Proteomics of protein trafficking by in vivo tissue-specific labeling

Abstract

Conventional approaches to identify secreted factors that regulate homeostasis are limited in their abilities to identify the tissues/cells of origin and destination. We established a platform to identify secreted protein trafficking between organs using an engineered biotin ligase (BirA*G3) that biotinylates, promiscuously, proteins in a subcellular compartment of one tissue. Subsequently, biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Applying this approach in Drosophila, we identify 51 muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, including CG2145 (human ortholog ENDOU) that binds directly to muscles and promotes activity. In addition, in mice, we identify 291 serum proteins secreted from conditional BirA*G3 embryo stem cell-derived teratomas, including low-abundance proteins with hormonal properties. Our findings indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease.

Spatiotemporally-resolved mapping of RNA binding proteins via functional proximity labeling reveals a mitochondrial mRNA anchor promoting stress recovery

“…Addition of a small molecule substrate to live cells results in catalytic generation of a reactive biotin species (such as biotin-phenoxyl radical for APEX or biotin-AMP for TurboID) from the active site of the promiscuous enzyme, which diffuses outward to covalently tag proximal endogenous proteins. PL methods have been used to map organelle proteomes (mitochondrion, ER, lipid droplets, stress granules) 6 , dynamic interactomes 7 , 8 , and in vivo secretomes 9 , 10 . Recently, PL was extended to spatial mapping of transcriptomes in living cells 11 – 13 .…”

Dynamic tracking and identification of tissue-specific secretory proteins in the circulation of live mice

“…Liu et al 24 generated ER-BioID mice and characterized muscle-derived serum protein changes with exercise training. Droujinine et al 25 showed protein trafficking by tissue-specific labeling in Drosophila using BirA*G3 and identified serum proteins secreted from mouse teratomas. All these studies used the KDEL signal peptide to position TurboID, BioID, or BirA*G3 to the ER lumen whereas our study utilizes the membrane-anchored Sec61b-TurboID which is not subject to secretion.…”

Horizontal Transfer of Histone H3 by Mammalian Cells

“…In hopes of identifying new secreted biomarkers controlled by the VHL tumor suppressor gene, we introduced the BirA biotin ligase fused to an ER localization signal (ER-BirA 7,8 ), or the corresponding empty expression vector (EV), into 786-O human VHL -/- clear cell renal carcinoma cells (ccRCCs) that reexpress the VHL in the presence of Doxycycline (Dox) (Fig. 1a,b and Extended Data Fig.…”

“…Here we show that histone H3 is horizontally transferred between viable cells. Using an ER-targeted biotin ligase to detect secreted proteins 7,8 , we serendipitously discovered that histone H3 is selectively secreted by autophagic cells relative to histones H2A, H2B, and H4. Specific H3 posttranslational modifications are enriched or depleted on secreted H3 relative to intracellular H3, suggesting that specific PTMs dictate H3’s ability to be secreted or the information it conveys to neighboring cells.…”