Regional increase in the expression of the BCAT proteins in Alzheimer's disease brain: Implications in glutamate toxicity

BACKGROUNDThe human branched chain aminotransferases (hBCATm, mitochondrial and hBCATc, cytosolic) are major contributors to brain glutamate production. This excitatory neurotransmitter is thought to contribute to neurotoxicity in neurodegenerative conditions such as Alzheimer's disease (AD) but the expression of hBCAT in this disease has not previously been investigated.OBJECTIVEThe objective of investigating hBCAT expression is to gain insight into potential metabolic pathways that may be dysregulated in AD brain, which would contribute to glutamate toxicity.METHODSWestern blot analysis and immunohistochemistry were used to determine the expression and localization of hBCAT in postmortem frontal and temporal cortex from AD and matched control brains.RESULTSWestern blot analysis demonstrated a significant regional increase in hBCATc expression in the hippocampus (↑ 36%; p-values of 0.012), with an increase of ↑ 160% reported for hBCATm in the frontal and temporal cortex (p-values = 4.22 × 10-4 and 2.79 × 10-5, respectively) in AD relative to matched controls, with evidence of post-translational modifications to hBCATm, more prominent in AD samples. Using immunohistochemistry, a significant increase in immunopositive labelling of hBCATc was observed in the CA1 and CA4 region of the hippocampus (p-values = 0.011 and 0.026, respectively) correlating with western blot analysis. Moreover, the level of hBCATm in the frontal and temporal cortex correlated significantly with disease severity, as indicated by Braak staging (p-values = 5.63 × 10-6 and 9.29 × 10-5, respectively).CONCLUSIONThe expression of the hBCAT proteins is significantly elevated in AD brain. This may modulate glutamate production and toxicity, and thereby play a role in the pathogenesis of the disease

The branched-chain aminotransferase proteins: Novel redox chaperones for protein disulfide isomerase-implications in Alzheimer's disease

Aims: The human branched-chain aminotransferase proteins (hBCATm and hBCATc) are regulated through oxidation and S-nitrosation. However, it remains unknown whether they share common redox characteristics to enzymes such as protein disulfide isomerase (PDI) in terms of regulating cellular repair and protein misfolding. Results: Here, similar to PDI, the hBCAT proteins showed dithiol-disulfide isomerase activity that was mediated through an S-glutathionylated mechanism. Site-directed mutagenesis of the active thiols of the CXXC motif demonstrates that they are fundamental to optimal protein folding. Far Western analysis indicated that both hBCAT proteins can associate with PDI. Co-immunoprecipitation studies demonstrated that hBCATm directly binds to PDI in IMR-32 cells and the human brain. Electron and confocal microscopy validated the expression of PDI in mitochondria (using Mia40 as a mitochondrial control), where both PDI and Mia40 were found to be co-localized with hBCATm. Under conditions of oxidative stress, this interaction is decreased, suggesting that the proposed chaperone role for hBCATm may be perturbed. Moreover, immunohistochemistry studies show that PDI and hBCAT are expressed in the same neuronal and endothelial cells of the vasculature of the human brain, supporting a physiological role for this binding. Innovation: This study identifies a novel redox role for hBCAT and confirms that hBCATm differentially binds to PDI under cellular stress. Conclusion: These studies indicate that hBCAT may play a role in the stress response of the cell as a novel redox chaperone, which, if compromised, may result in protein misfolding, creating aggregates as a key feature in neurodegenerative conditions such as Alzheimer's disease. © 2014 Mary Ann Liebert, Inc

BCAT-induced autophagy regulates Aβ load through an interdependence of redox state and PKC phosphorylation-implications in Alzheimer's disease

Leucine, nutrient signal and substrate for the branched chain aminotransferase (BCAT) activates the mechanistic target of rapamycin (mTORC1) and regulates autophagic flux, mechanisms implicated in the pathogenesis of neurodegenerative conditions such as Alzheimer's disease (AD). BCAT is upregulated in AD, where a moonlighting role, imparted through its redox-active CXXC motif, has been suggested. Here we demonstrate that the redox state of BCAT signals differential phosphorylation by protein kinase C (PKC) regulating the trafficking of cellular pools of BCAT. We show inter-dependence of BCAT expression and proteins associated with the P13K/Akt/mTORC1 and autophagy signalling pathways. In response to insulin or an increase in ROS, BCATc is trafficked to the membrane and docks via palmitoylation, which is associated with BCATc-induced autophagy through PKC phosphorylation. In response to increased levels of BCATc, as observed in AD, amyloid β (Aβ) levels accumulate due to a shift in autophagic flux. This effect was diminished when incubated with leucine, indicating that dietary levels of amino acids show promise in regulating Aβ load. Together these findings show that increased BCATc expression, reported in human AD brain, will affect autophagy and Aβ load through the interdependence of its redox-regulated phosphorylation offering a novel target to address AD pathology

Characterization of the Salmonella bacteriophage vB_SenS-Ent1

The bacteriophage vB_SenS-Ent1 (Ent1) is a member of the family Siphoviridae of tailed bacteriophages and infects a broad range of serovars of the enteric pathogen Salmonella enterica. The virion particle is composed of an icosahedral head 64 nm in diameter and a flexible, non-contractile tail of 116 × 8.5 nm possessing terminal fibres. The adsorption rate constant at 37 °C is 6.73 × 10−9 ml min−1. Latent and eclipse periods are 25 and 20 min, respectively, and the burst size is 35 progeny particles per cell after 35 min at 37 °C. Sequencing revealed a circularly permuted, 42 391 bp dsDNA genome containing 58 ORFs organized into four major transcriptional units. Comparisons with the genome sequences of other bacteriophages revealed a high level of nucleotide sequence identity and shared orthologous proteins with the Salmonella phages SETP3, SE2 and KS7 (SS3e) and the Escherichia phages K1G, K1H, K1ind1 and K1ind3