Lu, Y.-N. | bioRxiv Science

bioRxiv2025

Noncanonical amino acid incorporation enables minimally disruptive labeling of stress granule and TDP-43 proteinopathy

We report a minimally disruptive labeling strategy for stress granule protein, G3BP Stress Granule Assembly Factor 1 (G3BP1), and ALS-linked protein, TAR DNA-binding protein 43 (TDP-43), using the fluorescent noncanonical amino acid Anap. By integrating genetic code expansion (GCE) with rational site selection, we achieved precise incorporation of Anap that preserves protein structure and function. In live cells and neurons, Anap labeling faithfully recapitulated localization, stress-induced dynamics, and recovery behavior, outperforming conventional fluorescent tags and enabling physiologically relevant visualization of protein pathobiology.

cell biology↗

bioRxiv2024

Ubiquitin Ligase ITCH Regulates Life Cycle of SARS-CoV-2 Virus

SARS-CoV-2 infection poses a major threat to public health, and understanding the mechanism of viral replication and virion release would help identify therapeutic targets and effective drugs for combating the virus. Herein, we identified E3 ubiquitin-protein ligase Itchy homolog (ITCH) as a central regulator of SARS-CoV-2 at multiple steps and processes. ITCH enhances the ubiquitination of viral envelope and membrane proteins and mutual interactions of structural proteins, thereby aiding in virion assembly. ITCH-mediated ubiquitination also enhances the interaction of viral proteins to the autophagosome receptor p62, promoting their autophagosome-dependent secretion. Additionally, ITCH disrupts the trafficking of the protease furin and the maturation of cathepsin L, thereby suppressing their activities in cleaving and destabilizing the viral spike protein. Furthermore, ITCH exhibits robust activation during the SARS-CoV-2 replication stage, and SARS-CoV-2 replication is significantly decreased by genetic or pharmacological inhibition of ITCH. These findings provide new insights into the mechanisms of the SARS-CoV-2 life cycle and identify a potential target for developing treatments for the virus-related diseases.

molecular biology↗