Topel, H. | Search bioRxiv

bioRxiv2025

EctoShed: A novel Gene Delivery Platform for Functional Analysis of Adipocyte-Shed Proteoforms

ObjectiveThe proteolytic cleavage of membrane-bound proteins, ectodomain shedding, functionally expands the reservoire of proteins/peptides available for endocrine crosstalk, and metabolic regulation. However, the functional understanding of secreted proteoforms, including whether they act synergistically or antagonistically with their membrane precursors, is often unknown. We aimed to develop a novel viral vector based gene delivery platform enabling characterization of both membrane-bound and soluble proteoforms in adipocytes, independent of endogenous shedding. ResultsWe describe a novel platform, termed EctoShed, achieves expression of proteoforms of amine oxidase copper-containing 3 (AOC3) in adipocytes by capitalising on both the established lentiviral (LV) and AAV gene delivery systems, expressing full length or a soluble AOC3 mimic (m-sAOC3) isoforms. In vitro transduction of primary white adipocytes induced significant expression of both isoforms, retaining AOC3 enzymatic activity. In vivo delivery to inguinal white adipose tissue enabled depot-specific AOC3 expression and increased abundance of m-sAOC3 in the serum. Mice expressing m-sAOC3 exhibited reduced fat mass and fasting glucose levels. ConclusionEctoShed represents a versatile tool to dissect the functional roles of soluble proteoforms shed from adipocytes, enabling in vitro and in vivo applications in cardiometabolic health and disease.

molecular biology↗

bioRxiv2022

Cold-induced expression of a truncated Adenylyl Cyclase 3 acts as rheostat to brown fat function.

Promoting brown adipose tissue (BAT) activity has been recognized as innovative therapeutic approach to improve obesity and metabolic disease. Whilst the molecular circuitry underlying thermogenic activation of BAT is well understood, the processes underlying rheostatic regulation of BAT to maintain homeostasis and avoid excessive energy dissipation remain ill-defined. Increasing cyclic AMP (cAMP) biosynthesis is key for BAT activation. Here, we demonstrate that ADCY3, an adenylyl cyclase whose expression is induced during cold exposure and regulates cAMP homeostasis in thermogenic fat, is dispensable for BAT function in lean mice, but becomes critical during obesity. Furthermore, by combining RNA-seq with epigenomic H3K4me3 profiling, we detected a novel, cold-inducible promoter that generates a 5 truncated Adcy3-at mRNA isoform, Adcy3-at. Mice lacking only Adcy3-at, but not full-length Adcy3, displayed increased energy expenditure already under lean conditions and were protected against obesity and ensuing metabolic imbalances. Subcellularly, translated ADCY3-AT proteins are retained in the endoplasmic reticulum (ER), did not translocate to the cell membrane, and lacked enzymatic activity. By interacting with ADCY3, ADCY3-AT retained ADCY3 in the ER and, thereby, reduced the plasma membrane pool of ADCYs available for G-protein mediated cAMP synthesis. Thereby, ADCY3-AT acts as a signaling rheostat in BAT, limiting adverse consequences of uncurbed cAMP activity after long-term BAT activation. Adcy3-at induction was driven by a cold-induced, truncated isoform of the transcriptional cofactor PPARGC1A (PPARG Coactivator 1 Alpha, PPARGC1A-AT). Expression of Ppargc1a-at and Adcy3-at are evolutionary conserved, indicating that transcriptional rewiring by commissioning of alternative promoters is key for thermogenic fat function.

cell biology↗