Validating an artificial organelle: Studies of lipid droplet-specific proteins on adiposome platform

Abstract

HighlightsAn artificial organelle was validated for the targeting and regulation of LD proteins Binding affinity analysis of PLIN2 targeting on artificial LD was performed PtdIns and the 73 rd glutamic acid in PLIN2 affect the targeting of PLIN2 to LD In situ enzymatic activity of ATGL was demonstrated and measured via artificial LD

Artificial Lipid Droplets: Novel Effective Biomaterials to Protect Cells against Oxidative Stress and Lipotoxicity

“…In recent years, synthetic biology has developed rapidly and has been able to synthesize cell-like structures with ATPase activity [21]. An in vitro experimental system for LDs has been constructed and has identified the function of proteins such as microorganism lipid droplet small protein (MLDS) and ATGL [22][23][24]. A LDs-like structure is obtained by injecting triglycerides into a giant unilamellar vesicle (GUV), which confirms that surface tension affects the directionality of lipid droplet growth [25].…”

Protocol for using artificial lipid droplets to study the binding affinity of lipid droplet-associated proteins

“… (C) The diameter distribution of adiposomes determined using dynamic light scattering. Parts of the figure are reprinted with permission from ( Ma et al., 2021 ) and ( Wang et al., 2016 ). Store adiposomes at 2°C–8°C up to two weeks.…”

“…This method avoids the use of mechanical force, such as sonication, to incorporate proteins into the adiposomes. The artificial LDs are prepared using adiposomes to recruit recombinant LD-associated proteins onto their phospholipid monolayers ( Ma et al., 2021 ; Wang et al., 2016 ; Zhang et al., 2017 ). It allows LD-associated proteins to bind on the surface of adiposomes spontaneously and allows the binding to reach equilibrium, which is close to the natural pattern.…”

“…The isolation of natural LDs as well as the density of PLIN2 on them is described as a control of artificial LDs. The protocol is applicable to several LD-associated proteins ( Ma et al., 2021 ; Wang et al., 2016 ; Zhang et al., 2017 ). However, it is essential to confirm that the proteins are correctly expressed and purified in advance, since several LD-associated proteins are highly hydrophobic so that are easy to aggregate and precipitate in aqueous buffer, e.g., perilipin 1 (PLIN1), oleosins, and perilipin 2 (PLIN2) ( Gidda et al., 2016 ; Julien et al., 2021 ; Subramanian et al., 2004 ; Wang et al., 2016 ).…”

“…This protocol can be adapted to determine the binding properties of various lipid droplet-associated proteins. For complete details on the use and execution of this protocol, please refer to Ma et al. (2021) .…”