High-Confidence Interactome for RNF41 Built on Multiple Orthogonal Assays.

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Ring finger protein 41 (RNF41) functions as an E3 ubiquitin ligase, mediating the ubiquitination and degradation of key proteins such as ErbB3 receptors, BIRC6, and parkin.
• RNF41 influences intracellular trafficking of JAK2-associated cytokine receptors by ubiquitinating and inhibiting USP8, consequently destabilizing the ESCRT-0 complex.

Purpose of the Study:

• To comprehensively identify the protein partners of RNF41 using orthogonal proteomic approaches.
• To establish a high-confidence RNF41 interactome network and functionally validate novel interaction partners.

Main Methods:

• Employed affinity purification-mass spectrometry, BioID, and Virotrap to map the RNF41 protein complex.
• Integrated data with existing microarray MAPPIT and yeast two-hybrid (Y2H) datasets.
• Filtered results to generate a high-confidence interactome by selecting proteins identified through multiple methods.

Main Results:

• Established a comprehensive RNF41 interactome network with 175 candidate protein partners.
• Generated a high-confidence interactome map of 19 proteins identified by at least two orthogonal methods.
• Identified AP2S1 as a novel RNF41 interaction partner and validated its role in leptin and LIF receptor signaling.
• Demonstrated that RNF41 stabilizes and influences the cellular localization of AP2S1.

Conclusions:

• The study successfully elucidated the RNF41 interactome, revealing novel protein associations.
• AP2S1 is identified as a key RNF41-interacting protein involved in cytokine receptor signaling.
• RNF41 plays a critical role in regulating AP2S1 stability and subcellular localization, impacting cellular signaling pathways.