Catalytic Mechanism of Amyloid-β Peptide Degradation by Insulin Degrading Enzyme: Insights from QM/MM MP2 Calculation

Insulin degrading enzyme (IDE), a metalloprotease that degrades amyloid-β (Aβ) peptides and insulin, is associated with Alzheimer’s disease and diabetes. The mechanism of IDE catalyzed degrading of Aβ peptides, which is of fundamental importance in the design of therapeutic methods for Alzheimer’s disease, has not been fully understood. In this work, combined quantum mechanics and molecular mechanics (QM/MM) style Møller-Plesset second order perturbation theory (MP2) geometry optimization calculations are performed to investigate the catalytic mechanism of the Aβ40 Phe19-Phe20 peptide bond cleavage by human IDE. The analyses using QM/MM MP2 optimization suggest that a neutral water molecule is at the active site of the enzyme-substrate (ES) complex. The water molecule is in hydrogen bonding with the nearby anionic Glu111 of IDE, but not directly bound to the catalytic Zn ion. This is confirmed by QM/MM DFTB3 molecular dynamics simulation. Our studies also reveal that the hydrolysis of the Aβ40 Phe19-Phe20 peptide bond by IDE consists of four key steps. The neutral water is first activated by moving toward and binding to the Zn ion. A gem-diol intermediate is then formed by the activated neutral water molecule attacking the C atom of the Phe19-Phe20 peptide bond. The next is the protonation of the N atom of Phe19-Phe20 peptide bond to form an intermediate with an elongated C-N bond. The final step is the breaking of the Phe19-Phe20 C-N bond. The final step is the rate-determining step with a calculated Gibbs free energy of activation of 17.34 kcal/mol, in good agreement with the experimental value 16.7 kcal/mol. This mechanism provides the basis for the design of biochemical methods to modulate the activity of IDE in humans

Daughterless homodimer synergizes with Eyeless to induce Atonal expression and retinal neuron differentiation

AbstractClass I Basic Helix-Loop-Helix (bHLH) transcription factors form homodimers or heterodimers with class II bHLH proteins. While bHLH heterodimers are known to have diverse roles, little is known about the role of class I homodimers. In this manuscript, we show that a linked dimer of Daughterless (Da), the only Drosophila class I bHLH protein, activates Atonal (Ato) expression and retinal neuron differentiation synergistically with the retinal determination factor Eyeless (Ey). The HLH protein Extramacrocheate (Emc), which forms heterodimer with Da, antagonizes the synergistic activation from Da but not the Da–Da linked dimer with Ey. We show that Da directly interacts with Ey and promotes Ey binding to the Ey binding site in the Ato 3׳ enhancer. Interestingly, the Ey binding site in the Ato 3׳ enhancer contains an embedded E-box that is also required for the synergistic activation by Ey and Da. Finally we show that mammalian homologs of Ey and Da can functionally replace their Drosophila counterparts to synergistically activate the Ato enhancer, suggesting that the observed function is evolutionary conserved

Golgi Cisternal Unstacking Stimulates COPI Vesicle Budding and Protein Transport

The Golgi apparatus in mammalian cells is composed of flattened cisternae that are densely packed to form stacks. We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae. We established an assay to measure protein transport to the cell surface in post-mitotic cells in which the Golgi was unstacked. Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes. Vesicle budding from unstacked cisternae in vitro was significantly increased compared to stacked membranes. These results suggest that Golgi cisternal stacking can directly regulate vesicle formation and thus the rate of protein transport through the Golgi. The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning

Properties of Lactobacillus reuteri chitosan-calcium-alginate encapsulation under simulated gastrointestinal conditions

The protective effects of encapsulation on the survival of Lactobacillus reuteri and the retention of the bacterium’s probiotic properties under simulated gastrointestinal conditions were investigated. Viable counts and the remaining probiotic properties of calcium (Ca)-alginate encapsulated (A group), chitosan-Ca-alginate encapsulated (CA group), and unencapsulated, free L. reuteri (F group) were determined. Encapsulation improved the survival of L. reuteri subjected to simulated gastrointestinal conditions, with the greatest protective effect achieved in the CA group. The degree of cell membrane injury increased with increasing bile salt concentrations at constant pH, but the extent of injury was less in the encapsulated than in the free cells. Adherence rates were, in descending order: CA (0.524%) > A (0.360%) > F (0.275%). Lactobacillus reuteri cells retained their antagonistic activity toward Listeria monocytogenes even after incubation of the lactobacilli under simulated gastrointestinal conditions. Displacement of the pathogen by cells released from either of the encapsulation matrices was higher than that by free cells. The safety of L. reuteri was demonstrated in an in vitro invasion assay. [Int Microbiol 2015; 18(1):61-69]Keywords: Lactobacillus reuteri · Listeria monocytogenes · chitosan–calcium-alginate encapsulation · probiotic properties · simulated gastrointestinal condition

Interactions of Bordetella pertussis adenylyl cyclase toxin CyaA with calmodulin mutants and calmodulin antagonists: Comparison with membranous adenylyl cyclase I

The adenylyl cyclase (AC) toxin CyaA from Bordetella pertussis constitutes an important virulence factor for the pathogenesis of whooping cough. CyaA is activated by calmodulin (CaM) and compromises host defense by excessive cAMP production. Hence, pharmacological modulation of the CyaA/CaM interaction could constitute a promising approach to treat whooping cough, provided that interactions of endogenous effector proteins with CaM are not affected. As a first step toward this ambitious goal we examined the interactions of CyaA with wild-type CaM and four CaM mutants in which most methionine residues were replaced by leucine residues and studied the effects of the CaM antagonists calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). CyaA/CaM interaction was monitored by CaM-dependent fluorescence resonance energy transfer (FRET) between tryptophan residues in CyaA and 2′-(N-methylanthraniloyl)-3′-deoxy-adenosine 5′-triphosphate and catalytic activity. Comparison of the concentration/response curves of CaM and CaM mutants for FRET and catalysis revealed differences, suggesting a two-step activation mechanism of CyaA by CaM. Even in the absence of CaM, calmidazolium inhibited catalysis, and it did so according to a biphasic function. Trifluoperazine and W-7 did not inhibit FRET or catalysis. In contrast to CyaA, some CaM mutants were more efficacious than CaM at activating membranous AC isoform 1. The slope of CyaA activation by CaM was much steeper than of AC1 activation. Collectively, the two-step activation mechanism of CyaA by CaM offers opportunities for pharmacological intervention. The failure of classic CaM inhibitors to interfere with CyaA/CaM interactions and the different interactions of CaM mutants with CyaA and AC1 point to unique CyaA/CaM interactions

Distinct Interactions of 2′- and 3′-O-(N-Methyl)anthraniloyl-Isomers of ATP and GTP with the Adenylyl Cyclase Toxin of Bacillus anthracis, Edema Factor

Anthrax disease is caused by the spore-forming bacterium, Bacillus anthracis. Bacillus anthracis produces a calmodulin-activated adenylyl cyclase (AC) toxin, edema factor (EF). Through excessive cAMP accumulation EF disrupts host defence. In a recent study we showed that various 2′(3′)-O-N-(methyl)anthraniloyl (MANT)-substituted nucleoside 5′-triphosphates are potent inhibitors (Ki values in the 0.1-5 μM range) of purified EF. Upon interaction with calmodulin we observed efficient fluorescence resonance energy transfer (FRET) between tryptophan and tyrosine residues of EF and the MANT-group of MANT-ATP. Molecular modelling suggested that both the 2′- and 3′-MANT-isomers can bind to EF. The aim of the present study was to examine the effects of defined 2′- and 3′-MANT-isomers of ATP and GTP on EF. 3′-MANT-2′-deoxy-ATP inhibited EF more potently than 2′-MANT-3′-deoxy-ATP, whereas the opposite was the case for the corresponding GTP analogs. Calmodulin-dependent direct MANT-fluorescence and FRET was much larger with 2′-MANT-3′-deoxy-ATP and 2′-MANT-3′-deoxy-GTP compared to the corresponding 3′-MANT-2′-deoxy-isomers and the 2′(3′)-racemates. Ki values of MANT-nucleotides for inhibition of catalysis correlated with Kd values of MANT-nucleotides in FRET studies. Molecular modelling indicated different positioning of the MANT-group in 2′-MANT-3′-deoxy-ATP/GTP and 3′-MANT-2′-deoxy-ATP/GTP bound to EF. Collectively, EF interacts differentially with 2′-MANT- and 3′-MANT-isomers of ATP and GTP, indicative for conformational flexibility of the catalytic site and offering a novel approach for the development of potent and selective EF inhibitors. Moreover, our present study may serve as a general model of how to use MANT-nucleotide isomers for the analysis of the molecular mechanisms of nucleotide/protein interactions