ABSTRACT
The lipid droplet (LD) is a cellular organelle that consists of a neutral lipid core with a monolayer-phospholipid membrane and associated proteins. Recent LD studies demonstrate its importance in metabolic diseases and biofuel development. However, the mechanisms governing its formation and dynamics remain elusive. Therefore, we developed an in vitro system to facilitate the elucidation of these mechanisms. We generated sphere-shaped structures with a neutral lipid core and a monolayer-phospholipid membrane by mechanically mixing neutral lipids and phospholipids followed by a two-step purification. We named the nanodroplet "adiposome". We then recruited LD structure-like/resident proteins to the adiposome, including the bacterial MLDS, Caenorhabditis elegans MDT-28/PLIN-1, or mammalian perilipin-2. In addition, adipose triglyceride lipase (ATGL) and apolipoprotein A1 (apo A-I) were recruited to adiposome. We termed the functional protein-coated adiposomes, Artificial Lipid Droplets (ALDs). With this experimental system, different proteins can be recruited to build ALDs for some biological goals and potential usage in drug delivery.
Subject(s)
Adipose Tissue, Brown/chemistry , Lipid Droplets/chemistry , Liposomes/chemistry , Animals , Apolipoprotein A-I/analysis , Caenorhabditis elegans/chemistry , Caenorhabditis elegans Proteins/analysis , Drug Delivery Systems , Lipids/chemistry , Male , Mice , Mice, Inbred C57BL , Perilipin-2/analysis , Phospholipids/chemistryABSTRACT
Rhodococcus opacus strain PD630 (R. opacus PD630), is an oleaginous bacterium, and also is one of few prokaryotic organisms that contain lipid droplets (LDs). LD is an important organelle for lipid storage but also intercellular communication regarding energy metabolism, and yet is a poorly understood cellular organelle. To understand the dynamics of LD using a simple model organism, we conducted a series of comprehensive omics studies of R. opacus PD630 including complete genome, transcriptome and proteome analysis. The genome of R. opacus PD630 encodes 8947 genes that are significantly enriched in the lipid transport, synthesis and metabolic, indicating a super ability of carbon source biosynthesis and catabolism. The comparative transcriptome analysis from three culture conditions revealed the landscape of gene-altered expressions responsible for lipid accumulation. The LD proteomes further identified the proteins that mediate lipid synthesis, storage and other biological functions. Integrating these three omics uncovered 177 proteins that may be involved in lipid metabolism and LD dynamics. A LD structure-like protein LPD06283 was further verified to affect the LD morphology. Our omics studies provide not only a first integrated omics study of prokaryotic LD organelle, but also a systematic platform for facilitating further prokaryotic LD research and biofuel development.
Subject(s)
Lipid Metabolism , Rhodococcus/metabolism , Bacterial Proteins/metabolism , Gene Expression , Gene Expression Profiling , Genome, Bacterial , Genomics , Lipids , Organelles/metabolism , Organelles/ultrastructure , Proteomics , Rhodococcus/genetics , Rhodococcus/ultrastructure , Triglycerides/biosynthesis , Triglycerides/metabolismABSTRACT
Storage of cellular triacylglycerols (TAGs) in lipid droplets (LDs) has been linked to the progression of many metabolic diseases in humans, and to the development of biofuels from plants and microorganisms. However, the biogenesis and dynamics of LDs are poorly understood. Compared with other organisms, bacteria seem to be a better model system for studying LD biology, because they are relatively simple and are highly efficient in converting biomass to TAG. We obtained highly purified LDs from Rhodococcus sp. RHA1, a bacterium that can produce TAG from many carbon sources, and then comprehensively characterized the LD proteome. Of the 228 LD-associated proteins identified, two major proteins, ro02104 and PspA, constituted about 15% of the total LD protein. The structure predicted for ro02104 resembles that of apolipoproteins, the structural proteins of plasma lipoproteins in mammals. Deletion of ro02104 resulted in the formation of supersized LDs, indicating that ro02104 plays a critical role in cellular LD dynamics. The putative α helix of the ro02104 LD-targeting domain (amino acids 83-146) is also similar to that of apolipoproteins. We report the identification of 228 proteins in the proteome of prokaryotic LDs, identify a putative structural protein of this organelle, and suggest that apolipoproteins may have an evolutionarily conserved role in the storage and trafficking of neutral lipids.
