Next-generation cell lines for profiling different proteasome forms and their implications in cancer.

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• Proteasomes, crucial for intracellular protein degradation, exist in constitutive, immune, and intermediate forms.
• These proteasome subtypes differ in their catalytic β subunits, impacting cellular processes like metabolism, gene expression, and immune signaling.
• Understanding the specific biological functions of each proteasome subtype is essential but remains incompletely defined.

Purpose of the Study:

• To develop novel experimental systems for dissecting the distinct functions of proteasome subtypes.
• To enable visualization and discrimination between constitutive, intermediate, and immunoproteasomes within cells.
• To provide a platform for assessing the impact of compounds on proteasome activity in cancer models.

Main Methods:

• Generation of cancer cell lines (SW620B8-mCherry and TZM-blB8-mCherry) using CRISPR/Cas9 nickase technology.
• Insertion of fluorescent protein genes (eGFP, PS-CFP2) into genes encoding proteasome subunits (PSMB5, PSMB10) for chimeric subunit expression.
• Utilized microscopy techniques, including multiphoton microscopy, to visualize proteasome localization and behavior in vitro and in vivo.

Main Results:

• Successfully generated cell lines with robust expression and integration of fluorescently tagged proteasome subunits.
• Demonstrated visualization of intermediate and immunoproteasomes in TZM-blB8-mCherryB10-CFP cells and discrimination between constitutive and β5i-proteasomes in SW620B8-mCherryB5-GFP cells.
• Observed proteasome-enriched inclusions in tumors and suggested their potential release from cancer cells.

Conclusions:

• The generated cell lines serve as a novel platform for studying proteasome subtype functions.
• These tools facilitate the investigation of distinct proteasome properties and their roles in cellular processes.
• The developed system allows for the assessment of therapeutic compounds targeting the proteasome repertoire in cancer.