Regulation of DGKε Activity and Substrate Acyl Chain Specificity by Negatively Charged Phospholipids

Please cite this article in press as: Bozelli et al., Regulation of DGKε Activity and Substrate Acyl Chain Specificity by Negatively Charged Phospholipids, Biophysical Journal (2019), https://doi.org/10.1016/j.bpj.2019.09.008

Article

Regulation of DGKε Activity and Substrate Acyl Chain Specificity by Negatively Charged Phospholipids  Carlos Bozelli, Jr.,1 Jenny Yune,1 You H. Hou,1 Preet Chatha,1 Alexia Fernandes,1 Zihao Cao,1 Jose Yufeng Tong,2,3 and Richard M. Epand1,* 1 Department of Biochemistry and Biomedical Sciences, Health Sciences Centre, McMaster University, Hamilton, Ontario, Canada; 2Structural Genomics Consortium, Toronto, Ontario, Canada; and 3Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada

ABSTRACT Diacylglycerol kinase ε (DGKε) is a membrane-bound enzyme that catalyzes the ATP-dependent phosphorylation of diacylglycerol to form phosphatidic acid (PA) in the phosphatidylinositol cycle. DGKε lacks a putative regulatory domain and has recently been reported to be regulated by highly curved membranes. To further study the effect of other membrane properties as a regulatory mechanism of DGKε, our work reports the effect of negatively charged phospholipids on DGKε activity and substrate acyl chain specificity. These studies were conducted using purified DGKε and detergent-free phospholipid aggregates, which present a more suitable model system to access the impact of membrane physical properties on membrane-active enzymes. The structural properties of the different model membranes were studied by means of differential scanning calorimetry and 31P-NMR. It is shown that the enzyme is inhibited by a variety of negatively charged phospholipids. However, PA, which is a negatively charged phospholipid and the product of DGKε catalyzed reaction, showed a varied regulatory effect on the enzyme from being an activator to an inhibitor. The type of feedback regulation of DGKε by PA depends on the particular PA molecular species as well as the physical properties of the membrane that the enzyme binds to. In the presence of highly packed PA-rich domains, the enzyme is activated. However, its acyl chain specificity is only observed in liposomes containing 1,2-dioleoyl PA in the presence of Ca2þ. It is proposed that to endow the enzyme with its substrate acyl chain specificity, a highly dehydrated (hydrophobic) membrane interface is needed. The presence of an overlap of mechanisms to regulate DGKε ensures proper phosphatidylinositol cycle function regardless of the trigged stimulus and represents a sophisticated and specialized manner of membrane-enzyme regulation.

SIGNIFICANCE Phosphatidylinositol (PI) lipids play a role in a plethora of signaling pathways. Diacylglycerol kinase ε (DGKε) is a membrane-bound enzyme responsible in part for the enrichment of PI lipids with specific acyl chains. This enzyme does not have a regulatory domain. However, it is shown that the enzyme is very sensitive to the chemical and physical properties of the membrane it binds to. These membrane properties show a broad regulation effect on the enzyme from activation to inhibition. It is proposed that an overlap of different membrane properties is used to ensure proper DGKε function within the PI cycle, the major pathway for recycling of PI lipids upon stimulus.

INTRODUCTION Diacylglycerol kinases (DGKs) are lipid kinases that catalyze the ATP-dependent phosphorylation of diacylglycerol (DAG) to form phosphatidic acid (PA). Both DAG and PA are intermediates in lipid biosynthesis pathways as well as lipid signaling molecules involved in a variety of cellular Submitted July 18, 2019, and accepted for publication September 10, 2019. *Correspondence: [email protected] Editor: Ilya Levental. https://doi.org/10.1016/j.bpj.2019.09.008

processes (1,2). In multicellular organisms, DGKs provide a link between lipid metabolism and signaling. In mammals, 10 DGK isoforms have been identified, which differ in their level of expression, cellular localization, and function (3). With such a central role played by these enzymes, a tight regulation of their activity is needed, which otherwise could lead to cellular malfunction. Indeed, DGK’s dysfunction has been suggested to play a role in a number of pathological conditions (4,5). Based on various structural domains or motifs, mammalian DGKs are classified into five groups (6). These

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Bozelli et al.

structural domains or motifs are believed to play a role in the regulation of these enzymes. Of the 10 isoforms, only the ε (DGKε) does not bear any of these domains or motifs (6). DGKε is the only isoform reported to be permanently membrane bound. In a cell, DGKε has been reported to be associated with the plasma membrane and endoplasmic reticulum (7,8). This isoform has also been suggested to catalyze one of the steps of the phosphatidylinositol (PI) cycle, the major pathway to regenerate PI lipids, which play a role in a plethora of cellular functions (9–12). DGKε substrate acyl chain specificity is believed to be in part responsible for the enrichment of PI lipids with their mature (1-stearoyl-2-arachidonoyl) acyl chains (13,14). Hence, DGKε have an exquisite role within a cell, and understanding its regulation is of crucial importance. There are several mechanisms by which membrane proteins could be regulated by membrane properties. Some of the most acknowledge mechanisms are lipid binding, lateral lipid segregation (domains), lipid packing, and membrane curvature (15–17). The absence of a regulatory domain (motif) in DGKε, as well as the fact it is a membrane-bound enzyme, strongly suggests that membrane physical and chemical properties might be involved in the regulation of this enzyme. Indeed, recently it has been shown that DGKε activity and substrate acyl chain specificity are regulated by membrane shape (18). It was shown that the presence of membrane structures bearing negative Gaussian curvature activates the enzyme and endows it with substrate acyl chain specificity. Because the PI cycle could be triggered by a variety of different stimuli, it seems reasonable that DGKε could also be regulated by other membrane properties, which would lead to an intricate and specific cellular response. Here, the effect of negatively charged phospholipids on DGKε activity and substrate acyl chain specificity was studied. To address this, purified DGKε and liposomes were used. The use of this simple model system is not intended to model the complexity of the whole physiological situation (several proteins, interaction between proteins and/ or metabolites, posttranslational modification, levels of expression, membrane composed of several lipid species, its lipid asymmetry, etc.); but rather, it is to determine specifically the role of membrane physical and chemical properties on the regulation of the enzyme. It is demonstrated that a variety of negatively charged phospholipids inhibit DGKε activity, with the exception of its product PA. The enzyme is subjected to feedback regulation by its product; however, the specific response of the enzyme could vary from activation to inhibition. It is shown that different molecular species of PA could modulate DGKε activity via alterations of the membrane properties. These results indicate a sophisticated regulation of DGKε by the chemical and physical properties of the membrane it binds to. It is proposed that the specific mechanism that will regulate the enzyme will depend on the particular stimulus that triggered the PI cycle.

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MATERIALS AND METHODS Materials All lipids used in this study were purchased from Avanti Polar Lipids (Alabaster, AL). Ni-NTA agarose resin was from Qiagen (Hilden, Germany). g-32P-ATP was purchased from PerkinElmer (Waltham, MA). All reagents were used as received.

DGKε overexpression and purification Human full-length DGKε-His (6) was overexpressed in Sf21 or Sf9 insect cells using recombinant baculovirus for cell infection as described in (19). The enzyme was affinity purified with Ni-NTA resin. The enzyme purification procedure was as described in Bozelli et al. (18). Briefly, cell pellets overexpressing the enzyme were lysed using lysis buffer (20 mM TrisHCl (pH 8), 500 mM NaCl, 20% v/v glycerol, 10 mM imidazole, 1 mM b-glycerophosphate, 1 mM activated sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1:1000 dilution of Roche protease inhibitor cocktail tablet (Sigma-Aldrich, Ontario, Canada) in water, 2% v/v Triton X-100 (Sigma-Aldrich), 0.05% v/v b-mercaptoethanol), and the obtained lysate was clarified by centrifuging it at 13,000  g for 30 min at 4 C. The clarified lysate was incubated with Ni-NTA resin at a ratio of 15:1 (v/v) for 1 h on an inverting rotor at 4 C. After the binding step, the resin was washed three times for 5 min with wash buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20% v/v glycerol, 30 mM imidazole, 0.05% v/v Tween 20, 0.05% v/v b-mercaptoethanol) on an inverting rotor at 4 C. Purified enzyme was eluted using elution buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20% v/v glycerol, 300 mM imidazole, 0.05% v/v Tween 20, 0.05% b-mercaptoethanol) on an inverting rotor at 4 C for 10 min and either used fresh for the experiments or flash frozen with liquid nitrogen and stored at 80 C until used.

Model membrane preparation Lipid stock solutions were prepared from powder in chloroform/methanol, 2:1 (v/v). The concentration of the phospholipids was determined by measuring the amount of inorganic phosphate released after digestion by the method of Ames (20). Lipids at the desired molar ratio were deposited as a film on the wall of a glass test tube by solvent evaporation under a nitrogen flux. Final traces of solvent were removed for 2–3 h in a vacuum chamber attached to a liquid nitrogen trap. The lipid films were then suspended in liposome buffer (10 mM HEPES, 100 mM NaCl (pH 7.2)) by vortexing at room temperature to form MLVs (multilamellar vesicles). MLVs were then submitted to five freeze (dry ice in ethanol)/thaw (water bath at 40 C) cycles before use. The transfer of the lipid mixtures from the films to liposomes in solution was determined as the ratio between the phospholipids in the organic solutions and the total amount of phospholipid transferred. The total amount of phospholipid transferred was found to be within error of quantitative (94 5 4%) and essentially the same for the two lipid mixtures measured (POPC:POPA:DOG and POPC:DOPA:DOG, both at 50:40:10, mol%). We had demonstrated in earlier work that the activity of DGKε in MLVs was very similar to that in 100-nm-diameter large unilamellar vesicles (18). Model membranes were used for experimentation right after preparation.

Enzymatic activity assays Activity assays were performed using previously described methods (19). Briefly, the reaction mixture containing 50 mL of 20 mM model membranes, 50 mL of assay buffer (50 mM HEPES, 300 mM NaCl, 20 mM MgCl2, 4 mM EGTA (pH 7.2)), 20 mL of 10 mM dithiothreitol, and 35 mL of ddH2O were added to a salinized glass tube. Then, 25 mL of purified DGKε was added to the reaction mixture and incubated for 10 min at

Please cite this article in press as: Bozelli et al., Regulation of DGKε Activity and Substrate Acyl Chain Specificity by Negatively Charged Phospholipids, Biophysical Journal (2019), https://doi.org/10.1016/j.bpj.2019.09.008

Anionic Phospholipids Regulate DGKε FIGURE 1 Regulation of DGKε lipid kinase activity by negatively charged phospholipids. The normalized enzymatic activity is presented as a function of (A) POPS mol % and (B) the chemical structure of the negatively charged phospholipid. Enzymatic activity was normalized to values of pure POPC liposomes. In (A), data are presented as the mean 5 SEM (n ¼ 9). The red line represents data fitting with Eq. 1. In (B), data are presented as a box plot (n ¼ 5; *p < 0.01). Lipid composition for POPC was POPC/SAG (90:10, mol %); for the anionic lipids, it was POPC:anionic lipid:SAG (50:40:10, mol %). PS and PA had 1-palmitoyl-2oleoyl acyl chains. Cardiolipin (18:1)4 (CL) had tetralinoleoyl acyl chains. PI was from bovine liver, which is enriched in 1-stearoyl-2-arachidonyl acyl chains. Enzymatic activity assays were performed at 20 5 1 C. To see this figure in color, go online. 20 (5 1) C. The reaction was started by adding 20 mL of 5 mM ATP in the presence of trace amounts of g-32P ATP (50 mCi , mL1). All reactions were performed using a 10-min end point at 20 (51) C, after which the reaction was quenched with 2 ml of stop solution (1:1 v/v; chloroform/methanol, 0.25 mg , mL1 dihexadecyl phosphate). The organic phase was washed three times with 2 ml wash solution (7:1 ddH2O/CH3OH, 1% HClO4, 100 mM H3PO4). The radioactivity of the organic-soluble phase was measured using a liquid scintillation counter (Hidex 300, Turku, Finland). All experiments were conducted within the linear range of enzyme activity with regard to its amount and incubation time (data not shown). The enzyme concentration was not determined for each individual case; rather, it was normalized to the activity of the same preparation in that enzyme assay, and all comparisons were run side by side. It was shown previously that this purification procedure usually yields a purified enzyme in a 1–5 mM concentration range (18). Experiments in the presence of Ca2þ were conducted in absence of EGTA in the assay buffer. To quantify DGKε inhibition by phosphatidylserine (PS), enzyme activity as a function of PS (mol %) data were fitted with Eq. 1 as follows:

A0 þ ðAF  A0 Þ  PSn
Activity ¼ ; An50 þ PSn

(1)

where A0, AF, PS, A50, and n were, respectively, activity in absence of PS, activity at 100% PS, PS mol %, PS mol % for 50% inhibition, and the Hill coefficient.

31

P-NMR

The 31P-NMR spectra from 65 mM of the desired lipid mixture in 10 mM HEPES and 100 mM NaCl (pH 7.2) were obtained using a Bruker AVIII 700 MHz spectrometer (Billerica, MA) equipped with a 5-mm quadruple nuclei cryoprobe over 17-kHz sweep width in 32,768 data points. Block decay spectra were acquired using a 30-degree pulse, inverse gated proton decoupling, and 2 s total recycle time. Each spectrum is representative of two independent runs. Unless otherwise indicated, all NMR spectra were recorded at 25 C sample temperature, and the chemical shift scale was referenced to an external 85% (wt/vol) phosphoric acid standard set to 0 ppm. The spectra were generated by Fourier transform of the first 1024 free induction decay data points.

Differential scanning calorimetry The calorimetric curves from 5 mM of desired lipid mixtures were obtained in a VP-DSC (MicroCal, LLC, Northampton, MA). Lipids as MLVs and buffer were degassed before being loaded into the sample and reference

cells of the calorimeter, respectively. The cell volume was 0.514 ml. Samples were equilibrated in the calorimeter at 5 C, and successive heating and cooling scans (three cycles each) were carried out between 5 and 60 C at a scan rate of 20 C/h with a delay of 15 min between sequential scans for thermal equilibration. The resulting curves had their reference subtracted, baseline adjusted using a cubic line, and normalized by lipid concentration and volume. Each thermogram is representative of two independent runs, and only the third heating scan is presented.

Statistical analysis Statistical analysis was done by performing one-way analysis of variance test on the data set, and statistical significance was assumed for p-values <0.01.

RESULTS DGKε activity is inhibited by negatively charged lipids, except PA DGKε catalyzes the ATP-dependent phosphorylation of DAG to form PA. It has been shown that DGKε activity is modulated by negatively charged lipids; that is, these lipids strongly inhibit DGKε activity in vitro (21,22). However, those experiments were performed using mixed micelles, and it has been recently shown that the modulation of the enzyme activity is different between mixed micelles and liposomes (18). Because the latter more closely resemble a biological membrane, the effects of negatively charged lipids on DGKε enzymatic activity were evaluated using liposomes. DGKε is a membrane-bound enzyme reported to be present in the plasma membrane and endoplasmic reticulum (7,8). In these membranes, PS is one of the most abundant negatively charged lipids (23). Hence, the enzymatic activity of DGKε was measured as a function of increased 1-palmitoyl-2-oleoyl PS (POPS) content in liposomes having 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) as the host lipid (Fig. 1 A). The results showed that increasing POPS mol % gradually inhibited DGKε enzymatic activity. At 20 mol % POPS, the enzymatic activity was inhibited twofold, and the data fitting predicted that in a pure POPS

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liposome, the activity would be inhibited by 98%. This result is in agreement with the literature (21) and showed that regardless of the lipid carrier (mixed micelle versus bilayer), DGKε is inhibited by the negatively charged phospholipid POPS. To evaluate whether the inhibition of DGKε activity was sensitive to the chemical structure of the negatively charged phospholipid, its activity was measured in POPC liposomes containing 40 mol % of different negatively charged phospholipids (PI: liver PI, which is enriched with 1-stearoyl-2-archydonoyl acyl chains; CL: tetraoleoyl cardiolipin; PA: POPA) (Fig. 1 B). At this molar percentage, POPS, liver PI, and tetraoleoyl cardiolipin all inhibited 70% DGKε activity. However, surprisingly, 1-palmitoyl2-oleoyl PA (POPA), which is the product of DGKε catalyzed reaction, led to 4-fold increase in the enzyme activity; that is, POPA led to an activation of DGKε, suggesting a positive feedback regulation of the enzyme. The activation of DGKε by POPA is contrary to the inhibitory effect reported using mixed micelles (22) and highlights how sensitive DGKε is to the lipid environment. PA modulation of DGKε activity is dependent on the nature of its acyl chains The surprising finding of DGKε activation by its product, POPA, led to a further investigation to evaluate whether the PA effect is dependent on the nature of its acyl chains. Fig. 2 A presents DGKε relative activity for POPC liposomes containing 40 mol % of different PA molecular species. The results showed that DGKε activity is differently modulated according to the nature of the PA molecular

FIGURE 2 Feedback regulation of DGKε lipid kinase activity, but not its substrate acyl chain specificity, depends on PA molecular species. The normalized lipid kinase activity (A) and substrate acyl chain specificity (B) as a function of PA molecular species are shown. Activity values were normalized for the activity in pure POPC liposomes, and data are presented as a box plot (n ¼ 5 for POPA, 3 for SOPA and SAPA, and 4 for DOPA; *p <0.01). Substrate acyl chain specificity was measured by quantifying the ratio of enzymatic activity between its preferred substrate (SAG) and its poor substrate (DOG), and data are presented as a box plot (n ¼ 6 for POPA, 3 for SOPA and SAPA, and 4 for DOPA). Lipid composition was POPC:PA:SAG (50:40:10, mol %). PA molecular species were POPA (1-palmitoyl-2-oleoyl PA), SOPA (1-stearoyl-2-oleoyl PA), DOPA (1,2-dioleoyl PA), and SAPA (1-stearoyl-2-arachidonoyl PA). Enzymatic assays were performed at 20 5 1 C. To see this figure in color, go online.

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species; whereas POPA led to an 4-fold increase in DGKε activity, 1-stearoyl-2-oleyl PA (SOPA) and 1,2-dioleoyl PA (DOPA) essentially did not impact the enzyme activity when compared to pure POPC liposomes, and 1stearoyl-2-arachidonoyl PA (SAPA) inhibited the enzyme activity 10-fold. It is important to mention that DGKε bears substrate acyl chain specificity for 1-stearoyl-2-arachidonoyl glycerol (SAG) (13,14,18). When DGKε performs its enzymatic activity on SAG, SAPA is formed. The inhibition of DGKε by SAPA is 3-fold stronger than that observed for all the other negatively charged phospholipids (Fig. 1 B) and suggests a negative feedback regulation. This preferred inhibition by SAPA is in agreement with previous findings (22). The effect of different molecular species of PA on DGKε substrate acyl chain specificity was also evaluated. Fig. 2 B presents the ratio of activity for SAG (the preferred substrate) over DOG (1,2-dioleoyl glycerol, a poor substrate) for POPC liposomes containing different PA molecular species. The data showed that the presence of different molecular species of PA in POPC liposomes did not impact DGKε substrate acyl chain specificity to a great extent, even for POPA (compared to some cases in the presence of Ca2þ, shown below). PA-rich domains enhance DGKε activity but do not endow the enzyme with substrate acyl chain specificity Recently, it has been shown that highly curved membranes activate DGKε (18). The presence of highly curved membrane structures in POPA-containing membranes was evaluated by acquiring the static 31P-NMR spectrum of this sample, which is a well-established method to assess the morphology of membrane phases (Fig. 3 A). The static 31P-NMR of POPAcontaining MLVs showed a powder pattern with two intense peaks in the upfield region, which are ascribed to the phosphate groups of POPC and POPA (Table S1). This static 31PNMR spectrum is characteristic of lipids assembled as a lamellar phase (bilayer), which is in agreement with the fact that POPA is a lamellar-forming lipid in the conditions studied and excludes the possibility of highly curved membranes as the reason for DGKε activation by POPA. Because membrane curvature is not the reason for the POPA activation effect on DGKε, the possibility of lateral lipid segregation was investigated by differential scanning calorimetry (DSC) (Fig. S1; Table S2). The DSC thermogram of POPA-containing MLVs showed a single broad endothermic transition centered at 14.5 C, which is ascribed to the transition from gel to liquid crystal (fluid) phases (Table S2). The enzymatic assays were carried out at 20 C, and this data suggested the lipids were in the liquid crystal (fluid) phase during the enzymatic assays. However, it should be mentioned that DGKε is a lipid kinase, which needs Mg2þ as a cofactor. Mg2þ is known to bind PA (24,25). Hence, it was hypothesized that Mg2þ binding to

Please cite this article in press as: Bozelli et al., Regulation of DGKε Activity and Substrate Acyl Chain Specificity by Negatively Charged Phospholipids, Biophysical Journal (2019), https://doi.org/10.1016/j.bpj.2019.09.008

Anionic Phospholipids Regulate DGKε

FIGURE 3 Structural characterization of PAcontaining MLVs. (A) 31P-NMR of POPC:POPA: SAG (50:40:10, mol %) in absence (black) and presence of 5 mM Mg2þ (red) are shown. (B) 31 P-NMR spectra of POPC:POPA:SAG (black) and POPC:DOPA:SAG (blue) (50:40:10, mol %) in the presence of 5 mM Mg2þ and 10 mM Ca2þ are shown. To see this figure in color, go online.

POPA could trigger lateral lipid segregation. The static 31 P-NMR spectrum showed that POPA-containing liposomes were still arranged as a bilayer (lamellar phase) in the presence of Mg2þ (Fig. 3 A). In addition, the two peaks in the 31P-NMR were shifted upfield in the presence of Mg2þ, indicating the binding of Mg2þ to the phosphate groups of POPC and POPA (Table S1). Interestingly, the DSC thermograms showed the presence of two additional small endothermic transitions in the presence of Mg2þ, one centered at 21.8 C and another one at 47.2 C (Fig. S1; Table S2). These results indicate some segregated higher melting lipid domains in the presence of Mg2þ, which is likely due to Mg2þ binding to POPA leading to lipid lateral segregation. It is acknowledged that the binding of divalent cations (such as Mg2þ and Ca2þ) to PA decrease the apparent pK as of its phosphate headgroup leading to increased negative charge on the PA molecule (26). Thus, Mg2þ and Ca2þ (see below) not only promote the formation of POPA-rich domains, but these cations also increase the negative charge on the phosphate group of POPA. Both the formation of POPA-rich domains as well as the increased negative charge of PA may contribute to increased activity of DGKε. Mg2þ binds to PA at the phosphate headgroup. Thus, it would be expected that Mg2þ would bind to the other PA molecular species because all of them have the same headgroup, differing only by the nature of their acyl chains. However, if lateral lipid segregation is the reason for DGKε activation by POPA, compared to other PA species that show no activation (Fig. 2), then, the other PA molecular species should not present lateral lipid segregation. It should be mentioned that the POPA effect is quantitatively not very large. Mg2þ binding to SOPA was indicated by an 4 C increase in its gel-to-fluid phase transition temperature (Table S2). In the presence of Mg2þ, SOPA-containing liposomes presented a single broad endothermic transition centered at 17.3 C on DSC thermograms (Fig. S1). These results showed that Mg2þ binds to SOPA; however, it does not seem to impact greatly the acyl chain packing of SOPA-containing liposomes. Mg2þ also does not seem to

change significantly the acyl chain packing in DOPA-containing liposomes as suggested by the absence of any phase transition on DSC thermograms (Fig. S1). However, 31PNMR showed Mg2þ binding to DOPA-containing liposomes, which were assembled as a lamellar (bilayer) phase in the absence and presence of Mg2þ (Table S1). Altogether, these data support the hypothesis that POPA activation of DGKε is due to the presence of a small fraction of highly packed PA-rich domains. Ca2D induces changes in PA-containing liposomes physical properties impacting on DGKε activity and substrate acyl chain specificity To test the hypotheses that DGKε activity is enhanced by the presence of PA-rich domains, we used a system aimed to increase the lateral lipid segregation of PA-containing liposomes. It is well accepted that Ca2þ and Mg2þ interact differently with negatively charged phospholipids, with the former, generally, inducing larger changes on the membrane physical properties than the latter (25,27,28). Indeed, Ca2þ binding to PA leads to extensive lateral lipid segregation in mixed-model membranes (29–34). DGKε does not present a Ca2þ binding motif, and it has been shown that its activity and substrate acyl chain specificity is not altered by the presence of Ca2þ itself (6,18). Hence, the addition of Ca2þ to PA-containing liposomes was used as a strategy to increase the lateral lipid segregation caused by divalent cation-anionic lipid interaction. It should be mentioned though that because of the need of high lipid concentrations (5 mM) to measure the activity of the enzyme in the liposome system, we used supraphysiological concentrations of Ca2þ to observe the Ca2þ effect on the physical properties of the model membranes. In addition, because Mg2þ is cofactor for DGKε kinase activity, these experiments needed to be conducted in the presence of Mg2þ. The enzymatic activity as a function of PA molecular species in the presence of Ca2þ is presented (Fig. 4, A and B). The results clearly show an increase in the activity of the enzyme in the

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FIGURE 4 Calcium-PA interaction enhanced DGKε lipid kinase activity, but only calciumDOPA interaction endowed the enzyme with its substrate acyl chain specificity. Enzymatic activity normalized for values in (A) pure POPC liposomes and (B) PA-containing liposomes in the absence of Ca2þ as a function of PA molecular species is shown. (C) Substrate acyl chain specificity as measured by the enzymatic activity ratio between DGKε preferred substrate (SAG) and its poor substrate (DOG) as a function of PA molecular species in the presence of Ca2þ is shown. Data are presented as a box plot (in A and B, n ¼ 5, and in C, n ¼ 6 for POPA; for the remaining, n ¼ 3; *p <0.01). Raft lipid composition was POPC: egg sphingomyelin:cholesterol:SAG (30:30:30:10, mol %). Enzymatic assays were performed at 20 5 1 C. To see this figure in color, go online.

presence of Ca2þ for all PA molecular species (Fig. 4 B). On average, the presence of Ca2þ increased the activity 15fold for POPA and SOPA when normalized for the values of PA-containing membranes in the absence of Ca2þ (Fig. 4 B). The activity in DOPA-containing liposomes was slightly higher (35-fold increase); however, it was only statistically different from that of SAPA (10-fold increase). It should be recalled that SAPA in the absence of Ca2þ inhibits DGKε 10-fold. Hence, although Ca2þ enhanced the enzymatic activity for SAPA-containing membranes, it did not lead to an activation of the enzyme and only brought it to basal levels (Fig. 4 A). The effect on substrate acyl chain specificity for PA-containing liposomes was also evaluated in the presence of Ca2þ (Fig. 4 C). The results showed essentially no differences from the values obtained in absence of Ca2þ for POPA, SOPA, and SAPA (last one is not shown), revealing weak substrate acyl chain specificity in PA-containing liposomes in the presence of Ca2þ. This behavior is different from other cases studied of lipid effects on DGKε in which the specific activity of the enzyme changed together with changes in substrate acyl chain specificity. However, DOPA-containing liposomes were an exception to this and showed a strong (an average of a ninefold difference between SAG and DOG) substrate acyl chain specificity (Fig. 4 C). The effect of Ca2þ on the PA-containing membrane physical properties was evaluated by means of DSC (Fig. S1; Table S2) and 31P-NMR (Fig. 3 B; Table S1).

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DSC thermograms clearly indicated Ca2þ-induced lateral lipid segregation (Fig. S1; Table S2). This is supported by the presence of two broad endothermic transitions for SOPA-containing membranes, one below and another above the temperature of the activity assays. The DSC thermograms of POPA-containing membranes presented a rather complex behavior. For this case, two endothermic transitions below and three above the temperature of the activity assays were observed. Although the understanding of the complexity of this system is beyond the scope of this study, the DSC results seem to corroborate very well the presence of lateral lipid heterogeneities in the plane of the membrane in agreement with several previous studies (29–34). These are in very good agreement with the hypothesis of PA-rich domains enhancing DGKε activity, but not endowing the enzyme with substrate acyl chain specificity. The presence of these endothermic transitions above the activity assay temperature (20 C) suggests that these PA-rich domains are tightly packed because of Ca2þ binding to PA, which is in agreement with the literature (30,31). Moreover, the presence of lipid liquid-liquid immiscibility did not affect DGKε activity and substrate acyl chain specificity, as evinced by the basal levels of activity and weak substrate acyl chain specificity measured in raft-like mixtures (Fig. 4, A and C). Hence, DGKε seems to be activated by the presence of highly packed PA-rich domains, but not by coexisting liquid lipid domains. It should be pointed out that although the divalent cation-induced

Please cite this article in press as: Bozelli et al., Regulation of DGKε Activity and Substrate Acyl Chain Specificity by Negatively Charged Phospholipids, Biophysical Journal (2019), https://doi.org/10.1016/j.bpj.2019.09.008

Anionic Phospholipids Regulate DGKε

highly packed PA-rich domains very likely bear a net negative charge, the liquid lipid domains in the raft mixture bear no net charge. This might be another contributing factor for the differences observed in the two systems, that is, highly packed PA-rich domains and coexisting liquid lipid domains. DOPA-containing liposomes in the presence of Ca2þ do not present any phase transition on DSC thermograms in the temperature range measured (Fig. S1). However, fluorescence and electron microscopy studies reported PA-rich domains in the presence of Ca2þ for DOPA-containing liposomes (32,35). Although the presence of PA-rich domains is in agreement with the activation shown for the other PA molecular species, it alone does not seem to explain the strong acyl chain specificity observed in DOPA-containing liposomes in the presence of Ca2þ (Fig. 4 C), which was the only system in which this property of the enzyme was observed. These results point to different physical properties between DOPA-containing liposomes and liposomes containing other PA molecular species in the presence of Ca2þ. Recently, complex behavior of PCPA lipid mixtures dependent on their acyl chains has been reported (36). Differences between DOPA- and POPA-containing liposomes in the presence of Ca2þ are supported by 31 P-NMR (Fig. 3 B; Table S1). The static 31P-NMR spectra showed that both POPA- and DOPA-containing liposomes are arranged in a bilayer (lamellar) phase in presence of Ca2þ (Fig. 3 B). However, in POPA-containing liposomes, the 31P-NMR spectrum still showed two upfield peaks ascribed to the point of maximal intensity of the phosphatidylcholine (PC) and PA phosphorus powder pattern. The presence of Ca2þ increased their separation (Table S1), which is likely because of the formation of PA-rich domains and increased differences in the chemical microenvironment of the two phosphate groups in the absence and presence of Ca2þ. On the other hand, in DOPA-containing liposomes, the presence of Ca2þ decreased the separation of the two upfield peaks ascribed to PC and PA phosphate groups, which was driven mainly by a downshift of PC phosphate peak (Table S1). This result suggests that both phosphate groups experience a more similar chemical microenvironment in the presence of Ca2þ (Table S1). In addition, the DOPAcontaining liposomes presented a reduction in signal intensity suggesting an increased immobilization of the phosphate groups in the presence of Ca2þ. This behavior is unique for DOPA, among the lipids studied here. DOPA-Ca2þ is also unique in showing the greatest activation and substrate acyl chain specificity (Fig. 4). It is the only anionic lipid mixture that supports enzymatic properties similar to that of highly curved membranes (18). A common property between the highly curved membranes (rich in dioleoyl-phosphatidylethanolamine or Ca2þ-POPS) and the Ca2þ-DOPA system reported here is the low hydration of the membrane-water interface (i.e., a more hydrophobic interface) (18,36).

DISCUSSION In the study of membrane-active enzymes, micelles (or mixed micelles) are usually the system of first choice because of the low cost, ease of use, and compatibility with a number of biophysical techniques. However, these molecular aggregates lack the basic lipid bilayer arrangement of biological membranes as well as the presence of important membrane physical properties. In addition, the presence of the detergent itself may affect the properties of the enzyme. Hence, detergent-free phospholipid aggregates present a more suitable model system to access the impact of membrane physical properties on membraneactive enzymes. DGKε is a membrane-bound enzyme catalyzing one of the steps of the PI cycle, a major pathway for the recycling of PI lipids, which plays a role in a plethora of cell signaling pathways (6). Recently, marked differences have been reported in DGKε activity and substrate acyl chain specificity between mixed micelles and liposomes (18). In our work, the modulation of DGKε activity and substrate acyl chain specificity by negatively charged phospholipids was evaluated in a liposome system. In agreement with results reported in mixed micelles, most of the negatively charged phospholipids showed an inhibitory effect on DGKε activity (21). However, contrary to results reported in mixed micelles, the modulation of DGKε activity by PA, the product of the reaction catalyzed by DGKε, was dependent on the particular PA molecular species as well as on the physical properties of the PA-containing liposomes (22). It was shown that the presence of PA-rich domains enhances DGKε activity; however, it did not endow the enzyme with substrate acyl chain specificity, an important property of the enzyme to function in the PI cycle. The enzymatic activities of many isoforms of DGK are very sensitive to the presence of anionic lipids. In mammals, there are 10 known isoforms of DGKs, and several of these isoforms (a, b, g, z, i, q) have been shown to be strongly activated by negatively charged lipids in vitro (3,37). Instead of an activation effect, in vitro studies using mixed micelles have shown that negatively charged phospholipids strongly inhibit the activity of DGKε (21,22). It has been previously proposed that negatively charged phospholipids such as PS, PI, and phosphatidiylinositol-4,5-phosphate inhibit DGKε by a nonspecific electrostatic interaction with a cluster of positively charged residues in the enzyme N-terminus (residues 44–56) (21). The results presented here are in very good agreement with the literature showing that most negatively charged phospholipids inhibit DGKε in vitro. Moreover, the fact that these lipids inhibit the enzyme regardless if the lipid carrier is either a mixed micelle or a liposome supports the proposal of the inhibition of DGKε by anionic lipids being due to a nonspecific electrostatic interaction between the enzyme cationic cluster on the N-terminus and the negatively charged headgroup of these lipids.

Biophysical Journal 117, 1–10, December 17, 2019 7

Please cite this article in press as: Bozelli et al., Regulation of DGKε Activity and Substrate Acyl Chain Specificity by Negatively Charged Phospholipids, Biophysical Journal (2019), https://doi.org/10.1016/j.bpj.2019.09.008

Bozelli et al.

PA is the product of DGKε catalyzed reactions. PA is a negatively charged phospholipid reported to inhibit DGKε in vitro, which led to the proposition of a negative feedback regulation (22). However, contrary to other negatively charged phospholipids, PA inhibition was proposed to be via a competitive mechanism with the enzyme showing selectivity for PA acyl chains with the strongest inhibitory effect observed for SAPA. The results presented in this study show that the modulation of DGKε by PA is dependent on the membrane’s physical as well as lipid chemical features. It is shown that DGKε modulation by PA could vary greatly from being an activator to an inhibitor of the enzyme activity when assayed in a lipid bilayer (liposome) system. Although for the majority of the PA molecular species studied the data showed that the presence of tightly packed PA-rich domains activate the enzyme, DGKε is strongly inhibited by SAPA, and even the presence of Ca2þ did not lead to activation of enzyme. The strong inhibition observed for SAPA is likely due to a direct interaction with the enzyme catalytic site as reported in mixed micelles and the known presence of an encoded motif in the enzyme responsible to selectively bind 1-stearoyl-2-arachidonoyl acyl chains (13,22). On the other hand, the presence of highly packed PA-rich domains seems to change the membrane’s physical property in a way that the enzyme becomes more active. DAG mixes nonideally with phospholipids yielding DAG-rich and -poor domains even in the fluid phase (38). In the presence of lipid heterogeneities in the plane of membrane, DAG has been shown to decrease the line tension between lipid domains (39). Hence, one likely explanation for the PA-activation effect on DGKε would be that the presence of PA-rich domains leads to a dispersion of DAGrich domains because of the preferential partition of DAG at lipid domain boundaries, which ultimately increase its chemical activity and, therefore, DGKε enzymatic activity. The fact that the enzyme activation was not observed in raft-like lipid mixtures suggested that this effect is specific for the characteristics of the domain. The binding between the oxygen groups in PA headgroup and divalent cations leads to a doubly deprotonated PA headgroup (26). Therefore, in addition to inducing highly packed PA domains, the binding of divalent cations yields domains with more negative charge. Based on previous results and this study, it seems that the cationic cluster at the enzyme N-terminus favors the nonspecific interaction with negatively charged lipids (21). Hence, one possible explanation for differences between divalent cation-induced PA-rich domains and the raft-like mixtures is that the former presents a net negative charge favoring protein interaction with these domains, increasing its concentration at the membrane and, consequently, its enzymatic activity. One of the most remarkable properties of DGKε is its substrate acyl chain specificity, which plays an important role in enriching PI lipids with their mature (1-stearoyl-2arachidonoyl) acyl chains within the PI cycle. Although

8 Biophysical Journal 117, 1–10, December 17, 2019

for a long time it has been believed that this property was due solely to the presence of a conserved encoded motif in the enzyme, recent studies showed that this property is also dependent on the presence of highly curved membrane structures (13,18). In the studies presented here, the only system that endowed the enzyme with substrate acyl chain specificity was DOPA-containing liposomes in the presence of Ca2þ. Although the DSC results presented here did not show any transition in the temperature range studied, fluorescence and electron microscopy studies have reported Ca2þ-induced lateral lipid segregation in DOPA-containing liposomes (32,35). However, the presence of PA-rich domains per se does not seem sufficient to endow DGKε with substrate acyl chain specificity because only weak substrate acyl chain specificity was observed for the other PA molecular species although these data indicated the presence of highly packed PA-rich domains in the presence of Ca2þ. It is important to mention though that according to electron microscopy studies, the Ca2þ-induced PA-rich domain structures in DOPA-containing liposomes were sharply curved membrane surfaces embedded in fluid PC regions (32). The lamellar-to-hexagonal phase transition is a qualitative measure of lipids’ spontaneous curvature. For instance, whereas Ca2þ induces the lamellar-to-hexagonal phase transition of DOPA liposomes at pH 6, this is not observed for egg PA, which has the predominant acyl chain 1-palmitoyl-2-oleoyl (40,41). Hence, differences in the morphology of PA-rich domains would be one likely reason of the strong acyl chain specificity observed in DOPA-containing liposomes in the presence of Ca2þ. This suggestion is supported by differences in the 31P-NMR powder pattern of POPA- and DOPA-containing liposomes in the presence of Ca2þ reported here and is in very good agreement with recent findings showing that highly curved membranes positively allosterically regulate DGKε activity and substrate acyl chain specificity (18). Thus, it is conceivable that the different morphology of PA-rich domains of DOPA-containing membranes in the presence of Ca2þ might provide an energetically more favorable environment for the localization of polyunsaturated acyl chains (see below) such as the one found in SAG. This would allow the enzyme to differentiate the acyl chains of its DAG substrate. DGKε is 1 of 10 known isoforms of mammalian DGK. It is the smallest among the known DGK isoforms and it is the only isoform lacking a domain that could bind a putative allosteric modulator. Results of our work together with those from recent studies of DGKε in curved membranes (18) demonstrate the extraordinary sensitivity of this enzyme to the properties of the membrane to which it is bound. Both the specific activity of the enzyme as well as its substrate acyl chain specificity are dependent on the shape of the membrane (18) as well as on the species of anionic lipid that is present and whether this occurs in the presence of calcium. A unifying feature that might be common to both kinds of activating membranes is a dehydrated

Please cite this article in press as: Bozelli et al., Regulation of DGKε Activity and Substrate Acyl Chain Specificity by Negatively Charged Phospholipids, Biophysical Journal (2019), https://doi.org/10.1016/j.bpj.2019.09.008

Anionic Phospholipids Regulate DGKε

(hydrophobic) membrane-water interface. This more dehydrated membrane-water interface would be of particular importance for DGKε substrate acyl chain specificity. A number of biophysical studies have shown that in model membranes containing mixed (1-saturated-2-unsaturated) acyl chains phospholipids, polyunsaturated acyl chains are more frequently found at the membrane-water interface than saturated or monounsaturated acyl chains (42). It seems reasonable to propose that a dehydrated membrane-water interface would be a more energetically favorable environment for the localization of the polyunsaturated acyl chains in comparison to a more hydrated membrane-water interface. The preferred substrate of DGKε is SAG, which is a mixed acyl chain lipid containing a polyunsaturated acyl chain (arachidonoyl, 20:4) at position sn-2. Indeed, NMR and x-ray studies suggested a more interfacial localization SAG arachidonoyl acyl chain in comparison to its stearoyl acyl chain (43). DGKε has been reported to have a conserved encoded motif responsible for binding the arachidonoyl acyl chain (13). Although a membrane-bound enzyme, DGKε is not an integral membrane protein (8), which should nevertheless be able to distinguish the acyl chains of its lipid substrate. Membrane properties that favor an interfacial localization of the arachidonoyl acyl chain of the SAG seem to be those that endow the enzyme with its substrate acyl chain specificity. The fact that DGKε is so highly regulated by multiple factors suggests that this regulation plays an important role. DGKε activity will contribute to affect the rate of cycling of the PI cycle. The role of the PI cycle is to enrich PI species with 18:0/20:4 acyl chains (see for example (44)). Hence, a component to facilitate this enrichment is increased activity and substrate acyl chain specificity of DGKε. This is an important biological role suggested by the fact that there is a backup system in the form of the enzymes of the Lands cycle. The presence of an overlap of membrane properties able to regulate DGKε suggests a sophisticated and specific manner to engage DGKε in the PI cycle regardless of the stimulus that triggered the cycle.

dration (increased hydrophobicity) is a common factor for engaging DGKε in the PI cycle. SUPPORTING MATERIAL Supporting Material can be found online at https://doi.org/10.1016/j.bpj. 2019.09.008.

AUTHOR CONTRIBUTIONS J.C.B., J.Y., Y.H.H., P.C., A.F., and M.C. conducted and analyzed the kinase activity assays. J.C.B. performed and analyzed the 31P-NMR and DSC measurements. Y.H.H. and Y.T. overexpressed the DGKε. J.Y., Y.H.H., P.C., A.F., and Z.C. purified the DGKε. J.C.B. and R.M.E. designed the experiments, analyzed the data, and wrote the manuscript.

ACKNOWLEDGMENTS We appreciate the technical support of Alma Seitova, Ashley Hutchinson, and Yanjun Li (Structural Genomics Consortium) in the cloning and expression of DGKε. This work was supported by Canadian Natural Sciences and Engineering Research Council grant RGPIN-2018-05585 to R.M.E. The Structural Genomics Consortium is a registered charity of the United Kingdom (no. 1097737) that receives funds from AbbVie, Bayer, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada through the Ontario Genomics Institute (OGI-055), Innovative Medicines Initiative (European Union/European Federation of Pharmaceutical Industries and Associations) (Unrestricted Leveraging of Targets for Research Advancement and Drug Discovery grant 115766), Janssen, Merck Kommanditgesellschaft Auf Aktien, Merck Sharp & Dohme, Novartis, Ontario Ministry of Research, Innovation and Science, Pfizer, Sa˜o Paulo Research Foundation-FAPESP, Takeda, and the Wellcome Trust.

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In conclusion, this work showed that DGKε activity and substrate acyl chain specificity is very sensitive to the chemical and physical properties of the membrane it is bound to. Whereas negatively charged phospholipids inhibit DGKε activity, the regulation by its product, PA, varies greatly from activation to inhibition. The feedback regulation of DGKε by PA depends on the particular molecular species of PA as well as the membrane’s physical properties. Because the PI cycle could be triggered by a variety of stimulus, there should be an overlap of mechanisms to ensure the substrate acyl chain specificity of DGKε, which is an important property of the enzyme in the PI cycle. It is proposed that changes in the membrane that lead to interfacial dehy-

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