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1.
Proc Natl Acad Sci U S A ; 119(2)2022 01 11.
Article in English | MEDLINE | ID: mdl-34996868

ABSTRACT

Lipoprotein-associated phospholipase A2 (Lp-PLA2) associates with low- and high-density lipoproteins in human plasma and specifically hydrolyzes circulating oxidized phospholipids involved in oxidative stress. The association of this enzyme with the lipoprotein's phospholipid monolayer to access its substrate is the most crucial first step in its catalytic cycle. The current study demonstrates unequivocally that a significant movement of a major helical peptide region occurs upon membrane binding, resulting in a large conformational change upon Lp-PLA2 binding to a phospholipid surface. This allosteric regulation of an enzyme's activity by a large membrane-like interface inducing a conformational change in the catalytic site defines a unique dimension of allosterism. The mechanism by which this enzyme associates with phospholipid interfaces to select and extract a single phospholipid substrate molecule and carry out catalysis is key to understanding its physiological functioning. A lipidomics platform was employed to determine the precise substrate specificity of human recombinant Lp-PLA2 and mutants. This study uniquely elucidates the association mechanism of this enzyme with membranes and its resulting conformational change as well as the extraction and binding of specific oxidized and short acyl-chain phospholipid substrates. Deuterium exchange mass spectrometry coupled with molecular dynamics simulations was used to define the precise specificity of the subsite for the oxidized fatty acid at the sn-2 position of the phospholipid backbone. Despite the existence of several crystal structures of this enzyme cocrystallized with inhibitors, little was understood about Lp-PLA2's specificity toward oxidized phospholipids.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Allosteric Regulation , Binding Sites , Catalysis , Catalytic Domain , Fatty Acids , Humans , Hydrolysis , Lipoproteins, HDL/metabolism , Membranes , Molecular Dynamics Simulation , Phospholipids/metabolism , Substrate Specificity
2.
Elife ; 112022 01 07.
Article in English | MEDLINE | ID: mdl-34994688

ABSTRACT

The lissencephaly 1 gene, LIS1, is mutated in patients with the neurodevelopmental disease lissencephaly. The Lis1 protein is conserved from fungi to mammals and is a key regulator of cytoplasmic dynein-1, the major minus-end-directed microtubule motor in many eukaryotes. Lis1 is the only dynein regulator known to bind directly to dynein's motor domain, and by doing so alters dynein's mechanochemistry. Lis1 is required for the formation of fully active dynein complexes, which also contain essential cofactors: dynactin and an activating adaptor. Here, we report the first high-resolution structure of the yeast dynein-Lis1 complex. Our 3.1 Å structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1's ß-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1's ability to form fully active human dynein complexes and to regulate yeast dynein's mechanochemistry and in vivo function.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , Cytoplasmic Dyneins/genetics , Dyneins/genetics , Gene Expression Regulation , Microtubule-Associated Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Cytoplasmic Dyneins/metabolism , Dyneins/metabolism , Dyneins/ultrastructure , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism
3.
Chem Phys Lipids ; 239: 105103, 2021 09.
Article in English | MEDLINE | ID: mdl-34116047

ABSTRACT

Intracellular platelet activating-factor acetylhydrolase type II (PAF-AH II) is a 40-kDa monomeric enzyme. It was originally identified as an enzyme that hydrolyzes the acetyl group of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). As a member of phospholipase A2 super family, PAF-AH II has broad substrate specificity. It can hydrolyze phospholipids with relatively short-length or oxidatively modified sn-2 chains which endows it with various functions such as protection against oxidative stress, transacetylase activity and producing lipid mediators. PAF-AH II has been proven to be involved in several diseases such as allergic diseases, oxidative stress-induced injury and ischemia injury, thus it has drawn more attention from researchers. In this paper, we outline an entire summary of PAF-AH II, including its structure, substrate specificity, activity assay, inhibitors and biological activities.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Phospholipids/metabolism , 1-Alkyl-2-acetylglycerophosphocholine Esterase/antagonists & inhibitors , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Hydrolysis , Oxidation-Reduction , Phospholipids/chemistry , Substrate Specificity
4.
Med Res Rev ; 40(1): 79-134, 2020 01.
Article in English | MEDLINE | ID: mdl-31140638

ABSTRACT

Inflammation is thought to play an important role in the pathogenesis of vascular diseases. Lipoprotein-associated phospholipase A2 (Lp-PLA2) mediates vascular inflammation through the regulation of lipid metabolism in blood, thus, it has been extensively investigated to identify its role in vascular inflammation-related diseases, mainly atherosclerosis. Although darapladib, the most advanced Lp-PLA2 inhibitor, failed to meet the primary endpoints of two large phase III trials in atherosclerosis patients cotreated with standard medical care, the research on Lp-PLA2 has not been terminated. Novel pathogenic, epidemiologic, genetic, and crystallographic studies regarding Lp-PLA2 have been reported recently, while novel inhibitors were identified through a fragment-based lead discovery strategy. More strikingly, recent clinical and preclinical studies revealed that Lp-PLA2 inhibition showed promising therapeutic effects in diabetic macular edema and Alzheimer's disease. In this review, we not only summarized the knowledge of Lp-PLA2 established in the past decades but also emphasized new findings in recent years. We hope this review could be valuable for helping researchers acquire a much deeper insight into the nature of Lp-PLA2, identify more potent and selective Lp-PLA2 inhibitors, and discover the potential indications of Lp-PLA2 inhibitors.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , 1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , Animals , Disease , Genetic Variation , Humans , Lipoproteins/chemistry , Lipoproteins/metabolism , Small Molecule Libraries/pharmacology , Substrate Specificity/drug effects
5.
Chem Phys Lipids ; 225: 104814, 2019 12.
Article in English | MEDLINE | ID: mdl-31493387

ABSTRACT

Recombinant lipoprotein-associated phospholipase A2 (rLp-PLA2) expressed in HEK293 cells has a propensity to form oligomers in the absence of detergents. Dilution of rLp-PLA2 in the absence of detergents results in irreversible inactivation of the enzyme. The monomeric rLp-PLA2 may expose its hydrophobic interfacial binding region or substrate binding compartment to water and that may cause structural collapsing of the enzyme. Formation of self-aggregate, complex with binding partners or association with detergent micelles is to block the access of aqueous solvent to the hydrophobic substrate binding site and therefore prevents the structural collapsing. Dilution inactivation of the enzyme can be prevented in the presence of LDL or HDL suggesting that Lp-PLA2 association with lipoprotein particles (LDL and HDL) is necessary for Lp-PLA2 to maintain its enzymatic activity in human plasma. Formation of higher affinity complex gave better protection of rLp-PLA2 structure and activity. The method can be harnessed to detect the interaction between rLp-PLA2 and components of lipoprotein particles. Apo(a), ApoB 100 and ApoA1 were found to protect the enzyme from inactivation at roughly the similar level (˜80 ±â€¯5%) comparing to human serum albumin control (˜40%). One mg/ml pig brain phospholipid showed 100% protection under the same conditions.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , Detergents/chemistry , Lipids/chemistry , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Animals , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Structure , Swine
6.
Sci Rep ; 9(1): 5609, 2019 04 04.
Article in English | MEDLINE | ID: mdl-30948779

ABSTRACT

Long follow up is needed in prospective cohort study evaluation of plasma biomarkers for incident peripheral arterial disease (PAD) Middle-aged PAD-free individuals from the cardiovascular cohort of the Malmö Diet and Cancer study (n = 5550; 1991-94) were followed prospectively for a median time of 23.4 years. The plasma biomarkers lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and mass, proneurotensin, and CRP, were studied in relation to incidence of PAD until December 31st, 2016. The diagnosis of PAD could be validated and confirmed in 98%. Cox regression was used to calculate hazard ratios (HR) per 1 standard deviation increment of each respective log transformed plasma biomarker. Cumulative incidence of PAD was 4.4% (men 5.9%, women 3.3%). Adjusting for age, gender, smoking, body mass index, hypertension, diabetes mellitus, Lp-PLA2 activity (HR 1.33; 95% CI 1.17-1.52), Lp-PLA2 mass (HR 1.20; 95% CI 1.05-1.37) and CRP (HR 1.55; 95% CI 1.36-1.76) remained independently associated with incident PAD. The plasma biomarkers Lp-PLA2 activity and mass, and CRP were markers of PAD risk, implying that they might be useful biomarkers for subclinical atherosclerosis and atherosclerotic disease.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/analysis , Peripheral Arterial Disease/epidemiology , Peripheral Arterial Disease/metabolism , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Adult , Aged , Atherosclerosis , Biomarkers/blood , C-Reactive Protein/analysis , Cohort Studies , Female , Humans , Hypertension , Incidence , Longitudinal Studies , Male , Middle Aged , Peripheral Arterial Disease/blood , Proportional Hazards Models , Prospective Studies , Risk Assessment , Risk Factors
7.
Lab Med ; 50(3): 273-278, 2019 Jul 16.
Article in English | MEDLINE | ID: mdl-30770710

ABSTRACT

OBJECTIVE: To analyze the performance characteristics, stability, and clinical value of lipoprotein-associated phospholipase A2 (Lp-PLA2) using an enzymatic kinetic method. METHODS: The performance characteristics included reference intervals, precision, and accuracy. We assessed Lp-PLA2 stability by comparing Lp-PLA2 changes under different conditions. Lp-PLA2 was determined in the following groups: control individuals, patients with coronary heart disease (CHD), patients of different lipid subgroups within CHD, and patients with high total cholesterol (TC). Also, correlations between Lp-PLA2 and traditional cardiovascular risk factors were assessed. RESULTS: The mean (SD) reference interval of serum Lp-PLA2 activity was 451 (113) U per L with sex differences. Inter- and intra-assay precision revealed coefficients of variance (CVs) of 1.81% to 2.63% and 1.43% to 1.77%. The average bias was 0.33%. Lp-PLA2 activity was stable. In the CHD group, high-lipid subgroups, and high-TC group, Lp-PLA2 was elevated, and correlation was observed between Lp-PLA2 and traditional risk factors. CONCLUSION: Lp-PLA2 activity has important clinical value in CHD.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/blood , Clinical Laboratory Techniques/methods , Coronary Disease/diagnosis , Coronary Disease/pathology , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , Adolescent , Adult , Aged , Enzyme Stability , Female , Humans , Kinetics , Male , Middle Aged , Risk Assessment , Young Adult
8.
Anal Bioanal Chem ; 410(29): 7645-7653, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30283999

ABSTRACT

A facile and feasible photoelectrochemical (PEC) immunoassay based on plasmon-enhanced energy transfer between gold nanoparticles (AuNPs) and CdS quantum dots (QDs)/g-C3N4 nanosheets was developed for the ultrasensitive detection of lipoprotein-associated phospholipase A2 (Lp-PLA2). To construct such a sensing platform, the immunosensor was prepared by immobilizing Lp-PLA2 on a CdS QDs/g-C3N4-modified electrode. A competitive-type immunoreaction was utilized for Lp-PLA2 detection, with AuNP-labeled anti-Lp-PLA2 antibody used as the competitor. Introducing AuNPs with the specific antibody for the antigen target Lp-PLA2 led to heavy quenching of the photocurrent of CdS QDs/g-C3N4 due to the plasmon-enhanced energy transfer between AuNPs and CdS QDs. The quenching efficiency decreased with increasing target Lp-PLA2 concentration. Under optimal conditions, the PEC immunosensor presented a good photocurrent response to the target Lp-PLA2 in the dynamic linear range of 0.01-300 ng mL-1, with a low detection limit of 5.3 pg mL-1. Other biomarkers and natural enzymes did not interfere with response of this system. The reproducibility and accuracy of this method for the analysis of human serum specimens were evaluated, and the results given by the method developed here were found to closely correspond to the results obtained with commercial Lp-PLA2 ELISA kits. Importantly, this protocol offers promise for the development of exciton-plasmon interaction-based PEC detection systems. Graphical abstract ᅟ.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , Cadmium Compounds/chemistry , Gold/chemistry , Light , Metal Nanoparticles/chemistry , Quantum Dots , Sulfides/chemistry , Antibodies, Monoclonal/chemistry , Electrochemical Techniques/methods , Immunoassay/methods , Limit of Detection , Microscopy, Electron, Transmission , Reproducibility of Results
9.
J Cell Biochem ; 119(8): 7009-7021, 2018 08.
Article in English | MEDLINE | ID: mdl-29737567

ABSTRACT

Non-synonymous single nucleotide polymorphisms (nsSNPs) are genetic variations at single base resulting in an amino acid change which have been associated with various complex human diseases. The human Lipoprotein-associated phospholipase A2 (Lp-PLA2 ) gene harbours a rare Q281R polymorphism which was previously reported to cause loss of enzymatic function. Lp-PLA2 is an important enzyme which catalyzes the hydrolysis of polar phospholipids releasing pro-atherogenic and pro-inflammatory mediators involved in the pathogenesis of atherosclerosis. Our current study is aimed at elucidating the structural and functional consequences of Q281R polymorphism on Lp-PLA2 . The Q281R mutation is classified as deleterious and causes protein instability as deduced from evolutionary, folding free energy changes and Support vector machine (SVM)-based methods. A Q281R mutant structure was deciphered using homology modelling approach and was validated using phi and psi dihedral angles distribution, ERRAT, Verify_3D scores, Protein Structure Analysis (ProSA) energ,y and Z-score. A decreased hydrophobic interactions and weaker substrate binding affinity was observed in the mutant compared to the wild- type (WT) using molecular docking. Further, the mutant displayed enhanced structural flexibility particularly in the low density lipoprotein (LDL) binding domain, decreased solvent accessibility of catalytic residues-Phe274 and Ser273 and increased Cɑ distance between Phe274 and Leu153 and large conformational entropy change as inferred from all-atom molecular dynamics (MD) simulation and essential dynamics (ED) studies. Our results corroborate well with previous experimental studies and thus these aberrations in the Q281R mutant structure may help explain the molecular basis of loss of enzyme activity.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , 1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , Molecular Dynamics Simulation , Mutation, Missense , Protein Folding , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Amino Acid Substitution , Animals , Chickens , Dogs , Guinea Pigs , Humans , Mice , Polymorphism, Genetic , Protein Domains , Rats , Swine
10.
Int J Biol Macromol ; 112: 831-840, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29427679

ABSTRACT

Human Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an important biomarker for cardiovascular diseases and a therapeutically important drug target against Atherosclerosis. It has the ability to hydrolyze various oxidized low density lipoproteins (LDL) and generates potent pro-inflammatory signaling molecules. Both physiological and non-physiological oxidants have been reported to inhibit Lp-PLA2 activity. The mechanism of the enzyme inhibition due to oxidation of surface exposed Met117 at the structural level is not clearly understood. In the present work, molecular dynamics (MD) simulation and Essential dynamics (ED) has been used in tandem with molecular docking approach to understand the structural alteration in Lp-PLA2 upon Met117 oxidation. Further, the binding of substrate, Platelet-activating factor (PAF) with the wild type and oxidized form have also been investigated. Our results showed that Met117 oxidation caused enhanced flexibility and decreased compactness in oxidized state. PAF binding interaction with oxidized protein was mediated only through hydrophobic interactions. MD simulation studies revealed that the oxidized protein failed to firmly bind PAF. Our present findings will help understand the mechanism of Lp-PLA2 inhibition under oxidative stress.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Cardiovascular Diseases/metabolism , Methionine/metabolism , Platelet Activating Factor/metabolism , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , Apoproteins/chemistry , Biomarkers/metabolism , Humans , Hydrogen Bonding , Molecular Docking Simulation , Molecular Dynamics Simulation , Oxidation-Reduction , Principal Component Analysis , Substrate Specificity , Thermodynamics
11.
Bioorg Med Chem Lett ; 28(4): 787-792, 2018 02 15.
Article in English | MEDLINE | ID: mdl-29336874

ABSTRACT

Darapladib is one of the most potent Lp-PLA2 (Lipoprotein-associated phospholipase A2) inhibitor with an IC50 of 0.25 nM. We demonstrate that a crucial step of Darapladib synthesis was not correctly described in the literature, leading to the production of wrong regioisomers. Moreover we show that the inhibitory activity is directly linked to the position on N1 since compounds bearing alkylation on different sites have potentially less interaction within the active site of Lp-PLA2.


Subject(s)
Benzaldehydes/chemistry , Oximes/chemistry , Phospholipase A2 Inhibitors/chemistry , Thiouracil/chemistry , 1-Alkyl-2-acetylglycerophosphocholine Esterase/antagonists & inhibitors , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , Alkylation , Benzaldehydes/chemical synthesis , Benzaldehydes/pharmacology , Catalytic Domain , Humans , Isomerism , Oximes/chemical synthesis , Oximes/pharmacology , Phospholipase A2 Inhibitors/chemical synthesis , Phospholipase A2 Inhibitors/pharmacology
12.
Int J Biol Macromol ; 107(Pt B): 1956-1964, 2018 Feb.
Article in English | MEDLINE | ID: mdl-29051098

ABSTRACT

Protein tyrosine nitration (PTN) is a post translational event which results in the generation of 3-Nitrotyrosine (3-NT). High levels of 3-NT were reported in several human diseases such as Parkinson's disease, Alzheimer's disease, amylotrophic lateral sclerosis and coronary artery disease. It was reported that PTN at positions 307 and 335 of Lipoprotein-associated phospholipase A2 (Lp-PLA2) curtails its enzymatic activity but the mechanism of inhibition at the structure level is still incomprehensible. The present study is an in silico endeavor to understand nitrative stress induced structural changes in Lp-PLA2. Molecular docking studies revealed a decreased binding affinity of substrate, Platelet Activating Factor (PAF) with the nitrated forms of Lp-PLA2 (NT-Tyr307 and NT-Tyr335) compared to the wild type, due to differences in the hydrogen bond interaction patterns. Molecular dynamics (MD) simulation studies suggests higher flexibility of nitrated forms compared to wild type, disorientation of the catalytic triad and decreased molecular interactions of NT-Tyr307 and NT-Tyr335 with other residues of the protein. Essential dynamics (ED) further confirmed the enhanced structural flexibility of nitrated forms of Lp-PLA2. Our findings would help understand the molecular mechanism of nitrative stress induced inhibition of Lp-PLA2 which may further assist in designing of therapeutics having protective functions against PTN.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , Atherosclerosis/enzymology , Biomarkers/metabolism , Tyrosine/metabolism , Amino Acid Sequence , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Molecular Dynamics Simulation , Nitrosation , Platelet Activating Factor/metabolism , Principal Component Analysis , Substrate Specificity , Thermodynamics , Tyrosine/analogs & derivatives , Tyrosine/chemistry
13.
J Med Chem ; 60(24): 10231-10244, 2017 12 28.
Article in English | MEDLINE | ID: mdl-29193967

ABSTRACT

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a promising therapeutic target for atherosclerosis, Alzheimer's disease, and diabetic macular edema. Here we report the identification of novel sulfonamide scaffold Lp-PLA2 inhibitors derived from a relatively weak fragment. Similarity searching on this fragment followed by molecular docking leads to the discovery of a micromolar inhibitor with a 300-fold potency improvement. Subsequently, by the application of a structure-guided design strategy, a successful hit-to-lead optimization was achieved and a number of Lp-PLA2 inhibitors with single-digit nanomolar potency were obtained. After preliminary evaluation of the properties of drug-likeness in vitro and in vivo, compound 37 stands out from this congeneric series of inhibitors for good inhibitory activity and favorable oral bioavailability in male Sprague-Dawley rats, providing a quality candidate for further development. The present study thus clearly demonstrates the power and advantage of integrally employing fragment screening, crystal structures determination, virtual screening, and medicinal chemistry in an efficient lead discovery project, providing a good example for structure-based drug design.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacology , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , Administration, Oral , Animals , Biological Availability , Caco-2 Cells , Crystallography, X-Ray , Drug Evaluation, Preclinical/methods , Drug Stability , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacokinetics , Hepatocytes/drug effects , Humans , Male , Molecular Docking Simulation , Rats, Sprague-Dawley , Sulfonamides/administration & dosage , Sulfonamides/pharmacokinetics
14.
Cell ; 170(6): 1197-1208.e12, 2017 Sep 07.
Article in English | MEDLINE | ID: mdl-28886386

ABSTRACT

Regulation is central to the functional versatility of cytoplasmic dynein, a motor involved in intracellular transport, cell division, and neurodevelopment. Previous work established that Lis1, a conserved regulator of dynein, binds to its motor domain and induces a tight microtubule-binding state in dynein. The work we present here-a combination of biochemistry, single-molecule assays, and cryoelectron microscopy-led to the surprising discovery that Lis1 has two opposing modes of regulating dynein, being capable of inducing both low and high affinity for the microtubule. We show that these opposing modes depend on the stoichiometry of Lis1 binding to dynein and that this stoichiometry is regulated by the nucleotide state of dynein's AAA3 domain. The low-affinity state requires Lis1 to also bind to dynein at a novel conserved site, mutation of which disrupts Lis1's function in vivo. We propose a new model for the regulation of dynein by Lis1.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Dyneins/metabolism , Microtubule-Associated Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Cryoelectron Microscopy , Dyneins/chemistry , Humans , Microtubule-Associated Proteins/chemistry , Models, Molecular , Molecular Motor Proteins/metabolism , Protein Domains , Saccharomyces cerevisiae Proteins/chemistry , Sequence Alignment
15.
Biochem J ; 474(4): 597-609, 2017 02 15.
Article in English | MEDLINE | ID: mdl-27993970

ABSTRACT

Cyclic AMP (cAMP)-specific phosphodiesterase-4 (PDE4) enzymes underpin compartmentalised cAMP signalling by localising to distinct signalling complexes. PDE4 long isoforms can be phosphorylated by mitogen-activated protein kinase-activated protein kinase 2 (MK2), which attenuates activation of such enzymes through their phosphorylation by protein kinase A. Here we show that MK2 interacts directly with PDE4 long isoforms and define the sites of interaction. One is a unique site that locates within the regulatory upstream conserved region 1 (UCR1) domain and contains a core Phe141, Leu142 and Tyr143 (FLY) cluster (PDE4A5 numbering). Located with the second site is a critical core Phe693, Glu694, Phe695 (FQF) motif that is also employed in the sequestering of PDE4 long forms by an array of other signalling proteins, including the signalling scaffold ß-arrestin, the tyrosyl kinase Lyn, the SUMOylation E2 ligase UBC9, the dynein regulator Lis1 (PAFAH1B1) and the protein kinase Erk. We propose that the FQF motif lies at the heart of a multifunctional docking (MFD) site located within the PDE4 catalytic unit. It is clear from our data that, as well as aiding fidelity of interaction, the MFD site confers exclusivity of binding between PDE4 and a single specific partner protein from the cohort of signalling proteins whose interaction with PDE4 involves the FQF motif.


Subject(s)
Catalytic Domain , Cyclic Nucleotide Phosphodiesterases, Type 4/chemistry , Intracellular Signaling Peptides and Proteins/chemistry , Molecular Docking Simulation , Protein Serine-Threonine Kinases/chemistry , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , 1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Amino Acid Motifs , Animals , COS Cells , Chlorocebus aethiops , Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 4/genetics , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Gene Expression , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mitogen-Activated Protein Kinase 1/chemistry , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/chemistry , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , beta-Arrestins/chemistry , beta-Arrestins/genetics , beta-Arrestins/metabolism , src-Family Kinases/chemistry , src-Family Kinases/genetics , src-Family Kinases/metabolism
16.
J Med Chem ; 59(10): 5115-20, 2016 05 26.
Article in English | MEDLINE | ID: mdl-27078579

ABSTRACT

Lipoprotein-associated phospholipase A2 (Lp-PLA2) represents a promising therapeutic target for atherosclerosis and Alzheimer's disease. Here we reported the first crystal structures of Lp-PLA2 bound with reversible inhibitors and the thermodynamic characterization of complexes. High rigidity of Lp-PLA2 structure and similar binding modes of inhibitors with completely different scaffolds are revealed. It not only provides the molecular basis for inhibitory activity but also sheds light on the essential features of Lp-PLA2 recognition with reversible inhibitors.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/antagonists & inhibitors , Phospholipase A2 Inhibitors/pharmacology , Thermodynamics , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Dose-Response Relationship, Drug , Drug Design , Humans , Ligands , Models, Molecular , Molecular Structure , Phospholipase A2 Inhibitors/chemical synthesis , Phospholipase A2 Inhibitors/chemistry , Structure-Activity Relationship
17.
Comput Biol Chem ; 59 Pt A: 199-207, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26595893

ABSTRACT

INTRODUCTION: PLA2G7 encodes Lp-PLA2 having role in the formation of atherosclerotic plaques by catalyzing its substrate, phosphatydilcholine (PC), to be pro-inflammatory substances. The increased risk for coronary artery disease (CAD) in Asian population has been related with this enzyme. 279(Val→Phe) variant was reported to have a protective role against CAD due to, in part, secretion defect or loss of enzymatic function. Therefore, We study folding kinetics and enzyme-substrate interaction in 279(Val→Phe) by using clinical and computational biology approach. METHODS: Polymorphisms were detected by genotyping among 103 acute myocardial infarction patients and 37 controls. Folding Lp-PLA2 was simulated using GROMACS software by assessing helicity, hydrogen bond formation and stability. The interactions of Lp-PLA2 and its substrate were simulated using Pyrx software followed by molecular dynamics simulation using YASARA software. RESULT: Polymorphism of 279(Val→Phe) was represented by the change of nucleotide from G to T of 994th PLA2G7 gene. The folding simulation suggested a decreased percentage of α-helix, hydrogen bond formation, hydrogen bond stability and hydrophobicity in 279(Val→Phe). The PC did not interact with active site of 279(Val→Phe) as paradoxically observed in 279 valine. 279(Val→Phe) polymorphism is likely to cause unstable binding to the substrate and decrease the enzymatic activity as observed in molecular dynamics simulations. The results of our computational biology study supported a protected effect of 279(Val→Phe) Polymorphism showed by the odd ratio for MI of 0.22 (CI 95% 0.035-1.37) in this study. CONCLUSION: 279(Val→Phe) Polymorphism of Lp-PLA2 may lead to decrease the enzymatic activity via changes of folding kinetics and recognition to its substrate.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Phenylalanine/genetics , Polymorphism, Genetic/genetics , Protein Folding , Valine/genetics , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , Acute Disease , Case-Control Studies , Computational Biology , Coronary Artery Disease/diagnosis , Coronary Artery Disease/genetics , Coronary Artery Disease/metabolism , Genotype , Humans , Kinetics , Male , Middle Aged , Molecular Dynamics Simulation , Software , Substrate Specificity
18.
Prostaglandins Other Lipid Mediat ; 121(Pt B): 176-83, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26358846

ABSTRACT

Interleukin 1 beta (IL-1ß) induced platelet activating factor (PAF) synthesis in U-937 cells through stimulation of acetyl-CoA:lysoPAF-acetyltransferase (lyso PAF-AT) at 3 h and DTT-independentCDP-choline-1-alkyl-2-acetyl-sn-glycerol cholinophosphotransferase (PAF-CPT) at 0.5 h. The aim of this study was to investigate the effect of tyrosol (T), resveratrol (R) and their acetylated derivatives(AcDs) which exhibit enhanced bioavailability, on PAF synthesis in U-937 after IL-1ß stimulation. The specific activity of PAF enzymes and intracellular levels were measured in cell homogenates. T and R concentration capable of inducing 50% inhibition in IL-1ß effect on lyso PAF-AT was 48 µΜ ± 11 and 157 µΜ ± 77, for PAF-CPT 246 µΜ ± 61 and 294 µΜ ± 102, respectively. The same order of concentration was also observed on inhibiting PAF levels produced by IL-1ß. T was more potent inhibitor than R (p<0.05). AcDs of T retain parent compound inhibitory activity, while in the case of R only two AcDs retain the activity. The observed inhibitory effect by T,R and their AcDs, may partly explain their already reported beneficial role.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antioxidants/pharmacology , Down-Regulation/drug effects , Monocytes/drug effects , Phenylethyl Alcohol/analogs & derivatives , Platelet Activating Factor/antagonists & inhibitors , Stilbenes/pharmacology , 1-Alkyl-2-acetylglycerophosphocholine Esterase/antagonists & inhibitors , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Acetylation , Acetyltransferases/antagonists & inhibitors , Acetyltransferases/chemistry , Acetyltransferases/metabolism , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Antioxidants/chemical synthesis , Antioxidants/chemistry , Cell Line , Diacylglycerol Cholinephosphotransferase/antagonists & inhibitors , Diacylglycerol Cholinephosphotransferase/chemistry , Diacylglycerol Cholinephosphotransferase/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Monocytes/immunology , Monocytes/metabolism , Osmolar Concentration , Phenylethyl Alcohol/pharmacology , Platelet Activating Factor/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Resveratrol , Stilbenes/chemistry
19.
Fungal Genet Biol ; 82: 213-27, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26231681

ABSTRACT

LIS1 is a microtubule (Mt) plus-end binding protein that interacts with the dynein/dynactin complex. In humans, LIS1 is required for proper nuclear and organelle migration during cell growth. Although gene duplication is absent from Neurospora crassa, we found two paralogues of human LIS1. We named them LIS1-1 and LIS1-2 and studied their dynamics and function by fluorescent tagging. At the protein level, LIS1-1 and LIS1-2 were very similar. Although, the characteristic coiled-coil motif was not present in LIS1-2. LIS1-1-GFP and LIS1-2-GFP showed the same cellular distribution and dynamics, but LIS1-2-GFP was less abundant. Both LIS1 proteins were found in the subapical region as single fluorescent particles traveling toward the cell apex, they accumulated in the apical dome forming prominent short filament-like structures, some of which traversed the Spitzenkörper (Spk). The fluorescent structures moved exclusively in anterograde fashion along straight paths suggesting they traveled on Mts. There was no effect in the filament behavior of LIS1-1-GFP in the Δlis1-2 mutant but the dynamics of LIS1-2-GFP was affected in the Δlis1-1 mutant. Microtubular integrity and the dynein-dynactin complex were necessary for the formation of filament-like structures of LIS1-1-GFP in the subapical and apical regions; however, conventional kinesin (KIN-1) was not. Deletion mutants showed that the lack of lis1-1 decreased cell growth by ∼75%; however, the lack of lis1-2 had no effect on growth. A Δlis1-1;Δlis1-2 double mutant showed slower growth than either single mutant. Conidia production was reduced but branching rate increased in Δlis1-1 and the Δlis1-1;Δlis1-2 double mutants. The absence of LIS1-1 had a strong effect on Mt organization and dynamics and indirectly affected nuclear and mitochondrial distribution. The absence of LIS1-1 filaments in dynein mutants (ropy mutants) or in benomyl treated hyphae indicates the strong association between this protein and the regulation of the dynein-dynactin complex and Mt organization. LIS1-1 and LIS1-2 had a high amino acid homology, nevertheless, the absence of the coiled-coil motif in LIS1-2 suggests that its function or regulation may be distinct from that of LIS1-1.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , Fungal Proteins/genetics , Microtubule-Associated Proteins/genetics , Neurospora crassa/genetics , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , Amino Acid Sequence , Cell Nucleus/metabolism , Dynactin Complex , Dyneins/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Gene Expression , Humans , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/metabolism , Mitochondria/metabolism , Molecular Sequence Data , Mutation , Neurospora crassa/metabolism , Protein Binding , Protein Transport , Recombinant Fusion Proteins , Sequence Alignment
20.
Biochim Biophys Acta ; 1854(5): 469-75, 2015 May.
Article in English | MEDLINE | ID: mdl-25707358

ABSTRACT

The intracellular enzyme platelet-activating factor acetylhydrolase type-II (PAFAH-II) hydrolyzes platelet-activating factor and oxidatively fragmented phospholipids. PAFAH-II in its resting state is mainly cytoplasmic, and it responds to oxidative stress by becoming increasingly bound to endoplasmic reticulum and Golgi membranes. Numerous studies have indicated that this enzyme is essential for protecting cells from oxidative stress induced apoptosis. However, the regulatory mechanism of the oxidative stress response by PAFAH-II has not been fully resolved. Here, changes to the oligomeric state of human PAFAH-II were investigated as a potential regulatory mechanism toward enzyme trafficking. Native PAGE analysis in vitro and photon counting histogram within live cells showed that PAFAH-II is both monomeric and dimeric. A Gly-2-Ala site-directed mutation of PAFAH-II demonstrated that the N-terminal myristoyl group is required for homodimerization. Additionally, the distribution of oligomeric PAFAH-II is distinct within the cell; homodimers of PAFAH-II were localized to the cytoplasm while monomers were associated to the membranes of the endoplasmic reticulum and Golgi. We propose that the oligomeric state of PAFAH-II drives functional protein trafficking. PAFAH-II localization to the membrane is critical for substrate acquisition and effective oxidative stress protection. It is hypothesized that the balance between monomer and dimer serves as a regulatory mechanism of a PAFAH-II oxidative stress response.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Protein Multimerization/physiology , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , Cytoplasm/metabolism , HEK293 Cells , Humans , Models, Molecular , Oxidative Stress , Protein Transport , Tissue Distribution
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