P3-322: The Nicastrin peptidase-like domain is crucially involved in the assembly of the...
1
P3-321 THE CARBOXYL TERMINI OF GSK-3 ISOFORMS ARE ESSENTIAL FOR ACTIVITY Jessica L. Buescher 1 , Christopher J. Phiel 1,2 , 1 The Ohio State University, Columbus, OH, USA; 2 The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA. Contact e-mail: [email protected] Background: Glycogen synthase kinase-3 (GSK-3) isoforms, GSK-3 and GSK-3, are serine/threonine kinases involved in numerous cellular pro- cesses. GSK-3 isoforms have also been implicated in a wide array of disease processes including Alzheimer’s disease, cancer, diabetes, and mood disorders. Thus, pharmacological modulation of GSK-3 isoforms offers great potential for the treatment of a variety of disorders. Further- more, understanding the structural requirements governing GSK-3 activity is a fundamental step in the development of therapeutic agents. Objective: In order to determine the structural requirements of GSK-3 isoform activ- ity, we have performed structure-function analyses in cultured mammalian cells. Methods: The activity of GSK-3 mutant constructs was determined by examining the GSK-3-specific phosphorylation of the substrates: 1) -catenin, by measuring its transcriptional activity in the Wnt signaling pathway in luciferase reporter assays and 2) tau, a microtubule-associated protein, by immunoblot analysis. Results: Our findings demonstrate that while the amino termini are dispensible, the carboxyl termini are essential for GSK-3 activity. Interestingly, sequence analysis of the critical carboxyl termini regions demonstrates divergence between the otherwise highly homologous isoforms. Conclusions: We have identified a unique, non- catalytic region within both GSK-3 isoforms that is essential for kinase activity towards the substrates -catenin and tau. P3-322 THE NICASTRIN PEPTIDASE-LIKE DOMAIN IS CRUCIALLY INVOLVED IN THE ASSEMBLY OF THE -SECRETASE COMPLEX Lucia Chavez, Elke Maes, Alexandra Tolia, Bart De Strooper, KUL, Leuven, Belgium. Contact e-mail: [email protected] Background: The identification of Nicastrin (NCT) as the substrate re- ceptor of the -secretase complex raised the possibility of considering it as a drug target in AD. It has been reported that hNCT-E333 is crucial in the recognition mechanism by interacting with the substrate’s alpha amino terminal group. Methods: In the absence of structural information for the complex, we used the NCT similarity to peptidases with known crystal structure, as a guide to further explore and characterize NCT’s role. We performed alanine-scanning mutagenesis to further investigate the role of the NCT “peptidase-like” domain in the activity-specificity of the -secre- tase complex. We evaluated residues located in the putative substrate’s binding pocket of the NCT model. Nicastrin knock-out mouse fibroblast were rescued with mouse wild type NCT or mutants. Results: The expres- sion levels were similar among mutants and at a similar extent to the endogenous NCT. However, not all of them rescued NCT maturation. Unexpectedly, the E332A-NCT mutant severely affected NCT maturation and complex assembly, stabilizing only about 10% of the wt-NCT com- plex. We next investigated whether any of these substitutions influences the -secretase processing of APP or Notch substrates. In general, mutations the NCT-peptidase like domain slightly regulated the processing of these substrates. Interestingly, we observed that the specific activity of the E332A- -secretase complex was comparable with the wt enzyme suggest- ing that the substrate recognition mechanism is mainly unaffected. Con- clusions: In conclusion, our data show that the NCT-peptidase like domain is mainly implicated in the assembly of the complex, whereas its contri- bution to the activity-specificity of the complex is less obvious. Most importantly, our data strongly shows that the substrate recognition mech- anism is not critically dependent on the E332 residue, as previously reported. However, we cannot discard a dual mechanism for NCT-ectodo- main: initially involved in the complex assembly and that could lead to a contribution in substrate recognition. P3-323 BACE1 PROCESSING OF HUMAN NEUREGULIN 1 TYPE III 1A IN TRANSFECTED MAMMALIAN CELLS Maxine Chen, Matthew E. Kennedy, Schering-Plough Research Institute, Kenilworth, NJ, USA. Contact e-mail: [email protected] Background: BACE1 null mice exhibit reduced peripheral nerve my- elination similar to what is observed in NRG1 Type III het mice. BACE1 null mice also exhibit reduced processing of Neuregulin 1 (NRG1) type III 1a in vivo. Therefore, BACE1 processing of NRG1 type III 1a was proposed to be required for proper peripheral nerve myelination. Since BACE1 inhibition is the most attractive therapeutic strategy for Alzheimer’s Disease (AD) it is important to determine if BACE1 inhibitors impact the processing of NRG1 type III 1a and myelination in vivo. Methods: Previous biochemical studies with neu- regulins relied on rat cDNAs and the human NRG1 type III 1a cDNA has not been reported. As a first step to determining the impact of BACE1 inhibitors on NRG1 type III 1a processing, we cloned human NRG1 type III 1a from a human fetal brain cDNA library and ex- pressed it in a mammalian cell system. Results: Mutations in NRG1 type III 1a were identified which altered its subcellular localization. And in addition, similar to APP, co-expression of BACE1 also changed the subcellular localization of NRG1 type III 1a. Conclusions: Com- parisons between BACE1 processing of APP and NRG1 type III 1a and the effects of inhibitors will also be reported. P3-324 CHARACTERIZATION OF S-PALMITOYLATION IN GAMMA-SECRETASE SUBUNITS Haipeng Cheng 1 , Kulandaivelu S. Vetrivel 1 , Renaldo C. Drisdel 1 , Xavier Meckler 1 , Ping Gong 1 , Tong Li 2 , Meghan Carter 1 , Jaeyoon Leem 1 , Ying Chen 1 , Phuong Nguyen 3 , Philip C. Wong 2 , William N. Green 1 , Maria Z. Kounnas 3 , Gopal Thinakaran 1 , 1 Department of Neurobiology,University of Chicago, Chicago, IL, USA; 2 Department. of Pathology, The Johns Hopkins University, School of Medicine, Baltimore, MD, USA; 3 TorreyPines Therapeutics, La Jolla, CA, USA. Contact e-mail: [email protected] Background: Proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases generate beta-amyloid (A beta) peptides, which accumulate in the brains of individuals affected by Alzheimer’s disease (AD). Detergent resistant membrane microdomains rich in cholesterol, termed lipid rafts, are involved in gamma-cleavage of APP. Our previous studies showed that components of the gamma- secretase complex (PS1, nicastrin, APH-1 and PEN-2) and APP C- terminal fragments associate with lipid rafts. In this study we investi- gated the mechanisms underlying raft localization of gamma-secretase and the regulation of gamma-secretase activity on APP processing within lipid raft microdomains. Methods: Immunoprecipited proteins were treated with 1M of hydroxylamine or Tris HCl (as control) for 1h, then labeled with Btn-BMCC. Samples were reacted with Streptavidin- HRP to detect biotin labeled proteins, or subject to Western blotting to visualize total proteins. Lipid rafts were isolated from nearly confluent cells grown in two 100-mm dishes. Briefly, cells were scraped into 1.5 ml of lysis buffer containing 0.5% Lubrol WX. A discontinuous sucrose gradient was subject to ultracentrifugation at 39,000 rpm for 21 h at 4 °C. Twelve 1-ml fractions were analyzed by Western blotting. Results: We found that two subunits of the gamma-secretase complex, nicastrin and APH-1, undergo post-translational addition of a 16-carbon satu- rated fatty acid to cysteine residues through thioester linkage; this modification is termed S-palmitoylation. By site-directed mutagenesis we identified three sites that are modified by S-palmitoylation. Co- expression of S-palmitoylation deficient APH-1 and nicastrin in APH- 1a/b KO or nicastrin KO fibroblasts, respectively, results in their re- markable displacement, and minor loss of PS1 and PEN2 from lipid raft fractions. Moreover, S-palmitoylation contributes the stability of nas- T616 Poster Presentations P3:
Transcript of P3-322: The Nicastrin peptidase-like domain is crucially involved in the assembly of the...
P3-321 THE CARBOXYL TERMINI OF GSK-3 ISOFORMSARE ESSENTIAL FOR ACTIVITY
Jessica L. Buescher1, Christopher J. Phiel1,2, 1The Ohio StateUniversity, Columbus, OH, USA; 2The Research Institute at NationwideChildren’s Hospital, Columbus, OH, USA. Contact e-mail:[email protected]
Background: Glycogen synthase kinase-3 (GSK-3) isoforms, GSK-3� andGSK-3�, are serine/threonine kinases involved in numerous cellular pro-cesses. GSK-3 isoforms have also been implicated in a wide array ofdisease processes including Alzheimer’s disease, cancer, diabetes, andmood disorders. Thus, pharmacological modulation of GSK-3 isoformsoffers great potential for the treatment of a variety of disorders. Further-more, understanding the structural requirements governing GSK-3 activityis a fundamental step in the development of therapeutic agents. Objective:In order to determine the structural requirements of GSK-3 isoform activ-ity, we have performed structure-function analyses in cultured mammaliancells. Methods: The activity of GSK-3 mutant constructs was determinedby examining the GSK-3-specific phosphorylation of the substrates: 1)�-catenin, by measuring its transcriptional activity in the Wnt signalingpathway in luciferase reporter assays and 2) tau, a microtubule-associatedprotein, by immunoblot analysis. Results: Our findings demonstrate thatwhile the amino termini are dispensible, the carboxyl termini are essentialfor GSK-3 activity. Interestingly, sequence analysis of the critical carboxyltermini regions demonstrates divergence between the otherwise highlyhomologous isoforms. Conclusions: We have identified a unique, non-catalytic region within both GSK-3 isoforms that is essential for kinaseactivity towards the substrates �-catenin and tau.
P3-322 THE NICASTRIN PEPTIDASE-LIKE DOMAIN ISCRUCIALLY INVOLVED IN THE ASSEMBLY OFTHE �-SECRETASE COMPLEX
Lucia Chavez, Elke Maes, Alexandra Tolia, Bart De Strooper, KUL,Leuven, Belgium. Contact e-mail:[email protected]
Background: The identification of Nicastrin (NCT) as the substrate re-ceptor of the �-secretase complex raised the possibility of considering it asa drug target in AD. It has been reported that hNCT-E333 is crucial in therecognition mechanism by interacting with the substrate’s alpha aminoterminal group. Methods: In the absence of structural information for thecomplex, we used the NCT similarity to peptidases with known crystalstructure, as a guide to further explore and characterize NCT’s role. Weperformed alanine-scanning mutagenesis to further investigate the role ofthe NCT “peptidase-like” domain in the activity-specificity of the �-secre-tase complex. We evaluated residues located in the putative substrate’sbinding pocket of the NCT model. Nicastrin knock-out mouse fibroblastwere rescued with mouse wild type NCT or mutants. Results: The expres-sion levels were similar among mutants and at a similar extent to theendogenous NCT. However, not all of them rescued NCT maturation.Unexpectedly, the E332A-NCT mutant severely affected NCT maturationand complex assembly, stabilizing only about 10% of the wt-NCT com-plex. We next investigated whether any of these substitutions influences the�-secretase processing of APP or Notch substrates. In general, mutationsthe NCT-peptidase like domain slightly regulated the processing of thesesubstrates. Interestingly, we observed that the specific activity of theE332A- �-secretase complex was comparable with the wt enzyme suggest-ing that the substrate recognition mechanism is mainly unaffected. Con-clusions: In conclusion, our data show that the NCT-peptidase like domainis mainly implicated in the assembly of the complex, whereas its contri-bution to the activity-specificity of the complex is less obvious. Mostimportantly, our data strongly shows that the substrate recognition mech-anism is not critically dependent on the E332 residue, as previouslyreported. However, we cannot discard a dual mechanism for NCT-ectodo-main: initially involved in the complex assembly and that could lead to acontribution in substrate recognition.
P3-323 BACE1 PROCESSING OF HUMAN NEUREGULIN 1TYPE III �1A IN TRANSFECTED MAMMALIANCELLS
Maxine Chen, Matthew E. Kennedy, Schering-Plough ResearchInstitute, Kenilworth, NJ, USA. Contact e-mail:[email protected]
Background: BACE1 null mice exhibit reduced peripheral nerve my-elination similar to what is observed in NRG1 Type III het mice.BACE1 null mice also exhibit reduced processing of Neuregulin 1(NRG1) type III � 1a in vivo. Therefore, BACE1 processing of NRG1type III � 1a was proposed to be required for proper peripheral nervemyelination. Since BACE1 inhibition is the most attractive therapeuticstrategy for Alzheimer’s Disease (AD) it is important to determine ifBACE1 inhibitors impact the processing of NRG1 type III � 1a andmyelination in vivo. Methods: Previous biochemical studies with neu-regulins relied on rat cDNAs and the human NRG1 type III � 1a cDNAhas not been reported. As a first step to determining the impact ofBACE1 inhibitors on NRG1 type III � 1a processing, we cloned humanNRG1 type III � 1a from a human fetal brain cDNA library and ex-pressed it in a mammalian cell system. Results: Mutations in NRG1type III � 1a were identified which altered its subcellular localization.And in addition, similar to APP, co-expression of BACE1 also changedthe subcellular localization of NRG1 type III � 1a. Conclusions: Com-parisons between BACE1 processing of APP and NRG1 type III � 1aand the effects of inhibitors will also be reported.
P3-324 CHARACTERIZATION OF S-PALMITOYLATIONIN GAMMA-SECRETASE SUBUNITS
Haipeng Cheng1, Kulandaivelu S. Vetrivel1, Renaldo C. Drisdel1,Xavier Meckler1, Ping Gong1, Tong Li2, Meghan Carter1,Jaeyoon Leem1, Ying Chen1, Phuong Nguyen3, Philip C. Wong2,William N. Green1, Maria Z. Kounnas3, Gopal Thinakaran1,1Department of Neurobiology,University of Chicago, Chicago, IL, USA;2Department. of Pathology, The Johns Hopkins University, School ofMedicine, Baltimore, MD, USA; 3TorreyPines Therapeutics, La Jolla,CA, USA. Contact e-mail: [email protected]
Background: Proteolytic processing of amyloid precursor protein(APP) by beta- and gamma-secretases generate beta-amyloid (A beta)peptides, which accumulate in the brains of individuals affected byAlzheimer’s disease (AD). Detergent resistant membrane microdomainsrich in cholesterol, termed lipid rafts, are involved in gamma-cleavageof APP. Our previous studies showed that components of the gamma-secretase complex (PS1, nicastrin, APH-1 and PEN-2) and APP C-terminal fragments associate with lipid rafts. In this study we investi-gated the mechanisms underlying raft localization of gamma-secretaseand the regulation of gamma-secretase activity on APP processingwithin lipid raft microdomains. Methods: Immunoprecipited proteinswere treated with 1M of hydroxylamine or Tris HCl (as control) for 1h,then labeled with Btn-BMCC. Samples were reacted with Streptavidin-HRP to detect biotin labeled proteins, or subject to Western blotting tovisualize total proteins. Lipid rafts were isolated from nearly confluentcells grown in two 100-mm dishes. Briefly, cells were scraped into 1.5ml of lysis buffer containing 0.5% Lubrol WX. A discontinuous sucrosegradient was subject to ultracentrifugation at 39,000 rpm for 21 h at 4°C. Twelve 1-ml fractions were analyzed by Western blotting. Results:We found that two subunits of the gamma-secretase complex, nicastrinand APH-1, undergo post-translational addition of a 16-carbon satu-rated fatty acid to cysteine residues through thioester linkage; thismodification is termed S-palmitoylation. By site-directed mutagenesiswe identified three sites that are modified by S-palmitoylation. Co-expression of S-palmitoylation deficient APH-1 and nicastrin in APH-1a/b KO or nicastrin KO fibroblasts, respectively, results in their re-markable displacement, and minor loss of PS1 and PEN2 from lipid raftfractions. Moreover, S-palmitoylation contributes the stability of nas-
T616 Poster Presentations P3:
