TM184C is a GPCR-like regulator of intercellular exchange and autophagy

Key Points:

• The study utilized extensive distributed computing resources at the University of Miami to process the AlphaFold v.4 protein structure database, involving large-scale data redistribution, decompression, and job submission across computing clusters.
• A comprehensive computational pipeline was developed in Python to identify and analyze 7-transmembrane (7TM) protein folds, including GPCR-like structures, using TM-align and custom algorithms for structural alignment, topology scoring, and filtering.
• The team constructed an annotated catalogue of 7TMP hits by integrating UniProt and InterPro metadata, followed by GPCR-focused classification and sequence clustering, enabling detailed structural and functional analyses.
• Experimental validation involved CRISPR–Cas9 genome editing, lentiviral transduction for stable cell line generation, and multiple biochemical assays such as BRET, immunoprecipitation, and mass spectrometry to study protein expression, interactions, and post-translational modifications.
• Advanced microscopy techniques and custom Python-based image analysis software were employed to quantify protein localization, vesicle transfer, and intercellular connectivity, with rigorous statistical analysis ensuring reproducibility and data robustness.