Identification of novel deubiquitinases in stabilizing integrins

Yu, Kaikai (2024): Identification of novel deubiquitinases in stabilizing integrins. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

The extracellular matrix (ECM) serves as a structural framework for tissues and organs. Numerous matrix-adhesion molecules bind to the ECM, initiating a cascade of biochemical and mechanical signals which govern diverse cellular functions such as survival, polarity, proliferation, and differentiation. Integrins, heterodimeric transmembrane proteins with α and β subunits, are key adhesion molecules linking the ECM and the intracellular actin-cytoskeleton and transducing biochemical and mechanical signals. To dynamically modulate interactions between cells and the extracellular matrix (ECM), the spatial and temporal presence of receptors on the cell membrane and signaling transduction, integrins depend on the endosomal trafficking system. Integrins undergo internalization, enter the endosomal system which either recycles them back to the plasma membrane or routes them into the lysosomes for degradation. The ubiquitin system plays a central role in determining the fate of transmembrane proteins by orchestrating the delicate balance between their recycling and degradation. During my PhD thesis, I searched for the players in the ubiquitin system that regulates Itgb1-class integrins. I validated the novel deubiquitinase USP12 (and its paralog USP46) identified in genetic and biochemical screens. First, in collaboration with Prof. Bassermann from the Technical University of Munich, we established a CRISPR/Cas9-based genetic screen using gRNAs targeting human deubiquitinases in human haploid cell line HAP1. Using the surface Itgb1 level as a readout measured by flow cytometry, several deubiquintinases including USP46 was identified as Itgb1 stabilizer. Second, I found that the deubiquitinase complexes consisting of USP12/USP46-WDR48-WDR20 associate in proximity to Itga5 using mouse kidney fibroblasts expressing Itga5-TurboID using the proximity-dependent biotin identification (BioID) assay. Based on these results, I decided to determine how the USP12/USP46-WDR48-WDR20 deubiquitinase complex stabilizes Itgb1 at the cellular and molecular level. Third, I generated CRISPR/Cas9 knockouts of each component of the USP12/USP46-WDR48-WDR20 complex. USP12 and USP46 shared around 90% of protein homology and compensate each other’s function in regulating the surface level of Itgb1, whereas the double KO (dKO) of USP12/46 reduced both surface and total Itgb1 levels. A similar loss-of-function phenotype was observed when the adaptor proteins WDR20 and WDR48 were deleted in cells. Importantly, reconstituting the deleted components revealed that an active ternary USP12/USP46-WDR48-WDR20 complex was required for maintaining Itgb1 levels. The findings were observed in fibroblasts and confirmed in the MDA-MB-231 human breast cancer cell line. I also investigated the mechanism of how the DUB complex regulating integrin stability in cells. I found that the instability of Itgb1 caused by USP12/46 dKO was due to increased lysosomal degradation and reduced recycling, while transcription was unaffected. This regulation occurred independently of SNX17, a key regulator of Itgb1 trafficking. USP12 facilitated the removal of polyubiquitin chains from the cytosolic tail of Itgb1 in cells and in vitro. Consequently, this deubiquitination process impeded the recognition of Itgb1 by the ESCRT (endosomal sorting complex required for transport)-mediated sorting machinery, thereby mitigating degradation. A non-ubiquitinable α5β1 integrin with all cytosolic lysines being mutated to arginine, escaped the regulation of USP12/46 with resistance to ESCRT-mediated degradation. In silico data revealed that breast cancer patients with high USP12/46 expression levels have a poor prognosis and my experimental data also confirmed that loss of USP12/46 not only affected cell adhesion and spreading capacity of fibroblasts, but also impeded cell migration and invasion ability of breast cancer cells. Altogether, my PhD study identified that the USP12/USP46-WDR48-WDR20 deubiquitinase complex is a novel regulator of integrin stability by counteracting the ESCRT-ubiquitin degradation pathway and that this regulation facilitates cancer cell migration and invasion.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: ubiquitination, integrin, DUB, USP12/USP46, ESCRT
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 2. Oktober 2024
• 1. Bericht­erstatter:in: Fässler, Reinhard