PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores

Abstract

The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms.

Figure 1. PAT family members have similar predicted structural features

This cartoon compares the structure of ten members of the PAT family of lipid droplet proteins. The first seven shown (perilipin A through S3-12) are from mammals, the last three from non-mammalian species (flies and fungi, respectively). With the exception of S3-12, these proteins share a ~ 100 amino acid region of high sequence similarity near their N-termini (PAT domain, green). For this figure, we estimated the approximate extent of this domain based on the analysis of Lu and colleagues [30]. Despite the absence of a clear PAT domain, S3-12 retains sequence similarity to the other family members in other parts of the protein, including the 11-mer repeats. Numbers indicate mapped (perilipin A, LSD-1) or predicted (MPL1) PKA sites. For perilipin A, phosphorylation of those sites has been linked to specific functions of the protein, such as interactions with HSL and ATGL or droplet dispersion. All drawings are to scale with the exception of that for S3-12, a 1403 amino acid-protein with a 11-mer repeat that runs ~70 % of the length of the protein (from aa~100 to ~1060). X-ray crystallography has revealed a four-helix bundle domain in TIP47 (shown in light blue). Based on sequence similarity, similar domains have been predicted in ADRP and OXPAT; they are proposed to target the proteins to lipid droplets. A different set of potentially droplet-targeting helical segments has been predicted in LSD-1. The absence of features from a specific protein as depicted in this cartoon does not necessarily mean that these features do not exist, but rather that they have not yet been reported. Specifically, attempts to predict the structural features of LSD-1, LSD-2 and MPL1 are only in their infancy. For example, no published studies critically examine the presence or absence of 11-mer repeats in the non-mammalian family members. The reader is referred to a complementary depiction of the structural features of the mammalian PAT proteins in a recent review [25].

Figure 2. Regulation of lipolysis is conserved in the adipose tissues of mammals (A) and insects (B)

In both cases, circulating hormones (CA = catecholamines; AKH = adipokinetic hormone) stimulate G-protein coupled surface receptors (βAR = β-adrenergic receptor; AKHR = AKH receptor) resulting in the production of the second messenger cAMP. High cAMP levels activate Protein Kinase A (PKA) which in turn phosphorylates PAT proteins on the surface of lipid droplets: perilipin and LSD-1. In mammals, phosphorylated perilipin recruits the lipase HSL to the surface of lipid droplets, stimulating lipolysis. HSL is also a target of PKA. In insects, phosphorylated LSD-1 is thought to activate the lipase CG8552; the possible insect ortholog of HSL (CG11055) has yet to be functionally characterized. A second set of lipases from the ATGL/Brummer family cooperates in lipolysis. In mammals, both perilipin and ATGL interact with CGI-58; these interactions are modulated by perilipin phosphorylation (see Fig. 3 for a closer look at these mechanisms); the function of the insect ortholog (CG1882) is unknown, but this protein has been found associated with lipid droplets.

Figure 3. Phosphorylated perilipin A organizes lipolysis in murine adipocytes

(A and B) In the basal state (left) the lipolytic regulator perilipin A is found at the surface of the lipid droplet in a complex with CGI-58. ATGL may also be in this complex. ATGL activity is kept quiescent through the autoinhibitory C-terminus of the lipase. Upon lipolytic stimulation (right), PKA is activated and phosphorylates up to 6 serine residues on perilipin A (Ser81, 222, 276, 433, 492, and 517) and 2 on HSL (Ser659, and 660). This results in the following rearrangements. 1) CGI-58 dissociates from the droplet surface; it is unclear if ATGL remains associated with GCI-58 in the cytosol. 2) Phosphorylated HSL translocates to the droplet surface and associates with both perilipin A and AFABP. 3) ATGL interacts with perilipin A through phosphorylated Ser517, and lipolysis commences. 4) At a later time (several hours) droplets fragment and disperse, a phenomenon dependent on phosphorylation of perilipin A Ser492. The fragmented microdroplets become coated with ADRP and S3-12, as well as perilipin, and ADRP also associates with the remaining larger droplets. The depiction of perilipin A delineates the acidic loop region and amino- and carboxy-terminal segments for the sake of clarity, but is not meant to show discreetly folding domains. (C) Timeline of lipolysis. Please note that this line is not to scale.