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Cross-link MS analysis of human 26S proteasome in complex with RPN13:UCHL5 (with substrate).

ABSTRACT: This project investigates how the human 26S proteasome recognizes and processes proteins tagged with K11/K48-branched ubiquitin chains, which signal for rapid degradation during cell cycle progression and proteotoxic stress. Using cryo-electron microscopy (cryo-EM), the study uncovers a novel K11-linked ubiquitin binding site at the interface of RPN2 and RPN10, alongside known K48-specific sites. Structural insights reveal how these branched ubiquitin chains are preferentially recognized, enhancing proteasomal degradation efficiency. Complementary cross-linking mass spectrometry (XL-MS) identifies transient interactions between RPN13, RPN2, RPN10, and core proteasome subunits, further elucidating the dynamic coordination within the proteasome during substrate processing.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Piotr Draczkowski

LAB HEAD: Shang-Te Danny Hsu

PROVIDER: PXD064932 | Pride | 2025-09-22

REPOSITORIES: Pride

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			Action	DRS
	p26splussubstrate_MS2_MS3.raw	Raw
	p26splussubstrate_MS2_MS3_FAIMS405570charge3.raw	Raw
	p26splussubstrate_MS2_MS3_FAIMS405570charge3gs.raw	Raw
	p26splussubstrate_MS2_MS3_FAIMS405570chrage4.raw	Raw
	p26splussubstrate_MS2_MS3_FAIMS506075.raw	Raw

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Cross-link MS analysis of human 26S proteasome in complex with RPN13:UCHL5 (without substrate).

Project description:This project investigates how the human 26S proteasome recognizes and processes proteins tagged with K11/K48-branched ubiquitin chains, which signal for rapid degradation during cell cycle progression and proteotoxic stress. Using cryo-electron microscopy (cryo-EM), the study uncovers a novel K11-linked ubiquitin binding site at the interface of RPN2 and RPN10, alongside known K48-specific sites. Structural insights reveal how these branched ubiquitin chains are preferentially recognized, enhancing proteasomal degradation efficiency. Complementary cross-linking mass spectrometry (XL-MS) identifies transient interactions between RPN13, RPN2, RPN10, and core proteasome subunits, further elucidating the dynamic coordination within the proteasome during substrate processing.

2025-09-22 | PXD064931 | Pride

Enabling Photoactivated Cross-linking Mass Spectrometric Analysis of Protein Complexes by Novel MS-cleavable Cross-linkers

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26S proteasome is conserved, therapeutically relevant mediator of mutant p53 gain-of-function in cancer

Project description:Mutant p53 proteins, resulting form frequent TP53 tumor suppressor missense mutations, possess gain-of-function activities and are among the most widespread and robust oncoproteins in human tumors. They are potentially important but understudied therapeutic targets. No studies to date have distinguished common, therapeutically relevant mutant p53 gain-of-function effects, from effects specific to different mutant variants and cell backgrounds. Here we identify 26S proteasome machinery as the common downstream effector controlled by mutant p53s in Triple Negative Breast Cancer (TNBC - aggressive carcinomas with TP53 as the most frequently mutated locus) and conserved in other human cancers. We have identified this pathway using a combination of single-model, multi-method vertical analysis (whole cell proteome, RNA sequencing an ChIP sequencing) and multi-cell line, horizontal analysis of transcriptiomes. We found that different missense mutant p53s regardless of the cell background transcriptionaly activate whole 26S proteasome machinery. Proteasome activity is significantly increased in p53 mutant versus wild-type or knockdown/null status - in cellular and mouse models as well as in human breast tumors. Increased proteasome activity leads to inhibition of tumor suppressive pathways. The control of mutant p53 over proteasome transcription and activity results in the increased resistance to proteasome inhibitors. By combining the mutant p53 targeting agents and proteasome inhibitor we were able to overcome the “bounce-back” proteasome inhibitor resistance mechanism in mutant p53 bearing TNBC cells and xenografts in vivo.

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Effect of TRIP12, UBR5, or HOIP in gene expression

Project description:The protein ubiquitylation is under the equilibrium between ubiquitin conjugation and deconjugation. How substrates stabilized by deubiquitylation are directed for degradation remains unclear. Branched ubiquitin chains promote substrate degradation through the proteasome, but the underlying mechanisms are not fully understood. TRIP12 and UBR5 are HECT-type E3s specific for the K29 and K48 linkages, respectively. Here, we show that the deubiquitylase (DUB) OTUD5 is cooperatively modified by TRIP12 and UBR5, resulting in the conjugation of K29/K48 branched ubiquitin chains and accelerated proteasomal degradation. The TRIP12–OTUD5 antagonism regulates TNF-–induced NF-B signaling. Mechanistically, although OTUD5 readily cleaves K48 linkages, K29 linkages are resistant against OTUD5 activity. Consequently, K29 linkages overcome OTUD5 DUB activity to facilitate UBR5-dependent K48-linked chain branching. This mechanism is applicable to other TRIP12 substrates associated with OTUD5. These results reveal a unique cellular strategy in which the combination of DUB-resistant and proteasome-targeting ubiquitin linkages efficiently promotes the degradation of substrates protected by deubiquitylation, underscoring the role of branched ubiquitin chains in shifting the ubiquitin conjugation/deconjugation equilibrium.

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A combinatorial ubiquitin code degrades substrates protected by deubiquitylation

Project description:Reversibility of protein ubiquitylation play essential roles in cellular protein homeostasis. How substrates stabilized by deubiquitylation are directed for degradation remains largely elusive. Here, we show that the branched ubiquitin chains promote the degradation of the deubiquitylase (DUB) OTUD5. OTUD5 is sequentially modified by TRIP12 and UBR5, E3s specific for the K29 and K48 linkages, respectively, resulting in the conjugation of K29/K48 branched ubiquitin chains. The TRIP12-OTUD5 antagonism regulates TNF--induced NF-B signaling. Mechanistically, while OTUD5 readily cleaves K48-linkages, K29-linkages are resistant against OTUD5 activity. Consequently, K29-linkages overcome OTUD5 DUB activity to facilitate UBR5-dependent K48-linked chain branching. Regarding generality, this mechanism adopts to other TRIP12 substrates associated with OTUD5. These results uncover a cellular unique strategy in which the sequential addition of DUB-resistant and proteasome-targeting ubiquitin linkages efficiently promote degradation of substrates protected by deubiquitylation, underscoring the role of branched ubiquitin chains in the quality control of hard-to-degrade substrates.

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Project description:Proteins play a central role in most biological processes within the cell and deciphering how they interact is key to understand their function. Cross-linking coupled to mass spectrometry is an essential tool for elucidating protein-protein interactions. Despite its importance, we still know surprisingly little about the principles that underlie the process of chemical cross-link formation itself and how it is influenced by different physicochemical factors. To understand the molecular details of cross-link formation, we have set-up a comprehensive kinetic model and carried out simulations of protein cross-linking on large protein complexes. We dissect the contribution on the cross-link yield of parameters such as amino acid reactivity, cross-linker concentration, and hydrolysis rate. Our model can compute cross-link formation based solely on the structure of a protein complex, thereby enabling realistic predictions for a diverse set of systems. We quantitatively show how cross-links and mono-links are in direct competition and how the hydrolysis rate and abundance of cross-linker and proteins directly influence their relative formation. We show how cross-links and mono-links exist in a “all-against-all” competition due to their simultaneous formation, resulting in a non-intuitive network of interdependence. We show that this interdependence is locally confined and mainly limited to direct neighbors or residues in direct vicinity. These results enable us to identify the optimal cross-linker concentration at which the maximal number of cross-links are formed. Taken together, our study establishes a comprehensive kinetic model to quantitatively describe cross-link formation for protein-protein interactions.

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