Dioleoyl‐phosphatidic acid selectively binds to α‐synuclein and strongly induces its aggregation

Mizuno, Seitoku; Sasai, Hirotaka; Kume, Atsumi; Takahashi, Daisuke; Satoh, Mamoru; Kado, Satoshi; Sakane, Fumio

doi:10.1002/1873-3468.12592

Cited by 35 publications

(41 citation statements)

References 45 publications

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“…α‐Syn‐N most strongly bound to 18:1/18:1‐PA and considerably interacted with 16:0/16:0‐ and 16:0/18:1‐PA (Fig. ), consistent with our previous results using full‐length α‐Syn .…”

Section: Resultssupporting

confidence: 91%

“…). Consistent with a previous report using full‐length α‐Syn , α‐Syn‐N exhibited relatively broad selectivity for PA species, including 18:1/18:1‐PA, 16:0/18:1‐, and 16:0/16:0‐PA (Fig. A,B).…”

Section: Discussionsupporting

confidence: 91%

“…We first performed a liposome cosedimentation assay of purified 6 × His‐SUMO‐tagged α‐Syn and its deletion mutants (Fig. ), the N‐terminal region (α‐Syn‐N), the non‐amyloid‐β‐component (α‐Syn‐NAC), the C‐terminal region (α‐Syn‐C), and NAC and C (α‐Syn‐NAC‐C), using 18:1/18:1‐PA, to which full‐length α‐Syn strongly bound . As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To prepare 6 × His‐SUMO‐fused constructs, the cDNAs of α‐Syn (Met1–Ala140), α‐Syn‐N (Met1–Lys60), α‐Syn‐NAC (Glu61–Val95), α‐Syn‐C (Lys96–Ala140), and α‐Syn‐(NAC‐C) (Glu61–Ala140; Fig. ), flanked by NdeI and XhoI restriction sites, were amplified by PCR from the pET‐14b‐mouse α‐Syn and inserted into the p6 × His‐SUMO vector. The resulting plasmids were used to transform Escherichia coli strain Rosetta‐gami 2 (DE3) (Novagen‐Merck, Darmstadt, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we found that α‐synuclein (α‐Syn) strongly and selectively bound to PA . Therefore, we explored the possibility of α‐Syn as a promising PA sensor.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of α‐synuclein N‐terminal domain as a novel cellular phosphatidic acid sensor

et al. 2019

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Tracking the localization and dynamics of the intracellular bioactive lipid phosphatidic acid (PA) is important for understanding diverse biological phenomena. Although several PA sensors have been developed, better ones are still needed for comprehensive PA detection in cells. We recently found that a-synuclein (a-Syn) selectively and strongly bound to PA in vitro. Here, we revealed that the N-terminal region of a-Syn (a-Syn-N) specifically bound to PA, with a dissociation constant of 6.6 lM. a-Syn-N colocalized with PAproducing enzymes, diacylglycerol kinase (DGK) b at the plasma membrane (PM), myristoylated DGKf at the Golgi apparatus, phorbol ester-stimulated DGKc at the PM, and phospholipase D2 at the PM and Golgi but not with the phosphatidylinositol-4,5-bisphosphate-producing enzyme in COS-7 cells. However, a-Syn-N failed to colocalize with them in the presence of their inhibitors and/or their inactive mutants. These results indicate that a-Syn-N specifically binds to cellular PA and can be applied as an excellent PA sensor.

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Section: Resultssupporting

confidence: 91%

Section: Discussionsupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Recently, we found that α‐synuclein (α‐Syn) strongly and selectively bound to PA . Therefore, we explored the possibility of α‐Syn as a promising PA sensor.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of α‐synuclein N‐terminal domain as a novel cellular phosphatidic acid sensor

et al. 2019

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1‐Stearoyl‐2‐docosahexaenoyl‐phosphatidic acid interacts with and activates Praja‐1, the E3 ubiquitin ligase acting on the serotonin transporter in the brain

Lü

Murakami

et al. 2020

FEBS Letters

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Edited by Sandro SonninoSerotonin transporter (SERT) is involved in serotonergic system regulation and in the pathophysiology/therapeutics of serotonin-/SERT-related diseases such as obsessive-compulsive disorder, depression, autism, and schizophrenia. We recently revealed that diacylglycerol (DG) kinase (DGK) d induces ubiquitination/degradation of SERT in a DGK activity-dependent manner through Praja-1 E3 ubiquitin-protein ligase. However, it is still unclear how Praja-1 activity is regulated by DGKd. Here, we reveal that 1-stearoyl-2-docosahexaenoyl (18:0/22:6)-phosphatidic acid (PA) and 18:0/22:6-DG are simultaneously decreased and accumulated, respectively, in the DGKd-knockout mouse brain, indicating that DGKd selectively phosphorylates 18:0/22:6-DG to generate 18:0/22:6-PA. Moreover, we find that 18:0/22:6-PA selectively binds to Praja-1 and enhances its activity. These results strongly suggest that 18:0/ 22:6-PA generated by DGKd activates Praja-1 to degrade SERT in the brain.Keywords: diacylglycerol kinase; docosahexaenoic acid; E3 ubiquitinprotein ligase; obsessive-compulsive disorder; Praja-1; serotonin transporter Serotonin/5-hydroxytryptamine (5-HT) is known to be involved in anxiety, depression, compulsiveness, and impulsivity [1]. The serotonin/5-HT transporter (SERT) reuptakes 5-HT from the synaptic cleft into the presynaptic neuron for recycling and metabolic degradation [2,3]. Selective serotonin reuptake (SERT) inhibitors (SSRIs) are used for the treatment of obsessive-compulsive disorder (OCD) [2,3] and major depressive disorder [4]. Several studies using SERTdeficient rodents have demonstrated that SERT is implicated in neurodevelopmental disorders, such as depression, anxiety, and autism [5]. Therefore, hyperfunction of SERT, which reduces the amount of 5-HT in the synaptic cleft, causes various psychiatric disorders mentioned above. However, the regulatory mechanisms of SERT function are still obscure.Diacylglycerol (DG) kinase (DGK), which consists of ten isozymes (a-j), is a lipid-metabolizing enzyme that phosphorylates DG to generate phosphatidic acid (PA) [6][7][8][9][10]. DG and PA are established lipid second messengers that regulate a wide variety of physiological and pathological events.

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Diacylglycerol Kinase η Activity in Cells Using Protein Myristoylation and Cellular Phosphatidic Acid Sensor

Ishizaki

Murakami

Yamada

et al. 2021

Lipids

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Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid (PtdOH) and regulates the balance between two lipid second messengers: diacylglycerol and PtdOH. Several lines of evidence suggest that the η isozyme of DGK is involved in the pathogenesis of bipolar disorder. However, the detailed molecular mechanisms regulating the pathophysiological functions remain unclear. One reason is that it is difficult to detect the cellular activity of DGKη. To overcome this difficulty, we utilized protein myristoylation and a cellular PtdOH sensor, the N-terminal region of α-synuclein (α-Syn-N). Although DGKη expressed in COS-7 cells was broadly distributed in the cytoplasm, myristoylated (Myr)-AcGFP-DGKη and Myr-AcGFP-DGKη-KD (inactive (kinase-dead) mutant) were substantially localized in the plasma membrane. Moreover, DsRed monomer-α-Syn-N significantly colocalized with Myr-AcGFP-DGKη but not Myr-AcGFP-DGKη-KD at the plasma membrane. When COS-7 cells were osmotically shocked, all DGKη constructs were exclusively translocated to osmotic shock-responsive granules (OSRG). DsRed monomerα-Syn-N markedly colocalized with only Myr-AcGFP-DGKη at OSRG and exhibited a higher signal/background ratio (3.4) than Myr-AcGFP-DGKη at the plasma membrane in unstimulated COS-7 cells (2.5), indicating that α-Syn-N more effectively detects Myr-AcGFP-DGKη activity in OSRG. Therefore, these results demonstrated that the combination of myristoylation and the PtdOH sensor effectively detects DGKη activity in cells and that this method is convenient to examine the molecular functions of DGKη. Moreover, this method will be useful for the development of drugs targeting DGKη. Furthermore, the combination of myristoylation (intensive accumulation in membranes) and α-Syn-N can be applicable to assays for various cytosolic PtdOH-generating enzymes.

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Dioleoyl‐phosphatidic acid selectively binds to α‐synuclein and strongly induces its aggregation

Cited by 35 publications

References 45 publications

Characterization of α‐synuclein N‐terminal domain as a novel cellular phosphatidic acid sensor

Characterization of α‐synuclein N‐terminal domain as a novel cellular phosphatidic acid sensor

1‐Stearoyl‐2‐docosahexaenoyl‐phosphatidic acid interacts with and activates Praja‐1, the E3 ubiquitin ligase acting on the serotonin transporter in the brain

Diacylglycerol Kinase η Activity in Cells Using Protein Myristoylation and Cellular Phosphatidic Acid Sensor

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