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LcFAX-MS-Powered Biomolecular Condensate Analysis Reveals TRIM28-Mediated G3BP1/2 SUMOylation in Regulating Stress Granule Dynamics

ABSTRACT: Membraneless organelles are formed through liquid-liquid phase separation (LLPS) without the need for lipid membranes. Various membraneless organelles, such as stress granules (SGs), nucleoli, and P-bodies, play critical roles in regulating cellular homeostasis, and proteins undergoing LLPS have been linked to numerous human diseases. However, due to their transient, heterogeneous, and membraneless nature, capturing their states with preserved integrity is challenging. In this study, we introduce a low-concentration formaldehyde crosslinking (lcFAX) method that effectively stabilizes membraneless organelles, including arsenite (AS)-induced G3BP1 SGs and TDP43 formed nuclear bodies. Using lcFAX-MS, we identified hundreds of new SG proteins. Specifically, we demonstrate that TRIM28 SUMOylates G3BP1 at K287 and G3BP2 at K281, respectively, a crucial mechanism for regulating SG dynamics. Furthermore, lcFAX-seq provides insights into the RNA composition of SGs. Altogether, lcFAX is a powerful tool for stabilizing membraneless organelles, facilitating comprehensive proteomic and transcriptomic analyses that reveal new regulatory mechanisms. Notably, lcFAX-MS highlights the critical role of TRIM28-mediated SUMOylation in modulating SG dynamics.

ORGANISM(S): Homo Sapiens

SUBMITTER: Yaoyang Zhang

PROVIDER: PXD059295 | iProX | Fri Dec 27 00:00:00 GMT 2024

REPOSITORIES: iProX

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lcFAX-MS-Powered Biomolecular Condensate Analysis Reveals TRIM28-Mediated G3BP1/2 SUMOylation in Regulating Stress Granule Dynamics

Project description:Stress granules (SGs) are stress-induced membraneless organelles whose dynamics are tightly regulated by protein interactions and modifications. However, whether SUMOylation directly targets SG core proteins G3BP1/2 and which ligase is involved remains unclear. Capturing these key events while preserving SG integrity is challenging due to their transient and membraneless nature. To address this, we introduce a low-concentration formaldehyde crosslinking (lcFAX) method that stabilizes membraneless organelles, including G3BP1 SGs and TDP-43 nuclear bodies, enabling enhanced proteome identification. Importantly, we identify TRIM28 as a previously undefined SG-associated protein and reveal that TRIM28 SUMOylates G3BP1 at K287 and G3BP2 at K281, establishing a critical mechanism regulating SG dynamics that ultimately impacts cellular ROS and apoptosis. lcFAX-Seq also provides insights into the RNA composition of SGs. Altogether, lcFAX-MS uncovers the critical role of TRIM28-mediated SUMOylation in modulating SG dynamics, establishing lcFAX as a powerful tool for analyzing membraneless organelles and the underlying regulatory mechanisms.

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