Dynamic translocation of Inside-Out proteins to the cell surface underlies cellular adaptation to cancer-induced stress

• 成果类型: Article
• 署名作者: Slezak, Tomasz; O'Leary, Kelly M.; Avella, Tanya Guevara; Musial, Natalia; Li, Jinyang; Andrzejczak, Anna; Scott, Elizabeth F.; Le, Duc Anh; Kossiakoff, Anthony A.
• 署名单位: University of Chicago; Fahrenheit Universities; University of Gdansk; Fahrenheit Universities; Medical University Gdansk; Polish Academy of Sciences; Hirszfeld Institute of Immunology & Experimental Therapy of the Polish Academy of Sciences; University of Chicago
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2529493123
• 发表日期: 2026-03-31
• 页码: e2529493123
• 关键词: Inside-Out proteins; tumor-specific biomarkers; cell surface proteome; cancer immunotherapy; HEAT-SHOCK PROTEINS; MOONLIGHTING PROTEINS; tumor-cells; membrane; GRANULE; translation; expression; generation; proteomics; hsp70

摘要

Inside-Out (I-O) protein display, the noncanonical surface localization of intracellular proteins, represents an underexplored feature of tumor cell biology. Here, we map the molecular landscape and trafficking mechanisms that control the presentation of I-O proteins on cancer cell membranes. Employing APEX2-mediated proximity biotinylation and a custom antibody generation and validation platform, we identified approximately 140 high-confidence I-O proteins, primarily ribosomal, proteasomal, chaperone, and translation factors, notably enriched in protein families associated with stress-response pathways. Validation of 500 antibodies encompassing 40 I-O targets across seven tumor cell lines confirmed selective and robust surface localization, while in vivo imaging in mouse xenografts demonstrated pronounced and tumor-specific antibody accumulation. I-O proteins were absent on peripheral blood mononuclear cells (PBMCs) and in normal tissues, indicating cancer cell selectivity. Functional analyses revealed that I-O protein tethering to the membrane is dependent on heparan sulfate interactions; enzymatic removal of these glycans led to the clearance of I-O proteins from the cell surface. Notably, the removed proteins returned to baseline levels within 6 h, indicating a dynamic balance related to Endoplasmic Reticulum (ER)-Golgi trafficking and cellular stress. Nearly half of these I-O proteins overlapped with known stress granule (SG) components; however, stress elements that promote SG formation do not similarly affect surface display of I-O proteins. Furthermore, I-O proteins are present on standard cancer cell lines under lower stress levels needed to induce SG formation, suggesting parallel yet mechanistically distinct aspects of the stress response. These findings position I-O display as a paradigm in protein trafficking, different from traditional secretion pathways and closely linked to stress response.