Functional analysis of FSP27 protein regions for lipid droplet localization, caspase-dependent apoptosis, and dimerization with CIDEA

Abstract

MS, Smas CM.Functional analysis of FSP27 protein regions for lipid droplet localization, caspase-dependent apoptosis, and dimerization with CIDEA. Am J Physiol Endocrinol Metab 297: E1395-E1413, 2009. First published October 20, 2009 doi:10.1152/ajpendo.00188.2009.-The adipocytespecific protein FSP27, also known as CIDEC, is one of three cell death-inducing DFF45-like effector (CIDE) proteins. The first known function for CIDEs was promotion of apoptosis upon ectopic expression in mammalian cells. Recent studies in endogenous settings demonstrated key roles for CIDEs in energy metabolism. FSP27 is a lipid droplet-associated protein whose heterologous expression enhances formation of enlarged lipid droplets and is required for unilocular lipid droplets typical of white adipocytes in vivo. Here, we delineate relationships between apoptotic function and lipid droplet localization of FSP27. We demonstrate that ectopic expression of FSP27 induces enlarged lipid droplets in multiple human cell lines, which is indicative that its mechanism involves ubiquitously present, rather than adipocyte-specific, cellular machinery. Furthermore, promotion of lipid droplet formation in HeLa cells via culture in exogenous oleic acid offsets FSP27-mediated apoptosis. Using transient cotransfections and analysis of lipid droplets in HeLa cells stably expressing FSP27, we show that FSP27 does not protect lipid droplets from action of ATGL lipase. Domain mapping with eGFP-FSP27 deletion constructs indicates that lipid droplet localization of FSP27 requires amino acids 174 -192 of its CIDE C domain. The apoptotic mechanism of FSP27, which we show involves caspase-9 and mitochondrial cytochrome c, also requires this 19-amino acid region. Interaction assays determine the FSP27 CIDE C domain complexes with CIDEA, and Western blot reveals that FSP27 protein levels are reduced by coexpression of CIDEA. Overall, our findings demonstrate the function of the FSP27 CIDE C domain and/or regions thereof for apoptosis, lipid droplet localization, and CIDEA interaction.fat-specific protein 27; cell death-inducing DFF45-like effector A; adipose OBESITY AND ITS RELATED COMORBIDITIES are approaching epidemic levels (1, 9). The development of new therapeutic inroads to treat these conditions would be greatly facilitated by a full understanding of lipid homeostasis (35,36). Lipotoxicity is a highly detrimental outcome of the obese state, leading to derangement of cell function and/or cell death in various tissues (34,35,37). White adipocytes present in white adipose tissue are the major site storage of excess energy in the form of triacylglycerol, contained within intracellular lipid droplets (7,38). Efficient storage of excess fatty acids within adipocyte lipid droplets also serves to protect other cells and tissues from their lipotoxic effects (7, 38). Lipid droplets are highly dynamic organelles consisting of a neutral lipid core, a phospholipid monolayer, and a large number of lipid droplet-associated proteins (7, 38). The role for the vast majority ...

Differential Roles of Cell Death-inducing DNA Fragmentation Factor-α-like Effector (CIDE) Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes

“…Previous data showed that overexpression of CIDE proteins in many cell types resulted in a caspase-independent cell death (45). However, the rate of cell death was significantly reduced in CIDE-overexpressing cells when exogenous oleic acid was introduced (46). In the presence of lipid-rich medium, CIDE proteins localized to LDs promote LD fusion and lipid storage, resulting in reduced lipotoxicity and oxidative stress.…”

Fat-specific protein 27 modulates nuclear factor of activated T cells 5 and the cellular response to stress

“…FSP27 can form a complex with its family member Cidea ( 9 ), but the interaction of FSP27 with other cellular proteins has not been reported. In the current study, we sought to identify proteins that interact with FSP27 using a yeast two-hybrid system.…”

The role of lipid droplets in metabolic disease in rodents and humans

“…Cidec was the first of this family to be identified as an LD-associated protein (58,59). Subsequently, similar to PAT proteins, ectopic expression of Cide proteins promotes LD formation and reduces TAG hydrolysis (57,60,61). Relevant to their homology to proteins associated with apoptosis, ectopic expression of CIDE members enhances rates of apoptosis.…”

“…Relevant to their homology to proteins associated with apoptosis, ectopic expression of CIDE members enhances rates of apoptosis. However, when CIDE-expressing cells are incubated with oleic acid to promote TAG storage and LD formation, the rates of apoptosis are significantly reduced, presumably because sequestration of the Cide proteins on the droplets blocks their proapoptotic function (60). Cidea in lean mice is expressed predominately in brown adipose tissue (BAT), but not in white adipose tissue (WAT) (62).…”