Accumulating experimental evidence suggests that rather than simply “coating” and “shielding” the lipid droplet in white adipocytes, PLIN1 orchestrates the access of lipases to core lipids, particularly triglyceride and diglyceride hydrolases, each of which predominantly hydrolyzes a specific class of glycerolipids. Early work by Brasaemle and co-workers (12
) suggested that both the N and C termini of PLIN1 were required for optimal triglyceride storage, whereas central hydrophobic domains appeared to anchor PLIN1 to the LD surface. Hickenbottom et al.
) solved the three-dimensional structure of the C terminus of PLIN3 and, on the basis of sequence homology, suggested that the central hydrophobic domains appear to be embedded in a four-helix bundle that is not likely to be lipid-associated. They instead proposed that N-terminal amphipathic helices in all PLINs are likely to be responsible for lipid droplet targeting. HSL appears to interact directly with PLIN1 and to depend upon PLIN1 for localization to the lipid droplet (6
). These interactions require an intact N-terminal of PLIN1, although they might also involve some interaction with the C terminus (8
). Further investigations by the Greenberg and Zechner groups (5
) suggested that ATGL, rather than HSL, is the major determinant of basal lipolysis in adipocytes. These data also suggested that the effects of PLIN1 on basal lipolysis are predominantly mediated via inhibition of ATGL. Granneman et al.
) provided a molecular explanation for these observations by demonstrating the ability of WT PLIN1 to effectively sequester ABHD5 from ATGL, thereby inhibiting basal lipolysis.
We sought to shed further light on these findings by characterizing the molecular consequences of naturally occurring human PLIN1
mutations. We previously identified two different frame shift mutations affecting the C terminus of PLIN1 in patients with a novel familial partial lipodystrophy phenotype. Although the fact that the patients were already diabetic and at least relatively insulin-deficient precluded us from definitively characterizing lipolysis in vivo
in these patients. Their phenotype, particularly the overall reduction in fat mass, elevated liver fat content, and elevated plasma triglyceride levels, is consistent with increased basal lipolysis (1
). Increased basal lipolysis has been demonstrated in adipocytes from PLIN1-null mice (22
). We deliberately characterized the cellular effects of the PLIN1 mutants in cells in which endogenous PLIN1 is not expressed to allow us to compare the behavior of WT versus
mutant PLIN1. The fact that the PLIN1 mutants failed to suppress basal lipolysis prompted further molecular studies aimed at addressing the consequences of the mutants on ATGL activation. Subramanian et al.
) used several deletion mutants of mouse PLIN1 to identify a stretch of amino acids (382–429) indispensable for binding of ABHD5. Our data are consistent with these findings insofar as both the Leu-404fs and Val-398fs mutants alter the homologous region of human PLIN1 and fail to bind ABHD5 in transfected cells. Interestingly our data suggest that this does not prevent ABHD5 localization to the lipid droplet, which is consistent with the work of Gruber et al.
), who proposed that an N-terminal Trp-rich region of ABHD5 is the major requirement for LD localization. These data are also consistent with subsequent binding to ATGL, which appears to be contingent upon ABHD5 localization to the lipid droplet (19
). In addition, our data provide evidence that stabilization of ABHD5 requires the C-terminally intact perilipin.
The C terminus of mouse PLIN3 (TIP47) is the only PAT (P
IP47) domain-containing protein for which the crystal structure has been solved (11
). PLIN1 and PLIN3 belong to a gene/protein family of five members sharing remote homology. Conserved residues at the C terminus map to a hydrophobic cleft formed between a four-helix bundle and a terminal α/β fold. In PLIN3, the α/β fold is contributed in part by two α helices and two β sheets at the N terminus and two β sheets at the C terminus of the four-helix bundle (11
). The cleft was proposed to bind either a hydrophobic peptide, protein, or small monomeric lipid (11
). PLIN1a, unlike the other PLINs, contains in addition an extended C-terminal tail with two putative PKA phosphorylation sites (11
). Although the sequence similarity of PLINs in the C-terminal domain is low, their sequences can be aligned with confidence thanks to the modern profile-to-profile methods (29
) and availability of hundreds of homologous PLIN sequences from different species in the databases (supplemental Fig. 3A
). Despite clear homologies, many differences exist between the C termini, e.g
. 1) several insertions and deletions between the elements of the regular secondary structure, of which the most prominent is the stretch of acidic amino acids between positions 302 and 316; 2) the alteration of the predicted irregular helix 4 with prolines at positions 326 and 328; and 3) altered residues in the putative groove.
Based on the currently available structural data of PLIN3, work by Subramanian et al.
), and the available human PLIN1 mutants associated with lipolytic dysfunction, we propose an important role for this unique C-terminal region of PLIN1 in the control of ATGL-mediated lipolysis. The frame shift replaces the C-terminal tail and the two predicted β-strands with a long missense chain (supplemental Fig. 3B
) with no predicted secondary structure (31
ABHD5 is known to bind both PLIN5 and PLIN2 (24
). Recent work by Chong et al.
) demonstrates that the C terminus of PLIN2 (residues 172–425) forms an independently folding α-helical structure. In contrast to PLIN1, truncation of this C-terminal domain resulted in larger lipid droplets in mouse mammary epithelial cells. Together, these data and our findings suggest that a secondary structure, most likely unique to PLIN1, is required for efficiently sequestering ABHD5 and thereby promoting efficient fat packaging.
One of the more striking and unexpected findings of our work was the dramatic reduction in ABHD5 protein levels in cells expressing mutant forms of PLIN1. Our observations from cultured cells are consistent with similarly reduced protein levels of ABHD5 in white adipose tissue from PLIN1-null mice (supplemental Fig. 1
). These data suggest that as well as sequestering ABHD5 from ATGL and thereby inhibiting basal lipolysis, PLIN1 stabilizes ABHD5 protein levels, potentially priming the cell to activate ATGL in response to lipolytic stimuli. Alternatively, the decrease in ABHD5 protein level may be due to increased association with ATGL. The relatively lower levels of ABHD5 seen in cells expressing the PLIN1 mutants and in tissue from Plin
-null mice might also be contributing to the observed failure to further increase lipolysis in response to forskolin/IBMX in cultured cells or to in vivo
lipolytic stimuli in the knockout mice. In the case of the human mutants, coexpressing WT and mutant PLIN isoforms in cells significantly increases ABHD5 levels. An alternative potential explanation for the reduced protein levels of ABHD5 is the reduction in lipid droplet size and cellular triglyceride content observed in these cells. Protein levels of PLIN1 itself and other lipid droplet proteins such as PLIN2 are known to be stabilized by interaction with the lipid droplet (30
As well as providing a molecular explanation for the observed increase in basal lipolysis associated with two novel human PLIN1 mutants, these data largely corroborate the emerging impression of PLIN1 as an essential lipid droplet coat protein whose C terminus regulates ATGL activity and basal lipolysis indirectly by binding and stabilizing its coactivator, ABHD5 (see supplemental Fig. 4
for a schematic illustration of this concept). This scaffolding function is also critical for HSL activation, enabling PLIN1 to precisely regulate both tri- and diglyceride hydrolysis. Finally, molecular understanding of the consequences of the PLIN1 mutants together with the observed response to ATGL knockdown suggests that ATGL inhibitors, which are as yet unavailable, may be therapeutically useful.