Biology subjects

Dong, K. C.

Publications and source records attributed to Dong, K. C..

7 recordsLinked to original sources

bioRxiv2026

Proteasomal surveillance of exposed cysteines

Cysteine residues are critical for both the catalytic activity and structural stability of many proteins, yet their high chemical reactivity threatens proteome integrity. Whether cells have specialized mechanisms to recognize and eliminate proteins with aberrant or exposed cysteines is unknown. By systematically analyzing the stability of thousands of cysteine-containing peptides, here we identify two complementary surveillance mechanisms targeting proteins with exposed cysteine residues. First, a set of E3 ubiquitin ligases, including AMBRA1, TRPC4AP, FBXO3, and FBXO21, detects double cysteines and targets proteins for ubiquitin-dependent proteasomal degradation. Second, the PSMD14/Rpn11 subunit of the 26S proteasome recognizes exposed cysteines in a ubiquitin-independent manner. Both pathways function in quality control to clear misfolded proteins exposing cysteine residues, including zinc finger-containing proteins. Mutational escape from the ubiquitin-independent pathway through dysregulation of ornithine decarboxylase or FOS levels is responsible for two human diseases, Bachmann-Bupp Syndrome and osteoblastoma, respectively. Our findings reveal conserved mechanisms for degrading proteins with exposed cysteines and uncover an alternative mode of substrate recognition by the 26S proteasome.

cell biology↗

bioRxiv2026

Molecular basis of ubiquitin-independent recognition and degradation of ODC/Antizyme by the 26S proteasome

The ubiquitin-proteasome system represents the main pathway for targeted protein degradation in eukaryotic cells. The majority of substrates is recruited for degradation through ubiquitin modifications, and the underlying principles are well established. However, the requirements for ubiquitin-independent substrates are still poorly understood. Here, we reveal the mechanisms for the antizyme-mediated degradation of the yeast ornithine decarboxylase (yODC), the first reported ubiquitin-independent substrate of the 26S proteasome. Using biochemical studies and cryo-EM structure determination, we show how antizyme binding makes the yODC monomer prone for degradation by exposing an interface that is normally buried in the catalytically active ODC dimer. Together with a surface on antizyme, yODC forms a two-part interface that binds the N-terminal coiled coil of two ATPase subunits, Rpt4 and Rpt5, for delivery to the 26S proteasome motor. This positions the N-terminal unstructured region of yODC for insertion into the ATPase channel to initiate degradation, which we found does not depend on a specific sequence. Interestingly, binding of the globular yODC/antizyme complex to the Rpt4/Rpt5 coiled coil allosterically stabilizes a proteasome conformation that facilitates substrate engagement by the ATPase motor and may represent a primed pre-initiation state with a general role in ubiquitin-dependent and -independent degradation.

biochemistry↗

bioRxiv2025

Identification of novel ubiquitin receptors on the 26S proteasome by photo-crosslinking mass spectrometry

The 26S proteasome is the endpoint of the ubiquitin-proteasome system, an essential pathway for maintaining cellular homeostasis through targeted degradation of misfolded, damaged, and obsolete proteins. Substrates labeled with ubiquitin are directed to the 26S proteasome by binding to one or more ubiquitin receptors. However, ubiquitin-dependent degradation occurs even when the canonical receptor sites are mutated, suggesting the presence of additional, unidentified binding sites. Here we created photo-crosslinkable probes for ubiquitin interactions by incorporating the unnatural amino acid p-benzoyl-L-phenylalanine into ubiquitin. We show that these probes can be used to measure apparent affinities for known receptors and to reveal novel ubiquitin-binding sites on the yeast 26S proteasome. Through photo-crosslinking mass-spectrometry experiments we identified a groove on the top of the proteasome, formed by Rpn2, Rpn9, Rpn10, and Rpn12, that serves as an additional ubiquitin-binding interface. Our photo-crosslinkable probes thus serve as versatile tools for the characterization of ubiquitin-protein interactions and the identification of ubiquitin-binding domains.

biochemistry↗

bioRxiv2024

The deubiquitinase Rpn11 functions as an allosteric ubiquitin sensor to promote substrate engagement by the 26S proteasome

The 26S proteasome is the major compartmental protease in eukaryotic cells, responsible for the ATP-dependent turnover of obsolete, damaged, or misfolded proteins that are delivered for degradation through attached ubiquitin modifications. In addition to targeting substrates to the proteasome, ubiquitin was recently shown to promote degradation initiation by directly modulating the conformational switching of the proteasome, yet the underlying mechanisms are unknown. Here, we used biochemical, mutational, and single-molecule FRET-based approaches to show that the proteasomal deubiquitinase Rpn11 functions as an allosteric sensor and facilitates the early steps of degradation. After substrate recruitment to the proteasome, ubiquitin binding to Rpn11 interferes with conformation-specific interactions of the ubiquitin-receptor subunit Rpn10, thereby stabilizing the engagement-competent state of the proteasome and expediting substrate insertion into the ATPase motor for mechanical translocation, unfolding, and Rpn11-mediated deubiquitination. These findings explain how modifications with poly-ubiquitin chains or multiple mono-ubiquitins allosterically promote substrate degradation and allow up to four-fold faster turnover by the proteasome.

biochemistry↗

bioRxiv2024

Nub1 traps unfolded FAT10 for ubiquitin-independent degradation by the 26S proteasome

The ubiquitin-like modifier FAT10 targets hundreds of proteins in the mammalian immune system to the 26S proteasome for degradation. This degradation pathway requires the cofactor Nub1, yet the underlying mechanisms remain unknown. Here, we reconstituted a minimal in vitro system and revealed that Nub1 utilizes FAT10s intrinsic instability to trap its N-terminal ubiquitin-like domain in an unfolded state and deliver it to the 26S proteasome for engagement, allowing the degradation of FAT10-ylated substrates in a ubiquitin- and p97-independent manner. Through hydrogen-deuterium exchange, structural modeling, and site-directed mutagenesis, we identified the formation of a peculiar complex with FAT10 that activates Nub1 for docking to the 26S proteasome, and our cryo-EM studies visualized the highly dynamic Nub1 complex bound to the proteasomal Rpn1 subunit during FAT10 delivery and the early stages of ATP-dependent degradation. These studies thus identified a novel mode of cofactor-mediated, ubiquitin-independent substrate delivery to the 26S proteasome that relies on trapping partially unfolded states for engagement by the proteasomal ATPase motor.

biochemistry↗

bioRxiv2021

Ubiquitin modulates 26S proteasome conformational dynamics and promotes substrate degradation

The 26S proteasome is the major ATP-dependent protease in eukaryotic cells, where it catalyzes the degradation of thousands of proteins for general homeostasis and the control of vital processes. It specifically recognizes appropriate substrates through attached ubiquitin chains and uses its ATPase motor for mechanical unfolding and translocation into a proteolytic chamber. Here, we used single-molecule Forster Resonance Energy Transfer (FRET) measurements to provide unprecedented insights into the mechanisms of selective substrate engagement, ATP-dependent degradation, and the regulation of these processes by ubiquitin chains. Our assays revealed the proteasome conformational dynamics and allowed monitoring individual substrates as they progress through the central channel during degradation. We found that rapid transitions between engagement- and processing-competent conformations of the proteasome control substrate access to the ATPase motor. Ubiquitin-chain binding functions as an allosteric regulator to slow these transitions, stabilize the engagement-competent state, and facilitate degradation initiation. The global conformational transitions cease upon substrate engagement, and except for apparent motor slips when encountering stably folded domains, the proteasome remains in processing-competent states for substrate translocation and unfolding, which is further accelerated by ubiquitin chains. Our studies revealed the dependence of ATP-dependent substrate degradation on the conformational dynamics of the proteasome and its allosteric regulation by ubiquitin chains, which ensure substrate selectivity and prioritization in a crowded cellular environment.

biochemistry↗

bioRxiv2018

Deconvolution of substrate processing by the 26S proteasome reveals a selective kinetic gateway to degradation

The 26S proteasome is the principle macromolecular machine responsible for protein degradation in eukaryotes. However, little is known about the detailed kinetics and coordination of the underlying substrate-processing steps of the proteasome, and their correlation with observed conformational states. Here, we used reconstituted 26S proteasomes with unnatural amino acid-attached fluorophores in a series of FRET and anisotropy-based assays to probe substrate-proteasome interactions, the individual steps of the processing pathway, and the conformational state of the proteasome itself. We develop a complete kinetic picture of proteasomal degradation, which reveals that the engagement steps prior to substrate commitment are fast relative to subsequent deubiquitination, translocation and unfolding. Furthermore, we find that non-ideal substrates are rapidly rejected by the proteasome, which thus employs a kinetic proofreading mechanism to ensure degradation fidelity and substrate prioritization.

biochemistry↗