Inactivation of human DGAT2 by oxidative stress on cysteine residues

<div><p>Diacylglycerol acyltransferases (DGATs) have a crucial role in the biosynthesis of triacylglycerol (TG), the major storage form of metabolic energy in eukaryotic organisms. Even though DGAT2, one of two distinct DGATs, has a vital role in TG biosynthesis, little is known about the regulation of DGAT2 activity. In this study, we examined the role of cysteine and its oxidation in the enzymatic activity of human DGAT2 <i>in vitro</i>. Human DGAT2 activity was considerably inhibited not only by thiol-modifying reagents (NEM and IA) but also by ROS-related chemicals (H₂O₂ and β-lapachone), while human DGAT1 and GPAT1 were little affected. Particularly, ROS-related chemicals concomitantly induced intermolecular disulfide crosslinking of human DGAT2. Both the oxidative inactivation and disulfide crosslinking were almost completely reversed by the treatment with DTT, a disulfide-reducing agent. These results clearly demonstrated the significant role of ROS-induced intermolecular crosslinking in the inactivation of human DGAT2 and also suggested DGAT2 as a redox-sensitive regulator in TG biosynthesis.</p></div

Additional file 3: Figure S3. of Leucine-Rich Repeat Kinase 2 (LRRK2) phosphorylates p53 and induces p21WAF1/CIP1 expression

Increased phosphorylation of Thr in the p53 TXR sites in dopaminergic neurons differentiated from fibroblast-derived iPS cells that were obtained from the human G2019S carrier (WT/GS) and non-carrier (WT/WT). The indicated cell lysates were immunoprecipitated with the p53 antibody and the immunoprecipitates (IP: p53) were subjected to Western blot. Input indicates 20% of the cell lysates. Numbers below the gel figures are relative protein level of each TXR band ([p-TXR]/[total p53]) based on the densitometric analysis. The antibodies used for Western blot analysis were indicated in the right side of each blot. (DOC 57 kb

Additional file 1: Figure S1. of Leucine-Rich Repeat Kinase 2 (LRRK2) phosphorylates p53 and induces p21WAF1/CIP1 expression

LRRK2 phosphorylates p53 and moesin at TXR sites in the in vitro kinase assay. A. Phosphorylation of various p53 proteins by the Flag-tagged full length LRRK2 protein (Invitrogen) after the in vitro kinase assay. Phosphorylated p53 was detected by either autoradiography or Western blot with the p-TXR antibody. B. Western blot analysis after the in vitro kinase assay using moesin, various GST-ΔN-LRRK2 WT and mutant proteins, and cold ATP. (DOC 88 kb

Additional file 4: Figure S4. of Leucine-Rich Repeat Kinase 2 (LRRK2) phosphorylates p53 and induces p21WAF1/CIP1 expression

LRRK2 kinase inhibitor decreased p21 expression. Differentiated SH-SY5Y cells were treated with 1 μM LRRK2-IN-1 for 2 h (A-D) or 0.5 μM GSK2578215A (Tocris Biosciences, Bristol, United Kingdom) for 12 h (E). The p53 immunoprecipitates of the cell lysates were used to detect p-TXR level (A). The LRRK2-IN-1 treated cells were also used to determine relative amount of nuclear p53 (B) as in Fig. 3a. The cell lysates were used to detect p21 mRNA (C) and protein (D) levels as in Figs. 4 and 5. p21 expression level of GSK2578215A treated cells were also tested (E). Activities of LRRK2 kinase inhibitor treatments were confirmed by reduced level of LRRK2 p935 (pS935). – indicates vehicle treatment. Lamin B and LDH were used for nuclear and cytosolic markers, respectively. All experiments were repeated three times, and a representative result is shown with a bar graph. *: p <0.05; **: p <0.01. (DOC 188 kb

Additional file 5: Figure S5. of Leucine-Rich Repeat Kinase 2 (LRRK2) phosphorylates p53 and induces p21WAF1/CIP1 expression

Expression of T304/377D mutant increased cytotoxicity in rat primary neurons. The neuronal cells were transfected with vector, HA-p53, T304/377D and T304/377A. The cytotoxicity was measured by LDH assay. n = 6. **: p <0.01; ***: p <0.001. (DOC 49 kb

Multimeric complex of human DGAT2 formed by H₂O₂-induced disulfide crosslinking in human cells.

<p>Huh-7 cells were transfected with plasmid overexpressing human DGAT2 for 47 hours and further incubated with indicated concentrations of H₂O₂ for 1 hour. Cell extracts were harvested in a way described in Materials and Methods section and subjected to Western blot analysis using anti-DGAT2 antibody (A). The amount of monomeric human DGAT2 proteins presented as redDGAT2 in (A) was quantified and the amount of relative redDGAT2 protein was calculated by setting the values from samples treated with PBS to 100% (B). The mean values and standard deviations were determined from three independent experiments. Asterisks indicate non-specific bands.</p

Susceptibility of human DGAT2 activity to cysteine-specific modifying reagents.

<p>(A) Membrane extracts from human DGAT2-overexpressing Sf9 insect cells were treated with indicated concentrations of NEM or IA. Human DGAT2 activity was measured by using the conventional extraction-based <i>in vitro</i> DGAT assay. The relative DGAT2 activity in percentage was calculated by setting the value from DMSO-treated sample to 100%. (B) Selective inhibitory effect of NEM on human DGAT2 activity compared to that on human DGAT1 and GPAT1. Membrane extracts from human DGAT2-, DGAT1-, or GPAT1-overexpressing Sf9 insect cells were treated with indicated concentrations of NEM or DMSO. Human DGAT1, DGAT2, and GPAT1 activity was measured by using the conventional extraction-based <i>in vitro</i> assays which are described in detail in the Materials and Methods section. The relative enzyme activity in percentage was calculated by setting the value from DMSO-treated sample to 100%. The mean values and standard deviations were determined from four independent assays.</p

Multimeric complex of human DGAT2 formed by ROS-induced intermolecular disulfide crosslinking <i>in vitro</i>.

<p>Membrane extracts from human DGAT2-overexpressing Sf9 insect cells were treated with H₂O₂ (A) or β-lapachone (B) in the presence or absence of 20 mM DTT and subjected to Western blot analysis using anti-DGAT2 antibody. The amount of monomeric human DGAT2 proteins presented as redDGAT2 in (A) and (B) was quantified and the amount of relative redDGAT2 protein was calculated by setting the values from samples treated with PBS (C) or DMSO (D) to 100%. Asterisk indicates a non-specific band.</p

Significant role of cysteines in human DGAT2 activity.

<p>(A) The location of cysteine residues in human DGAT2 was depicted by asterisks. The transmembrane domains (TMD) and the highly conserved domain are indicated by black and grey squares, respectively. Black vertical line indicates the HPHG motif. (B) The amount of newly synthesized TG in HEK293 cells overexpressing flag-tagged wild-type, mutants (C87A, C96A, C99A, C172A, C214A and C312A) with single cysteine to alanine substitution and mutant (C0) human DGAT2 with all cysteines to alanine substitution. Wild-type and mutant human DGAT2 were overexpressed in HEK293 cells for 42 hours and incubated in the presence of [¹⁴C] glycerol for additional 6 hours. Newly synthesized [¹⁴C] TG was extracted and measured by TLC analysis. The relative TG synthesis in percentage was calculated by setting the value from pcDNA3 vector-transfected cells to 100%. The mean values and standard deviations were determined from three independent experiments. (C) The immunoblots of wild-type and mutant human DGAT2. Flag-tagged wild-type and mutant human DGAT2 were overexpressed in HEK293 cells for 48 hours. Cell extracts were harvested and subjected to Western blot analysis using anti-flag antibody. Magoh protein was examined as a loading control. (D) The normalized human DGAT2 activities of wild-type and mutant human DGAT2. Relative human DGAT2 activities were determined by dividing the amount of newly synthesized TG in panel B by the relative protein amount in panel C and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181076#pone.0181076.s002" target="_blank">S2 Fig</a>. The normalized activity of wild-type DGAT2-transfected cells was defined as 100%.</p

Inhibitory effect of ROS and ROS generator on human DGAT2 catalytic activity.

<p>Membrane extracts from human DGAT2-overexpressing Sf9 insect cells were treated with indicated concentrations of H₂O₂ (A) or β-lapachone (B) in the presence or absence of 20 mM DTT. Human DGAT2 activity was measured by using the conventional extraction-based <i>in vitro</i> assays which are described in detail in the Materials and Methods section. The activities of membrane extracts treated with PBS (instead of H₂O₂) or DMSO (instead of β-lapachone) in the absence of DTT were defined as 100%. The mean values and standard deviations were determined from four independent experiments.</p