Abstract Alzheimer disease (AD) is associated with increased amyloidogenic processing of amyloid precursor protein (APP) to β-amyloid peptides (Aβ), cholinergic neuron loss with decreased choline ...acetyltransferase (ChAT) activity, and cognitive dysfunction. Both 69-kDa ChAT and 82-kDa ChAT are expressed in cholinergic neurons in human brain and spinal cord with 82-kDa ChAT localized predominantly to neuronal nuclei, suggesting potential alternative functional roles for the enzyme. By gene microarray analysis, we found that 82-kDa ChAT-expressing IMR32 neural cells have altered expression of genes involved in diverse cellular functions. Importantly, genes for several proteins that regulate APP processing along amyloidogenic and non-amyloidogenic pathways are differentially expressed in 82-kDa ChAT-containing cells. The predicted net effect based on observed changes in expression patterns of these genes would be decreased amyloidogenic APP processing with decreased Aβ production. This functional outcome was verified experimentally as a significant decrease in BACE1 protein levels and activity and a concomitant reduction in the release of endogenous Aβ1–42 from neurons cultured from brains of AD-model APP/PS1 transgenic mice. The expression of 82-kDa ChAT in neurons increased levels of GGA3, which is involved in trafficking BACE1 to lysosomes for degradation. shRNA-induced decreases in GGA3 protein levels attenuated the 82-kDa ChAT-mediated decreases in BACE1 protein and activity and Aβ1–42 release. Evidence that 82-kDa ChAT can enhance GGA3 gene expression is shown by enhanced GGA3 gene promoter activity in SN56 neural cells expressing this ChAT protein. These studies indicate a novel relationship between cholinergic neurons and APP processing, with 82-kDa ChAT acting as a negative regulator of Aβ production. This decreased formation of Aβ could result in protection for cholinergic neurons, as well as protection of other cells in the vicinity that are sensitive to increased levels of Aβ. Decreasing levels of 82-kDa ChAT due to increasing age or neurodegeneration could alter the balance towards increasing Aβ production, with this potentiating the decline in function of cholinergic neurons.
J. Neurochem. (2011) 117, 538–553.
In Alzheimer’s disease, the amyloid‐β peptide (Aβ) interacts with distinct proteins at the cell surface to interfere with synaptic communication. Recent data have ...implicated the prion protein (PrPC) as a putative receptor for Aβ. We show here that Aβ oligomers signal in cells in a PrPC‐dependent manner, as might be expected if Aβ oligomers use PrPC as a receptor. Immunofluorescence, flow cytometry and cell surface protein biotinylation experiments indicated that treatment with Aβ oligomers, but not monomers, increased the localization of PrPC at the cell surface in cell lines. These results were reproduced in hippocampal neuronal cultures by labeling cell surface PrPC. In order to understand possible mechanisms involved with this effect of Aβ oligomers, we used live cell confocal and total internal reflection microscopy in cell lines. Aβ oligomers inhibited the constitutive endocytosis of PrPC, but we also found that after Aβ oligomer‐treatment PrPC formed more clusters at the cell surface, suggesting the possibility of multiple effects of Aβ oligomers. Our experiments show for the first time that Aβ oligomers signal in a PrPC‐dependent way and that they can affect PrPC trafficking, increasing its localization at the cell surface.
The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. We surveyed Hsf1 activity in a genome-wide ...loss-of-function library in Saccaromyces cerevisiae as well as ∼78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC, and Hsf1 senses an RQC-mediated translation-stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.
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► Comprehensive characterization of the stresses sensed by Hsf1 ► Characterization of a complex that targets ribosomes stalled at translation ► An autoregulatory loop regulates activity of the complex ► Discovery of a translation-stress signaling pathway from the ribosome to Hsf1
A ribosome-bound complex designated RQC associates with 60S ribosomal subunits containing stalled polypeptides to trigger their degradation.
Carbonic anhydrases (CAs) and metallothioneins (MTs) are both families of zinc metalloproteins central to life, however, they coordinate and interact with their Zn
ion cofactors in completely ...different ways. CAs and MTs are highly sensitive to the cellular environment and play key roles in maintaining cellular homeostasis. In addition, CAs and MTs have multiple isoforms with differentiated regulation. This review discusses current literature regarding these two families of metalloproteins in carcinogenesis, with a dialogue on the association of these two ubiquitous proteins in vitro in the context of metalation. Metalation of CA by Zn-MT and Cd-MT is described. Evidence for protein-protein interactions is introduced from changes in metalation profiles of MT from electrospray ionization mass spectrometry and the metalation rate from stopped-flow kinetics. The implications on cellular control of pH and metal donation is also discussed in the context of diseased states.
We previously demonstrated that nerve cell lines selected for resistance to amyloid β (Aβ) peptide exhibit elevated aerobic glycolysis in part due to increased expression of pyruvate dehydrogenase ...kinase 1 (PDK1) and lactate dehydrogenase A (LDHA). Here, we show that overexpression of either PDK1 or LDHA in a rat CNS cell line (B12) confers resistance to Aβ and other neurotoxins. Treatment of Aβ-sensitive cells with various toxins resulted in mitochondrial hyperpolarization, immediately followed by rapid depolarization and cell death, events accompanied by increased production of cellular reactive oxygen species (ROS). In contrast, cells expressing either PDK1 or LDHA maintained a lower mitochondrial membrane potential and decreased ROS production with or without exposure to toxins. Additionally, PDK1- and LDHA-overexpressing cells exhibited decreased oxygen consumption but maintained levels of ATP under both normal culture conditions and following Aβ treatment. Interestingly, immunoblot analysis of wild type mouse primary cortical neurons treated with Aβ or cortical tissue extracts from 12-month-old APPswe/PS1dE9 transgenic mice showed decreased expression of LDHA and PDK1 when compared with controls. Additionally, post-mortem brain extracts from patients with Alzheimer disease exhibited a decrease in PDK1 expression compared with nondemented patients. Collectively, these findings indicate that key Warburg effect enzymes play a central role in mediating neuronal resistance to Αβ or other neurotoxins by decreasing mitochondrial activity and subsequent ROS production. Maintenance of PDK1 or LDHA expression in certain regions of the brain may explain why some individuals tolerate high levels of Aβ deposition without developing Alzheimer disease.
Background: Aerobic glycolysis promotes resistance against Aβ toxicity.
Results: Increased LDHA and PDK1 expression attenuates mitochondrial activity and confers resistance to Aβ. These proteins are down-regulated in a transgenic Alzheimer disease (AD) mouse model, and PDK1 is decreased in AD brain.
Conclusion: PDK and LDHA are central mediators of Aβ resistance.
Significance: Drugs that augment aerobic glycolysis may enhance brain cell survival in AD patients.
Aberrant angiogenesis is an essential step for the progression of solid tumors. Thus anti-angiogenic therapy is one of the most promising approaches to control tumor growth. In this study, we ...examined the ability of 20(R)-ginsenoside Rg3 (Rg3), one of the active compounds present in ginseng root, to interfere with the various steps of angiogenesis. Rg3 was found to inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) with an IC50 of 10 nM in Trypan blue exclusion assay. Rg3 (1-10(3) nM) also dose dependently suppressed the capillary tube formation of HUVEC on the Matrigel in the presence or absence of 20 ng/ml vascular endothelial growth factor (VEGF). The VEGF-induced chemoinvasion of HUVEC and ex vivo microvascular sprouting in rat aortic ring assay were both significantly attenuated by Rg3. In addition, Rg3 (150 and 600 nM) remarkably abolished the basic fibroblast growth factor (bFGF)-induced angiogenesis in an in vivo Matrigel plug assay. The Matrix metalloproteinases (MMPs), such as MMP-2 and MMP-9, which play an important role in the degradation of basement membrane in angiogenesis and tumor metastasis present in the culture supernatant of Rg3-treated aortic ring culture were found to decrease in their gelatinolytic activities. Taken together, these data underpin the anti-tumor property of Rg3 through its angiosuppressive activity.
Copper is a ubiquitous trace metal of vital importance in that it serves as a cofactor in many metalloenzymes. Excess copper becomes harmful if not sequestered appropriately in the cell. As a metal ...ion chaperone, metallothionein (MT) has been proposed as a key player in zinc and copper homeostasis within the cell. The underlying mechanisms by which MT sequesters and transfers copper ions, and subsequently achieves its proposed biological function remain unknown. Using a combination of electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and emission spectroscopy, we report that the Cu(i) to human apo-MT1a binding mechanism is highly pH-dependent. The 20 relative K
-values for the binding of 1 to 20 Cu(i) to the 20 cysteines of MT were obtained from computational simulation of the experimental mass spectral results. These data identified the pH-dependent formation of three sequential but completely different Cu-S
clusters, as a function of Cu(i) loading. These data provide the first overall sequence for Cu(i) binding in terms of domain specificity and transient binding site structures. Under cooperative binding at pH 7.4, a series of four clusters form: Cu
S
, followed by Cu
S
(β), then a second Cu
S
(α), and finally Cu
S
(α) (x = up to 11). Upon further addition of Cu(i), a mixture of species is formed in a non-cooperative mechanism, saturating the 20 cysteines of MT1a. Using benzoquinone, a cysteine modifier, we were able to confirm that Cu
S
formed solely in the N-terminal β-domain, as well as confirming the existence of the presumed Cu
S
cluster in the α-domain. Based on the results of ESI-MS and computational simulation we were able to identify Cu:MT speciation that resulted in specific emission and CD spectral properties.
Anthropogenic sources of xenobiotic metals with no physiological benefit are increasingly prevalent in the environment. The platinum group metals (Pd, Pt, Rh, Ru, Os, and Ir) are found in marine and ...plant species near urban sources, and are known to bioaccumulate, introducing these metals into the human food chain. Many of these metals are also being used in innovative cancer therapy, which leads to a direct source of exposure for humans. This paper aims to further our understanding of nontraditional metal metabolism via metallothionein, a protein involved in physiologically important metal homeostasis. The aggressive reaction of metallothionein and dirhodium(II) tetraacetate, a common synthetic catalyst known for its cytotoxicity, was studied in detail in vitro. Optical spectroscopic and equilibrium and time-dependent mass spectral data were used to define binding constants for this robust reaction, and molecular dynamics calculations were conducted to explain the observed results.
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