Characterization of N-terminal processing of group VIA phospholipase A<sub>2</sub> and of potential cleavage sites of amyloid precursor protein constructs by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests

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Mouse macrophages undergo ER stress and apoptosis upon free cholesterol loading (FCL). We recently generated iPLA 2 ␤-null mice, and here we demonstrate that iPLA 2 ␤-null macrophages have reduced sensitivity to FCL-induced apoptosis, although they and wild-type (WT) cells exhibit similar increases in the transcriptional regulator CHOP. iPLA 2 ␤-null macrophages are also less sensitive to apoptosis induced by the sarcoplasmic reticulum Ca 2؉ -ATPase inhibitor thapsigargin and the scavenger receptor A ligand fucoidan, and restoring iPLA 2 2 ␤ modulates mitochondrial cytochrome c release, and we find that thapsigargin and fucoidan induce mitochondrial phospholipid loss and cytochrome c release into WT macrophage cytosol and that these events are blunted in iPLA 2 ␤-null cells. Immunoblotting studies indicate that iPLA 2 ␤ associates with mitochondria in macrophages subjected to ER stress. AA incorporation into glycerophosphocholine lipids is unimpaired in iPLA 2 ␤-null macrophages upon electrospray ionization-tandem mass spectrometry analyses, and their complex lipid composition is similar to WT cells. These findings suggest that iPLA 2 ␤ participates in ER stress-induced macrophage apoptosis caused by FCL or thapsigargin but that deletion of iPLA 2 ␤ does not impair macrophage arachidonate incorporation or phospholipid composition.

Section: Discussionmentioning

confidence: 99%

Attenuated Free Cholesterol Loading-induced Apoptosis but Preserved Phospholipid Composition of Peritoneal Macrophages from Mice That Do Not Express Group VIA Phospholipase A2

Bao

Lei

et al. 2007

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“…Data were processed with Masslynx 3.5 software. Multipoint calibration was performed using selected fragment ions produced by CAD of Glu-fibrinopeptide B. MS/MS spectra were processed by Masslynx software to generate a peak list file as described previously (26).…”

Section: Methodsmentioning

confidence: 99%

Modulation of the Regulatory Activity of Bacterial Two-component Systems by SlyA

Song

Weatherspoon

Qin

et al. 2008

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Activation of the transcriptional regulator SlyA by the PhoP/PhoQ two-component system controls intracellular expression of numerous factors influencing Salmonella virulence. By dissecting the SlyA regulon using stable isotope labeling with amino acids in cell culture analysis, we found that SlyA enhances overall transcription of PhoP-activated loci. This amplification of cellular responses to Mg 2؉ occurs when SlyA binds to the phoPQ promoter thereby activating phoP autoregulation via a positive feedback mechanism. SlyA footprints a DNA region located one helical turn upstream of the PhoP box, which overlaps the H-NS-binding motif required for signal-dependent phoP repression in high Mg 2؉ conditions. Therefore, binding of SlyA likely antagonizes H-NS and facilitates the interaction of PhoP to its own promoter, subsequently activating the phoPQ operon. Establishment of this regulatory circuit allows SlyA to exert its effect on the PhoP/PhoQ system specifically in Salmonella, which may confer an additional transcriptional regulation. Thus, our results provide a molecular mechanism that determines SlyA-dependent activation of PhoP-regulated genes in modulating Salmonella virulence. Evidence from this study also suggests a function of SlyA as a mediator in signal transduction from the PhoP/PhoQ system to other bacterial two-component systems in Salmonella.

“…Recombinant His-iPLA 2 ␤-FLAG was purified by immobilized metal affinity chromatography on cobalt-based TALON columns as described (34,38).…”

Section: Ceramide Analyses By Electrospray Ionization Tandem Mass Spementioning

confidence: 99%

“…Because iPLA 2 ␤ undergoes proteolytic processing that affects its organellar association (35,38,56,57), a His-iPLA 2 ␤-FLAG fusion protein with N-terminal polyhistidine and C-terminal FLAG tags was expressed in INS-1 cells that were incubated with palmitate and then disrupted. Mitochondria were isolated, and their proteins were analyzed by SDS-PAGE and immunoblotting with antibodies against polyhistidine or FLAG or against mitochondrial COX IV (Fig.…”

Section: Overexpression Of Ipla 2 ␤ Reduces the Sensitivity Of Ins-1mentioning

confidence: 99%

Group VIA Phospholipase A2 Mitigates Palmitate-induced β-Cell Mitochondrial Injury and Apoptosis

Song

Wohltmann

Tan

et al. 2014

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Background: Lipid-induced ␤-cell loss contributes to type 2 diabetes mellitus (T2DM). Results: Palmitate-induced ␤-cell lipid oxidation, mitochondrial dysfunction, and apoptosis correlate inversely with expression of iPLA 2 ␤, which associates with mitochondria, generates monolysocardiolipin, and lowers oxidized phospholipid content. Conclusion: iPLA 2 ␤ mitigates palmitate-induced ␤-cell mitochondrial injury and apoptosis and may facilitate repair of oxidized lipids. Significance: Understanding lipid-induced ␤-cell loss could lead to T2DM therapies.