ApoJ regulates endothelial lipase activity and stability.

Figures

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  FIGURE 1. ApoJ Co‐purifies with EL; (a) Coomassie stained gel (left) and corresponding anti‐EL Western blot (right) shows the purification of EL. An unknown band shows up around 75 kDa. (b) Phospholipase activity assay using purified EL and the PED‐A1 liposome mix substrate shows a significantly higher EL phospholipase activity compared to buffer control. Data is from three independent trials. Significance is assessed by a two tailed Student's t‐test. (c) Filtered LC–MS/MS results for the unknown band (panel A). The results were filtered to include only proteins with molecular weights around 75 kDa. (d) Coomassie stained gel of control purification using only affinity tags and of purified EL using the same purification scheme. (e) Western blot of EL with the purified tags and of second elution of purified EL from the strep‐tactin purification. (f) ApoJ Western blot of the purified tags and of the second elution of purified EL that demonstrate that ApoJ is the unknown band. (g) Size‐exclusion chromatography (SEC) of mouse plasma, and Western blots of ApoA1 (top), ApoJ (middle), and EL (bottom) showing the colocalization of HDL with both EL and ApoJ.

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  FIGURE 2. ApoJ supports the phospholipase activity of EL; (a): Phospholipase (PED‐A1) activity assay of conditioned media from Expi293 cells expressing EL alone (baseline ApoJ) or co‐expressing EL and ApoJ (still contains baseline ApoJ). (b) Quantification of EL concentration (μM) in Expi293 conditioned media with baseline ApoJ expression versus EL co‐expressed with ApoJ. (c) Western blot and quantification of ApoJ expression in HEK293 stable cell lines expressing EL, transfected with either scrambled or ApoJ (CLU)‐targeting siRNA. (d) Quantification of EL concentration (μM) in HEK293 stable cell lines transfected with either scrambled or ApoJ (CLU)‐targeting siRNA. (e) Phospholipase (PED‐A1) activity assay of conditioned media from HEK293 stable cell lines transfected with either scrambled or ApoJ siRNA. Data was normalized across multiple independent biological replicates using Normalization by Optimal Alignment method as previously described (Degasperi et al., 2014). (f) Phospholipase (PED‐A1) activity assay using 50 nM EL expressed in Expi293 cells with baseline ApoJ. The PED‐A1 liposome mix was mixed with or without 500 nM of purified ApoJ. Data is from independent trials, and…

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  FIGURE 3. ApoJ Protects the solubility of EL, LPL, and HL, but not PL; (a) Solubility of EL, (b) LPL, (c) HL, and (d) PL in the pre‐ and post‐ultracentrifugation in the presence or absence of excess co‐expressed ApoJ above baseline. Solubility is determined by taking the ratio between the post‐ultracentrifugation supernatant (supe) and the pre‐ultracentrifugation media (input). Data is from three independent trials, and significance is assessed by a two‐tailed Student's t‐test.

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  FIGURE 4. ApoJ Interacts at the Tryptophan Loop Region and The Lid Region of EL; (a) Peptide array results mapped onto the alphafold predicted structure of EL. The dashed circles on the EL structures highlight its lid region and the solid circles highlight its tryptophan loop region. (b) The Protein Data Bank (PDB) structure of LPL (6OAU), (c) The alphafold predicted structure of HL, and (d) The PDB structure of PL (1LPA). The colors are a representation of the integrated density of negative control corrected ApoJ interaction with each spot on the peptide array. Darker blue represents a greater density.

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  FIGURE 5. Mutating EL Abolishes the Effect of ApoJ on EL; (a) The tryptophan loop and lid region of EL were mutated. The lid region of EL was switched to lid region of PL (top) and the tryptophan residues in the loop were mutated to leucine residues (bottom). (b) Switching the lid region of EL with the lid region of PL abolished the solubilizing effect of excess ApoJ on EL. (c) Switching the lid region of EL with the lid region of PL also abolished the effect of ApoJ on the phospholipase A1 activity of EL from conditioned media. (d) Mutating the tryptophan residues to leucine abolished the effect of ApoJ on the solubility of EL. (e) Mutating the tryptophan loop to leucine also abolished ApoJ's effect on the phospholipase A1 activity of EL from conditioned media. Data is from three independent trials, and significance is assessed by a two‐tailed Student's t‐test.

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  FIGURE 6. ApoJ Does Not Transport EL; It Helps EL Metabolize Lipoprotein‐like Substrates; (a) Diagram of the four density layers of the sucrose‐based floatation discontinuous gradient experiment along plot showing the HDL samples (e.g., HDL only, HDL with EL, HDL with ApoJ, and HDL, EL with ApoJ). HDL preferably floats up to the 15% sucrose density layer in all conditions. (b) Plot showing the ApoJ samples (e.g., ApoJ alone, ApoJ with EL, ApoJ with HDL, and ApoJ, EL with HDL). ApoJ preferably floats to the 15% sucrose layer alone or with EL, but it becomes significantly denser when HDL is present. (c) Plot showing the EL samples (e.g., EL alone, EL with ApoJ, EL with HDL, and EL, ApoJ with HDL). EL preferably floats to the 52% sucrose layer alone or with ApoJ present, but it prefers the 15% sucrose significantly more with HDL and significantly less with ApoJ. (d) Enzyme‐coupled Amplex Ultrared activity assay of EL with 0.2% intralipid or with 0.2% intralipid incorporated with 250 nM Lipid‐rich ApoJ as substrate. EL is significantly more active with ApoJ incorporated intralipid. Data is from three independent trials, and significance is assessed by a two tailed Student's t‐test.

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  FIGURE 7. Chaperone‐assisted enzyme activation Model; (1) Lipid‐rich ApoJ shields the lid and tryptophan loop of EL preventing aggregation. (2) Lipid‐rich ApoJ primes EL by stabilizing the lid region and tethers the enzyme to the lipoprotein substrate during hydrolysis.