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

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.

Probing the Diacylglycerol Binding Site of Presynaptic Munc13-1.

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.

SNARE Complex-Associated Proteins and Alcohol.

Alcohol addiction causes major health problems throughout the world, causing numerous deaths and incurring a huge economic burden to society. To develop an intervention for alcohol addiction, it is necessary to identify molecular target(s) of alcohol and associated molecular mechanisms of alcohol action. The functions of many central and peripheral synapses are impacted by low concentrations of ethanol (EtOH). While the postsynaptic targets and mechanisms are studied extensively, there are limited studies on the presynaptic targets and mechanisms. This article is an endeavor in this direction, focusing on the effect of EtOH on the presynaptic proteins associated with the neurotransmitter release machinery. Studies on the effects of EtOH at the levels of gene, protein, and behavior are highlighted in this article.

Effect of ethanol on Munc13-1 C1 in Membrane: A Molecular Dynamics Simulation Study.

BACKGROUND: EtOH has a significant effect on synaptic plasticity. Munc13-1 is an essential presynaptic active zone protein involved in priming the synaptic vesicle and releasing neurotransmitter in the brain. It is a peripheral membrane protein and binds to the activator, diacylglycerol (DAG)/phorbol ester at its membrane-targeting C1 domain. Our previous studies identified Glu-582 of C1 domain as the alcohol-binding residue (Das, J. et al, J. Neurochem., 126, 715-726, 2013). METHODS: Here, we describe a 250 ns molecular dynamics (MD) simulation study on the interaction of EtOH and the activator-bound Munc13-1 C1 in the presence of varying concentrations of phosphatidylserine (PS). RESULTS: In this study, Munc13-1 C1 shows higher conformational stability in EtOH than in water. It forms fewer hydrogen bonds with phorbol 13-acetate in the presence of EtOH than in water. EtOH also affected the interaction between the protein and the membrane and between the activator and the membrane. Similar studies in a E582A mutant suggest that these effects of EtOH are mostly mediated through Glu-582. CONCLUSIONS: EtOH forms hydrogen bonds with Glu-582. While occupancy of the EtOH molecules at the vicinity (4Å) of Glu-582 is 34.4%, the occupancy in the E582A mutant is 26.5% of the simulation time. In addition, the amount of PS in the membrane influences the conformational stability of the C1 domain and interactions in the ternary complex. This study is important in providing the structural basis of EtOH's effects on synaptic plasticity.

Munc13 Is a Molecular Target of Bryostatin 1.

Bryostatin 1 is a natural macrolide shown to improve neuronal connections and enhance memory in mice. Its mechanism of action is largely attributed to the modulation of novel and conventional protein kinase Cs (PKCs) by binding to their regulatory C1 domains. Munc13-1 is a C1 domain-containing protein that shares common endogenous and exogenous activators with novel and conventional PKC subtypes. Given the essential role of Munc13-1 in the priming of synaptic vesicles and neuronal transmission overall, we explored the potential interaction between bryostatin 1 and Munc13-1. Our results indicate that in vitro bryostatin 1 binds to both the isolated C1 domain of Munc13-1 ( Ki = 8.07 ± 0.90 nM) and the full-length Munc13-1 protein ( Ki = 0.45 ± 0.04 nM). Furthermore, confocal microscopy and immunoblot analysis demonstrated that in intact HT22 cells bryostatin 1 mimics the actions of phorbol esters, a previously established class of Munc13-1 activators, and induces plasma membrane translocation of Munc13-1, a hallmark of its activation. Consistently, bryostatin 1 had no effect on the Munc13-1H567K construct that is insensitive to phorbol esters. Effects of bryostatin 1 on the other Munc13 family members, ubMunc13-2 and bMunc13-2, resembled those of Munc13-1 for translocation. Lastly, we observed an increased level of expression of Munc13-1 following a 24 h incubation with bryostatin 1 in both HT22 and primary mouse hippocampal cells. This study characterizes Munc13-1 as a molecular target of bryostatin 1. Considering the crucial role of Munc13-1 in neuronal function, these findings provide strong support for the potential role of Munc13s in the actions of bryostatin 1.

Critical Role of Trp-588 of Presynaptic Munc13-1 for Ligand Binding and Membrane Translocation.

Munc13-1 is a presynaptic active-zone protein essential for neurotransmitter release and presynaptic plasticity in the brain. This multidomain scaffold protein contains a C1 domain that binds to the activator diacylglycerol/phorbol ester. Although the C1 domain bears close structural homology with the C1 domains of protein kinase C (PKC), the tryptophan residue at position 22 (588 in the full-length Munc13-1) occludes the activator binding pocket, which is not the case for PKC. To elucidate the role of this tryptophan, we generated W22A, W22K, W22D, W22Y, and W22F substitutions in the full-length Munc13-1, expressed the GFP-tagged constructs in Neuro-2a cells, and measured their membrane translocation in response to phorbol ester treatment by imaging of the live cells using confocal microscopy. The extent of membrane translocation followed the order, wild-type > W22K > W22F > W22Y > W22A > W22D. The phorbol ester binding affinity of the wild-type Munc13-1C1 domain and its mutants was phosphatidylserine (PS)-dependent following the order, wild-type > W22K > W22A ≫ W22D in both 20% and 100% PS. Phorbol ester affinity was higher for Munc13-1 than the C1 domain. While Munc13-1 translocated to the plasma membrane, the C1 domain translocated to internal membranes in response to phorbol ester. Molecular dynamics (80 ns) studies reveal that Trp-22 is relatively less flexible than the homologous Trp-22 of PKCδ and PKCθ. Results are discussed in terms of the overall negative charge state of the Munc13-1C1 domain and its possible interaction with the PS-rich plasma membrane. This study shows that Trp-588 is an important structural element for ligand binding and membrane translocation in Munc13-1.

Structural determinants of phorbol ester binding activity of the C1a and C1b domains of protein kinase C theta.

The PKC isozymes represent the most prominent family of signaling proteins mediating response to the ubiquitous second messenger diacylglycerol. Among them, PKCθ is critically involved in T-cell activation. Whereas all the other conventional and novel PKC isoforms have twin C1 domains with potent binding activity for phorbol esters, in PKCθ only the C1b domain possesses potent binding activity, with little or no activity reported for the C1a domain. In order to better understand the structural basis accounting for the very weak ligand binding of the PKCθ C1a domain, we assessed the effect on ligand binding of twelve amino acid residues which differed between the C1a and C1b domains of PKCθ. Mutation of Pro9 of the C1a domain of PKCθ to the corresponding Lys9 found in C1b restored in vitro binding activity for [3H]phorbol 12,13-dibutyrate to 3.6 nM, whereas none of the other residues had substantial effect. Interestingly, the converse mutation in the C1b domain of Lys9 to Pro9 only diminished binding affinity to 11.7 nM, compared to 254 nM in the unmutated C1a. In confocal experiments, deletion of the C1b domain from full length PKCθ diminished, whereas deletion of the C1a domain enhanced 5-fold (at 100 nM PMA) the translocation to the plasma membrane. We conclude that the Pro168 residue in the C1a domain of full length PKCθ plays a critical role in the ligand and membrane binding, while exchanging the residue (Lys240) at the same position in C1b domain of full length PKCθ only modestly reduced the membrane interaction.

Polyphenol compounds and PKC signaling.

BACKGROUND: Naturally occurring polyphenols found in food sources provide huge health benefits. Several polyphenolic compounds are implicated in the prevention of disease states, such as cancer. One of the mechanisms by which polyphenols exert their biological actions is by interfering in the protein kinase C (PKC) signaling pathways. PKC belongs to a superfamily of serine-threonine kinase and are primarily involved in phosphorylation of target proteins controlling activation and inhibition of many cellular processes directly or indirectly. SCOPE OF REVIEW: Despite the availability of substantial literature data on polyphenols' regulation of PKC, no comprehensive review article is currently available on this subject. This article reviews PKC-polyphenol interactions and its relevance to various disease states. In particular, salient features of polyphenols, PKC, interactions of naturally occurring polyphenols with PKC, and future perspective of research on this subject are discussed. MAJOR CONCLUSIONS: Some polyphenols exert their antioxidant properties by regulating the transcription of the antioxidant enzyme genes through PKC signaling. Regulation of PKC by polyphenols is isoform dependent. The activation or inhibition of PKC by polyphenols has been found to be dependent on the presence of membrane, Ca(2+) ion, cofactors, cell and tissue types etc. Two polyphenols, curcumin and resveratrol are in clinical trials for the treatment of colon cancer. GENERAL SIGNIFICANCE: The fact that 74% of the cancer drugs are derived from natural sources, naturally occurring polyphenols or its simple analogs with improved bioavailability may have the potential to be cancer drugs in the future.

Resveratrol inhibits phorbol ester-induced membrane translocation of presynaptic Munc13-1.

BACKGROUND: Resveratrol (1) is a naturally occurring polyphenol that has been implicated in neuroprotection. One of resveratrol's several biological targets is Ca2+-sensitive protein kinase C alpha (PKCα). Resveratrol inhibits PKCα by binding to its activator-binding C1 domain. Munc13-1 is a C1 domain-containing Ca2+-sensitive SNARE complex protein essential for vesicle priming and neurotransmitter release. METHODS: To test if resveratrol could also bind and inhibit Munc13-1, we studied the interaction of resveratrol and its derivatives, (E)-1,3-dimethoxy-5-(4-methoxystyryl)benzene, (E)-5,5'-(ethene-1,2-diyl)bis(benzene-1,2,3-triol), (E)-1,2-bis(3,4,5-trimethoxyphenyl)ethane, and (E)-5-(4-(hexadecyloxy)-3,5-dihydroxystyryl)benzene-1,2,3-triol with Munc13-1 by studying its membrane translocation from cytosol to plasma membrane in HT22 cells and primary hippocampal neurons. RESULTS: Resveratrol, but not the derivatives inhibited phorbol ester-induced Munc13-1 translocation from cytosol to membrane in HT22 cells and primary hippocampal neurons, as evidenced by immunoblot analysis and confocal microscopy. Resveratrol did not show any effect on Munc13-1H567K, a mutant which is not sensitive to phorbol ester. Binding studies with Munc13-1 C1 indicated that resveratrol competes with phorbol ester for the binding site. Molecular docking and dynamics studies suggested that hydroxyl groups of resveratrol interact with phorbol-ester binding residues in the binding pocket. CONCLUSIONS AND SIGNIFICANCE: This study characterizes Munc13-1 as a target of resveratrol and highlights the importance of dietary polyphenol in the management of neurodegenerative diseases.

Fokker-Planck equation for the non-Markovian Brownian motion in the presence of a magnetic field.

In the present study, we have proposed the Fokker-Planck equation in a simple way for a Langevin equation of motion having ordinary derivative (OD), the Gaussian random force and a generalized frictional memory kernel. The equation may be associated with or without conservative force field from harmonic potential. We extend this method for a charged Brownian particle in the presence of a magnetic field. Thus, the present method is applicable for a Langevin equation of motion with OD, the Gaussian colored thermal noise and any kind of linear force field that may be conservative or not. It is also simple to apply this method for the colored Gaussian noise that is not related to the damping strength.

Selective Modulation of Protein Kinase C α over Protein Kinase C ε by Curcumin and Its Derivatives in CHO-K1 Cells.

Members of the protein kinase C (PKC) family of serine/threonine kinases regulate various cellular functions, including cell growth, differentiation, metabolism, and apoptosis. Modulation of isoform-selective activity of PKC by curcumin (1), the active constituent of Curcuma L., is poorly understood, and the literature data are inconsistent and obscure. The effect of curcumin (1) and its analogues, 4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl oleate (2), (9Z,12Z)-4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl octadeca-9,12-dienoate (3), (9Z,12Z,15Z)-4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl octadeca-9,12,15-trienoate (4), and (1E,6E)-1-[4-(hexadecyloxy)-3-methoxyphenyl]-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (5), and didemethylcurcumin (6) on the membrane translocation of PKCα, a conventional PKC, and PKCε, a novel PKC, has been studied in CHO-K1 cells, in which these PKC isoforms are endogenously expressed. Translocation of PKC from the cytosol to the membrane was measured using immunoblotting and confocal microscopy. 1 and 6 inhibited the TPA-induced membrane translocation of PKCα but not of PKCε. Modification of the hydroxyl group of curcumin with a long aliphatic chain containing unsaturated double bonds in 2-4 completely abolished this inhibition property. Instead, 2-4 showed significant translocation of PKCα but not of PKCε to the membrane. No membrane translocation was observed with 1, 6, or the analogue 5 having a saturated long chain for either PKCα or PKCε. 1 and 6 inhibited TPA-induced activation of ERK1/2, and 2-4 activated it. ERK1/2 is the downstream readout of PKC. These results show that the hydroxyl group of curcumin is important for PKC activity and the curcumin template can be useful in developing isoform specific PKC modulators for regulating a particular disease state.

Curcumin Inhibits Protein Kinase Cα Activity by Binding to Its C1 Domain.

Curcumin is a polyphenolic nutraceutical that acts on multiple biological targets, including protein kinase C (PKC). PKC is a family of serine/threonine kinases central to intracellular signal transduction. We have recently shown that curcumin selectively inhibits PKCα, but not PKCε, in CHO-K1 cells [Pany, S. (2016) Biochemistry 55, 2135-2143]. To understand which domain(s) of PKCα is responsible for curcumin binding and inhibitory activity, we made several domain-swapped mutants in which the C1 (combination of C1A and C1B) and C2 domains are swapped between PKCα and PKCε. Phorbol ester-induced membrane translocation studies using confocal microscopy and immunoblotting revealed that curcumin inhibited phorbol ester-induced membrane translocation of PKCε mutants, in which the εC1 domain was replaced with αC1, but not the PKCα mutant in which αC1 was replaced with the εC1 domain, suggesting that αC1 is a determinant for curcumin's inhibitory effect. In addition, curcumin inhibited membrane translocation of PKCε mutants, in which the εC1A and εC1B domains were replaced with the αC1A and αC1B domains, respectively, indicating the role of both αC1A and αC1B domains in curcumin's inhibitory effects. Phorbol 13-acetate inhibited the binding of curcumin to αC1A and αC1B with IC50 values of 6.27 and 4.47 µM, respectively. Molecular docking and molecular dynamics studies also supported the higher affinity of curcumin for αC1B than for αC1A. The C2 domain-swapped mutants were inactive in phorbol ester-induced membrane translocation. These results indicate that curcumin binds to the C1 domain of PKCα and highlight the importance of this domain in achieving PKC isoform selectivity.

Alcohol binding in the C1 (C1A+C1B) domain of protein kinase C epsilon.

BACKGROUND: Alcohol regulates the expression and function of protein kinase C epsilon (PKCε). In a previous study we identified an alcohol binding site in the C1B, one of the twin C1 subdomains of PKCε (Das et al., Biochem. J., 421, 405-13, 2009). METHODS: In this study, we investigated alcohol binding in the entire C1 domain (combined C1A and C1B) of PKCε. Fluorescent phorbol ester, SAPD and fluorescent diacylglycerol (DAG) analog, dansyl-DAG were used to study the effect of ethanol, butanol, and octanol on the ligand binding using fluorescence resonance energy transfer (FRET). To identify alcohol binding site(s), PKCεC1 was photolabeled with 3-azibutanol and 3-azioctanol, and analyzed by mass spectrometry. The effects of alcohols and the azialcohols on PKCε were studied in NG108-15 cells. RESULTS: In the presence of alcohol, SAPD and dansyl-DAG showed different extent of FRET, indicating differential effects of alcohol on the C1A and C1B subdomains. Effects of alcohols and azialcohols on PKCε in NG108-15 cells were comparable. Azialcohols labeled Tyr-176 of C1A and Tyr-250 of C1B. Inspection of the model structure of PKCεC1 reveals that these residues are 40Šapart from each other indicating that these residues form two different alcohol binding sites. CONCLUSIONS: The present results provide evidence for the presence of multiple alcohol-binding sites on PKCε and underscore the importance of targeting this PKC isoform in developing alcohol antagonists.

Identification of the activator-binding residues in the second cysteine-rich regulatory domain of protein kinase Cθ (PKCθ).

PKC (protein kinase C) θ is predominantly expressed in T-cells and is critically involved in immunity. Design of PKCθ-selective molecules to manage autoimmune disorders by targeting its activator-binding C1 domain requires the knowledge of its structure and the activator-binding residues. The C1 domain consists of twin C1 domains, C1A and C1B, of which C1B plays a critical role in the membrane translocation and activation of PKCθ. In the present study we determined the crystal structure of PKCθC1B to 1.63 Å (1 Å=0.1 nm) resolution, which showed that Trp(253) at the rim of the activator-binding pocket was orientated towards the membrane, whereas in PKCδC1B the homologous tryptophan residue was orientated away from the membrane. This particular orientation of Trp(253) affects the size of the activator-binding pocket and the membrane affinity. To further probe the structural constraints on activator-binding, five residues lining the activator-binding site were mutated (Y239A, T243A, W253G, L255G and Q258G) and the binding affinities of the PKCθC1B mutants were measured. These mutants showed reduced binding affinities for phorbol ester [PDBu (phorbol 12,13-dibutyrate)] and diacylglycerol [DOG (sn-1,2-dioctanoylglycerol), SAG (sn-1-stearoyl 2-arachidonyl glycerol)]. All five full-length PKCθ mutants exhibited reduced phorbol-ester-induced membrane translocation compared with the wild-type. These results provide insights into the PKCθ activator-binding domain, which will aid in future design of PKCθ-selective molecules.

The pre-synaptic Munc13-1 binds alcohol and modulates alcohol self-administration in Drosophila.

Munc13-1 is a pre-synaptic active-zone protein essential for neurotransmitter release and involved in pre-synaptic plasticity in brain. Ethanol, butanol, and octanol quenched the intrinsic fluorescence of the C1 domain of Munc13-1 with EC50 s of 52 mM, 26 mM, and 0.7 mM, respectively. Photoactive azialcohols photolabeled Munc13-1 C1 exclusively at Glu-582, which was identified by mass spectrometry. Mutation of Glu-582 to alanine, leucine, and histidine reduced the alcohol binding two- to five-fold. Circular dichroism studies suggested that binding of alcohol increased the stability of the wild-type Munc13-1 compared with the mutants. If Munc13-1 plays some role in the neural effects of alcohol in vivo, changes in the activity of this protein should produce differences in the behavioral responses to ethanol. We tested this prediction with a loss-of-function mutation in the conserved Dunc-13 in Drosophila melanogaster. The Dunc-13(P84200) /+ heterozygotes have 50% wild-type levels of Dunc-13 mRNA and display a very robust increase in ethanol self-administration. This phenotype is reversed by the expression of the rat Munc13-1 protein within the Drosophila nervous system. The present studies indicate that Munc13-1 C1 has binding site(s) for alcohols and Munc13-1 activity is sufficient to restore normal self-administration to Drosophila mutants deficient in Dunc-13 activity. The pre-synaptic Mun13-1 protein is a critical regulator of synaptic vesicle fusion and may be involved in processes that lead to ethanol abuse and addiction. We studied its interaction with alcohol and identified Glu-582 as a critical residue for ethanol binding. Munc13-1 can functionally complement the Dunc13 haploinsufficient ethanol self-administration phenotype in Drosophila melanogaster, indicating that this protein participates in alcohol-induced behavioral plasticity.

Comparison of the ligand binding site of C1 domains: a molecular dynamics simulation study of the C1 domain-phorbol 13-acetate-membrane system.

C1 domains are lipid-binding structural units of about 50 residues. Typical C1 domains associate with the plasma membrane and bind to diacylglycerol/phorbol ester during the activation of the proteins containing these domains. Although the overall structure of the C1 domains are similar, there are differences in their primary sequence and in the orientation of the ligand/lipid binding residues. To gain structural insights into the ligand/lipid binding, we performed molecular docking of phorbol 13-acetate into the C1 domain and 1.0 µs molecular dynamics simulation on the C1 domain-ligand-lipid ternary system for PKCθ C1A, PKCδ C1B, PKCßII C1B, PKCθ C1B, Munc13-1 C1, and ßII-Chimaerin C1. We divided these C1 domains into three types based on the orientations of Gln-27 and Trp/Tyr-22. In type 1, Trp/Tyr-22 is outside and Gln-27 is inside the ligand binding pocket. In type 2, both Trp/Tyr-22 and Gln-27 are outside the ligand binding pocket, and in type 3, Trp/Tyr-22 is inside and Gln-27 is outside the pocket. The type 1 C1 domains showed higher ligand binding and higher membrane binding with a shorter distance between the C1 domain and the membrane than the type 2 and type 3. For ligand binding, Pro-11 plays a major role in the type 1 and 2, and Gly-23 in the type 1 and type 3 C1 domains. This study elucidates the role of Gln-27, Trp-22, Pro-11 and Gly-23 in ligand/lipid binding in typical C1 domains and bears significance in developing selective modulators of C1 domain-containing proteins.Communicated by Ramaswamy H. Sarma.

Structural and functional characterization of an anesthetic binding site in the second cysteine-rich domain of protein kinase Cδ*.

Elucidating the principles governing anesthetic-protein interactions requires structural determinations at high resolutions not yet achieved with ion channels. Protein kinase C (PKC) activity is modulated by general anesthetics. We solved the structure of the phorbol-binding domain (C1B) of PKCδ complexed with an ether (methoxymethylcycloprane) and with an alcohol (cyclopropylmethanol) at 1.36-Å resolution. The cyclopropane rings of both agents displace a single water molecule in a surface pocket adjacent to the phorbol-binding site, making van der Waals contacts with the backbone and/or side chains of residues Asn-237 to Ser-240. Surprisingly, two water molecules anchored in a hydrogen-bonded chain between Thr-242 and Lys-260 impart elasticity to one side of the binding pocket. The cyclopropane ring takes part in π-acceptor hydrogen bonds with the amide of Met-239. There is a crucial hydrogen bond between the oxygen atoms of the anesthetics and the hydroxyl of Tyr-236. A Tyr-236-Phe mutation results in loss of binding. Thus, both van der Waals interactions and hydrogen-bonding are essential for binding to occur. Ethanol failed to bind because it is too short to benefit from both interactions. Cyclopropylmethanol inhibited phorbol-ester-induced PKCδ activity, but failed to do so in PKCδ containing the Tyr-236-Phe mutation.

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

Munc13-1 is a presynaptic active zone protein that plays a critical role in priming the synaptic vesicle and releasing neurotransmitters in the brain. Munc13-1 acts as a scaffold and is activated when diacylglycerol (DAG)/phorbol ester binds to its C1 domain in the plasma membrane. Our previous studies showed that bryostatin 1 activated the Munc13-1, but resveratrol inhibited the phorbol ester-induced Munc13-1 activity. To gain structural insights into the binding of the ligand into Munc13-1 C1 in the membrane, we conducted 1.0 µs molecular dynamics (MD) simulation on Munc13-1 C1-ligand-lipid ternary system using phorbol 13-acetate, bryostatin 1 and resveratrol as ligands. Munc13-1 C1 shows higher conformational stability and less mobility along membrane with phorbol 13-acetate and bryostatin 1 than with resveratrol. Bryostatin 1 and phorbol ester remained in the protein active site, but resveratrol moved out of Munc13-1 C1 during the MD simulation. While bryostatin 1-bound Munc13-1 C1 showed two different positioning in the membrane, phorbol 13-acetate and resveratrol-bound Munc13-1 C1 only showed one positioning. Phorbol 13-acetate formed hydrogen bond with Ala-574 and Gly-589. Bryostatin 1 had more hydrogen bonds with Trp-588 and Arg-592 than with other residues. Resveratrol formed hydrogen bond with Ile-590. This study suggests that different ligands control Munc13-1 C1's mobility and positioning in the membrane differently. Ligand also has a critical role in the interaction between Munc13-1 C1 and lipid membrane. Our results provide structural basis of the pharmacological activity of the ligands and highlight the importance of membrane in Munc13-1 activity.Communicated by Ramaswamy H. Sarma.

Differential Expression of Presynaptic Munc13-1 and Munc13-2 in Mouse Hippocampus Following Ethanol Drinking.

The Munc13 family of proteins is critically involved in synaptic vesicle priming and release in glutamatergic neurons in the brain. Munc13-1 binds to alcohol and, in Drosophila, modulates sedation sensitivity and self-administration. We examined the effect of alcohol consumption on the expression of Munc13-1 and Munc13-2, NMDA receptor subunits GluN1, GluN2A and GluN2B in the hippocampus-derived HT22 cells, hippocampal primary neuron culture, and wild-type and Munc13-1+/- male mouse hippocampus after ethanol consumption (Drinking in the Dark (DID) paradigm). In HT22 cells, Munc13-1 was upregulated following 25 mM ethanol treatment for 24 h. In the primary neuronal culture, however, the expression of both Munc13-1 and Munc13-2 increased after ethanol exposure. While Munc13-1 was upregulated in the hippocampus, Munc13-2 was downregulated following DID. This differential effect was found in the CA1 subfield of the hippocampus. Although Munc13-1+/- mice had approximately 50% Munc13-1 expression compared to wild-type, it was nonetheless significantly increased following DID. Munc13-1 and Munc13-2 were expressed in vesicular glutamate transporter1 (VGLUT1) immunoreactive neurons in the hippocampus, but ethanol did not alter the expression of VGLUT1. The NMDA receptor subunits, GluN1, GluN2A and GluN2B were upregulated in the hippocampal primary culture and in the CA1. Ethanol exerts a differential effect on the expression of Munc13-1 and Munc13-2 in the CA1 in male mice. Our study also found that ethanol's effect on Munc13 expression is dependent on the experimental paradigm, and both Munc13-1 and Munc13-2 could contribute to the ethanol-induced augmentation of glutamatergic neurotransmission.

Modulation of proteasome activity by curcumin and didemethylcurcumin.

Modulation of proteasome function by pharmacological interventions and molecular biology tools is an active area of research in cancer biology and neurodegenerative diseases. Curcumin (diferuloylmethane) is a naturally occurring polyphenol that affects multiple signaling pathways. Curcumin shows anti-inflammatory, antioxidant, anti-angiogenic, or anti-apoptotic properties. Recent research suggests that the therapeutic efficacy of curcumin may be due to its activity as a potent inhibitor of the proteasome. Using in vitro cell culture and molecular docking methods, here we show that both curcumin and its synthetic polyphenolic derivative (didemethylcurcumin, CUIII) modulated proteasome activity in a biphasic manner. Curcumin and CUIII increased proteasome activity at nanomolar concentrations, but inhibited proteasome activity at micromolar concentrations. Curcumin was more effective than CUIII in increasing relative proteasome activity at nanomolar concentrations. Also, curcumin was more effective than CUIII in inhibiting relative proteasome activity at micromolar concentrations. Docking simulations of curcumin and didemethylcurcumin binding to the 20S proteasome catalytic subunit estimated Kd values of 0.0054 µM and 1.3167 µM, respectively. These values correlate well with the results of the effectiveness of modulation by curcumin compared to CUIII. The small size of CUIII allows it to dock to the narrow cavity of the active site, but the binding interaction is not strong compared to curcumin. These results indicate that curcumin and its didemethyl derivative can be used to modulate proteasome activity and suggest that curcumin and its didemethyl derivative may be useful in treating two different disease classes: neurodegeneration and cancer.Communicated by Ramaswamy H. Sarma.