Abstract The mechanism of unconventional protein secretion remains an unresolved issue. Here, we describe an unconventional protein secretion pathway for galectin-3 that is mediated by phase separation and condensation. Using four lysosomal damage models, we observed a rapid, pronounced release of galectin-3 in large, non-exosomal particles. This secretion is driven by glycoprotein-induced galectin-3 phase separation and is independent of pyroptosis and secretory autophagy. During phase separation, the S-face of galectin-3 carbohydrate recognition domain binds glycoproteins that triggers galectin-3 N-terminal tail release and condensation. These condensates then recruit ALG-2 via the exposed N-terminal tail. ALG-2 directs the condensates to the endoplasmic reticulum–late endosome interface. After translocation into late endosomes, galectin-3 condensates are secreted into the extracellular milieu by SNARE-dependent vesicular transport. This mechanism of exporting phase-separated protein condensates may serve as a clean-up response to membrane damage.

Immune checkpoint inhibitors (ICIs), including PD-L1 inhibitors, have been approved by the FDA for the treatment of cancers; however, only a small number of cancer patients benefit from these ICIs. Furthermore, the development of drug resistance to this type of treatment is often inevitable. The mechanisms of resistance to PD-L1 inhibitors can be attributed, in part, to an incomplete understanding of the regulation of PD-L1 protein expression. In this study, we identified the role of the E3 ligase GP78, also known as the Autocrine Motility Factor Receptor (AMFR), in the regulation of PD-L1 protein levels. We show that GP78 physically interacts with PD-L1, which is confirmed by IP and Western blotting and is supported by molecular modelling using AlphaFold2. Our modeling studies predict that the interface amino acids of the Ig1 domain of PD-L1 interact with the RING domain and a β-hairpin preceding the CUE domain of GP78. The crystal structure of the PD-1/PD-L1 complex reveals that the interaction with PD-1 is mediated by the Ig1 domain of PD-L1. Furthermore, proteasomal degradation of PD-L1 has been observed via GP78-mediated K48-linked ubiquitination, indicating a key regulatory role for GP78 in the downregulation of PD-L1. Because GP78 expression is inversely correlated with PD-L1 levels in cancer, these findings may have clinical implications for predicting tumor immune evasion and patient response to PD-1/PD-L1 blockade therapies. Taken together, these findings identify a previously unknown mechanism by which GP78 targets PD-L1 for ubiquitination and subsequent degradation in cancer cells, and suggest that blocking the interaction between PD-L1 and PD-1 by an E3 ligase is a novel strategy to improve immunotherapies for cancer patients.

Since 1976, Surveillance Epidemiology End Results (SEER) began collecting ethnicity data for the National Cancer Institute. The incidence of prostate cancer (PCa) among African American men (AAM) has been 60–70

PDF file, 1740K, Microvascular density in A375SM NT and Gal-3 shRNA tumors

Supplementary Tables 1-2 from Racial Disparity in Breast Cancer and Functional Germ Line Mutation in Galectin-3 (rs4644): A Pilot Study

Supplementary Figure 2 from Phosphoglucose Isomerase/Autocrine Motility Factor Mediates Epithelial and Mesenchymal Phenotype Conversions in Breast Cancer

Supplementary Methods and materials, Figure Legends 1-2 from Phosphoglucose Isomerase/Autocrine Motility Factor Mediates Epithelial and Mesenchymal Phenotype Conversions in Breast Cancer

Supplementary Table 2 from Phosphorylation of Galectin-3 Contributes to Malignant Transformation of Human Epithelial Cells via Modulation of Unique Sets of Genes

Supplementary Figure 1 from Phosphoglucose Isomerase/Autocrine Motility Factor Mediates Epithelial and Mesenchymal Phenotype Conversions in Breast Cancer

Anti-CRD of Gal-3 inhibits osteoclast cell fusion.

Materials and Methods for Tissue microarray, Immunofluorescence, Quantitative Real-Time PCR, Proteomics analysis, Co-immunoprecipitation, Co-culture.

PDF file, 1706K, Expression status of LPA receptors in SB-2, WM2664, and A375SM melanoma cells.

Results of immunohistochemistry in breast and prostate cancer patients with metastasis.

Mouse osteoclast precursor expresses and secretes endogenous Gal-3.

PDF file, 1483K, Transcriptional analysis of autotaxin after silencing galectin-3 by nuclear run-on and dual luciferase assays

<p>PDF file - 500K, Supplementary Figure S1. gp78/AMFR is not responsible for AMF-induced p-AKT.</p>

PDF file, 1717K, qRT-PCR of autotaxin and nfat1 mRNA after silencing nfat1 in A375SM melanoma cells

PDF file - 21K, Supplementary Figure S4. AMF secretion is associated with motility in T47D and MDA-MB-231 breast cancer cells.

Supplementary Figure 1 from Down-Regulation of Phosphoglucose Isomerase/Autocrine Motility Factor Results in Mesenchymal-to-Epithelial Transition of Human Lung Fibrosarcoma Cells