Degradation of gamma secretase activating protein by the ubiquitin–proteasome pathway

A major hallmark feature of Alzheimer's disease is the accumulation of amyloid β (Aβ), whose formation is regulated by the γ‐secretase complex and its activating protein (also known as γ‐secretase activating protein, or GSAP). Because GSAP interacts with the γ‐secretase without affecting the cleavage of Notch, it is an ideal target for a viable anti‐Aβ therapy. GSAP derives from a C‐terminal fragment of a larger precursor protein of 98 kDa via a caspase 3‐mediated cleavage. However, the mechanism(s) involved in its degradation remain unknown. In this study, we show that GSAP has a short half‐life of approximately 5 h. Neuronal cells treated with proteasome inhibitors markedly prevented GSAP protein degradation, which was associated with a significant increment in Aβ levels and γ‐secretase cleavage products. In contrast, treatment with calpain blocker and lysosome inhibitors had no effect. In addition, we provide experimental evidence that GSAP is ubiquitinated. Taken together, our findings reveal that GSAP is degraded through the ubiquitin–proteasome system. Modulation of the GSAP degradation pathway may be implemented as a viable target for a safer anti‐Aβ therapeutic approach in Alzheimer's disease.

     

Trpm2 enhances physiological bioenergetics and protects against pathological oxidative cardiac injury: Role of Pyk2 phosphorylation

The mechanisms by which Trpm2 channels enhance mitochondrial bioenergetics and protect against oxidative stress-induced cardiac injury remain unclear. Here, the role of proline-rich tyrosine kinase 2 (Pyk2) in Trpm2 signaling is explored. Activation of Trpm2 in adult myocytes with H₂O₂ resulted in 10- to 21-fold increases in Pyk2 phosphorylation in wild-type (WT) myocytes which was significantly lower (~40%) in Trpm2 knockout (KO) myocytes. Pyk2 phosphorylation was inhibited (~54%) by the Trpm2 blocker clotrimazole. Buffering Trpm2-mediated Ca²⁺ increase with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) resulted in significantly reduced pPyk2 in WT but not in KO myocytes, indicating Ca²⁺ influx through activated Trpm2 channels phosphorylated Pyk2. Part of phosphorylated Pyk2 translocated from cytosol to mitochondria which has been previously shown to augment mitochondrial Ca²⁺ uptake and enhance adenosine triphosphate generation. Although Trpm2-mediated Ca²⁺ influx phosphorylated Ca²⁺-calmodulin kinase II (CaMKII), the CaMKII inhibitor KN93 did not significantly affect Pyk2 phosphorylation in H₂O₂-treated WT myocytes. After ischemia/reperfusion (I/R), Pyk2 phosphorylation and its downstream prosurvival signaling molecules (pERK1/2 and pAkt) were significantly lower in KO-I/R when compared with WT-I/R hearts. After hypoxia/reoxygenation, mitochondrial membrane potential was lower and superoxide level was higher in KO myocytes, and were restored to WT values by the mitochondria-targeted superoxide scavenger MitoTempo. Our results suggested that Ca²⁺ influx via tonically activated Trpm2 phosphorylated Pyk2, part of which translocated to mitochondria, resulting in better mitochondrial bioenergetics to maintain cardiac health. After I/R, Pyk2 activated prosurvival signaling molecules and prevented excessive increases in reactive oxygen species, thereby affording protection from I/R injury.     

Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

Annexin V and Sytox Green are widely used markers to evaluate apoptosis in various cell types using flow cytometry and fluorescent microscopy. Recently, a novel fluoroprobe MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells and was validated for confocal microscopy and flow cytometry. This protocol describes simultaneous measurements of mitochondrial superoxide generation with apoptotic markers (Annexin V and Sytox Green) by both flow cytometry and confocal microscopy in endothelial cell lines. The advantages of the described flow cytometry method over other cell-based techniques are the tremendous speed (1-2 h), exquisite precision and the possibility of simultaneous quantitative measurements of mitochondrial superoxide generation and apoptotic (and other) markers, with maximal preservation of cellular functions. This method combined with fluorescent microscopy may be very useful to reveal important spatial-temporal changes in mitochondrial superoxide production and execution of programmed cell death in virtually any cell type.     

Nitration of the mitochondrial complex I subunit NDUFB8 elicits RIP1- and RIP3-mediated necrosis

Nitric oxide (NO) and other reactive nitrogen species target multiple sites in the mitochondria to influence cellular bioenergetics and survival. Kinetic imaging studies revealed that NO from either activated macrophages or donor compounds rapidly diffuses to the mitochondria, causing a dose-dependent progressive increase in NO-dependent DAF fluorescence, which corresponded to mitochondrial membrane potential loss and initiated alterations in cellular bioenergetics that ultimately led to necrotic cell death. Cellular dysfunction is mediated by an elevated 3-nitrotyrosine signature of the mitochondrial complex I subunit NDUFB8, which is vital for normal mitochondrial function as evidenced by selective knockdown via siRNA. Overexpression of mitochondrial superoxide dismutase substantially decreased NDUFB8 nitration and restored mitochondrial homeostasis. Further, treatment of cells with either necrostatin-1 or siRNA knockdown of RIP1 and RIP3 prevented NO-mediated necrosis. This work demonstrates that the interaction between NO and mitochondrially derived superoxide alters mitochondrial bioenergetics and cell function, thus providing a molecular mechanism for reactive oxygen and nitrogen species-mediated alterations in mitochondrial homeostasis.     

Apoptosis in the monkey small intestinal epithelium: structural and functional alterations in the mitochondria

Our earlier studies have shown apoptosis in the villus tip cells of the monkey small intestinal epithelium. Because mitochondria have been implicated in the apoptotic process, this study looked at the function and lipid composition of mitochondria isolated from apoptotic villus tip cells and compared it with middle and crypt cells. Decreased MTT reduction and respiratory control ratio, increased swelling and altered mitochondrial enzyme activities were seen in the villus tip cell mitochondria when compared to other cells. The lipid composition of the villus tip mitochondria were different from the other mitochondria. A decrease in phosphatidylethanolamine and phosphatidyl-inositol and an increase in phosphatidic acid was seen in these mitochondria. Fatty acid composition analysis showed more unsaturated fatty acids in the free fatty acid and phospholipid fraction in villus tip cell mitochondria as compared to other cells. These studies suggest that in the monkey small intestinal epithelium, apoptotic process is associated with functional and structural alterations in the mitochondria.     

Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis

Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca2+ ([Ca2+]i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca2+]i store depletion, but not extracellular Ca2+ chelation, prevented [Ca2+]i elevation in response to O2*- that was inositol 1,4,5-trisphosphate (InsP3) dependent, and cells lacking the three InsP3 receptor (InsP3R) isoforms failed to display the [Ca2+]i transient. Importantly, the O2*--triggered Ca2+ mobilization preceded a loss in mitochondrial membrane potential that was independent of other oxidants and mitochondrially derived ROS. Activation of apoptosis occurred selectively in response to O2*- and could be prevented by [Ca2+]i buffering. This study provides evidence that O2*- facilitates an InsP3R-linked apoptotic cascade and may serve a critical function in I/R injury and inflammation.     

STIM proteins: dynamic calcium signal transducers

Stromal interaction molecule (STIM) proteins function in cells as dynamic coordinators of cellular calcium (Ca(2+)) signals. Spanning the endoplasmic reticulum (ER) membrane, they sense tiny changes in the levels of Ca(2+) stored within the ER lumen. As ER Ca(2+) is released to generate primary Ca(2+) signals, STIM proteins undergo an intricate activation reaction and rapidly translocate into junctions formed between the ER and the plasma membrane. There, STIM proteins tether and activate the highly Ca(2+)-selective Orai channels to mediate finely controlled Ca(2+) signals and to homeostatically balance cellular Ca(2+). Details are emerging on the remarkable organization within these STIM-induced junctional microdomains and the identification of new regulators and alternative target proteins for STIM.     

Blockade of MCU-Mediated Ca2+ Uptake Perturbs Lipid Metabolism via PP4-Dependent AMPK Dephosphorylation

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Purification and characterization of butyrate-induced protein phosphatase involved in apoptosis of Ehrlich ascites tumor cells

Short chain fatty acids including butyrate exhibit wide variety of biological effects towards cell growth, morphology and gene expression. In this report, we study the mechanism by which butyrate (BuA) modulates the expression of protein phosphatase when treated to the cells. As a model system, we used Ehrlich Ascites Tumor (EAT) cells in which BuA-treatment induces expression of a protein phosphatase enzyme. Subsequently, BuA-induced protein phosphatase has been biochemically purified and characterized. Further, pretreatment of caspase-3 inhibitor abolished the activity of BuA-induced protein phosphatase indicating the involvement of caspase-3 in the activation of BuA-induced protein phosphatase. In addition, the relationship between BuA-induced protein phosphatase and apoptosis has been verified. Activation of endonuclease-II has been shown in BuA-treated EAT cells and that activity was completely inhibited by sodium orthovanadate, a tyrosine phosphatase inhibitor suggesting that endonuclease-II may serve as a possible down-stream target for BuA-induced protein phosphatase. Together, the data suggest that activation of protein phosphatase may be an early and essential step in BuA-mediated apoptotic signaling pathway in EAT cells.