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{"title"=>"Turnover of C99 is controlled by a crosstalk between ERAD and ubiquitin-independent lysosomal degradation in human neuroglioma cells", "type"=>"journal", "authors"=>[{"first_name"=>"Hianara A.", "last_name"=>"Bustamante", "scopus_author_id"=>"56023761000"}, {"first_name"=>"Andrés", "last_name"=>"Rivera-Dictter", "scopus_author_id"=>"43061406200"}, {"first_name"=>"Viviana A.", "last_name"=>"Cavieres", "scopus_author_id"=>"56024856300"}, {"first_name"=>"Vanessa C.", "last_name"=>"Muñoz", "scopus_author_id"=>"55928550600"}, {"first_name"=>"Alexis", "last_name"=>"González", "scopus_author_id"=>"56892825300"}, {"first_name"=>"Yimo", "last_name"=>"Lin", "scopus_author_id"=>"55647116900"}, {"first_name"=>"Gonzalo A.", "last_name"=>"Mardones", "scopus_author_id"=>"6602407146"}, {"first_name"=>"Patricia V.", "last_name"=>"Burgos", "scopus_author_id"=>"18436143800"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"24376644", "doi"=>"10.1371/journal.pone.0083096", "sgr"=>"84893359728", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "scopus"=>"2-s2.0-84893359728", "issn"=>"19326203", "pui"=>"372245337"}, "id"=>"9976cee2-c543-3cf0-af82-58b72a76e4f0", "abstract"=>"Alzheimer's disease (AD) is characterized by the buildup of amyloid-β peptides (Aβ) aggregates derived from proteolytic processing of the β-amyloid precursor protein (APP). Amyloidogenic cleavage of APP by β-secretase/BACE1 generates the C-terminal fragment C99/CTFβ that can be subsequently cleaved by γ-secretase to produce Aβ. Growing evidence indicates that high levels of C99/CTFβ are determinant for AD. Although it has been postulated that γ-secretase-independent pathways must control C99/CTFβ levels, the contribution of organelles with degradative functions, such as the endoplasmic reticulum (ER) or lysosomes, is unclear. In this report, we investigated the turnover and amyloidogenic processing of C99/CTFβ in human H4 neuroglioma cells, and found that C99/CTFβ is localized at the Golgi apparatus in contrast to APP, which is mostly found in endosomes. Conditions that localized C99/CTFβ to the ER resulted in its degradation in a proteasome-dependent manner that first required polyubiquitination, consistent with an active role of the ER associated degradation (ERAD) in this process. Furthermore, when proteasomal activity was inhibited C99/CTFβ was degraded in a chloroquine (CQ)-sensitive compartment, implicating lysosomes as alternative sites for its degradation. Our results highlight a crosstalk between degradation pathways within the ER and lysosomes to avoid protein accumulation and toxicity.", "link"=>"http://www.mendeley.com/research/turnover-c99-controlled-crosstalk-between-erad-ubiquitinindependent-lysosomal-degradation-human-neur", "reader_count"=>26, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Researcher"=>5, "Student > Ph. D. Student"=>8, "Student > Postgraduate"=>1, "Student > Master"=>4, "Student > Bachelor"=>5, "Lecturer"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Researcher"=>5, "Student > Ph. D. Student"=>8, "Student > Postgraduate"=>1, "Student > Master"=>4, "Student > Bachelor"=>5, "Lecturer"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>3, "Medicine and Dentistry"=>3, "Agricultural and Biological Sciences"=>14, "Neuroscience"=>1, "Arts and Humanities"=>1, "Psychology"=>1, "Chemistry"=>2, "Computer Science"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>3}, "Neuroscience"=>{"Neuroscience"=>1}, "Chemistry"=>{"Chemistry"=>2}, "Psychology"=>{"Psychology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>14}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}, "Arts and Humanities"=>{"Arts and Humanities"=>1}}, "reader_count_by_country"=>{"Chile"=>1, "Germany"=>1, "Russia"=>1}, "group_count"=>0}

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• http://doi.org/10.3390/ijms21228880
• http://doi.org/10.1016/j.mehy.2019.109516
• http://doi.org/10.1016/j.biomaterials.2017.07.002
• http://doi.org/10.3390/ijms19103081
• http://doi.org/10.1111/jnc.15143
• http://doi.org/10.1155/2014/431858
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• http://doi.org/10.3233/JAD-160051
• http://doi.org/10.1093/hmg/ddu449
• http://doi.org/10.1074/jbc.M117.818138
• http://doi.org/10.1371/journal.pone.0136313
• http://doi.org/10.3389/fncel.2018.00126
• http://doi.org/10.1021/ja503535m
• http://doi.org/10.1111/jnc.13873
• http://doi.org/10.1096/fj.201700359
• http://doi.org/10.1096/fj.201600713R
• http://doi.org/10.1016/j.bbrc.2017.03.060
• http://doi.org/10.3389/fncel.2018.00505
• http://doi.org/10.1371/journal.pone.0108845
• http://doi.org/10.1016/j.neuroscience.2019.01.010
• http://doi.org/10.1016/j.ejphar.2017.07.019

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• {"files"=>["https://ndownloader.figshare.com/files/1324030"], "description"=>"<p>(A–B) H4 cells stably expressing GFP-tagged APP-F/P-D/A (A) or C99-F/P-D/A (B) were left untreated or treated for 16 h either with 1 µM DAPT, 100 µM CQ, or with a combination of 1 µM DAPT and 100 µM CQ. Cellular extracts were analyzed by immunoblot with anti-GFP antibody. The positions of molecular mass markers are indicated on the left. (C) Densitometric quantification of the levels of APP and C99 shown in A and B. Bars represent the mean ± SD (APP n = 7; C99 n = 6). *<i>P</i><0.05. (D) Confocal fluorescence microscopy of H4 cells stably expressing GFP-tagged APP-F/P-D/A or C99-F/P-D/A left untreated (Control) or treated with 100 µM CQ for 16 h. Bar, 10 µm.</p>", "links"=>[], "tags"=>["app", "c99"], "article_id"=>883084, "categories"=>["Biological Sciences"], "users"=>["Hianara A. Bustamante", "Andrés Rivera-Dictter", "Viviana A. Cavieres", "Vanessa C. Muñoz", "Alexis González", "Yimo Lin", "Gonzalo A. Mardones", "Patricia V. Burgos"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0083096.g003", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Different_response_of_APP_and_C99_to_CQ_/883084", "title"=>"Different response of APP and C99 to CQ.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-20 02:52:58"}
• {"files"=>["https://ndownloader.figshare.com/files/1324026"], "description"=>"<p>(A) Schematic representation of GFP-tagged APP and C99 indicating their topological domains and the position of the HA tag, the Aβ peptide, the proteolytic cleavage sites (α, β and γ), and the AICDγ fragment. (B) Fluorescence microscopy analysis of H4 human neuroglioma cells transiently expressing APP-GFP or C99-GFP. Bar, 10 µm. (C–E) H4 cells transiently expressing C99-GFP were left untreated or treated for 16 h with 1 µM DAPT, labeled for 4 hr at 20°C with 1 mCi/ml [³⁵S]-methionine-cysteine, and chased at 37°C for the indicated times. C99 and Aβ species were immunoprecipitated from cell lysates with anti-GFP antibody (C), or from the culture medium with 6E10 antibody (E), respectively. Proteins were analyzed on 10%–20% Tricine gels and fluorography. The positions of molecular mass markers are indicated on the left. (D) Densitometric quantification of the levels of C99, C83, and AICDγ shown in C.</p>", "links"=>[], "tags"=>["localization", "proteolytic"], "article_id"=>883080, "categories"=>["Biological Sciences"], "users"=>["Hianara A. Bustamante", "Andrés Rivera-Dictter", "Viviana A. Cavieres", "Vanessa C. Muñoz", "Alexis González", "Yimo Lin", "Gonzalo A. Mardones", "Patricia V. Burgos"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0083096.g001", "stats"=>{"downloads"=>2, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Intracellular_localization_and_proteolytic_processing_of_C99_/883080", "title"=>"Intracellular localization and proteolytic processing of C99.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-20 02:52:58"}
• {"files"=>["https://ndownloader.figshare.com/files/1324039"], "description"=>"<p>(A) Schematic representation of GFP-tagged C99 indicating its topological domains, the position of the Aβ peptide, the γ-secretase cleavage site, the AICDγ fragment, and the sequence of the cytosolic tail highlighting the substitutions in its three tyrosine residues (bold underline). (B) Immunoblot analysis of H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-3Y/A-F/P-D/A (C99-3Y/A). Cells were left untreated or treated with 1 µM DAPT for 16 h and subsequently analyzed by immunoblot with anti-GFP antibody. Immunoblot with anti-β-actin was used as loading control. The positions of molecular mass markers are indicated on the left. (C) H4 cells stably expressing C99-3Y/A were left untreated or treated for 16 h either with 1 µM MG132, 100 µM CQ, or with a combination of 1 µM MG132 and 100 µM CQ. Cells were biotinylated on the cell surface with Sulfo-NHS-LC-Biotin and soluble extracts pulled down with NeutrAvidin-agarose. Total and biotinylated proteins were analyzed by immunoblot with anti-GFP antibody. Immunoblot with anti-β-actin or anti-transferrin receptor (TfR) antibodies was used as loading control for total or biotinylated proteins, respectively. The positions of molecular mass markers are indicated on the left. (D) Densitometric quantification of C99-3Y/A left untreated or treated for 16 h either with 100 µM CQ, 1 µM MG132, or with a combination of 100 µM CQ and 1 µM MG132. Bars represent the mean ± SD (n = 3). *<i>P</i><0.05; **<i>P</i><0.01.</p>", "links"=>[], "tags"=>["c99", "cq", "cytosolic", "tyrosine"], "article_id"=>883093, "categories"=>["Biological Sciences"], "users"=>["Hianara A. Bustamante", "Andrés Rivera-Dictter", "Viviana A. Cavieres", "Vanessa C. Muñoz", "Alexis González", "Yimo Lin", "Gonzalo A. Mardones", "Patricia V. Burgos"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0083096.g007", "stats"=>{"downloads"=>2, "page_views"=>26, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Accumulation_of_C99_in_response_to_MG132_CQ_and_lack_of_its_cytosolic_tyrosine_residues_/883093", "title"=>"Accumulation of C99 in response to MG132, CQ and lack of its cytosolic tyrosine residues.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-20 02:52:58"}
• {"files"=>["https://ndownloader.figshare.com/files/1324038"], "description"=>"<p>(A) H4 cells stably expressing GFP-tagged C99-F/P-D/A were left untreated or treated for 16 h either with 100 µM CQ, 1 µM MG132, or with a combination of 100 µM CQ and 1 µM MG132. Cells were biotinylated on the cell surface with Sulfo-NHS-LC-Biotin and soluble extracts pulled down with NeutrAvidin-agarose. Total and biotinylated proteins were analyzed by immunoblot with anti-GFP antibody. Immunoblot with anti-β-actin or anti-transferrin receptor (TfR) antibodies was used as loading control for total or biotinylated proteins, respectively. The positions of molecular mass markers are indicated on the left. (B) Confocal fluorescence microscopy of H4 cells stably expressing GFP-tagged C99-F/P-D/A treated for 16 h either with 1 µM MG132 or with a combination of 1 µM MG132 and 100 µM CQ. Bar, 10 µm. (C) Densitometric quantification of the levels of C99 and AICDγ shown in A. Bars represent the mean ± SD (n = 4). *<i>P</i><0.05; **<i>P</i><0.01.</p>", "links"=>[], "tags"=>["c99", "mg132"], "article_id"=>883092, "categories"=>["Biological Sciences"], "users"=>["Hianara A. Bustamante", "Andrés Rivera-Dictter", "Viviana A. Cavieres", "Vanessa C. Muñoz", "Alexis González", "Yimo Lin", "Gonzalo A. Mardones", "Patricia V. Burgos"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0083096.g006", "stats"=>{"downloads"=>2, "page_views"=>39, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Accumulation_of_C99_at_the_cell_surface_in_response_to_MG132_and_CQ_/883092", "title"=>"Accumulation of C99 at the cell surface in response to MG132 and CQ.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-20 02:52:58"}
• {"files"=>["https://ndownloader.figshare.com/files/1324032"], "description"=>"<p>H4 cells stably expressing GFP-tagged C99-F/P-D/A were treated as follows: (A) with increasing concentrations of MG132 for 4 h; (B) left untreated or treated either with 1 µM DAPT for 16 h, 1 µM MG132 for 4 h, or 1 µM DAPT for 12 h followed by a combination of 1 µM DAPT and 1 µM MG132 for 4 h; (C) left untreated or treated for 4 h either with 5 µg/ml BFA, 1 µM MG132, or a combination of 1 µM MG132 and 5 µg/ml BFA; or (D) pretreated with 5 µg/ml BFA without or with 1 µM MG132 for 4 h followed by CHX-chase for 0–60 min without or with 1 µM MG132. (E) H4 cells stably expressing GFP-tagged APP-F/P-D/A were left untreated or treated for 4 h either with 5 µg/ml BFA, or a combination of 5 µg/ml BFA and 1 µM MG132. Cellular extracts were analyzed by immunoblot with anti-GFP antibody (A–E), or WO2 monoclonal antibody to detect C99 in cells expressing GFP-tagged APP-F/P-D/A (E). Immunoblot with anti-β-actin antibody was used as loading control. The positions of molecular mass markers are indicated on the left. (F) Densitometric quantification of the levels of C99 shown in E. Bars represent the mean ± SD (n = 4). *<i>P</i><0.05.</p>", "links"=>[], "tags"=>["degraded", "redistribution", "endoplasmic"], "article_id"=>883086, "categories"=>["Biological Sciences"], "users"=>["Hianara A. Bustamante", "Andrés Rivera-Dictter", "Viviana A. Cavieres", "Vanessa C. Muñoz", "Alexis González", "Yimo Lin", "Gonzalo A. Mardones", "Patricia V. Burgos"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0083096.g004", "stats"=>{"downloads"=>3, "page_views"=>27, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_C99_is_degraded_after_redistribution_to_the_endoplasmic_reticulum_/883086", "title"=>"C99 is degraded after redistribution to the endoplasmic reticulum.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-20 02:52:58"}
• {"files"=>["https://ndownloader.figshare.com/files/1324049", "https://ndownloader.figshare.com/files/1324050", "https://ndownloader.figshare.com/files/1324051", "https://ndownloader.figshare.com/files/1324052", "https://ndownloader.figshare.com/files/1324053", "https://ndownloader.figshare.com/files/1324054", "https://ndownloader.figshare.com/files/1324055"], "description"=>"<div><p>Alzheimer’s disease (AD) is characterized by the buildup of amyloid-β peptides (Aβ) aggregates derived from proteolytic processing of the β-amyloid precursor protein (APP). Amyloidogenic cleavage of APP by β-secretase/BACE1 generates the C-terminal fragment C99/CTFβ that can be subsequently cleaved by γ-secretase to produce Aβ. Growing evidence indicates that high levels of C99/CTFβ are determinant for AD. Although it has been postulated that γ-secretase-independent pathways must control C99/CTFβ levels, the contribution of organelles with degradative functions, such as the endoplasmic reticulum (ER) or lysosomes, is unclear. In this report, we investigated the turnover and amyloidogenic processing of C99/CTFβ in human H4 neuroglioma cells, and found that C99/CTFβ is localized at the Golgi apparatus in contrast to APP, which is mostly found in endosomes. Conditions that localized C99/CTFβ to the ER resulted in its degradation in a proteasome-dependent manner that first required polyubiquitination, consistent with an active role of the ER associated degradation (ERAD) in this process. Furthermore, when proteasomal activity was inhibited C99/CTFβ was degraded in a chloroquine (CQ)-sensitive compartment, implicating lysosomes as alternative sites for its degradation. Our results highlight a crosstalk between degradation pathways within the ER and lysosomes to avoid protein accumulation and toxicity.</p></div>", "links"=>[], "tags"=>["c99", "controlled", "crosstalk", "erad", "ubiquitin-independent", "lysosomal", "degradation", "neuroglioma"], "article_id"=>883097, "categories"=>["Biological Sciences"], "users"=>["Hianara A. Bustamante", "Andrés Rivera-Dictter", "Viviana A. Cavieres", "Vanessa C. Muñoz", "Alexis González", "Yimo Lin", "Gonzalo A. Mardones", "Patricia V. Burgos"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0083096.s001", "https://dx.doi.org/10.1371/journal.pone.0083096.s002", "https://dx.doi.org/10.1371/journal.pone.0083096.s003", "https://dx.doi.org/10.1371/journal.pone.0083096.s004", "https://dx.doi.org/10.1371/journal.pone.0083096.s005", "https://dx.doi.org/10.1371/journal.pone.0083096.s006", "https://dx.doi.org/10.1371/journal.pone.0083096.s007"], "stats"=>{"downloads"=>12, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Turnover_of_C99_is_Controlled_by_a_Crosstalk_between_ERAD_and_Ubiquitin_Independent_Lysosomal_Degradation_in_Human_Neuroglioma_Cells_/883097", "title"=>"Turnover of C99 is Controlled by a Crosstalk between ERAD and Ubiquitin-Independent Lysosomal Degradation in Human Neuroglioma Cells", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-12-20 02:52:58"}
• {"files"=>["https://ndownloader.figshare.com/files/1324028"], "description"=>"<p>(A) Schematic representation of GFP-tagged C99, C83 and C31 indicating their topological domains, and the position of the Aβ peptide, the p3 peptide, the proteolytic cleavage sites (α, γ and caspase), and the AICDγ fragment. (B) H4 cells transiently expressing wild-type C99-GFP (WT) or C99-GFP with either the D87A mutation, the F38P mutation, or both (F/P-D/A), were left untreated or treated with 1 µM DAPT for 16 h. Cellular extracts were analyzed by immunoblot with anti-GFP antibody. The positions of molecular mass markers are indicated on the left.</p>", "links"=>[], "tags"=>["proteolytically", "cleaved"], "article_id"=>883082, "categories"=>["Biological Sciences"], "users"=>["Hianara A. Bustamante", "Andrés Rivera-Dictter", "Viviana A. Cavieres", "Vanessa C. Muñoz", "Alexis González", "Yimo Lin", "Gonzalo A. Mardones", "Patricia V. Burgos"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0083096.g002", "stats"=>{"downloads"=>2, "page_views"=>35, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_C99_is_proteolytically_cleaved_in_different_sites_/883082", "title"=>"C99 is proteolytically cleaved in different sites.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-20 02:52:58"}
• {"files"=>["https://ndownloader.figshare.com/files/1324041"], "description"=>"<p>(A) (<i>i</i>) A small fraction of newly-synthesized APP in the endoplasmic reticulum (ER) can be a substrate of BACE1 that generates C99. Ubiquitinated (Ub) C99 can be a substrate of the endoplasmic reticulum-associated protein degradation (ERAD) pathway to ultimately be degraded by the proteasome. (<i>ii</i>) En route through the secretory pathway, a fraction of APP at the Golgi apparatus can also be a substrate of BACE1 that generates C99, which subsequently can be a substrate of γ-secretase (γ-sec) activity that generates Aβ peptides and cytosolic AICDγ, a proteolytic processing that can be inhibited by DAPT. (<i>iii</i>) Finally, within endo/lysosomal compartments APP can be degraded by acid hydrolases. (B) (<i>i</i>) Upon MG132 inhibition, ubiquitinated C99 accumulates within the ER. Ubiquitinated C99 can exit the ER and reach the Golgi apparatus. (<i>ii</i>) Both ubiquitinated C99 and C99 generated from APP can be cleaved at the Golgi apparatus by γ-secretase activity. Upon Brefeldin A (BFA) treatment, C99 can be relocated from the Golgi apparatus to the ER where it can be also cleaved by γ-secretase activity. (<i>iii</i>) Both APP and the excess of C99 can be degraded by acid hydrolases. (C) (<i>i</i>) Upon MG132 treatment, and (<i>ii</i>) the generation of an excess of C99 at the Golgi apparatus, (<i>iii</i>) chloroquine (CQ) treatment results in accumulation of both APP and C99 within endo/lysosomal compartments. For simplicity, other APP metabolites, such as sAPPβ, which is the other product of BACE1 activity on APP, or the C31 fragment, are not depicted.</p>", "links"=>[], "tags"=>["turnover", "routes"], "article_id"=>883095, "categories"=>["Biological Sciences"], "users"=>["Hianara A. Bustamante", "Andrés Rivera-Dictter", "Viviana A. Cavieres", "Vanessa C. Muñoz", "Alexis González", "Yimo Lin", "Gonzalo A. Mardones", "Patricia V. Burgos"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0083096.g009", "stats"=>{"downloads"=>0, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Proposed_processing_and_turnover_routes_of_C99_/883095", "title"=>"Proposed processing and turnover routes of C99.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-20 02:52:58"}
• {"files"=>["https://ndownloader.figshare.com/files/1324040"], "description"=>"<p>(A) H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-5K/R-F/P-D/A (C99-5K/R) were left untreated or treated for 16 h either with 100 µM CQ, 1 µM MG132, or with a combination of 100 µM CQ and 1 µM MG132. After cells were biotinylated on the cell surface with Sulfo-NHS-LC-Biotin and soluble extracts pulled down with NeutrAvidin-agarose, total and biotinylated proteins were analyzed by immunoblot with anti-GFP antibody. Immunoblot with anti-β-actin or anti-transferrin receptor (TfR) antibodies was used as loading control for total or biotinylated proteins, respectively. The positions of molecular mass markers are indicated on the left. (B) Confocal fluorescence microscopy of H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-5K/R-F/P-D/A (C99-5K/R) treated with a combination of 1 µM MG132 and 100 µM CQ for 16 h. Bar, 10 µm. (C) Densitometric quantification of the ratio AICDγ/C99 of H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-5K/R-F/P-D/A (C99-5K/R) left untreated or treated for 16 h with 1 µM MG132. (D) Densitometric quantification of the levels of C99 in H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-5K/R-F/P-D/A (C99-5K/R) left untreated or treated for 16 h with 1 µM MG132, or with a combination of 100 µM CQ and 1 µM MG132. (E) Densitometric quantification of the levels of C99 at the plasma membrane (PM) in H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-5K/R-F/P-D/A (C99-5K/R) left untreated or treated for 16 h with 1 µM MG132, or with a combination of 100 µM CQ and 1 µM MG132. Bar represents the mean ± SD (n = 3). *<i>P</i><0.05; **<i>P</i><0.01.</p>", "links"=>[], "tags"=>["c99", "acidic", "compartments"], "article_id"=>883094, "categories"=>["Biological Sciences"], "users"=>["Hianara A. Bustamante", "Andrés Rivera-Dictter", "Viviana A. Cavieres", "Vanessa C. Muñoz", "Alexis González", "Yimo Lin", "Gonzalo A. Mardones", "Patricia V. Burgos"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0083096.g008", "stats"=>{"downloads"=>4, "page_views"=>25, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Degradation_of_C99_in_acidic_compartments_is_independent_of_its_ubiquitination_/883094", "title"=>"Degradation of C99 in acidic compartments is independent of its ubiquitination.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-20 02:52:58"}
• {"files"=>["https://ndownloader.figshare.com/files/1324037"], "description"=>"<p>(A) Schematic representation of GFP-tagged C99 indicating its topological domains, the position of the Aβ peptide, the γ-secretase cleavage site, the AICDγ fragment, and the sequence of the cytosolic tail highlighting the substitutions in its five lysine residues (bold underline). (B–D) H4 cells stably expressing GFP-tagged C99-F/P-D/A (C99) or C99-5K/R-F/P-D/A (C99-5K/R) were processed as follows: (B) transfected with HA-tagged ubiquitin and left untreated or treated with 1 µM MG132 for 4 h, and after denaturation soluble extracts immunoprecipitated with anti-GFP antibody; (C) incubated with 150 µg/ml CHX and 40 µg/ml chloramphenicol for 0–30 min; or (D) pretreated with 5 µg/ml BFA for 1 h before further incubation with BFA and the combination of CHX and chloramphenicol for 0–30 min. Proteins were analyzed by immunoblot with HRP-conjugated anti-HA antibody (B; C99-Ub-HA), or anti-GFP antibody (B–D). Immunoblot with anti-β-actin antibody was used as loading control. The positions of molecular mass markers are indicated on the left. (E–F) Confocal fluorescence microscopy of cells stably expressing GFP-tagged C99 (E) or C99-5K/R (F) left untreated (Control) or treated with 5 µg/ml BFA for 1 h. Bars, 10 µm.</p>", "links"=>[], "tags"=>["c99", "redistribution", "endoplasmic", "reticulum", "requires", "polyubiquitination", "cytosolic", "lysine"], "article_id"=>883091, "categories"=>["Biological Sciences"], "users"=>["Hianara A. Bustamante", "Andrés Rivera-Dictter", "Viviana A. Cavieres", "Vanessa C. Muñoz", "Alexis González", "Yimo Lin", "Gonzalo A. Mardones", "Patricia V. Burgos"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0083096.g005", "stats"=>{"downloads"=>0, "page_views"=>23, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Degradation_of_C99_after_redistribution_to_the_endoplasmic_reticulum_requires_polyubiquitination_of_its_cytosolic_lysine_residues_/883091", "title"=>"Degradation of C99 after redistribution to the endoplasmic reticulum requires polyubiquitination of its cytosolic lysine residues.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-20 02:52:58"}

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Relative Metric

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F1000Prime