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An Integrated Platform for In vivo Imaging, Compositional Profiling, and Interactome Mapping of the Aggregated Proteome in Live Cells

ABSTRACT: Protein misfolding and aberrant aggregation are fundamental to neurodegenerative diseases, but studying these dynamic, heterogeneous complexes within their native cellular environment remains a major challenge. We present an integrated chemical proteomics platform that enables concurrent in situ imaging, compositional profiling, and interactome mapping of the aggregated proteome in live cells. The platform combines a novel aggregate-targeting photosensitizer probe (D6) for photocatalytic proximity labeling with a biocompatible, mass-spectrometry-cleavable crosslinker, trehalose disuccinimidyl ester (TDS). This allows for the covalent capture of transient protein conformations and interactions within aggregates prior to their isolation and analysis. Applying this method to a cellular model of proteostasis stress, we identified 1,165 enriched aggregate constituents and mapped 739 protein-protein interactions (67.8% novel), revealing a densely interconnected network centered on the chaperone HSPA1B (HSP70). Functional validation demonstrated that pharmacological activation of HSP70, the central hub of this network, directly promotes the disassembly and clearance of mutant huntingtin aggregates. Our work provides a comprehensive spatial and functional atlas of the aggregated proteome and establishes a generalizable platform for uncovering the mechanisms and therapeutic targets of protein conformational diseases.

ORGANISM(S): Homo Sapiens

SUBMITTER: Lihua Zhang

PROVIDER: PXD078039 | iProX | Thu Apr 30 00:00:00 BST 2026

REPOSITORIES: iProX

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Project description:Protein aggregation is a hallmark of many neurodegenerative diseases. In order to cope with misfolding and aggregation, cells have evolved an elaborate network of molecular chaperones, composed of different families. But while chaperoning mechanisms for different families are well established, functional and regulatory diversification within chaperone families is still largely a mystery. Here we decided to explore chaperone functional diversity, through the lens of pathological aggregation. We revealed that different naturally-occurring isoforms of DNAJ chaperones showed differential effects on different types of aggregates. We performed a chaperone screen for modulators of two neurodegeneration-related aggregating proteins, the Huntington’s disease-related HTT-polyQ, and the ALS-related mutant FUS (mutFUS). The screen identified known modulators of HTT-polyQ aggregation, confirming the validity of our approach. Surprisingly, modulators of mutFUS aggregation were completely different than those of HTT-polyQ. Interestingly, different naturally-occurring isoforms of DNAJ chaperones had opposing effects on HTT-polyQ vs. mutFUS aggregation. We identified a complex of the full length (FL) DNAJB14 and DNAJB12 isoforms which substantially alleviated mutFUS aggregation, in an HSP70-dependent manner. Their naturally occurring short isoforms were unable to form the complex, nor to interact with HSP70, and lost their ability to reduce mutFUS aggregation. In contrast, the short isoform of DNAJB12 significantly alleviated HTT-polyQ aggregation, while DNAJB12-FL aggravated HTT-polyQ aggregation. Finally, we demonstrated that full-length DNAJB14 ameliorated mutFUS aggregation compared to DNAJB14-short in primary neurons. Together, our data unraveled distinct molecular properties required for aggregation protection in different neurodegenerative diseases, and revealed a new layer of complexity of the chaperone network elicited by naturally occurring J-protein isoforms, highlighting functional diversity among the DNAJ family.

Project description:This model is from the article: Modelling the Role of the Hsp70/Hsp90 System in the Maintenance of Protein Homeostasis Proctor CJ, Lorimer IAJ PLoS ONE2011; 6(7): e22038. doi:10.1371/journal.pone.0022038, Abstract: Neurodegeneration is an age-related disorder which is characterised by the accumulation of aggregated protein and neuronal cell death. There are many different neurodegenerative diseases which are classified according to the specific proteins involved and the regions of the brain which are affected. Despite individual differences, there are common mechanisms at the sub-cellular level leading to loss of protein homeostasis. The two central systems in protein homeostasis are the chaperone system, which promotes correct protein folding, and the cellular proteolytic system, which degrades misfolded or damaged proteins. Since these systems and their interactions are very complex, we use mathematical modelling to aid understanding of the processes involved. The model developed in this study focuses on the role of Hsp70 (IPR00103) and Hsp90 (IPR001404) chaperones in preventing both protein aggregation and cell death. Simulations were performed under three different conditions: no stress; transient stress due to an increase in reactive oxygen species; and high stress due to sustained increases in reactive oxygen species. The model predicts that protein homeostasis can be maintained during short periods of stress. However, under long periods of stress, the chaperone system becomes overwhelmed and the probability of cell death pathways being activated increases. Simulations were also run in which cell death mediated by the JNK (P45983) and p38 (Q16539) pathways was inhibited. The model predicts that inhibiting either or both of these pathways may delay cell death but does not stop the aggregation process and that eventually cells die due to aggregated protein inhibiting proteasomal function. This problem can be overcome if the sequestration of aggregated protein into inclusion bodies is enhanced. This model predicts responses to reactive oxygen species-mediated stress that are consistent with currently available experimental data. The model can be used to assess specific interventions to reduce cell death due to impaired protein homeostasis. Note: Simulations were performed under three different conditions: 1) normal condition (no stress), 2) moderate stress due to an increase in reactive oxygen species (ROS) i.e. ROS levels were increased by a factor of 4 at time=4hours for a period of 1 hour (not 2 hours as mentioned in the figure 5 legend of the reference publication. This is a typo in the paper and is clarified by the author) and 3) high stress due to sustained increase in reactive oxygen species (ROS) (here ROS increases with time). The model that corresponds to the normal condition is submitted as a main model in the BioModels Database. The other two models, that corresponds to the moderate stress conditions and high stress conditions are available in SBML format as supporting files [go to Curation tab]. Supplementary figures S3 (normal condition), S4 (moderate stress condition) and S6 (high stress condition) are reproduced here.

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An Integrated Platform for In vivo Imaging, Compositional Profiling, and Interactome Mapping of the Aggregated Proteome in Live Cells

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