Co-immunoprecipitation for identifying protein-protein interaction on lipid droplets.

The lipid droplet (LD) is a conserved organelle that exists in almost all organisms, ranging from bacteria to mammals. Dysfunctions in LDs are linked to a range of human metabolic syndromes. The formation of protein complexes on LDs is crucial for maintaining their function. Investigating how proteins interact on LDs is essential for understanding the role of LDs. We have developed an effective method to uncover protein-protein interactions and protein complexes specifically on LDs. In this method, we conduct co-immunoprecipitation (co-IP) experiments using LD proteins extracted directly from isolated LDs, rather than utilizing proteins from cell lysates. To elaborate, we begin by purifying LDs with high-quality and extracting LD-associated proteins. Subsequently, the co-IP experiment is performed on these LD-associated proteins directly, which would enhance the co-IP experiment specificity of LD-associated proteins. This method enables researchers to directly unveil protein complexes on LDs and gain deeper insights into the functional roles of proteins associated with LDs.

Polydopamine-armored zeolitic imidazolate framework-8-incorporated zwitterionic hydrogel with multifunctional properties for infected wound healing.

Diabetic skin wound healing is compromised by bacterial infections, oxidative stress, and vascular disruption, leading to delayed recovery and potential complications. This study developed an antibacterial, antioxidant, and adhesive hydrogel dressing that promotes rapid bacterial-infected diabetic wound healing using the biological macromolecule of polydopamine (PDA). This hydrogel comprised PDA-armored zeolitic imidazolate framework-8 nanoparticles (PDA@ZIF-8 NPs) combined with a second armor of zwitterionic polymer network (poly(acrylamide-co-sulfobetaine methacrylate); PAS), realizing low concentration Zn2+ release, good adhesion (14.7 kPa for porcine skin), and improved tensile strength (83.2 kPa). The hydrogel exhibited good antibacterial efficacy against both Staphylococcus aureus (S. aureus, 92.8 %), Escherichia coli (E. coli, 99.6 %) and methicillin-resistant S. aureus (MRSA, 99.2 %), which was attributed to the properties of the incorporated PDA@ZIF-8 NPs. Notably, in vitro, the PDA@ZIF-8 PAS hydrogel not only promoted fibroblast proliferation and migration but also facilitated endothelial cell angiogenesis. In vivo, the PDA@ZIF-8 PAS hydrogel retained its Zn2+-releasing function and effectively suppressed bacterial growth in infected wounds, thereby accelerating the regeneration of both normal and diabetic wounds. This multiarmored hydrogel is a promising sustained-release carrier for functional metal ions and drugs, making it applicable for not only skin healing, but potentially the regeneration of other complex tissues.

Yeast perilipin Pet10p/Pln1p interacts with Erg6p in ergosterol metabolism.

Lipid droplets (LD) are highly dynamic organelles specialized for the regulation of energy storage and cellular homeostasis. LD consist of a neutral lipid core surrounded by a phospholipid monolayer membrane with embedded proteins, most of which are involved in lipid homeostasis. In this study, we focused on one of the major LD proteins, sterol C24-methyltransferase, encoded by ERG6. We found that the absence of Erg6p resulted in an increased accumulation of yeast perilipin Pet10p in LD, while the disruption of PET10 was accompanied by Erg6p LD over-accumulation. An observed reciprocal enrichment of Erg6p and Pet10p in pet10Δ and erg6Δ mutants in LD, respectively, was related to specific functional changes in the LD and was not due to regulation on the expression level. The involvement of Pet10p in neutral lipid homeostasis was observed in experiments that focused on the dynamics of neutral lipid mobilization as time-dependent changes in the triacylglycerols (TAG) and steryl esters (SE) content. We found that the kinetics of SE hydrolysis was reduced in erg6Δ cells and the mobilization of SE was completely lost in mutants that lacked both Erg6p and Pet10p. In addition, we observed that decreased levels of SE in erg6Δpet10Δ was linked to an overexpression of steryl ester hydrolase Yeh1p. Lipid analysis of erg6Δpet10Δ showed that PET10 deletion altered the composition of ergosterol intermediates which had accumulated in erg6Δ. In conclusion, yeast perilipin Pet10p functionally interacts with Erg6p during the metabolism of ergosterol.

LET-767 determines lipid droplet protein targeting and lipid homeostasis.

Lipid droplets (LDs) are composed of a core of neutral lipids wrapped by a phospholipid (PL) monolayer containing several hundred proteins that vary between different cells or organisms. How LD proteins target to LDs is still largely unknown. Here, we show that RNAi knockdown or gene mutation of let-767, encoding a member of hydroxysteroid dehydrogenase (HSD), displaced the LD localization of three well-known LD proteins: DHS-3 (dehydrogenase/reductase), PLIN-1 (perilipin), and DGAT-2 (diacylglycerol O-acyltransferase 2), and also prevented LD growth in Caenorhabditis elegans. LET-767 interacts with ARF-1 (ADP-ribosylation factor 1) to prevent ARF-1 LD translocation for appropriate LD protein targeting and lipid homeostasis. Deficiency of LET-767 leads to the release of ARF-1, which further recruits and promotes translocation of ATGL-1 (adipose triglyceride lipase) to LDs for lipolysis. The displacement of LD proteins caused by LET-767 deficiency could be reversed by inhibition of either ARF-1 or ATGL-1. Our work uncovers a unique LET-767 for determining LD protein targeting and maintaining lipid homeostasis.