Crystal structure of a lipin/Pah phosphatidic acid phosphatase

Abstract

Lipin/Pah phosphatidic acid phosphatases (PAPs) generate diacylglycerol to regulate triglyceride synthesis and cellular signaling. Inactivating mutations cause rhabdomyolysis, autoinflammatory disease, and aberrant fat storage. Disease-mutations cluster within the conserved N-Lip and CLip regions that are separated by 500-residues in humans. To understand how the N-Lip and CLip combine for PAP function, we determined crystal structures of Tetrahymena thermophila Pah2 (Tt Pah2) that directly fuses the N-Lip and CLip. Tt Pah2 adopts a two-domain architecture where the N-Lip combines with part of the CLip to form an immunoglobulin-like domain and the remaining CLip forms a HAD-like catalytic domain. An N-Lip CLip fusion of mouse lipin-2 is catalytically active, which suggests mammalian lipins function with the same domain architecture as Tt Pah2. HDX-MS identifies an N-terminal amphipathic helix essential for membrane association. Disease-mutations disrupt catalysis or destabilize the protein fold. This illustrates mechanisms for lipin/Pah PAP function, membrane association, and lipin-related pathologies.

Regulation of Signaling and Metabolism by Lipin-mediated Phosphatidic Acid Phosphohydrolase Activity

“…A canonical haloacid dehalogenase catalytic site is contained in the C-terminal domain and N-terminal amphipathic helices and a polybasic domain facilitate membrane interaction [ 4 , 9 ] ( Figure 2 A). Recent crystallization studies have suggested that these conserved termini interact to form an immunoglobulin-like domain that is enabled by the variable regions in the middle of the protein [ 12 ] ( Figure 2 B). There is also evidence that lipin proteins form hetero- and homo-oligomers in their native state [ 13 ], although the importance of oligomer formation is still unclear.…”

The Torsin/ NEP1R1-CTDNEP1/ Lipin axis regulates nuclear envelope lipid metabolism for nuclear pore complex insertion

“…This includes the cell cycle (Bahmanyar et al, 2014), mechanical stress (Romani et al, 2019), nutrition (Peterson et al, 2011), anoxia (MacVicar et al, 2019, membrane composition (Eaton et al, 2013;Karanasios et al, 2010), and when cells alter morphology (Yang et al, 2020). Lipin is a large > 100kD soluble enzyme (Khayyo et al, 2020) that receives extensive post-translational modifications: acetylation, sumoylation, ubiquitination, and phosphorylation have all been described (Harris et al, 2007;Li et al, 2018;Liu and Gerace, 2009;O'Hara et al, 2006;Shimizu et al, 2017).…”

“…Lipin PA phosphatase activity requires that the N-terminus inserts into the membrane as an amphipathic helix (Khayyo et al, 2020). This event is inhibited by phosphorylation, which reduces Lipin PA phosphatase activity (Eaton et al, 2013;Huffman et al, 2002;Karanasios et al, 2010;O'Hara et al, 2006).…”

A review of phosphatidate phosphatase assays

“…Whereas yeast PAP is also known as Pah1, the mouse and human forms of PAP, which are encoded by the Lpin 1, 2, and 3 and LPIN 1, 2, and 3 genes, respectively, are also known as lipin (10,11). In organisms studied thus far, PAP activity is dependent on the DXDX(T/V) catalytic motif in the haloacid dehalogenase (HAD)-like domain (10,13,14). The enzyme is extensively modified posttranslationally by phosphorylation, which acutely regulates its catalytic activity, subcellular localization, and protein stability (15)(16)(17).…”

“…In the radioactive PAP assay, the substrate PA contains a radiolabel in the phosphate moiety (e.g., 32 P) or in the glycerol backbone or the fatty acyl moieties (e.g., 14 synthesized from DAG and [ -32 P]ATP by DAG kinase (Fig. 2, reaction 1 (88,89)).…”