Probing the Diacylglycerol Binding Site of Presynaptic Munc13-1

You, Youngki; Katti, Sachin; Yu, Binhan; Igumenova, Tatyana I.; Das, Joydip

doi:10.1021/acs.biochem.1c00165

Cited by 4 publications

(8 citation statements)

References 73 publications

(171 reference statements)

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“…The extent of 1,2-dioctanoyl-sn-glycerol (DOG)-and phorbol esterinduced membrane translocation was in the order WT > I590A > W588A > R592A and WT > W588A > I590A > R592A, respectively. 64 In the present study for both AJH-836 and JH-131e-153, reduction of translocations was observed at both 1 and 10 μM. For JH-131e-153, at 10 μM, the order was WT > I590 ≈ R592A ≈ W588A.…”

Section: ■ Discussionsupporting

confidence: 53%

“…This different orientation of tryptophan may cause different ligand− protein interactions in Munc13-1 and PKCδ. Our recent study 64 using NMR suggests that Trp-588 can change its orientation upon ligand binding. Also, while the crystallization of AJH-836 with PKCδ C1B was done in the presence of lipids, water, and ions, our docking studies were performed only with the protein and the ligand.…”

Section: ■ Discussionmentioning

confidence: 99%

“…In our earlier study, we mutated Trp-588, Ile-590, and Arg-592 residues to the corresponding alanine residue, measured the ligand-induced membrane translocation activity of these mutants, and concluded that these residues are important for DAG/phorbol ester binding (Figure 6A). 64 To compare between DAG/phorbol ester and DAG-lactones in Munc13-1 activation, we used these three mutants here for measuring DAG-lactone-induced membrane translocation. We found that like in the case of DAG/phorbol ester, DAG-lactone-induced membrane translocation was reduced for all three mutants as compared to the wild-type (WT) protein, suggesting the involvement of these residues in DAG-lactone binding.…”

Section: ■ Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…In our previous study, we found that Trp-588, Ile-590, and Arg-592 residues of the ligand-binding site of Munc13-1 C1 are critical for interacting with diacylglycerol and phorbol ester. 64 To understand whether these residues are also involved in the interaction with DAG-lactones or not, these three residues of Munc13-1 C1 were mutated to alanine and their membrane translocation properties were measured. The HT22 cells expressing Munc13-1 were treated with JH-131e-153 and AJH-836 for 30 min and imaged by confocal microscopy (Figure 3).…”

Section: Effect Of Dag-lactones On the Membrane Translocation Of Munc...mentioning

confidence: 99%

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Activation of Munc13-1 by Diacylglycerol (DAG)-Lactones

Das,

You,

Mathukumalli

et al. 2023

Biochemistry

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Munc13-1 is a key protein necessary for vesicle fusion and neurotransmitter release in the brain. Diacylglycerol (DAG)/phorbol ester binds to its C1 domain in the plasma membrane and activates it. The C1 domain of Munc13-1 and protein kinase C (PKC) are homologous in terms of sequence and structure. In order to identify small-molecule modulators of Munc13-1 targeting the C1 domain, we studied the effect of three DAG-lactones, (R,Z)-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-yl)methyl pivalate (JH-131e-153), (E)-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-yl)methyl pivalate (AJH-836), and (E)-(2-(hydroxymethyl)-4-(4-nitrobenzylidene)-5-oxotetrahydrofuran-2-yl)methyl 4-(dimethylamino)benzoate (130C037), on Munc13-1 activation using the ligand-induced membrane translocation assay. JH-131e-153 showed higher activation than AJH-836, and 130C037 was not able to activate Munc13-1. To understand the role of the ligand-binding site residues in the activation process, three alanine mutants were generated. For AJH-836, the order of activation was wild-type (WT) Munc13-1 > R592A > W588A > I590A. For JH-131e-153, the order of activation was WT > I590 ≈ R592A ≈ W588A. Overall, the Z isomer of DAG-lactones showed higher potency than the E isomer and Trp-588, Ile-590, and Arg-592 were important for its binding. When comparing the activation of Munc13-1 and PKC, the order of activation for JH-131e-153 was PKCα > Munc13-1 > PKCε and for AJH-836, the order of activation was PKCε > PKCα > Munc13-1. Molecular docking supported higher binding of JH-131e-153 than AJH-836 with the Munc13-1 C1 domain. Our results suggest that DAG-lactones have the potential to modulate neuronal processes via Munc13-1 and can be further developed for therapeutic intervention for neurodegenerative diseases.

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Section: ■ Discussionsupporting

confidence: 53%

Section: ■ Discussionmentioning

confidence: 99%

Section: ■ Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Effect Of Dag-lactones On the Membrane Translocation Of Munc...mentioning

confidence: 99%

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Activation of Munc13-1 by Diacylglycerol (DAG)-Lactones

Das,

You,

Mathukumalli

et al. 2023

Biochemistry

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Triglycerides Stabilize Water/Organic Interfaces of Changing Area via Conformational Flexibility

Kinard,

Wrenn

2024

Langmuir

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The role of triglycerides (TGs) in both natural and synthetic biological membranes has long been the subject of study, involving metabolism, disease, and colloidal synthesis. TGs have been found to be critical components for successful liposomal encapsulation via a water/oil/water double emulsion, which this work endeavors to explain. TGs can occupy multiple positions in biological membranes. The glycerol backbone can reside at the water/organic interface, adjacent to phospholipid headgroups ("m" conformation), typically with relatively low (<3%) solubility. The glycerol backbone can also occupy hydrophobic regions, where it is isolated from water ("h" or "oil" conformation). This can occur in either midmembrane positions or phospholipid-coated lipid droplets (LDs). These conformations can be distinguished using 13 C-nuclear magnetic resonance spectroscopy (NMR), which determines the degree of hydration of the TG backbone. Using this method, it was revealed that TGs transition from "m" to "h" conformation as the organic solvent is removed via evaporation. A new transitional TG backbone position has been identified with a level of hydration between "m" and "h". These results suggest that TGs can temporarily coat and stabilize the large water/organic interfaces present after emulsification. As the organic solvent is removed and interfaces shrink, the TGs recede into midmembrane spaces or bud off into LDs, which are confirmed via transmission electron microscopy (TEM) and can be removed via centrifugation. Encapsulation efficiency is found to be inversely related to both the saturation and length of the TG acyl chains, indicating that membrane fluidization is a key property arising from the presence of TGs. Beyond clarification of a mechanism for high-efficiency liposomal encapsulation, these results implicate TGs as components that are able to stabilize biological membrane transitions involving a changing interfacial area and curvature. This role for TGs may be of use in the formulation of drug delivery systems as well as in the investigation of membrane transitions in life sciences.

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Molecular dynamics simulation studies on binding of activator and inhibitor to Munc13-1 C1 in the presence of membrane

You

Das

2021

Journal of Biomolecular Structure and Dynamics

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Probing the Diacylglycerol Binding Site of Presynaptic Munc13-1

Cited by 4 publications

References 73 publications

Activation of Munc13-1 by Diacylglycerol (DAG)-Lactones

Activation of Munc13-1 by Diacylglycerol (DAG)-Lactones

Triglycerides Stabilize Water/Organic Interfaces of Changing Area via Conformational Flexibility

Molecular dynamics simulation studies on binding of activator and inhibitor to Munc13-1 C1 in the presence of membrane

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