Project description:APEX2 RNA proximity labeling is a powerful method for determining localized RNAs in vivo. APEX2 RNA proximity labeling was adapted to bacterial cells, using an APEX2 fusion to the core scaffold of BR-bodies, RNase E. As a control, APEX2 was also fused to a variant of RNase E that lacks its C-terminal IDR and is unable to form BR-bodies, RNase E delta CTD. RNA proximity labeling was performed, and we observed a similar pattern of enriched RNAs to density centrifugation isolated BR-bodies (Al-Husini et al. Mol Cell 2020).
Project description:Proximity proteomics of GAL9 upon lysosomal damage by LLOMe or GPN. Proximity labeling was performed in SILAC labelled HEK293T cells stably expressing APEX2-GAL9.
Project description:When exposed to excess fatty acids, specific cell types produce nuclear lipid droplets (nLDs) that associate with promyelocytic leukemia (PML) protein to form Lipid Associated PML Structures (LAPS) that are enriched in lipid biosynthetic enzymes but deficient in canonical proteins associated with PML nuclear bodies (PML NBs). To identify the PML interactome during lipid stress, we employed proximity-dependent biotin identification (BioID) in U2OS cells expressing PMLI and PMLII fused to the ascorbate peroxidase APEX2 and cultured in the absence or presence of oleate to enhance lipid droplet formation. The resulting interactome included proteins enriched under oleate-treated conditions, such mitogen activated protein kinase-activated protein kinase 2 (MK2), ESCRT proteins and the COPII vesicle transport proteins Sec23B, Sec24A and USO1. COPII proteins co-localized with both PML-NBs and LAPS but were selectively enriched in PML-NBs following oleate treatment. The nuclear localization of USO1 was uniquely dependent on PML expression. Thus, the APEX2-PML proximity interactome implicates PML domains in the nuclear function of a non-canonical network of COPII vesicle trafficking proteins.
Project description:APEX2-mediated proximity labeling was performed with 4 endosomal RABs allowed an efficient and comprehensive identification of their protein neighbors. APEX2-mediated proximity labeling was performed in triplicate. Protein enrichments obtained from all RABs were compared to APEX2-nude proximity labeling results. Finally, bioinformatic and biochemical approaches were used to confirm functional validity of RAB protein neighbor’s identification.
Project description:Lipid droplet (LD) function is regulated by a complement of integral and peripheral proteins that associate with the LD phospholipid monolayer. Defining the composition of the LD proteome has remained a challenge due to the presence of contaminating proteins in LD-enriched buoyant fractions. To overcome this limitation, we developed a proximity labeling strategy that exploits LD-targeted APEX2 to biotinylate LD proteins in living cells. Application of this approach to U2OS and Huh7 cells identified the vast majority of previously validated LD proteins, excluded common contaminating proteins, and revealed new LD proteins.
Project description:Proximity labeling (PL) characterizes protein interactome in living cells. However, exogenous H2O2 required for engineered peroxidase, like APEX2, is cytotoxic, while biotin ligases-based PL have poor temporal resolution. Here, we showed that SOPP3, an engineered photosensitizer, could trigger APEX2 mediated PL within seconds under blue light illumination, without exogenous H2O2 or cytotoxicity. This new PL technology paves an avenue to characterize molecular dynamics of key biomedical events with high spatial-temporal resolution, efficiency, and flexible applicability.