Dataset Information

A genetic model for in vivo proximity labeling of the mammalian secretome

ABSTRACT: Yang R, Meyer AS, Droujinine I, Udeshi ND, Hu Y, Guo J, McMahon J, Carey DK, Xu C, Qiao F, Sha J, Qin S, Rocco D, Wohlschlegel J, Ting AY, Carr SA, Perrimon N, McMahon AP. 2022. Organ functions are highly specialized and interdependent. Tissue-specific secreted factors play a critical role in organ development, and homeostatic regulation, through serum trafficking and inter-organ communication. Enzyme-catalyzed proximity labeling is a powerful tool to label proteins within a specific cellular compartment. Here, we validated a BirA*G3 mouse strain enabling CRE-dependent promiscuous biotinylation of proteins within the endoplasmic reticulum. When intercrossed to a broadly active Sox2-Cre strain, widespread labeling of proteins was observed within the secretory pathway. Streptavidin affinity purification and peptide mapping by quantitative mass spectrometry (MS) revealed organ-specific secretory profiles and serum trafficking. As expected, secretory proteomes were highly enriched for signal peptide-containing proteins, and revealed conventional and non-conventional secretory processes, and ectodomain shedding. Lower-abundance proteins with hormone-like properties were recovered and validated using orthogonal approaches. Hepatocyte specific activation of BirA*G3 highlighted liver specific biotinylated secretome profiles. The model system will enhance an understanding of secretory protein interplay in development, and healthy and diseased adult states.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Exploris 480

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Steven A. Carr

PROVIDER: MSV000088848 | MassIVE | Wed Feb 16 08:51:00 GMT 2022

REPOSITORIES: MassIVE

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A genetic model for <i>in vivo</i> proximity labelling of the mammalian secretome.

Yang Rui R Meyer Amanda S AS Droujinine Ilia A IA Udeshi Namrata D ND Hu Yanhui Y Guo Jinjin J McMahon Jill A JA Carey Dominique K DK Xu Charles C Fang Qiao Q Sha Jihui J Qin Shishang S Rocco David D Wohlschlegel James J Ting Alice Y AY Carr Steven A SA Perrimon Norbert N McMahon Andrew P AP

Open biology 20220810 8

Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ communication. Enzyme-catalysed proximity labelling enables the identification of proteins within a specific cellular compartment. Here, we report a <i>BirA*G3</i> mouse strain that enables CRE-dependent promiscuous biotinylation of proteins trafficking through the endoplasmic reticulum. When broadly activated throughout the mous ...[more]

PMID: 35946312

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Project description:Droujinine IA, Wang D, Meyer AS, Udeshi ND, Hu Y, Rocco D, McMahon JA, Yang R, Guo J, Mu L, Carey DK, Svinkina T, Zeng R, Branon T, Tabatabai A, Bosch JA, Asara JM, Ting AY, Carr SA, McMahon AP, Perrimon N. 2021 Secreted interorgan communication factors encode key regulators of homeostasis. However, there are long-standing questions surrounding their origins/destinations, mechanisms of interactions, and the number of proteins involved. Progress has been hindered by the lack of methodologies for the large-scale identification and characterization of these factors, as conventional approaches cannot identify low-abundance factors or the origins and destinations of secreted proteins. We established an in vivo proteomic platform to investigate cell-specific secretomes and secreted protein trafficking between organs, whereby the engineered promiscuous biotin ligase BirA*G3 (a relative of TurboID) biotinylates all proteins in a subcellular compartment of one tissue, and biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Using this platform in Drosophila, we identified 51 putative muscle-secreted proteins from heads and 269 fat body (FB)-secreted proteins from legs/muscle organ (here defined as muscle tissue and innervations/neuromuscular junctions (NMJs)), of which 60-70% have human orthologs. Among these were FB signaling proteins that have known receptors and/or were previously demonstrated to bind to muscle organs with specific patterns. We demonstrate, in particular, that a conserved FB-derived novel interorgan communication factor CG2145 (human ortholog: ENDOU) promotes muscle activity and binds directly and with a specific pattern to the muscle organ (near muscles/neurons), but not to other organs. These results suggest that our approach can identify specific interactions and remote action in muscles by FB-produced proteins. To examine the potential of this approach in mammalian systems, a conditional BirA*G3 allele was generated through targeting of mouse embryo stem cells (ESCs), and biotinylation analyzed in ESC-derived teratomas and host serum samples. Quantitative tandem mass tag mass spectrometry identified biotin-dependent labelling of secreted proteins shared between the tumor and serum samples. Among 291 streptavidin-enriched blood serum proteins from BirA*G3 tumors were several low-abundance proteins with hormonal properties. Our findings indicate that the communication network of secreted proteins is vast, and we provide a resource for candidate interorgan communication factors. The BirA*G3 approach has broad potential across different model systems to identify cell secretomes and novel mediators of interorgan communication in healthy or diseased states.

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