FIG 3 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
![Radioactivity profile of 3 H incorporation into 8-and 12–14-kDa A-immunoreactive species. Cells expressing wild type APP were harvested after 4 h of metabolic labeling in SGal-ura containing [ 35 S]methionine and [ 3 H]phenylalanine. Lysates were sequentially immunoprecipitated with 369 (preclearing) followed by 4G8. The proteins recovered by 4G8 were subjected to SDS-PAGE and transferred to nitrocellulose. The 8-and 12–14-kDa bands were excised and eluted from the nitrocellulose after which Edman degradation was performed. Fractions were diluted in liquid scintillation fluid, and the tritium incorporation was measured by liquid scintillation spectrometry. Radioactivity was present in fractions 4, 19, and 20 of both species, as would be expected for a peptide bearing A at its N terminus. CPM, counts/minute.](publication/12734563/figure/fig2/AS:460816924319745@1486878724704/Radioactivity-profile-of-3-H-incorporation-into-8-and-12-14-kDa-A-immunoreactive-species.png)
Radioactivity profile of 3 H incorporation into 8-and 12–14-kDa A-immunoreactive species. Cells expressing wild type APP were harvested after 4 h of metabolic labeling in SGal-ura containing [ 35 S]methionine and [ 3 H]phenylalanine. Lysates were sequentially immunoprecipitated with 369 (preclearing) followed by 4G8. The proteins recovered by 4G8 were subjected to SDS-PAGE and transferred to nitrocellulose. The 8-and 12–14-kDa bands were excised and eluted from the nitrocellulose after which Edman degradation was performed. Fractions were diluted in liquid scintillation fluid, and the tritium incorporation was measured by liquid scintillation spectrometry. Radioactivity was present in fractions 4, 19, and 20 of both species, as would be expected for a peptide bearing A at its N terminus. CPM, counts/minute.
Source publication
The Alzheimer's amyloid-β precursor protein (βAPP) is a type 1 membrane-spanning protein from which the Alzheimer's disease
amyloid-β peptide (Aβ) is proteolytically derived. To date, attempts to identify the enzymes responsible for Aβ generation
have failed. Here we report the accumulation of Aβ-immunoreactive peptides in yeast expressing human βA...
Similar publications
The Alzheimer's disease amyloid peptide Abeta has a heterogeneous COOH terminus, as variants 40 and 42 residues long are found in neuritic plaques and are secreted constitutively by cultured cells. The proteolytic activity that liberates the Abeta COOH terminus from the beta-amyloid precursor protein is called gamma-secretase. It could be one prote...
β-Secretase, the rate-limiting enzymatic activity in the production of the amyloid-β (Aβ) peptide, is a major target of Alzheimer's disease (AD) therapeutics. There are two forms of the enzyme: β-site Aβ precursor protein cleaving enzyme (BACE) 1 and BACE2. Although BACE1 increases in late-stage AD, little is known about BACE2. We conducted a detai...
Citations
... Since the secretases responsible for APP processing in humans are still not completely identified, the discovery of secretases in yeast may help in the identification of new human secretases by homology. Though a possible β-secretase-like enzymatic activity was also reported in yeast [42], this finding was not supported by other works. Overcoming the lack of β-secretase-like activity, a fragment derived from the APP cleaved by β-secretase (C99) was expressed in yeast [43]. ...
As a model organism Saccharomyces cerevisiae has greatly contributed to our understanding of many fundamental aspects of cellular biology in higher eukaryotes. More recently, engineered yeast models developed to study endogenous or heterologous proteins that lay at the root of a given disease have become powerful tools for unraveling the molecular basis of complex human diseases like neurodegeneration. Additionally, with the possibility of performing target-directed large-scale screenings, yeast models have emerged as promising first-line approaches in the discovery process of novel therapeutic opportunities against these pathologies.
In this paper, several yeast models that have contributed to the uncovering of the etiology and pathogenesis of several neurodegenerative diseases are described, including the most common forms of neurodegeneration worldwide, Alzheimer's, Parkinson's, and Huntington's diseases. Moreover, the potential input of these cell systems in the development of more effective therapies in neurodegeneration, through the identification of genetic and chemical suppressors, is also addressed.
... The first approach was used in the study of Friedreich's ataxia, where the yeast protein Yfh1 is the frataxin homologue (Babcock et al., 1997; Foury and Cazzalini, 1997; Koutnikova et al., 1997; Wilson and Roof, 1997; reviewed in Puccio and Koenig, 2000), and of amyotrophic lateral sclerosis (ALS), based on Sod1 and Sen1, the yeast homologues of copper–zinc dismutase and senataxin (Nishida et al., 1994; Finkel et al., 2010). The second approach was applied in the discovery of central molecular aspects of Huntington's disease, Parkinson's disease and Alzheimer's disease (Greenfield et al., 1999; Krobitsch and Lindquist, 2000; Muchowski et al., 2000 Muchowski et al., , 2002 Willingham et al., 2003; ). The heterologous proteinexpression approach was also used in studies on ALS, where another potential causative protein, TDP-43, was expressed in yeast cells (Johnson et al., 2008). ...
... Zellkompartimenten des Sekretorischen Wegs vom ER [18,20,21], TGN [21,22] bis hin zu sekretorischen post-Golgi-Vesikeln [23], Endosomen [24][25][26][27], Lysosomen und Autophagosomen [28] und an der Plasma Membran [29][30][31] nachgewiesen. Andere Studien wiederum schlossen eine Aβ-Produktion im ER [32][33][34] und TGN [23] aus. ...
... Zellkompartimenten des Sekretorischen Wegs vom ER [18,20,21], TGN [21,22] bis hin zu sekretorischen post-Golgi-Vesikeln [23], Endosomen [24][25][26][27], Lysosomen und Autophagosomen [28] und an der Plasma Membran [29][30][31] nachgewiesen. Andere Studien wiederum schlossen eine Aβ-Produktion im ER [32][33][34] und TGN [23] aus. ...
... Es wurde eine stabil NotchΔE exprimierende HEK 293-Zelllinie verwendet. [18,20,21], Golgi und TGN [21][22][23] sowie in sekretorischen Vesikeln [35,36]. ...
... Each of these four g-secretase components has been found necessary for the enzymatic activity of the complex with deficiency in any of them dramatically impairing g-secretase activity. Coexpression of the four components in the yeast Saccharomyces cerevisiae has been found to be necessary and sufficient to reconstitute g-secretase activity, which is not endogenous to yeast [111,112]. In addition to the four critical components, several other factors have been proposed as additional g-secretase components. ...
An important pathological feature of Alzheimer's disease (AD) is the presence of extracellular senile plaques in the brain. Senile plaques are composed of aggregations of small peptides called β-amyloid (Aβ). Multiple lines of evidence demonstrate that overproduction/aggregation of Aβ in the brain is a primary cause of AD and inhibition of Aβ generation has become a hot topic in AD research. Aβ is generated from β-amyloid precursor protein (APP) through sequential cleavages first by β-secretase and then by γ-secretase complex. Alternatively, APP can be cleaved by α-secretase within the Aβ domain to release soluble APPα and preclude Aβ generation. Cleavage of APP by caspases may also contribute to AD pathologies. Therefore, understanding the metabolism/processing of APP is crucial for AD therapeutics. Here we review current knowledge of APP processing regulation as well as the patho/physiological functions of APP and its metabolites.
... These genes are the homologs of the human genes involved in Friedreich's ataxia (Puccio & Koenig, 2000) and amyotrophic lateral sclerosis (Leitch et al., 2009), respectively, and these studies strongly contributed toward the clarification of the pathogenic mechanisms underlying these disorders. As an example of the second approach, human genes underlying the histopatho-logical features of various neurodegenerative disorders were expressed in yeast, leading to the discovery of central molecular aspects of HD, PD and AD (Greenfield et al., 1999;Krobitsch & Lindquist, 2000;Muchowski et al., 2000Muchowski et al., , 2002Willingham et al., 2003), as we will show below. ...
The budding yeast, Saccharomyces cerevisiae, is the best-studied eukaryotic cell, at both genetic and physiological levels. As a eukaryote, yeast shares highly conserved molecular and cellular mechanisms with human cells. Thus, this simple fungus is an invaluable model to study the fundamental molecular mechanisms involved in several human diseases. In the particular case of neurodegenerative disorders, yeast models have been able to recapitulate several important features of complex and devastating disorders, such as Huntington's and Parkinson's diseases. Once validated, these models have also been used to accelerate the identification of both novel therapeutic targets and compounds with therapeutic potential. Here, we review the recent contributions of this simple, but powerful model organism toward our understanding of neurodegeneration.
... This suggests that there is hardly any active g-secretase in the ER. The Golgi/TGN was postulated to be a major site of g-secretase activity (8,9,40); however, our results of experiments using monensin, 20 C, and TA argue against that. Moreover, in an immunoelectron microscopy study, the Golgi/TGN was devoid of PS1 (41). ...
Alzheimer's disease is characterized by brain deposition of extracellular amyloid beta-peptide (Abeta)-containing plaques. The cellular site of gamma-secretase activity, which releases Abeta and the corresponding amyloid precursor protein intracellular domain (AICD), remains controversial. Proposed cleavage sites range from the endoplasmic reticulum (ER), the Golgi apparatus, and the cell surface to endosomal compartments. We now used C99-green fluorescent protein (GFP), a fluorescent reporter substrate for gamma-secretase activity and monitored AICD production in living cells. C99-GFP is efficiently cleaved by gamma-secretase, and AICD-GFP is released into the cytosol. Inhibiting gamma-secretase results in accumulation of C99-GFP in early endosomes. By blocking selective transport steps along the secretory pathway, we demonstrate that gamma-secretase does not cleave its substrates in the ER, the Golgi/trans-Golgi network, or in secretory vesicles. In contrast, inhibition of endocytosis did not inhibit cleavage of C99-GFP. Similar results were obtained for another gamma-secretase substrate, NotchDeltaE. Our results suggest that intracellular domains are generated by gamma-secretase at the plasma membrane and/or early endosomes.
... This reaction is catalyzed by the glycosylphosphatidylinositol-linked yeast aspartyl proteases, MKC7 and YAP3 (56,57). A more recent study demonstrates the presence of beta -secretase-like enzymatic activity in yeast as well (58). Given these findings along with the availability of mutant yeast strains and the wellcharacterized yeast secretory pathway, yeast may become an attractive system for studying beta APP processing and Abeta generation. ...
In molecular neurobiology, perhaps no molecule has been as thoroughly examined as Alzheimer's beta-amyloid precursor protein (beta-APP). In the years since the cDNA encoding beta-APP was cloned, the protein has been the subject of unparalleled scrutiny on all levels. From molecular genetics and cellular biology to neuroanatomy and epidemiology, no scientific discipline has been left unexplored - and with good reason. beta-amyloid (Abeta) is the main constituent of the amyloidogenic plaques which are a primary pathological hallmark of Alzheimer's disease, and bta-APP is the protein from which Abeta is cleaved and released. Unraveling the molecular events underlying Abeta generation has been, and remains, of paramount importance to scientists in our field. In this review we will trace the progress that has been made in understanding the molecular and cellular basis of beta-APP trafficking and processing, or alternatively stated, the molecular basis for Abeta generation. Imperative to a complete understanding of Abeta generation is the delineation of its subcellular localization and the identification of proteins that play either direct or accessory roles in Abeta generation. We will focus on the regulation of beta-APP cleavage through diverse signal transduction mechanisms and discuss possible points of therapeutic intercession in what has been postulated to be a seminal molecular step in the cascade of events terminating in the onset of dementia, loss of neurons, and eventual death from Alzheimer's disease.
... This reaction is catalyzed by the glycosyl-phosphatidylinositol-linked yeast aspartyl proteases, MKC7 and YAP3 (56,57). A more recent study demonstrates the presence of β-secretase-like enzymatic activity in yeast as well (58). Given these findings along with the availability of mutant yeast strains and the well-characterized yeast secretory pathway, yeast may become an attractive system for studying βAPP processing and Aβ generation. ...
Yeast models of neurodegenerative diseases associated with protein misfolding and protein aggregation have given unique insights into the underlying genetic and cellular pathomechanisms. These yeast models recapitulate central aspects of protein misfolding and the ensuing toxicity, such as interference with cellular protein quality control, concentration-dependent formation of insoluble, often amyloid-like aggregates and the associated toxicity. Advanced age is undoubtedly the highest and most common risk factor for most neurodegenerative diseases. Since yeast has served as a superb model to study cellular aspects of aging, we outline strategies to study how aging modulates protein misfolding and its toxicity, thereby opening new avenues to continue the success of yeast as powerful models to study neurodegenerative diseases.
Amyloid-β peptide (Aβ), the main component of Alzheimer's disease (AD) senile plaques, has been found to accumulate within the lysosomal compartment of AD neurons. We have previously shown that in differentiated SH-SY5Y neuroblastoma cells cultured under normal conditions, the majority of Aβ is localized extralysosomally, while oxidative stress significantly increases intalysosomal Aβ content through activation of macroautophagy. It is, however, not clear which cellular compartments contain extralysosomal Aβ in intact SH-SY5Y cells, and how oxidative stress influences the distribution of extralysosomal Aβ. Using confocal laser scanning microscopy and immunoelectron microscopy, we showed that in differentiated neuroblastoma cells cultured under normal conditions Aβ (Aβ40, Aβ42, and Aβ oligomers) is colocalized with both membrane-bound organelles (endoplasmic reticulum, Golgi complexes, multivesicular bodies/late endosomes, lysosomes, exocytotic vesicles and mitochondria) and non-membrane-bound cytosolic structures. Neuroblastoma cells stably transfected with AβPP Swedish KM670/671NL double mutation showed enlarged amount of Aβ colocalized with membrane compartments. Suppression of exocytosis by 5 nM tetanus toxin resulted in a significant increase of the amount of cytosolic Aβ as well as Aβ colocalized with exocytotic vesicles, endoplasmic reticulum, Golgi complexes, and lysosomes. Hyperoxia increased Aβ localization in the endoplasmic reticulum, Golgi apparatus, mitochondria, and lysosomes, but not in the secretory vesicles. These results indicate that in SH-SY5Y neuroblastoma cells intracellular Aβ is not preferentially localized to any particular organelle and, to a large extent, is secreted from the cells. Challenging cells to hyperoxia, exocytosis inhibition, or Aβ overproduction increased intracellular Aβ levels but did not dramatically changed its localization pattern.
