Munc13 Is a Molecular Target of Bryostatin 1

Blanco, Francisco Arrieta; Czikora, Ágnes; Kedei, Noémi; You, Youngki; Mitchell, Gary; Pany, Satyabrata; Ghosh, Alip; Blumberg, Peter M.; Das, Joydip

doi:10.1021/acs.biochem.9b00427

Cited by 9 publications

(5 citation statements)

References 69 publications

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“…The docking site is similar to those of our previous docking studies for resveratrol and bryostatin-1. 58,59 However, in PKCδ C1B, the phorbol 13-acetate formed three hydrogen bonds with Thr-242 and Leu-251 that are located deep inside the binding pocket (Figure 2B). The phorbol 13-acetate binding sites of Munc13-1 C1 and PKCδ C1B (PDB entry 1PTQ) are slightly different because of the different orientation of Trp-588.…”

Section: ■ Resultsmentioning

confidence: 99%

Probing the Diacylglycerol Binding Site of Presynaptic Munc13-1

You

Katti

et al. 2021

Biochemistry

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Munc13-1 is a presynaptic active zone protein that acts as a master regulator of synaptic vesicle priming and neurotransmitter release in the brain. It has been implicated in the pathophysiology of several neurodegenerative diseases. Diacylglycerol and phorbol ester activate Munc13-1 by binding to its C1 domain. The objective of this study is to identify the structural determinants of ligand binding activity of the Munc13-1 C1 domain. Molecular docking suggested that residues Trp-588, Ile-590, and Arg-592 of Munc13-1 are involved in ligand interactions. To elucidate the role of these three residues in ligand binding, we generated W588A, I590A, and R592A mutants in full-length Munc13-1, expressed them as GFP-tagged proteins in HT22 cells, and measured their ligand-induced membrane translocation by confocal microscopy and immunoblotting. The extent of 1,2-dioctanoyl-sn-glycerol (DOG)-and phorbol ester-induced membrane translocation decreased in the following order: wild type > I590A > W588A > R592A and wild type > W588A > I590A > R592A, respectively. To understand the effect of the mutations on ligand binding, we also measured the DOG binding affinity of the isolated wild-type C1 domain and its mutants in membrane-mimicking micelles using nuclear magnetic resonance methods. The DOG binding affinity decreased in the following order: wild type > I590A > R592A. No binding was detected for W588A with DOG in micelles. This study shows that Trp-588, Ile-590, and Arg-592 are essential determinants for the activity of Munc13-1 and the effects of the three residues on the activity are ligand-dependent. This study bears significance for the development of selective modulators of Munc13-1.

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Section: ■ Resultsmentioning

confidence: 99%

Probing the Diacylglycerol Binding Site of Presynaptic Munc13-1

You

Katti

et al. 2021

Biochemistry

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“…Of relevance, Bryostatin 1 was discovered to target UNC13A in vitro. 99 Bryostatin 1 is a natural compound isolated from a specific genus of moss animals, which is a diglyceride (DAG) homologue. DAG interacts both with protein kinase C (PKC) domain as well as the C1-domain of Munc13-1, which were both found to have similar structures.…”

Section: Other Potential Therapeutic Strategiesmentioning

confidence: 99%

“…Of relevance, Bryostatin 1 was discovered to target UNC13A in vitro 99. Bryostatin 1 is a natural compound isolated from a specific genus of moss animals, which is a diglyceride (DAG) homologue.…”

Section: Potential Treatments Targeting Unc13amentioning

confidence: 99%

UNC13Ain amyotrophic lateral sclerosis: from genetic association to therapeutic target

Willemse

Harley

Eijk

et al. 2023

J Neurol Neurosurg Psychiatry

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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with limited treatment options and an incompletely understood pathophysiology. Although genomewide association studies (GWAS) have advanced our understanding of the disease, the precise manner in which risk polymorphisms contribute to disease pathogenesis remains unclear. Of relevance, GWAS have shown that a polymorphism (rs12608932) in theUNC13Agene is associated with risk for both ALS and frontotemporal dementia (FTD). Homozygosity for the C-allele at rs12608932 modifies the ALS phenotype, as these patients are more likely to have bulbar-onset disease, cognitive impairment and FTD at baseline as well as shorter survival. UNC13A is expressed in neuronal tissue and is involved in maintaining synaptic active zones, by enabling the priming and docking of synaptic vesicles. In the absence of functional TDP-43, risk variants inUNC13Alead to the inclusion of a cryptic exon inUNC13Amessenger RNA, subsequently leading to nonsense mediated decay, with loss of functional protein. Depletion ofUNC13Aleads to impaired neurotransmission. Recent discoveries have identifiedUNC13Aas a potential target for therapy development in ALS, with a confirmatory trial with lithium carbonate inUNC13Acases now underway and future approaches with antisense oligonucleotides currently under consideration. ConsideringUNC13Ais a potent phenotypic modifier, it may also impact clinical trial outcomes. This present review describes the path from the initial discovery ofUNC13Aas a risk gene in ALS to the current therapeutic options being explored and how knowledge of its distinct phenotype needs to be taken into account in future trials.

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“…In order to develop modulators of Munc13-1 for regulating the related disease states, we studied various ligands for their effects on Munc13-1 activity. − Here, we studied the effect of three DAG-lactones, JH-131e-153, AJH-836, and 130C037 (Figure ), on the activation of Munc13-1 and its mutants. In addition to the different stereochemistry around the double bond ( E versus Z ), it is important to recognize that only JH-131e-153 is enantiopure ( R -enantiomer), while the other two DAG-lactones are racemates.…”

Section: Introductionmentioning

confidence: 99%

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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Munc13 Is a Molecular Target of Bryostatin 1

Cited by 9 publications

References 69 publications

Probing the Diacylglycerol Binding Site of Presynaptic Munc13-1

Probing the Diacylglycerol Binding Site of Presynaptic Munc13-1

UNC13Ain amyotrophic lateral sclerosis: from genetic association to therapeutic target

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

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