scholarly journals A cellular complex of BACE1 and γ-secretase sequentially generates Aβ from its full-length precursor

2019 ◽  
Vol 218 (2) ◽  
pp. 644-663 ◽  
Author(s):  
Lei Liu ◽  
Li Ding ◽  
Matteo Rovere ◽  
Michael S. Wolfe ◽  
Dennis J. Selkoe
Keyword(s):  
Molecular Weight ◽  
Cultured Cells ◽  
Amyloid Β ◽  
Full Length ◽  
Aβ Peptides ◽  
Whole Cells ◽  
Sequential Processing ◽  
Catalytically Active ◽  
Cellular Complex

Intramembrane proteolysis of transmembrane substrates by the presenilin–γ-secretase complex is preceded and regulated by shedding of the substrate’s ectodomain by α- or β-secretase. We asked whether β- and γ-secretases interact to mediate efficient sequential processing of APP, generating the amyloid β (Aβ) peptides that initiate Alzheimer’s disease. We describe a hitherto unrecognized multiprotease complex containing active β- and γ-secretases. BACE1 coimmunoprecipitated and cofractionated with γ-secretase in cultured cells and in mouse and human brain. An endogenous high molecular weight (HMW) complex (∼5 MD) containing β- and γ-secretases and holo-APP was catalytically active in vitro and generated a full array of Aβ peptides, with physiological Aβ42/40 ratios. The isolated complex responded properly to γ-secretase modulators. Alzheimer’s-causing mutations in presenilin altered the Aβ42/40 peptide ratio generated by the HMW β/γ-secretase complex indistinguishably from that observed in whole cells. Thus, Aβ is generated from holo-APP by a BACE1–γ-secretase complex that provides sequential, efficient RIP processing of full-length substrates to final products.

scholarly journals Development and Characterization of a Novel Membrane Assay for Full-Length BACE-1 at pH 6.0

2012 ◽  
Vol 18 (3) ◽  
pp. 277-285 ◽  
Author(s):  
Arman Saric ◽  
Lars zur Brügge ◽  
Dirk Müller-Pompalla ◽  
Thomas Rysiok ◽  
Solenne Ousson ◽  
...  
Keyword(s):  
Amyloid Precursor Protein ◽  
Amyloid Β ◽  
Full Length ◽  
Rational Drug Design ◽  
Precursor Protein ◽  
Catalytic Active Site ◽  
Catalytically Active ◽  
Intracellular Compartments ◽  
Bace 1

β-Site amyloid precursor protein cleaving enzyme–1 (BACE-1) is a transmembrane aspartic protease that mediates the initial cleavage of the amyloid precursor protein (APP), leading to the generation of amyloid-β (Aβ) peptides that are thought to be causative of Alzheimer’s disease (AD). Consequently, inhibition of BACE-1 is an attractive therapeutic approach for the treatment of AD. In general, in vitro biochemical assays to monitor BACE-1 activity have used the extracellular domain of the protein that contains the catalytic active site. This form of BACE-1 is catalytically active at acidic pH and cleaves APP-based peptide substrates at the β-site. However, this form of BACE-1 does not mimic the natural physiology of BACE-1 and shows minimal activity at pH 6.0, which is more representative of the pH within the intracellular compartments where BACE-1 resides. Moreover, high-throughput screens with recombinant BACE-1 at pH 4.5 have failed to identify tractable leads for drug discovery, and hence, BACE-1 inhibitor development has adopted a rational drug design approach. Here we describe the development and validation of a novel membrane assay comprising full-length BACE-1 with measurable activity at pH 6.0, which could be used for the identification of novel inhibitors of BACE-1.

scholarly journals Lithium ions display weak interaction with amyloid-beta (Aβ) peptides and have minor effects on their aggregation

2021 ◽  
Author(s):  
Elina Berntsson ◽  
Suman Paul ◽  
Faraz Vosough ◽  
Sabrina B. Sholts ◽  
Jüri Jarvet ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Amyloid Β ◽  
Underlying Mechanism ◽  
Aβ Peptides ◽  
Beneficial Effects ◽  
Incurable Disease ◽  
Age Related ◽  
Aβ Aggregation ◽  
In Vitro Interactions

Alzheimer’s disease (AD) is an incurable disease and the main cause of age-related dementia worldwide, despite decades of research. Treatment of AD with lithium (Li) has showed promising results, but the underlying mechanism is unclear. The pathological hallmark of AD brains is deposition of amyloid plaques, consisting mainly of amyloid-β (Aβ) peptides aggregated into amyloid fibrils. The plaques contain also metal ions of e.g. Cu, Fe, and Zn, and such ions are known to interact with Aβ peptides and modulate their aggregation and toxicity. The interactions between Aβ peptides and Li+ ions have however not been well investigated. Here, we use a range of biophysical techniques to characterize in vitro interactions between Aβ peptides and Li+ ions. We show that Li+ ions display weak and non-specific interactions with Aβ peptides, and have minor effects on Aβ aggregation. These results indicate that possible beneficial effects of Li on AD pathology are not likely caused by direct interactions between Aβ peptides and Li+ ions.

scholarly journals Pathogenicity of Vibrio alginolyticusfor Cultured Gilt-Head Sea Bream (Sparus aurataL.)

1998 ◽  
Vol 64 (11) ◽  
pp. 4269-4275 ◽  
Author(s):  
M. Carmen Balebona ◽  
Manuel J. Andreu ◽  
M. Angeles Bordas ◽  
Irene Zorrilla ◽  
Miguel A. Moriñigo ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Biological Activities ◽  
Embryo Cell ◽  
Sea Bream ◽  
Fish Cell ◽  
Whole Cells ◽  
Hydrolytic Activities ◽  
Extracellular Products

ABSTRACT The in vivo and in vitro pathogenic activities of whole cells and extracellular products of Vibrio alginolyticus for cultured gilt-head sea bream were evaluated. The 50% lethal doses ranged from 5.4 × 104 to 1.0 × 106 CFU/g of body weight. The strains examined had the ability to adhere to skin, gill, and intestinal mucus of sea bream and to cultured cells of a chinook salmon embryo cell line. In addition, the in vitro ability ofV. alginolyticus to adhere to mucus and skin cells of sea bream was demonstrated by scanning electron microscopy. The biological activities of extracellular products of V. alginolyticus were hydrolytic activities; the products were able to degrade sea bream mucus. V. alginolyticus was cytotoxic for fish cell lines and lethal for sea bream. Moreover, the extracellular products could degrade sea bream tissues. However, experiments performed with the bath immersion inoculation technique demonstrated that V. alginolyticus should be considered a pathogen for sea bream only when the mucus layer is removed and the skin is damaged.

Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen

1987 ◽  
Vol 7 (9) ◽  
pp. 3221-3230
Author(s):  
N Beauchemin ◽  
S Benchimol ◽  
D Cournoyer ◽  
A Fuks ◽  
C P Stanners
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Carcinoembryonic Antigen ◽  
Full Length ◽  
Cdna Clones ◽  
Base Pairs ◽  
Amino Terminal ◽  
Isolation And Characterization

Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.

Sulforaphane Switches Cyclin E Isoform Expression and Blocks Leukemic Cell Cycling.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4405-4405
Author(s):  
Laura M. Gorham ◽  
Gigi Frye ◽  
Michelle Miranda ◽  
Richard A. Steinman
Keyword(s):  
Molecular Weight ◽  
Cyclin E ◽  
Leukemic Cell ◽  
Full Length ◽  
Leukemic Cells ◽  
Pharmacokinetic Studies ◽  
Leukemic Cell Lines ◽  
Myeloid Leukemic Cell

Abstract Sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables, has been shown to inhibit the growth of prostate cancer cells in vitro and in vivo. We were interested in exploring potential antileukemic effects of SFN. The viability of multiple myeloid leukemic cell lines was decreased by 25uM SFN. Pharmacokinetic studies reported in rats suggest that this serum concentration can be achieved through oral dosing. Lower SFN concentrations (1–5 uM) inhibited leukemic cell growth without affecting cell viability. Synchronized HL-60 cells exposed to 25uM SFN were blocked at the G1/S phase transition. Kinetic analysis of cell cycle proteins demonstrated that the G1/S block arose from downmodulation of cyclins D3 and E rather than upregulation of cdk-inhibitors. Interestingly, we found that HL-60 cells expressed a low molecular weight (LMW, 36 kD) variant of cyclin E rather than (50 kD) full-length cyclin E. Treatment with SFN for as little as 2 hours caused a decrease in expression of the LMW cyclin E and induced the expression of a higher molecular weight (~50 kD) cyclin E isoform. Because LMW cyclin E has been associated with increased cdk2 activity and p27 resistance compared to full-length cyclin E, we postulate that SFN-mediated cyclin E isoform-switching contributed to growth inhibition of these leukemic cells. The signaling pathway through which SFN altered cyclin E expression appeared to be distinct from MEK/ERK and JNK pathways that have been implicated in the apoptotic effects of SFN. Given that cyclin E overexpression and, particularly, LMW cyclin E expression are correlated with poor prognosis in multiple cancers, the mechanism through which SFN decreases LMW cyclin E expression in these leukemic cells could have therapeutic significance.

scholarly journals Apolipoprotein J and Alzheimer's amyloid β solubility

1996 ◽  
Vol 316 (2) ◽  
pp. 671-679 ◽  
Author(s):  
Etsuro MATSUBARA ◽  
Claudio SOTO ◽  
Sam GOVERNALE ◽  
Blas FRANGIONE ◽  
Jorge GHISO
Keyword(s):  
Biological Fluids ◽  
Amyloid Β ◽  
Density Lipoprotein ◽  
Aβ Peptides ◽  
Normal Individuals ◽  
Apolipoprotein J ◽  
Major Carrier

Apolipoprotein J (apoJ) has been found associated with soluble amyloid β (sAβ) in plasma and cerebrospinal fluid in normal individuals and co-deposited with fibrillar Aβ in Alzheimer's cerebrovascular and parenchymal lesions. Although studies in vitro and in vivo indicate that apoJ is a major carrier protein for sAβ, its role in the fibrillogenesis process is not known. We report herein that apoJ in its native high-density lipoprotein lipidic environment is fully active to interact with Aβ peptides. Furthermore, apoJ prevents aggregation and polymerization of synthetic Aβ in vitro. The interaction was stable for at least 14 days at 37 °C in physiologic buffers, and the peptide retrieved after complex dissociation at low pH retained its inherent aggregation properties. In addition, the binding to apoJ protects synthetic Aβ from proteolytic degradation; both Aβ1–42 and Aβ1–40 were more resistant to proteolysis by trypsin and chymotrypsin when complexed to apoJ. The data suggest that the interaction may preclude sAβ aggregation in biological fluids and point to a protecting role of apoJ for complexed Aβ species.

scholarly journals Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen.

1987 ◽  
Vol 7 (9) ◽  
pp. 3221-3230 ◽  
Author(s):  
N Beauchemin ◽  
S Benchimol ◽  
D Cournoyer ◽  
A Fuks ◽  
C P Stanners
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Carcinoembryonic Antigen ◽  
Full Length ◽  
Cdna Clones ◽  
Base Pairs ◽  
Amino Terminal ◽  
Isolation And Characterization

Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.

scholarly journals Tumor-Specific Proteolytic Processing of Cyclin E Generates Hyperactive Lower-Molecular-Weight Forms

2001 ◽  
Vol 21 (18) ◽  
pp. 6254-6269 ◽  
Author(s):  
Donald C. Porter ◽  
Ning Zhang ◽  
Christopher Danes ◽  
Mollianne J. McGahren ◽  
Richard M. Harwell ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Tumor Cells ◽  
Serine Proteases ◽  
Cyclin E ◽  
S Phase ◽  
Proteolytic Processing ◽  
Full Length ◽  
Lower Molecular Weight ◽  
Normal Cells

ABSTRACT Cyclin E is a G1 cyclin essential for S-phase entry and has a profound role in oncogenesis. Previously this laboratory found that cyclin E is overexpressed and present in lower-molecular-weight (LMW) isoforms in breast cancer cells and tumor tissues compared to normal cells and tissues. Such alteration of cyclin E is linked to poor patient outcome. Here we report that the LMW forms of cyclin E are hyperactive biochemically and they can more readily induce G1-to-S progression in transfected normal cells than the full-length form of the protein can. Through biochemical and mutational analyses we have identified two proteolytically sensitive sites in the amino terminus of human cyclin E that are cleaved to generate the LMW isoforms found in tumor cells. Not only are the LMW forms of cyclin E functional, as they phosphorylate substrates such as histone H1 and GST-Rb, but also their activities are higher than the full-length cyclin E. These nuclear localized LMW forms of cyclin E are also biologically functional, as their overexpression in normal cells increases the ability of these cells to enter S and G2/M. Lastly, we show that cyclin E is selectively cleaved in vitro by the elastase class of serine proteases to generate LMW forms similar to those observed in tumor cells. These studies suggest that the defective entry into and exit from S phase by tumor cells is in part due to the proteolytic processing of cyclin E, which generates hyperactive LMW isoforms whose activities have been modified from that of the full-length protein.

scholarly journals Kindlin2-mediated phase separation underlies integrin adhesion formation

2020 ◽  
Author(s):  
Yujie Li ◽  
Ting Zhang ◽  
Huadong Li ◽  
Haibin Yang ◽  
Ruihong Lin ◽  
...  
Keyword(s):  
Phase Separation ◽  
Biochemical Characterization ◽  
Cultured Cells ◽  
Adhesion Formation ◽  
Mechanistic Explanation ◽  
Full Length ◽  
Regulation Mechanism ◽  
Dynamic Regulation ◽  
Interaction Control

AbstractFormation of cell-extracellular matrix adhesion requires assembly of the transmembrane receptor integrins and their intracellular activators, kindlin and talin proteins in minutes. The mechanisms governing the rapid formation and dynamics of the adhesion remain enigmatic. Here, we reported that the dimerized-kindlin2 underwent phase separation with clustered-integrin in solution and on lipid bilayer. The kindlin2/integrin condensate can further enrich other components for the adhesion complex assembly. The full-length structure of kindlin2 was solved and revealed that the kindlin2 dimers can further pack with each other to form a higher oligomer. Disrupting the intermolecular interaction between the kindlin2 dimer inhibits the phase formation on 2D membrane in vitro and impaired the adhesion formation, integrin activation, and cell spreading in cultured cells. We also determined the full-length structure of kindlin2 in its monomeric conformation. Structural analysis and biochemical characterization indicate that the interdomain interaction control the monomer-dimer transition of kindlin2, providing a regulation mechanism of the kindlin2-mediated phase separation. Our findings not only provide a mechanistic explanation for the formation and dynamic regulation of the integrin-based adhesion, but also shed light on understanding of how the clustered receptors participate in assembly of the functional membrane domains via phase separation.

scholarly journals Fluorescent indolizine derivative YI-13 detects amyloid-β monomers, dimers, and plaques in the brain of 5XFAD Alzheimer transgenic mouse model

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243041
Author(s):  
DaWon Kim ◽  
Jeong Hwa Lee ◽  
Hye Yun Kim ◽  
Jisu Shin ◽  
Kyeonghwan Kim ◽  
...  
Keyword(s):  
Mouse Model ◽  
Transgenic Mouse ◽  
Neurodegenerative Disorder ◽  
Amyloid Β ◽  
Transgenic Mouse Model ◽  
Aβ Oligomers ◽  
Aβ Peptides ◽  
Soluble Aβ Oligomers ◽  
The Brain

Alzheimer disease (AD) is a neurodegenerative disorder characterized by the aberrant production and accumulation of amyloid-β (Aβ) peptides in the brain. Accumulated Aβ in soluble oligomer and insoluble plaque forms are considered to be a pathological culprit and biomarker of the disorder. Here, we report a fluorescent universal Aβ-indicator YI-13, 5-(4-fluorobenzoyl)-7,8-dihydropyrrolo[1,2-b]isoquinolin-9(6H)-one, which detects Aβ monomers, dimers, and plaques. We synthesized a library of 26 fluorescence chemicals with the indolizine core and screen them through a series of in vitro tests utilizing Aβ as a target and YI-13 was selected as the final imaging candidate. YI-13 was found to stain and visualize insoluble Aβ plaques in the brain tissue, of a transgenic mouse model with five familial AD mutations (5XFAD), by a histochemical approach and to label soluble Aβ oligomers within brain lysates of the mouse model under a fluorescence plate reader. Among oligomers aggregated from monomers and synthetic dimers from chemically conjugated monomers, YI-13 preferred the dimeric Aβ.

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