic substrate-proteasome interactions from ubiquitin recognition to processive substrate translocation, during which ATP hydrolysis sequentially navigate through all six ATPase subunits. Three principle modes of coordinated ATP hydrolysis are observed, featuring hydrolytic events in two oppositely positioned ATPases, in two consecutive ATPases, and in one ATPase at a time. They regulate deubiquitylation, translocation initiation and processive unfolding of substrates, respectively. A collective power stroke in the ATPase motor is generated by synchronized ATP binding and ADP release in the substrate-engaging and disengaging ATPases, respectively. It is amplified largely in the substrate-disengaging ATPase, and propagated unidirectionally by coordinated ATP hydrolysis in the third consecutive ATPase.

biochemistry↗

bioRxiv2018

Structural insights into the conformational plasticity of the full-length trimeric HIV-1 envelope glycoprotein precursor

The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer mediates viral entry into cells and is the major target for the host antibody response. In infected cells, the mature Env [(gp120/gp41)3] is produced by cleavage of a trimeric gp160 precursor. Proteolytic cleavage decreases Env conformational flexibility, allowing the mature Env to resist antibody binding to conserved elements. The conformational plasticity of the Env precursor skews the humoral immune response towards the elicitation of ineffectual antibodies, contributing to HIV-1 persistence in the infected host. The structural basis for the plasticity of the Env precursor remains elusive. Here we use cryo-electron microscopy to visualize two coexisting conformational states of the full-length Env precursor at nominal resolutions of 5.5 and 8.0 [A]. The State-P2 conformation features a three-helix bundle of the gp41 heptad repeat region in the core, but has disordered membrane-interactive regions. State-P1 trimers lack the three-helix bundle and instead retain ordered transmembrane and membrane-proximal external regions embracing a central cavity. Our structural data shed light on the unusual plasticity of the Env precursor and provide new clues to Env immunogen discovery.

microbiology↗

bioRxiv2017

Nucleotide-Driven Triple-State Remodeling Of The AAA-ATPase Channel In The Activated Human 26S Proteasome

The proteasome is a sophisticated ATP-dependent molecular machine responsible for protein degradation in all eukaryotic cells. It remains elusive how conformational changes of the AAA-ATPase unfoldase in the regulatory particle (RP) control the gating of substrate-translocation channel to the proteolytic chamber of the core particle (CP). Here we report three alternative states of the ATP-{gamma}S-bound human proteasome, in which the CP gate is asymmetrically open, visualized by cryo-EM at near-atomic resolutions. Only four nucleotides are stably bound to the AAA-ATPase ring in the open-gate states. Concerted nucleotide exchange gives rise to a back-and-forth wobbling motion of the AAA-ATPase channel, coincident with remarkable transitions of their pore loops between the spiral staircase and saddle-shaped circle topologies. Gate opening in the CP is thus controlled with nucleotide-driven remodeling of the AAA-ATPase unfoldase. These findings demonstrate an elegant mechanism of allosteric coordination among sub-machines within the holoenzyme that is crucial for substrate translocation.

biochemistry↗