Abstract
Alpha-beta hydrolase domain-containing 5 (ABHD5), the defective gene in human Chanarin-Dorfman syndrome, is a highly conserved regulator of adipose triglyceride lipase (ATGL)-mediated lipolysis that plays important roles in metabolism, tumor progression, viral replication, and skin barrier formation. The structural determinants of ABHD5 lipolysis activation, however, are unknown. We performed comparative evolutionary analysis and structural modeling of ABHD5 and ABHD4, a functionally distinct paralog that diverged from ABHD5 ~500 million years ago, to identify determinants of ABHD5 lipolysis activation. Two highly conserved ABHD5 amino acids (R299 and G328) enabled ABHD4 (ABHD4 N303R/S332G) to activate ATGL in Cos7 cells, brown adipocytes, and artificial lipid droplets. The corresponding ABHD5 mutations (ABHD5 R299N and ABHD5 G328S) selectively disrupted lipolysis without affecting ATGL lipid droplet translocation or ABHD5 interactions with perilipin proteins and ABHD5 ligands, demonstrating that ABHD5 lipase activation could be dissociated from its other functions. Structural modeling placed ABHD5 R299/G328 and R303/G332 from gain-of-function ABHD4 in close proximity on the ABHD protein surface, indicating they form part of a novel functional surface required for lipase activation. These data demonstrate distinct ABHD5 functional properties and provide new insights into the functional evolution of ABHD family members and the structural basis of lipase regulation.
Highlights
The mobilization of free fatty acids (FFA) from stored triglyceride is a fundamental cellular process that is mediated in many tissues by the functional interaction of alpha-beta hydrolase domain-containing 5 (ABHD5) with adipose triglyceride lipase (ATGL)
We identified two highly conserved Alpha-beta hydrolase domain-containing 5 (ABHD5) amino acids (R299 and G328) that are necessary for ATGL activation by ABHD5 and sufficient to enable that activity in ABHD4 (ABHD4 N303R/S332G)
We found that ABHD4 N303R/S332G significantly stimulated ATGL-dependent lipolysis compared to ABHD4, which was inactive compared to ATGL lysate alone, though lipolysis activation was less than that observed with partially-purified ABHD5 (Fig. 3e)
Summary
The mobilization of free fatty acids (FFA) from stored triglyceride is a fundamental cellular process that is mediated in many tissues by the functional interaction of alpha-beta hydrolase domain-containing 5 (ABHD5) with adipose triglyceride lipase (ATGL). The lipase-activating function of ABHD5 is repressed by its binding to perilipin 1 (PLIN1), a lipid droplet (LD) scaffold. ABHD5 is the direct target of endogenous and synthetic ligands that modulate its lipase-activating function by regulating its interactions with inhibitory PLIN proteins[9]. ABHD5 binds ATGL8,15 and PLIN proteins indirectly regulate that interaction[13,16,17,18], the molecular basis of ATGL activation by ABHD5 remains unclear. Analysis of lipolysis-inactive ABHD5 mutants demonstrated that ATGL activation was dissociable from ATGL translocation to the LD surface and from ABHD5 interactions with PLIN proteins and synthetic ABHD5 ligands. Structural modeling based on shape analysis identified a novel functional surface in ABHD5 and gain-of-function ABHD4 that contains the residues critical for ATGL activation
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