EctoShed: a novel gene delivery platform for functional analysis of adipocyte-shed proteoforms

The proteolytic cleavage of membrane-bound proteins, ectodomain shedding, functionally expands the reservoir 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. To this end, we elected amine oxidase copper-containing 3 (AOC3) as a target for validation.

We describe a novel platform, termed 'EctoShed', achieving adipocyte-specific expression of different proteoforms of AOC3 - full-length and membrane-bound or a soluble AOC3 mimic (m-sAOC3) - by capitalising on the established lentiviral and adeno-associated virus gene delivery systems. In vitro transduction of primary white adipocytes induced significant expression of both isoforms, whilst retaining AOC3 enzymatic activity. In vivo delivery to inguinal white adipose tissue enabled depot-specific AOC3 expression and increased abundance of m-sAOC3 in serum. Mice expressing m-sAOC3 exhibited reduced fat mass and fasting glucose levels. Thus, EctoShed is a novel tool to dissect the functional roles of soluble proteoforms secreted from adipocytes, with broad in vitro and in vivo applications in cardiometabolic health.