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

Abstract

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.