Proteomics of protein trafficking byin vivotissue-specific labeling

Abstract

Secreted interorgan communication factors encode key regulators of homeostasis. However, long-standing questions surround their origins/destinations, mechanisms of interactions, and the number of proteins involved. Progress has been hindered by the lack of methodologies for these factors' largescale identification and characterization, as conventional approaches cannot identify low-abundance factors and the origins and destinations of secreted proteins. We established an in vivo platform to investigate secreted protein trafficking between organs proteome-wide, whereby 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, we identified 51 putative muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, of which 60-70% have human orthologs. We demonstrate, in particular, that conserved fat body-derived novel interorgan communication factors CG31326, CG2145, and CG4332 promote muscle activity. Our results indicate that the communication network of secreted proteins is vast, and we identified systemic functions for a number of these factors.This approach is widely applicable to studies in interorgan, local and intracellular protein trafficking networks, non-conventional secretion, and to mammalian systems, under healthy or diseased states. One Sentence SummaryWe developed an in vivo platform to investigate protein trafficking between organs proteomewide, provide a resource for interorgan communication factors, and determined conserved adipokines that affect muscles. Main TextLocal tissue homeostasis is becoming increasingly well-understood. However, the physiological importance and presence of secreted interorgan communication factors is only beginning to be documented from experiments in Drosophila and vertebrates. Secreted factors acting directly or indirectly between organs encode key regulators of systemic homeostasis (1). These factors traffic, or translocate, intracellularly from their production sites within cells (2) to distal organs through blood (1). For instance, Droujinine et al 3 adipokines including leptin and adiponectin encode adipose tissue-derived systemic metabolic regulators(1). In addition, myokines such as irisin (cleaved form of FNDC5) and interleukin-6 are secreted by muscles to control metabolism in adipose tissue (1). Despite their importance, identification of interorgan communication factors is technically challenging, and a number of published results were later determined to be irreproducible or controversial (1,(3)(4)(5). Also, origins and/or destinations of factors including glucagon-like peptide 1 (GLP-1), ghrelin, leptin, cholecystokinin (CCK), and growth differentiation factor 11 (GDF-11) need to be clarified (1, 5). Moreover, because large-scale screening

Physiological and Pathological Regulation of Peripheral Metabolism by Gut-Peptide Hormones in Drosophila

“…In vivo trafficking of hormones is difficult to achieve due to limited genetic tools. Recent studies, which engineered a promiscuous biotin ligase, BirA, to specifically label secreted proteins including peptide prohormones (Stevens et al, 2019;Droujinine et al, 2020), are very promising to address the limitation (Figure 4B). They used a fused BirA to biotinylate all proteins in the muscle ER and detected biotin-labeled proteins in the blood to identify potential myokines.…”

“…They used a fused BirA to biotinylate all proteins in the muscle ER and detected biotin-labeled proteins in the blood to identify potential myokines. Moreover, they further detected biotin-labeled proteins in the other organs to characterize in vivo trafficking of these myokines from skeletal muscle to the fat body ( Droujinine et al, 2020 ). Genetic validation is required to confirm the physiological outputs of gut-peptide hormone-induced interorgan communication.…”

An American Physiological Society cross-journal Call for Papers on “Inter-Organ Communication in Homeostasis and Disease”

“…Physiological Genomics will consider research articles and reviews that use high throughput screens, single cell technologies, genetics or -omics technologies, such as secretomics, to investigate intercellular communication among any organs and in any species. Recent studies in this area have established in vivo platforms to identify secreted proteins in Drosophila and discovered fatbody-derived proteins that promote muscle activity (86). As another example, Hudry and colleagues used RNA-Seq to discover that carbohydrate metabolism in the Drosophila intestine exhibits sex differences that are controlled by signaling from the adjacent male gonad (87).…”

Functional proximity mapping of RNA binding proteins uncovers a mitochondrial mRNA anchor that promotes 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 or biotin-AMP) 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) 19 , dynamic interactomes 20,21 , and in vivo secretomes 22,23 . Recently, PL has been extended to spatial mapping of transcriptomes in living cells [24][25][26] .…”