1224 CNS & Neurological Disorders - Drug Targets, 2014, 13, 1224-1231
Protein Interactions Between the C-Terminus of Aβ-Peptide and Phospholipase A
2- A Structure Biology Based Approach to Identify Novel Alzheimer’s Therapeutics
Zeenat Mirza
*,1,2, Vikram G. Pillai
2,3and Mohammad A. Kamal
11King Fahd Medical Research Center, King Abdulaziz University, P.O. Box: 80216, Jeddah - 21589, Kingdom of Saudi Arabia
2Department of Biophysics, All India Institute of Medical Sciences, New Delhi - 110029, India
3Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Abstract: Amyloid β (Aβ) polypeptide plays a key role in determining the state of protein aggregation in Alzheimer’s disease. The hydrophobic C-terminal part of the Aβ peptide is critical in triggering the transformation from α-helical to β- sheet structure. We hypothesized that phospholipase A2 (PLA2) may inhibit the aggregation of Aβ peptide by interacting with the peptide and keeping the two peptide chains apart. In order to examine the nature of interactions between PLA2
and Aβ peptide, we prepared and crystallized complex of Naja naja sagittifera PLA2 with the C-terminal hepta-peptide Val-Gly-Gly-Val-Val-Ile-Ala. The X-ray intensity data were collected to 2.04 Å resolution and the structure was determined by molecular replacement and refined to the crystallographic R factor of 0.186. The structural analysis revealed that the peptide binds to PLA2 at the hydrophobic substrate binding cavity forming at least eight hydrogen bonds and approximately a two dozen Van der Waals interactions. The number and nature of interactions indicate that the affinity between PLA2 and the hepta-peptide is greater than the affinity between two Aβ peptide chains. Therefore, PLA2
is proposed as a probable ligand to prevent the aggregation of Aβ peptides.
Keywords: Alzheimer’s disease, Aβ peptide, C-terminus, co-crystallization, phospholipase A2, VGGVVIA.
1. INTRODUCTION
Alzheimer’s disease (AD) is a significant social, economic and health burden of present century [1].
Deregulated amyloid β (Aβ) homeostasis and extracellular plaque formation by oligomerization and Aβ deposits is the prime player of AD’s neuropathology [2-6]. Alternate form of Aβ peptides having length of either 40 or 42 amino acids are cleaved from the large amyloid precursor protein (APP) by enzymatic action of β-secretase and γ-secretase [7]. Small soluble Aβ oligomers have also been linked to neuronal toxicity and synaptic dysfunction [8]. Regardless of the remarkable number of investigations, structural conformation of the culprit peptide that causes neurotoxicity remains undetermined. The peptide sequence prior to cleavage is part of the single transmembrane APP domain and adopts a helical structure in vivo, but in the plaques has a β conformation and it is strongly suggested that this conformational transition is a decisive step determining destiny of Aβ(1-42) under physiological or diseased scenario [9].
Among the two most abundant forms of Aβ in the human body (Aβ40 and Aβ42), Aβ40 is more abundant (90%) than Aβ42 under physiological conditions [10], however in
*Address correspondence to this author at the King Fahd Medical Research Center, P.B. No. 80216, King Abdulaziz University, Jeddah -21589, Saudi Arabia; Tel: +966-553017824, +966-12-6401000, Ext: 72074; Fax: +966- 12-6952076; E-mail: [email protected], [email protected]
amyloid deposits, Aβ42 is the dominant alloform. Genetic evidence shows that neurotoxic Aβ42 aggregates more readily and is associated strongly with a risk for AD than is Aβ40 [11, 12]. It has been shown that in vitro Aβ42 aggregates into fibrils faster than Aβ40 and Aβ42 fibrils are considerably more toxic to neurons than Aβ40 [13]. Aβ peptide in AD patient’s brain displays significant N- and C-terminal heterogeneity.
Several evidences suggest that both N-terminal truncations and C-terminal extensions assist to precipitate amyloid plaque formation [14].
To address the question how two amino acid residue (IA) differences contribute to the structural conformation, Yun et al. have done in silico predictions of Aβ40 versus Aβ42 folding and assembly. They have found that folding is driven primarily by effective hydrophobic attraction among hydrophobic regions of Aβ [15]. Also Aβ42 and Aβ40 folded monomer structures are different with Aβ42 having an additional turn at G37-G38. Aβ40 assembly is dominated by intermolecular attraction between pairs of central hydrophobic clusters (L17-A21). Aβ42 assembly is dominated by intermolecular attraction among pairs of C- terminal regions (V39-A42) as well as mid-hydrophobic regions (I31-M35). The additional two hydrophobic amino acids significantly augment its aggregation propensity [16], directing rapid formation of small Aβ oligomers, bigger intermediate assemblies akin to protofibrils, and in due course approximately 8-nm amyloid fibrils present profusely in neuritic plaques and amyloidic microvessels.
The Aβ structure solved in hexafluoroisopropanol and high water content by NMR and molecular-dynamics
1996-3181/14 $58.00+.00 © 2014 Bentham Science Publishers
Aβ-Peptide and Phospholipase A2 CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 7 1225
simulations illustrated that while going from strong nonpolar to polar solutions, longer N-terminal helix is effectively retained, but the shorter C-terminal helix gets disordered indicating localization of residues seeding β conformation.
Observation of this conformational transition directly is difficult since Aβ peptides have very limited water-solubility and when shifted from organic to aqueous solutions, they exhibit strong aggregating tendency and precipitate [9].
Bitan et al. have shown that Aβ42 but not Aβ40 forms paranuclei (pentamers/hexamers) and multiples of paranuclei (~12-18-mers) and the authors proposed that C-terminal’s Ile41 and Ala42 play a key role in Aβ42 paranuclei formation [17]. Rational designing of aggregation inhibitors require knowledge of molecular structure [18]; because of high aggregation tendency of Aβ, it has not been possible to obtain complete structural information by X-ray crystallography. Hence, in the present study, the carboxyl terminus peptide fragment of Aβ was structurally analyzed to have an insight into the structure adopted by it.
Phospholipase A2 (PLA2) are hydrolases that specifically cleave the acyl ester bond at the sn-2 position of triglyceride backbone of the membrane phospholipids to produce a lyso- phospholipid and a free fatty acid (mainly arachidonic acid) [19]. PLA2 activity and arachidonic acid-based signalling are vital for normal brain development and synaptic plasticity and studies have implicated varied roles of PLA2 in brain where it contributes to neuronal growth and inflammatory response [20-22]. Overall chemical nature and contour of the substrate – binding site in low molecular weight PLA2s are alike [23]. The structural difference of PLA2s from different sources is very little and their amino acid sequences show a great deal of homology. The primary structure of extracellular or intracellular PLA2s from mammalian pancreas and snake venom is also highly conserved [24].
Mammalian pancreatic PLA2 (group IB) is structurally more similar to PLA2 from the old world snakes like the Indian cobra (group IA) [25] and both are of approximately same molecular weight and have common disulphide linkages.
Additionally, snake venom is a very rich natural source of extra-cellular, soluble PLA2 required for native purification and crystallographic studies. Structural analysis reveals that these enzymes have a conserved globular structure with species specific variations seen at the active site, calcium binding loop, hydrophobic channel, the C-terminal domain and the quaternary conformational state. Binding of PLA2 to Aβ may reduce the concentration of free Aβ in the brain, thereby reducing its oligomerization feasibility and intensity and hence may have potential therapeutic benefit in AD.
This study involves the reductionist fragment based approach to understand the structure adopted by the C- terminal fragment of Alzheimer's Aβ peptide in its complex with PLA2. In the current communication, we report the structure determined by X-ray crystallography of C-terminal hydrophobic sequence Val-Gly-Gly-Val-Val-Ile-Ala (VGGVVIA) of Aβ-peptide with a Group I PLA2 purified from Andaman Cobra sub-species Naja naja sagittifera venom at 2.04 Å resolution (PDB Code: 3GCI). This is to our knowledge the first study to structurally establish interaction between amyloid-β peptide C-terminus fragment and hydrophobic substrate binding site of PLA2 involving H bond and van der Waals interactions.
2. MATERIALS AND METHODS 2.1. Purification of Monomeric PLA2
Lyophilized Andaman cobra (Naja naja sagittifera) venom samples purchased from Irula Snake-Catchers Industrial Cooperative Society, Chennai, India were dissolved at 100mg/mL concentration in Tris-HCl/ NaCl buffer, pH 7.0. Size-exclusion fractionation on pre- equilibrated Sephadex G-100 (Sigma, St. Louis, MO) column (100 x 2 cm) was performed initially with a flow rate of 6 ml/hour. The eluted peak having a molecular weight of 14 kDa as estimated on SDS-PAGE and showing PLA2 activity was pooled for further purification steps. After desalting by dialysis it was loaded on the carboxymethyl cellulose C-50 column (Pharmacia, Sweden). The column was washed with the above buffer; unbound fractions were collected and dialyzed against 50 mM ammonium acetate buffer, pH 6.0. The sample was loaded on a pre-equilibrated Affi- gel Cibacron blue F3GA (Bio-Rad) column for affinity chromatography using pH based elution method. The column was first washed with 50 mM ammonium acetate buffer, pH 6.0 to eliminate unbound proteins; followed by elution using 50 mM ammonium bicarbonate buffer, pH 8.0. These fractions showed PLA2 activity and had a molecular weight of ~14 kDa. The samples were pooled, desalted by ultrafiltration and lyophilized. Purity was checked by matrix assisted laser desorption-ionization – time of flight (MALDI- TOF) (Kratos, Shimadzu, Japan), N- terminal sequencing and by enzymatic activity measurements using a PLA2 Assay Kit (Cayman Chemical Company, USA) [26]. On MALDI- TOF it showed a molecular weight of 13791.50 Da.
2.2. Co-Crystallization
Purified samples of PLA2 were dissolved to a final protein concentration of 2.5 mg/mL in 10 mM sodium phosphate buffer, pH 6.0 containing 1mM CaCl2. The peptide VGGVVIA was commercially purchased from GenScript Corporation, NJ, USA. The hepta-peptide dissolved in 30% acetonitrile was added to the protein solution at 10X molar concentration and mixed well. Five µl drops of the above mixture were equilibrated in a vapour diffusion hanging drop method against the same buffer containing 35% ethanol as a precipitant. Various cuboidal shaped colorless crystals of dimensions up to 0.4 x 0.25 x 0.3 mm were obtained after about 20 days.
2.3. Data Collection and Processing
The crystal of the PLA2 and peptide VGGVVIA complex obtained was mounted carefully on a goniometer. It was stable in the X-ray beam and intensity data collection was done at 258 K. Mar345 imaging plate scanner (Marresearch GmbH, Germany) mounted on a Rigaku RU – 300 rotating anode X-ray generator operating at 100mA and 50kV using CuKα radiation obtained using Osmic Blue confocal optics (Rigaku, USA) were used. Indexing, intensity integration and data scaling (data reduction) were done using DENZO, AUTOMAR and SCALEPACK programs from HKL package [27]. The space group was found to be tetragonal with approximate cell dimensions of a=b=42.6 Å, c=65.8 Å.
The data had an overall Rsym of 11.5% with an overall
completeness of 97.6 % for resolution to 2.04 Å. The data collection statistics are given in Table 1.
Table 1. Data collection statistics.
Space Group System
P41
Tetragonal Unit-cell parameters (Ǻ)
a=b c
42.68 65.82
Vm (Ǻ3/Da) 2.3
Solvent Content (%) 46.7
Resolution range (Ǻ) 42.6 - 2.04
No. of observed reflections 33,620
No. of unique reflections 7596
Overall completeness (%) 97.6
Completeness in the highest shell (2.09 - 2.04 Ǻ)(%)
96.7
Overall Rsym (%) 11.5
Rsym in the highest shell (2.09 - 2.04 Ǻ)(%)
30.5
Overall I / σ(I) 7.8
I / σ(I) in the highest shell (2.09 - 2.04 Ǻ)
2.1
2.4. Structure Determination and Refinement
The structure determination of PLA2 – peptide complex was done by molecular replacement with the model of native PLA2 (PDB entry: 1MF4 as search model) [28]. The rotation and translation functions were calculated. The solution was transformed from Eulerian coordinates to orthogonal coordinates using the program AMoRe from the CCP4 software suite [29, 30]. It yielded a solution with R-factor of 32.4 % and a correlation coefficient of 62.3 %.
The refinement was done with CCP4 program’s - REFMAC 5.2.0019 [30, 31]. In each step |2Fo-Fc| and |Fo-Fc| maps were calculated to improve the structure using the graphics program “O” [32] on Silicon Graphics O2 work station. The R-factors dropped gradually and the individual B-factors were refined. The positions of 95 water molecules were determined from |Fo-Fc| map using ARP/wARP package [33, 34].
In addition, a calcium ion and a continuous electron density at 2.0 σ cut off was observed in the proximity of the previously known binding cavity indicating the presence of the peptide VGGVVIA. In the further steps of refinement, the coordinates of the peptide molecule were fitted into the characteristic electron density. Final refinement cycles using all 7596 reflections reduced R-factor to 0.186 and Rfree to 0.221 in the resolution range of 42.6-2.04 Å. In the final
|2Fo-Fc| electron density map, main chain was continuous when contoured at 2.0σ level. The atomic coordinates of this structure were deposited in Protein Data Bank (PDB) with accession code 3GCI. All molecular figures were prepared with PyMol (www.pymol.org).
3. RESULTS
3.1. Quality of the Final Model
The photograph of the PLA2-peptide complex crystal is shown in Fig. (1) and the characteristic electron density map contoured at 2.0σ cut off is shown in Fig. (2). The final model had 906 atoms of protein, 43 atoms of peptide molecule, 1 calcium ion and 93 water molecules. Final |2Fo - Fc| electron density map was distinct and without any breaks for both the backbone and the protein side chains. Overall geometry was good with r.m.s. deviations in bond lengths and bond angles being 0.01 Å and 1.2° respectively. The refinement statistics are given in Table 2. Ramachandran plot [35] calculated using PROCHECK [36], indicated that 91.6% of the dihedral angles were present in the most favorable regions, while the remaining 8.4% were present in the additionally allowed regions (Fig. 3).
Fig. (1). Crystal photograph of PLA2 in complex with the peptide VGGVVIA. The crystal diffracted upto 2.04 Å resolution.
Fig. (2). Difference Fourier |Fo-Fc| map contoured at 2.0 σ cut off.
3.2. Overall PLA2 Structure
The overall structure of PLA2 contains three long helices, a short two stranded antiparallel β-sheet (β-wing) and two
Aβ-Peptide and Phospholipase A2 CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 7 1227
helical short turns with a calcium-binding loop (Fig. 4). The two antiparallel helices (H2 and H3) form the core of the protein structure. The inner surface topology of helix H1 has well conserved hydrophobic residues which make sort of channel for providing access to the apolar catalytic site (Fig.
5). The hydrophobic channel is further complemented by residue 19 which is situated in the short turn following the helix H1, residues 30, 31 and 32 present within the calcium- binding loop and residue 69 sited before the first strand of the β-wing. Also, there are seven disulphide bridges present in the structure. The overall folding of PLA2 observed in the complex with the peptide is similar to that of model group I PLA2 [28] with an r.m.s. shift of 0.2 Å for the Cα positions.
The Ca+2 ion is considered generally essential for catalytic activities of secretory PLA2s [37, 38]. In the present structure, the Ca+2 ion, stabilizes the conformation of the calcium binding loop. A structurally conserved solvent water molecule (OW94) completes the coordination sphere of the Ca+2 ion, forming pentagonal bipyramidal geometry. The electron density of Ca+2 is shown in Fig. (6).
Table 2. Refinement statistics.
PDB Code 3GCI
Resolution range (Ǻ) 42.6 – 2.04
Number of reflections 7596
RCryst (%) 18.6
RFree (5 % data) (%) 22.1
Number of protein atoms 906
Number of peptide atoms 43
Number of Water Molecules 95
Number of Calcium ions 1
R. m. s. deviations
Bond length (Ǻ2) 0.01
Bond angles (o) 1.2
Dihedral angles (°) 14.5
Overall G factor 0.14
Mean B factor (Ǻ2)
Main chain atoms 23.3
Side chains and water molecules 27.5
Overall 25.5
Ramachandran plot statistics
Residues in the most allowed region (%) 91.4 Residues in the additionally allowed region (%) 8.6
3.3. Structure of Peptide
The structure of PLA2 in the complex remains unchanged from the previous group I structure taken from PDB [28]. All seven residues of the peptide can be traced clearly from their electron densities Fig. (2). The interaction of the peptide with the protein is shown in (Fig. 7). Kinetic studies showed that the peptide VGGVVIA binds to PLA2 in a competitive manner with a binding constant of 5.2 x 10-7 M. The torsion angles are listed in Table 3. Most of the peptide residue’s torsional angles are found to be in the core area of the Ramachandran plot with
only one residue in additionally allowed region. The structure of the peptide is given in Fig. (8).
Fig. (3). Ramachandran plot of the main chain torsion angles (φ,ψ) for the final refined model calculated with the program PROCHECK. Non-glycine residues are identified by squares.
Fig. (4). Overall diagram of PLA2 with peptide VGGVVIA.
Fig. (5). Surface diagram representation of the binding cavity and the hydrophobic channel with the bound peptide VGGVVIA in the pocket.
Fig. (6). Difference Fourier |Fo-Fc| map of calcium binding contoured at 3.0 σ cut off. Calcium coordinated interactions are indicated by dotted lines. Residue Ala7 of peptide is indicated by green.
4. DISCUSSION
Structural analysis reveals that PLA2 enzyme has an overall conserved globular structure with minor species specific variations seen at the active site, calcium binding loop, hydrophobic channel, the C-terminal domain and the quaternary conformational state. Electron density was viewed in the difference Fourier |Fo - Fc| map in the complex
Table 3. Torsion angles of the hepta-peptide VGGVVIA.
Most of the torsion angle values lie in the α-helical region of the Ramachandran Plot.
Residue Phi (φ°) Psi (Ψ°)
P2 Gly 95.9 59.1
P3 Gly -163.1 -161.6
P4 Val -102.2 -88.3
P5 Val -157.3 128.9
P6 Ile -106.1 -30.0
Fig. (7). The interaction involving peptide (green) and residues of PLA2.
Fig. (8). The structure adopted by peptide fragment (VGGVVIA) in the complex with PLA2.
structure (Fig. 2) which allowed the interpretation of one molecule of the hepta-peptide as well as the detailed description of its interactions with PLA2. The peptide was positioned well in the hydrophobic substrate binding channel
Aβ-Peptide and Phospholipase A2 CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 7 1229
of the enzyme (Fig. 5). This hepta-peptide interacts directly with the PLA2 active site residues (Fig. 7) as well as with many of the substrate recognition residues in the binding channel, at least through eight hydrogen bonds and about a two dozen Van der Waals interactions. The carboxyl oxygen of the terminal alanine of the hepta-peptide makes direct hydrogen bond with active site residue Asp49 Oδ1. It is also involved in interaction with the active site residue through a water molecule OW88 which is hydrogen bonded to Asp49.
The same carbonyl atom of peptide is also involved in the coordination with calcium. This peptide interacts with active site residue Asp49 with at least three hydrogen bonds. The OXT atom of terminal alanine hydrogen bonded to Lys69 of the protein. Backbone carboxyl of Ile6 is hydrogen bonded to Gly30 (Table 4). In addition, the peptide is involved in van der Waals interaction with most of the residues lining the hydrophobic substrate-binding channel (Table 5). Even Lys6 of the protein shows interactions with nonpolar residues of the peptide; suggesting that overriding non-polar environment of the binding cavity had an influence on disrupting the secondary structure of the peptidic ligand.
Table 4. Hydrogen bonds between PLA2 and peptide VGGVVIA.
Atoms of Peptide Protein Atoms Distance (Ǻ)
Ala7O Wat 88O 2.92
Asp 49Oδ1 2.96
Gly 30N 3.17
Ala7OXT Wat 94O 2.70
Lys 64Nζ 2.99
Ile6O Gly 30N 3.18
Val1N Wat 107O 2.83
Val1O Wat 36O 2.97
In an AD affected brain both Aβ40 and Aβ42 are seen and they share common aggregation features, but the 42-residue peptide is much more amyloidogenic and strongly associated with neurodegenerative pathology [39]. The hydrophobic C- terminal part of the Aβ peptide is critical in triggering the transformation from α-helical to β-sheet structure. In a combined CD and NMR study, Aβ42 peptide was found to be significantly structured at the C-terminal region [39-42] as compared to N-terminus and the central hydrophobic core, suggesting different aggregation propensity of Aβ40 andAβ42
peptides [40]. Potential ability of Aβ-binding proteins to bind and sequester Aβ may possibly influence its clearance and aggregation tendency [41]. The selected peptide fragment displays more non-polar interactions than polar interactions. The observed peptide conformation must have been dictated by the protein ligand interactions. This exemplifies the strength of the multiple interactions between the enzyme and peptidic ligand and also their possible effect on the adopted conformation of the peptide. We speculate that higher affinity between Aβ and PLA2 has therapeutic potential of decreasing the Aβ-Aβ interaction thereby reducing the amyloid aggregation and plaque formation in AD. This study extends our further knowledge about AD [42-44].
Table 5. Van Der Waals interactions between PLA2 and peptide VGGVVIA.
Atoms of Peptide Protein Atoms Distance (Ǻ)
Val1Cα Tyr 3Cβ 3.62
Val1Cβ Tyr 3Cβ 3.95
Tyr 3Cβ 3.97
Leu 2C 4.00
Leu 2Cβ 3.82
Val1Cγ2 Tyr 3Cβ 3.70
Ile6Cα Ala 23Cα 3. 98
Ile6Cδ1 Lys 6Cα 3.88
Lys 6Cβ 3.69
Trp 19Cε3 3.77
Ile6Cγ2 Trp 19Cε3 3.69
Ala 23Cα 3.87
Ala 23Cβ 3.90
Ile6C Gly 30Cα 3.73
Ile6Cγ1 Trp 19Cα3 3.50
Trp 19Cζ3 3.42
Ala7Cα His 48CE1 3.34
Ala7Cβ Leu 2Cδ1 3.78
Tyr 52Cε2 3.51
CONCLUSION
Aβ polypeptide structure plays a significant role in determining the protein aggregation in AD. Structural characterization of the Aβ peptide C-terminus is of vital significance in appreciating the molecular mechanism of Aβ oligomerization. The present work provides a structural evidence to show that Aβ may directly interact with PLA2 in physiological systems. The number of interactions indicates that the affinity between PLA2 and the hepta-peptide is greater than the affinity between two Aβ peptide chains. We speculate that phospholipase A2 (PLA2) may inhibit the aggregation of Aβ peptide by interacting with the peptide and keeping the two peptide chains apart. Binding of PLA2
to Aβ peptide may lessen the free Aβ concentration in the brain, thereby reducing its oligomerization feasibility and intensity and hence may have potential therapeutic benefit in AD. Therefore, PLA2 is proposed as a good molecule for therapeutic intervention to prevent or lessen Aβ peptides aggregation. This notion needs further systematic investigations and may provide a viable therapeutic strategy for AD in the future.
AUTHORS’ CONTRIBUTION
ZM conceived and designed research (project conception, development of overall research plan, and study oversight).
ZM and VGP were involved in hands-on conduct of the experiments, data collection and analysis. ZM was involved
in manuscript writing. MAK helped in revising and improving the final content of this paper.
LIST OF ABBREVIATIONS AD = Alzheimer’s disease
Aβ = Amyloid β
APP = Amyloid precursor protein
MALDI-TOF = Matrix assisted laser desorption-ionization –
time of flight
PDB = Protein Data Bank PLA2 = Phospholipase A2
VGGVVIA = Val-Gly-Gly-Val-Val-Ile-Ala
CONFLICT OF INTEREST
The authors have declared that no competing interests exist.
ACKNOWLEDGEMENTS
Authors acknowledge the substantial intellectual and financial support from all the faculties and the facilities of Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India. ZM was recipient of Junior and Senior Research Fellowship from Council of Scientific and Industrial Research, Govt. of India. ZM and MAK would also like to acknowledge Deanship of Scientific Research (DSR) and King Fahd Medical Research Center (KFMRC), King Abdulaziz University (Jeddah, Saudi Arabia) for providing the necessary research and support facilities.
REFERENCES
[1] Mielke MM, Vemuri P, Rocca WA. Clinical epidemiology of Alzheimer's disease: assessing sex and gender differences. Clin Epidemiol 2014; 6: 37-48.
[2] Masters CL, Simms G, Weinman NA, et al. Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci USA 1985; 82(12): 4245-9.
[3] Selkoe DJ. Physiological production of the β-amyloid protein and the mechanism of Alzheimer's disease. Trends Neurosci 1993;
16(10): 403-9.
[4] Yao F, Zhang R, Tian H, Li X. Studies on the Interactions of Copper and Zinc Ions with beta-Amyloid Peptides by a Surface Plasmon Resonance Biosensor. Int J Mol Sci 2012; 13(9): 11832- 43.
[5] Reale MA, Di Nicola M, Velluto L, et al. Selective Acetyl- and Butyrylcholinesterase Inhibitors Reduce amyloid-beta Ex Vivo Activation of Peripheral Chemo-Cytokines from Alzheimer's Disease Subjects: Exploring the Cholinergic Anti-Inflammatory Pathway. Curr Alzheimer Res 2014; 11(6): 608-22.
[6] Mirza Z, Ali A, Ashraf GM, et al. Proteomics Approaches to Understand Linkage Between Alzheimer's Disease and Type 2 Diabetes Mellitus. CNS Neurol Disord Drug Targets 2014; 13(2):
213-25.
[7] Selkoe DJ. Translating cell biology into therapeutic advances in Alzheimer's disease. Nature 1999; 399(6738 Suppl): A23-31.
[8] Xia W, Yang T, Shankar G, et al. A specific enzyme-linked immunosorbent assay for measuring beta-amyloid protein oligomers in human plasma and brain tissue of patients with Alzheimer disease. Arch Neurol 2009; 66(2): 190-9.
[9] Tomaselli S, Esposito V, Vangone P, et al. The alpha-to-beta conformational transition of Alzheimer's Abeta-(1-42) peptide in aqueous media is reversible: a step by step conformational analysis
suggests the location of beta conformation seeding. Chem Biochem 2006; 7(2): 257-67.
[10] Kuo YM, Emmerling MR, Vigo-Pelfrey C, et al. Water-soluble Abeta (N-40, N-42) oligomers in normal and Alzheimer disease brains. J Biol Chem 1996; 271(8): 4077-81.
[11] Burdick D, Soreghan B, Kwon M, et al. Assembly and aggregation properties of synthetic Alzheimer's A4/beta amyloid peptide analogs. J Biol Chem 1992; 267(1): 546-54.
[12] Haass C, Hung AY, Selkoe DJ, Teplow DB. Mutations associated with a locus for familial Alzheimer's disease result in alternative processing of amyloid beta-protein precursor. J Biol Chem 1994;
269(26): 17741-8.
[13] Urbanc B, Cruz L, Yun S, et al. In silico study of amyloid beta- protein folding and oligomerization. Proc Natl Acad Sci USA 2004; 101(50): 17345-50.
[14] Lee EB, Skovronsky DM, Abtahian F, Doms RW, Lee VM.
Secretion and intracellular generation of truncated Abeta in beta- site amyloid-beta precursor protein-cleaving enzyme expressing human neurons. J Biol Chem 2003; 278(7): 4458-66.
[15] Yun S, Urbanc B, Cruz L, et al. Role of electrostatic interactions in amyloid beta-protein (A beta) oligomer formation: a discrete molecular dynamics study. Biophys J 2007; 92(11): 4064-77.
[16] Jarrett JT, Berger EP, Lansbury PT, Jr. The carboxy terminus of the beta amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer's disease. Biochemistry 1993; 32(18): 4693-7.
[17] Bitan G, Kirkitadze MD, Lomakin A, et al. Amyloid beta -protein (Abeta) assembly: Abeta 40 and Abeta 42 oligomerize through distinct pathways. Proc Natl Acad Sci USA 2003; 100(1): 330-5.
[18] Tjernberg LO, Tjernberg A, Bark N, et al. Assembling amyloid fibrils from designed structures containing a significant amyloid beta-peptide fragment. Biochem J 2002; 366(Pt 1): 343-51.
[19] Farooqui AA, Ong WY, Horrocks LA, Farooqui T. Brain Cytosolic Phospholipase A2: Localization, Role, and Involvement in Neurological Diseases. Neuroscientist 2000; 6(3): 169-80.
[20] Negre-Aminou P, Pfenninger KH. Arachidonic acid turnover and phospholipase A2 activity in neuronal growth cones. J Neurochem 1993; 60(3): 1126-36.
[21] St-Gelais F, Menard C, Congar P, Trudeau LE, Massicotte G.
Postsynaptic injection of calcium-independent phospholipase A2
inhibitors selectively increases AMPA receptor-mediated synaptic transmission. Hippocampus 2004; 14(3): 319-25.
[22] Reale M, Kamal MA, Velluto L, et al. Relationship between inflammatory mediators, Abeta levels and ApoE genotype in Alzheimer disease. Curr Alzheimer Res 2012; 9(4): 447-57.
[23] Singh RK, Singh N, Jabeen T, et al. Crystal structure of the complex of group I PLA2 with a group II-specific peptide Leu-Ala- Ile-Tyr-Ser (LAIYS) at 2.6 Å resolution. J Drug Targeting 2005;
13(6): 367-74.
[24] Wery JP, Schevitz RW, Clawson DK, et al. Structure of recombinant human rheumatoid arthritic synovial fluid phospholipase A2 at 2.2 Å resolution. Nature 1991; 352(6330): 79- 82.
[25] Burke JE, Dennis EA. Phospholipase A2 structure/function, mechanism, and signaling. J Lipid Res 2009; 50 Suppl: S237-42.
[26] Jabeen T, Singh N, Singh RK, et al. Crystal structure of a heterodimer of phospholipase A2 from Naja naja sagittifera at 2.3 Å resolution reveals the presence of a new PLA2-like protein with a novel Cys 32–Cys 49 disulphide bridge with a bound sugar at the substrate-binding site. Prot Struct Func Bioinform 2006; 62(2):
329-37.
[27] Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. In: Carter CW Jr, Ed. Methods in Enzymology. Academic Press; 1997; Vol. 276: pp. 307-26.
[28] Singh RK, Vikram P, Makker J, et al. Design of specific peptide inhibitors for group I phospholipase A2: structure of a complex formed between phospholipase A2 from Naja naja sagittifera (group I) and a designed peptide inhibitor Val-Ala-Phe-Arg-Ser (VAFRS) at 1.9 Å resolution reveals unique features. Biochemistry 2003;
42(40): 11701-6.
[29] Navaza J. AMoRe: an automated package for molecular replacement. Acta Crystallogr Sect A 1994; 50(2): 157-63.
[30] 4 CCPN. The CCP4 suite: programs for protein crystallography.
Acta Crystallogr 1994; 50(Pt 5): 760-3.
Aβ-Peptide and Phospholipase A2 CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 7 1231 [31] Murshudov GN, Vagin AA, Dodson EJ. Refinement of
macromolecular structures by the maximum-likelihood method.
Acta Crystallogr 1997; 53(Pt 3): 240-55.
[32] Jones TA, Zou JY, Cowan SW, Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr 1991; 47(Pt 2):
110-9.
[33] Morris RJ, Perrakis A, Lamzin VS. ARP/wARP and automatic interpretation of protein electron density maps. Methods Enzymol 2003; 374: 229-44.
[34] Langer G, Cohen SX, Lamzin VS, Perrakis A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat Protoc 2008; 3(7): 1171-9.
[35] Ramachandran GN, Sasisekharan V. Conformation of polypeptides and proteins. Adv Prot Chem 1968; 23: 283-438.
[36] Laskowski RA, MacArthur MW, Moss DS, Thornton JM.
PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 1993; 26(2): 283-91.
[37] Berg OG, Yu BZ, Rogers J, Jain MK. Interfacial catalysis by phospholipase A2: determination of the interfacial kinetic rate constants. Biochemistry 1991; 30(29): 7283-97.
[38] Yu BZ, Berg OG, Jain MK. The divalent cation is obligatory for the binding of ligands to the catalytic site of secreted phospholipase A2. Biochemistry 1993; 32(25): 6485-92.
[39] Iwatsubo T, Odaka A, Suzuki N, et al. Visualization of Aβ42 (43) and Aβ40 in senile plaques with end-specific Aβ monoclonals:
evidence that an initially deposited species is Aβ42 (43). Neuron 1994; 13(1): 45-53.
[40] Lim KH, Collver HH, Le YT, Nagchowdhuri P, Kenney JM.
Characterizations of distinct amyloidogenic conformations of the Abeta (1-40) and (1-42) peptides. Biochemical and biophysical research communications. 2007; 353(2): 443-9.
[41] Bates KA, Verdile G, Li QX, et al. Clearance mechanisms of Alzheimer's amyloid-beta peptide: implications for therapeutic design and diagnostic tests. Mol Psychiatry 2009; 14(5): 469-86.
[42] Yu QS, Reale M, Kamal MA, et al. Synthesis of the Alzheimer drug Posiphen into its primary metabolic products (+)-N1- norPosiphen, (+)-N8-norPosiphen and (+)-N1, N8-bisnorPosiphen, their inhibition of amyloid precursor protein, alpha-Synuclein synthesis, interleukin-1beta release, and cholinergic action.
Antiinflammatory Antiallergy Agents Med Chem 2013; 12(2): 117- 28.
[43] Mirza Z, Kamal MA, Abuzenadah AM, Al-Qahtani MH, Karim S.
Establishing Genomic/Transcriptomic Links between Alzheimer's Disease and Type II Diabetes Mellitus by Meta-Analysis Approach. CNS Neurol Disord Drug Targets 2014; 13(3): 501-6.
[44] Karim S, Mirza Z, Kamal MA, et al. An Association of Virus Infection with Type 2 Diabetes and Alzheimer's Disease. CNS Neurol Disord Drug Targets 2014; 13(3): 429-39.
Received: March 31, 2014 Revised: April 15, 2014 Accepted: April 16, 2014
PMID: 25230229
