P4-201: Inhibitor pulldown of γ-secretase and associated components from human and rat brain
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the -secretase. Conclusions: Our studies revealed an important role of -secretase in the regulation of membrane lipid composition and ho- meostasis. P4-200 ACTIVE -SECRETASE IS ENRICHED IN SYNAPTIC MEMBRANES PREPARED FROM RAT BRAIN Susanne Frykman, Jarmila Nahalkova, Ji-Yeun Hur, Jenny Frånberg, Homira Behbahani, Bengt Winblad, Lars Tjernberg, Karolinska Institutet, Huddinge, Sweden. Contact e-mail: [email protected] Background: Synaptic degeneration is the first pathological hallmark in Alzheimer’s disease and the one that correlates best with disease progression. The mechanism for synaptic degeneration is unknown but a strong candidate as a causative agent is amyloid -peptide (A) oligomers that may cause Ca 2 dysregulation and oxidative stress. A is produced by the processing of the amyloid precursor protein (APP) by two sequential proteolytic events. The last cleavage is performed by a large membrane-bound protein complex called -secretase consisting of at least for components. In addition to APP, -secretase cleaves a large number of substrates which complicates its potential use as a theurapeutical target. In order to design specific -secretase inhibitors/ regulators it is important to increase the knowledge about this enzyme. Several studies have determined the subcellular localisation of -secre- tase to the late secretory-endosomal pathways using different cell-lines. However, the localization in brain has not been reported. Since the synapses seem to be particularly vulnerable for A toxicity we wanted to examine if -secretase is localized to the synapses. Methods: We prepared synaptic membranes and synaptic vesicles using sucrose gra- dients and synaptosomal lysis, and assayed these fractions for subcel- lular markers, -secretase components and -secretase activity as mea- sured by AICD and A production. Results: The synaptic membrane fraction was enriched in PSD-95, a post-synaptic membrane marker, but also contained endosomal markers. The -secretase activity was dra- matically enriched in this fraction, as assayed by endogenous AICD and A production. It is possible that the -secretase in synaptic membranes could be differently regulated compared to other parts of the brain and we speculate that synaptic membrane preparations could be useful for finding -secretase inhibitors/ regulators that specifically interacts with the synaptic A production. Conclusions: Gamma-secretase activity is enriched in synaptic membranes and high local production of A might contribute to the synaptic degeneration seen in Alzheimer’s disease. P4-201 INHIBITOR PULLDOWN OF -SECRETASE AND ASSOCIATED COMPONENTS FROM HUMAN AND RAT BRAIN Yasuhiro Teranishi 1 , Hedvig Welander 1 , Jenny Frånberg 1 , Ji-Yeun Hur 1 , Mikio Aoki 1,2 , Hiroyuki Nakagawa 2 , Jun Sakai 2 , Bengt Winblad 1 , Lars Tjernberg 1 , 1 Karolinska Institutet, Novum, KASPAC, Huddinge, Sweden; 2 Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan. Contact e-mail: [email protected] Background: The deposition of the A is a pathogenic event in Alzheimer’s disease and -secretase is responsible for the final cleavage of amyloid precursor protein to generate A. -Secretase is a trans- membrane (TM) aspartyl protease complex which catalyzes the cleav- age of type I TM proteins, including APP and Notch. Four proteins: PS, Nct, Aph-1 and Pen-2 are necessary and sufficient for an active -secre- tase complex, but little is known about how -secretase is regulated. Other -secretase associated proteins may affect -secretase activity, and studies in cell lines have shown that two TM proteins, TMP21 and CD147, associate with the -secretase complex and decrease A pro- duction. Methods: To investigate whether there are -secretase asso- ciate proteins in brain, we designed and synthesized a -secretase- inhibitor with hydrophilic long linker and a cleavable biotin group (GCB) for effective and specific pulldown of -secretase. As a starting material, we used a microsome fraction prepared from fresh rat brain. The microsomal pellet was dissolved in CHAPSO, the supernatant was incubated with GCB, and streptavidin beads were used for pulldown. Results: The specificity of the pulldown was confirmed by competition using a non-biotinylated inhibitor. Elution by DTT clearly reduced nonspecific binding compared to elution by SDS sample buffer. By western blotting the recovery was estimated to be around 20% for PS1-NTF, PS1-CTF and Aph-1, and around 5% for Nct and Pen-2. We are satisfied with the recovery, since only a fraction of the total amount of the individual -secretase components are present in an active complex. Next, we prepared microsome from frozen human brain, and found that the recovery of the -secretase components was similar as in the case of rat brain. The captured proteins from rat brain samples were digested by trypsin and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using the MASCOT algo- rithm and the NCBInr data base we identified PS-1, NCT, and TMP21. In addition to the known -secretase components, we found several TM-proteins. We are currently validating whether these proteins are truly -secretase associate proteins. Conclusions: We suggest that GCB-pulldown is an effective method for finding -secretase associate proteins. P4-202 ACTIVE -SECRETASE IS LOCALIZED TO DETERGENT RESISTANT MEMBRANES IN HUMAN BRAIN Ji-Yeun Hur, Hedvig Welander, Homira Behbahani, Mikio Aoki, Jenny Frånberg, Bengt Winblad, Susanne Frykman, Lars O. Tjernberg, Karolinska Institutet, Huddinge, Sweden. Contact e-mail: [email protected] Background: Characteristic hallmarks of Alzheimer’s disease (AD) are the deposition of amyloid -peptide (A) in senile plaques, and neurofi- brillary tangles containing the hyperphosphorylated tau protein in the brain. An initial cleavage by -secretase on the amyloid precursor protein (APP) produces soluble APP (-APPs) and a membrane bound C-terminal frag- ment (C99) which is cleaved by -secretase, generating the APP intracel- lular domain (AICD) and A. -Secretase is an aspartyl protease complex, containing at least four transmembrane proteins. Studies in cell lines show that -secretase is partially localized to lipid rafts. Lipid rafts are enriched in cholesterol and sphingolipids and are considered to be dynamic plat- forms for cell signaling, membrane protein sorting and transport. Lipid rafts are biochemically studied by treatment with detergents such as Triton X-100 at 4 °C followed by centrifugation. As a result, the insoluble parts of the lipid membranes are isolated and are called detergent resistant membranes (DRMs). However, different detergents give different results and further studies in intact cells or tissue sections are needed to determine whether the proteins found in DRMs are associated with lipid rafts. Meth- ods: Here, we studied -secretase in DRMs prepared from rat and human brain. DRMs were isolated from brain by sucrose gradient centrifugation. The fractions were immunoblotted using antibodies against -secretase components, BACE1, and APP. The DRM fraction was immunoprecipi- tated with anti-Nicastrin antibody and analyzed. To investigate whether the -secretase complex in DRMs is active, we incubated fractions in the absence or presence of 1 M of -secretase inhibitor L-685,458, AICD production was measured. At last, co-localization of -secretase and lipid rafts was shown by confocal microscopy. Results: DRMs from the brain were enriched in -secretase components and activity. Interestingly, the majority of BACE1, APP, and APP C-terminal fragments were distributed to non-raft fractions. Co-immunoprecipitation studies showed that the -secretase components in DRMs are associated even at high concentra- tions of CHAPSO (2%). AICD was also produced in DRMs in the absence of L-685,458. Finally, co-localization of -secretase and lipid rafts was shown in human brain sections. Conclusions: We conclude that the active -secretase complex is localized to lipid rafts in brain. T730 Poster Presentations P4:
Transcript of P4-201: Inhibitor pulldown of γ-secretase and associated components from human and rat brain
the �-secretase. Conclusions: Our studies revealed an important role of�-secretase in the regulation of membrane lipid composition and ho-meostasis.
P4-200 ACTIVE �-SECRETASE IS ENRICHED INSYNAPTIC MEMBRANES PREPARED FROM RATBRAIN
Susanne Frykman, Jarmila Nahalkova, Ji-Yeun Hur, Jenny Frånberg,Homira Behbahani, Bengt Winblad, Lars Tjernberg, KarolinskaInstitutet, Huddinge, Sweden. Contact e-mail: [email protected]
Background: Synaptic degeneration is the first pathological hallmarkin Alzheimer’s disease and the one that correlates best with diseaseprogression. The mechanism for synaptic degeneration is unknown buta strong candidate as a causative agent is amyloid �-peptide (A�)oligomers that may cause Ca2� dysregulation and oxidative stress. A�is produced by the processing of the amyloid precursor protein (APP)by two sequential proteolytic events. The last cleavage is performed bya large membrane-bound protein complex called �-secretase consistingof at least for components. In addition to APP, �-secretase cleaves alarge number of substrates which complicates its potential use as atheurapeutical target. In order to design specific �-secretase inhibitors/regulators it is important to increase the knowledge about this enzyme.Several studies have determined the subcellular localisation of �-secre-tase to the late secretory-endosomal pathways using different cell-lines.However, the localization in brain has not been reported. Since thesynapses seem to be particularly vulnerable for A� toxicity we wantedto examine if �-secretase is localized to the synapses. Methods: Weprepared synaptic membranes and synaptic vesicles using sucrose gra-dients and synaptosomal lysis, and assayed these fractions for subcel-lular markers, �-secretase components and �-secretase activity as mea-sured by AICD and A� production. Results: The synaptic membranefraction was enriched in PSD-95, a post-synaptic membrane marker, butalso contained endosomal markers. The �-secretase activity was dra-matically enriched in this fraction, as assayed by endogenous AICD andA� production. It is possible that the �-secretase in synaptic membranescould be differently regulated compared to other parts of the brain andwe speculate that synaptic membrane preparations could be useful forfinding �-secretase inhibitors/ regulators that specifically interacts withthe synaptic A� production. Conclusions: Gamma-secretase activity isenriched in synaptic membranes and high local production of A� mightcontribute to the synaptic degeneration seen in Alzheimer’s disease.
P4-201 INHIBITOR PULLDOWN OF �-SECRETASE ANDASSOCIATED COMPONENTS FROM HUMANAND RAT BRAIN
Yasuhiro Teranishi1, Hedvig Welander1, Jenny Frånberg1,Ji-Yeun Hur1, Mikio Aoki1,2, Hiroyuki Nakagawa2, Jun Sakai2,Bengt Winblad1, Lars Tjernberg1, 1Karolinska Institutet, Novum,KASPAC, Huddinge, Sweden; 2Dainippon Sumitomo Pharma Co., Ltd.,Osaka, Japan. Contact e-mail: [email protected]
Background: The deposition of the A� is a pathogenic event inAlzheimer’s disease and �-secretase is responsible for the final cleavageof amyloid precursor protein to generate A�. �-Secretase is a trans-membrane (TM) aspartyl protease complex which catalyzes the cleav-age of type I TM proteins, including APP and Notch. Four proteins: PS,Nct, Aph-1 and Pen-2 are necessary and sufficient for an active �-secre-tase complex, but little is known about how �-secretase is regulated.Other �-secretase associated proteins may affect �-secretase activity,and studies in cell lines have shown that two TM proteins, TMP21 andCD147, associate with the �-secretase complex and decrease A� pro-duction. Methods: To investigate whether there are �-secretase asso-ciate proteins in brain, we designed and synthesized a �-secretase-inhibitor with hydrophilic long linker and a cleavable biotin group(GCB) for effective and specific pulldown of �-secretase. As a starting
material, we used a microsome fraction prepared from fresh rat brain.The microsomal pellet was dissolved in CHAPSO, the supernatant wasincubated with GCB, and streptavidin beads were used for pulldown.Results: The specificity of the pulldown was confirmed by competitionusing a non-biotinylated inhibitor. Elution by DTT clearly reducednonspecific binding compared to elution by SDS sample buffer. Bywestern blotting the recovery was estimated to be around 20% forPS1-NTF, PS1-CTF and Aph-1, and around 5% for Nct and Pen-2. Weare satisfied with the recovery, since only a fraction of the total amountof the individual �-secretase components are present in an activecomplex. Next, we prepared microsome from frozen human brain, andfound that the recovery of the �-secretase components was similar as inthe case of rat brain. The captured proteins from rat brain samples weredigested by trypsin and analyzed by liquid chromatography coupled totandem mass spectrometry (LC-MS/MS). Using the MASCOT algo-rithm and the NCBInr data base we identified PS-1, NCT, and TMP21.In addition to the known �-secretase components, we found severalTM-proteins. We are currently validating whether these proteins aretruly �-secretase associate proteins. Conclusions: We suggest thatGCB-pulldown is an effective method for finding �-secretase associateproteins.
P4-202 ACTIVE �-SECRETASE IS LOCALIZED TODETERGENT RESISTANT MEMBRANES INHUMAN BRAIN
Ji-Yeun Hur, Hedvig Welander, Homira Behbahani, Mikio Aoki,Jenny Frånberg, Bengt Winblad, Susanne Frykman, Lars O. Tjernberg,Karolinska Institutet, Huddinge, Sweden. Contact e-mail:[email protected]
Background: Characteristic hallmarks of Alzheimer’s disease (AD) arethe deposition of amyloid �-peptide (A�) in senile plaques, and neurofi-brillary tangles containing the hyperphosphorylated tau protein in the brain.An initial cleavage by �-secretase on the amyloid precursor protein (APP)produces soluble APP (�-APPs) and a membrane bound C-terminal frag-ment (C99) which is cleaved by �-secretase, generating the APP intracel-lular domain (AICD) and A�. �-Secretase is an aspartyl protease complex,containing at least four transmembrane proteins. Studies in cell lines showthat �-secretase is partially localized to lipid rafts. Lipid rafts are enrichedin cholesterol and sphingolipids and are considered to be dynamic plat-forms for cell signaling, membrane protein sorting and transport. Lipidrafts are biochemically studied by treatment with detergents such as TritonX-100 at 4 °C followed by centrifugation. As a result, the insoluble partsof the lipid membranes are isolated and are called detergent resistantmembranes (DRMs). However, different detergents give different resultsand further studies in intact cells or tissue sections are needed to determinewhether the proteins found in DRMs are associated with lipid rafts. Meth-ods: Here, we studied �-secretase in DRMs prepared from rat and humanbrain. DRMs were isolated from brain by sucrose gradient centrifugation.The fractions were immunoblotted using antibodies against �-secretasecomponents, BACE1, and APP. The DRM fraction was immunoprecipi-tated with anti-Nicastrin antibody and analyzed. To investigate whether the�-secretase complex in DRMs is active, we incubated fractions in theabsence or presence of 1 �M of �-secretase inhibitor L-685,458, AICDproduction was measured. At last, co-localization of �-secretase and lipidrafts was shown by confocal microscopy. Results: DRMs from the brainwere enriched in �-secretase components and activity. Interestingly, themajority of BACE1, APP, and APP C-terminal fragments were distributedto non-raft fractions. Co-immunoprecipitation studies showed that the�-secretase components in DRMs are associated even at high concentra-tions of CHAPSO (2%). AICD was also produced in DRMs in the absenceof L-685,458. Finally, co-localization of �-secretase and lipid rafts wasshown in human brain sections. Conclusions: We conclude that the active�-secretase complex is localized to lipid rafts in brain.
T730 Poster Presentations P4:
![Inhibition of γ-Secretase Leads to an Increase in Presenilin-1 · defective 1 (APH1), and presenilin enhancer 2 (PEN2) [7]. γ-Secretase acts an aspartyl protease, which catalytic](https://static.fdocument.org/doc/165x107/5fcf13aeec1c843f815764d3/inhibition-of-secretase-leads-to-an-increase-in-presenilin-1-defective-1-aph1.jpg)
