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{"title"=>"Analysis of Proteolytic Processes and Enzymatic Activities in the Generation of Huntingtin N-Terminal Fragments in an HEK293 Cell Model", "type"=>"journal", "authors"=>[{"first_name"=>"Andrew T.N.", "last_name"=>"Tebbenkamp", "scopus_author_id"=>"16204188200"}, {"first_name"=>"Keith W.", "last_name"=>"Crosby", "scopus_author_id"=>"55520073500"}, {"first_name"=>"Zoe B.", "last_name"=>"Siemienski", "scopus_author_id"=>"55521074100"}, {"first_name"=>"Hilda H.", "last_name"=>"Brown", "scopus_author_id"=>"54782222300"}, {"first_name"=>"Todd E.", "last_name"=>"Golde", "scopus_author_id"=>"7005214095"}, {"first_name"=>"David R.", "last_name"=>"Borchelt", "scopus_author_id"=>"7005319619"}, {"first_name"=>"Xiao Jiang", "last_name"=>"Li", "scopus_author_id"=>"36012660600"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84870933532", "doi"=>"10.1371/journal.pone.0050750", "pui"=>"366250513", "pmid"=>"23236391", "scopus"=>"2-s2.0-84870933532", "issn"=>"19326203", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)"}, "id"=>"1bdd3cc3-2a9c-36b5-9245-7b6d83adb309", "abstract"=>"BACKGROUND: N-terminal fragments of mutant huntingtin (htt) that terminate between residues 90-115, termed cleavage product A or 1 (cp-A/1), form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD). These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments.\\n\\nRESULTS: Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like), were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400-600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115-124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1.\\n\\nCONCLUSIONS: Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.", "link"=>"http://www.mendeley.com/research/analysis-proteolytic-processes-enzymatic-activities-generation-huntingtin-nterminal-fragments-hek293", "reader_count"=>26, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Researcher"=>4, "Student > Ph. D. Student"=>10, "Student > Postgraduate"=>1, "Other"=>2, "Student > Master"=>6, "Student > Bachelor"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Researcher"=>4, "Student > Ph. D. Student"=>10, "Student > Postgraduate"=>1, "Other"=>2, "Student > Master"=>6, "Student > Bachelor"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>4, "Agricultural and Biological Sciences"=>14, "Medicine and Dentistry"=>2, "Neuroscience"=>3, "Physics and Astronomy"=>1, "Computer Science"=>1, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Neuroscience"=>{"Neuroscience"=>3}, "Physics and Astronomy"=>{"Physics and Astronomy"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>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"=>4}}, "reader_count_by_country"=>{"United States"=>1, "China"=>1, "United Kingdom"=>1, "France"=>1, "Germany"=>1}, "group_count"=>1}

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• http://doi.org/10.1111/febs.12418
• http://doi.org/10.1016/j.neuropharm.2018.03.009
• http://doi.org/10.1016/j.neuron.2016.02.003
• http://doi.org/10.1152/ajplung.00217.2014
• http://doi.org/10.1116/1.4984009
• http://doi.org/10.3389/fmicb.2019.02622
• http://doi.org/10.1074/jbc.M117.782300
• http://doi.org/10.15252/embj.201490808
• http://doi.org/10.1021/acs.jproteome.6b00991

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• {"id"=>"281179572874121216", "text"=>"#PLOSONE: Analysis of Proteolytic Processes & Enzymatic Activities in the Generation of Huntingtin N-Terminal Fragments http://t.co/RVGGQLCm", "created_at"=>"2012-12-18T23:30:08Z", "user"=>"PLOSCurrentsHD", "user_name"=>"PLOS Currents: HD", "user_profile_image"=>"http://a0.twimg.com/profile_images/2429987946/24tz480s344rbdlfci12_normal.jpeg"}

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Figshare

• {"files"=>["https://ndownloader.figshare.com/files/528080"], "description"=>"<p>HEK293 and mouse L cells [thymidine kinase negative (TK⁻)] were transiently transfected with httN171-18Q constructs. After 48 hours, the cells were harvested and analyzed by immunoblot with the htt antibody 2B4. Only the HEK293 cells produce a cleavage product. The image shown is representative of at least 3 repetitions.</p>", "links"=>[], "tags"=>["cells", "cleave", "htt"], "article_id"=>198567, "categories"=>["Biological Sciences", "Biochemistry", "Neuroscience", "Genetics"], "users"=>["Andrew T. N. Tebbenkamp", "Keith W. Crosby", "Zoe B. Siemienski", "Hilda H. Brown", "Todd E. Golde", "David R. Borchelt"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0050750.g001", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_HEK293_cells_but_not_mouse_L_cells_cleave_htt_to_produce_a_cp_A_1_like_fragment_/198567", "title"=>"HEK293 cells but not mouse L cells cleave htt to produce a cp-A/1 like fragment.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-12-07 02:22:47"}
• {"files"=>["https://ndownloader.figshare.com/files/286257", "https://ndownloader.figshare.com/files/286302", "https://ndownloader.figshare.com/files/286318", "https://ndownloader.figshare.com/files/286372", "https://ndownloader.figshare.com/files/286428", "https://ndownloader.figshare.com/files/286463", "https://ndownloader.figshare.com/files/286508", "https://ndownloader.figshare.com/files/286527", "https://ndownloader.figshare.com/files/286574", "https://ndownloader.figshare.com/files/286605", "https://ndownloader.figshare.com/files/286643", "https://ndownloader.figshare.com/files/286681", "https://ndownloader.figshare.com/files/286720"], "description"=>"<div><h3>Background</h3><p>N-terminal fragments of mutant huntingtin (htt) that terminate between residues 90–115, termed cleavage product A or 1 (cp-A/1), form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD). These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments.</p> <h3>Results</h3><p>Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like), were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400–600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115–124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1.</p> <h3>Conclusions</h3><p>Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.</p> </div>", "links"=>[], "tags"=>["proteolytic", "processes", "enzymatic", "activities", "huntingtin", "n-terminal", "fragments", "hek293", "model"], "article_id"=>116424, "categories"=>["Biological Sciences", "Biochemistry", "Neuroscience", "Genetics"], "users"=>["Andrew T. N. Tebbenkamp", "Keith W. Crosby", "Zoe B. Siemienski", "Hilda H. Brown", "Todd E. Golde", "David R. Borchelt"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0050750.s001", "https://dx.doi.org/10.1371/journal.pone.0050750.s002", "https://dx.doi.org/10.1371/journal.pone.0050750.s003", "https://dx.doi.org/10.1371/journal.pone.0050750.s004", "https://dx.doi.org/10.1371/journal.pone.0050750.s005", "https://dx.doi.org/10.1371/journal.pone.0050750.s006", "https://dx.doi.org/10.1371/journal.pone.0050750.s007", "https://dx.doi.org/10.1371/journal.pone.0050750.s008", "https://dx.doi.org/10.1371/journal.pone.0050750.s009", "https://dx.doi.org/10.1371/journal.pone.0050750.s010", "https://dx.doi.org/10.1371/journal.pone.0050750.s011", "https://dx.doi.org/10.1371/journal.pone.0050750.s012", "https://dx.doi.org/10.1371/journal.pone.0050750.s013"], "stats"=>{"downloads"=>14, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Analysis_of_Proteolytic_Processes_and_Enzymatic_Activities_in_the_Generation_of_Huntingtin_N_Terminal_Fragments_in_an_HEK293_Cell_Model__/116424", "title"=>"Analysis of Proteolytic Processes and Enzymatic Activities in the Generation of Huntingtin N-Terminal Fragments in an HEK293 Cell Model", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-12-07 01:47:04"}
• {"files"=>["https://ndownloader.figshare.com/files/528117"], "description"=>"<p>A, HEK293 cells were non-transfected (NTf) or transfected with cDNA encoding htt N171-18Q. Cell lysates were analyzed by immunoblots incubated with antibodies 2B4 or 1H6. B, HEK293 cells were transfected with htt cDNAs shown at the top of the figure, followed by lysis and SDS-PAGE. Arrowheads identify substrates or cleavage products that are generated at approximately equally levels for short and expanded polyglutamine lengths. The lack of an effect of polyglutamine length on cleavage efficiency was observed for both N171 and N586 based constructs. Immunoblots were probed with the htt81-90 antibody (1∶3000). The images shown are representative of at least 3 repetitions of the experiment. EH = endogenous htt.</p>", "links"=>[], "tags"=>["cleavage"], "article_id"=>198613, "categories"=>["Biological Sciences", "Biochemistry", "Neuroscience", "Genetics"], "users"=>["Andrew T. N. Tebbenkamp", "Keith W. Crosby", "Zoe B. Siemienski", "Hilda H. Brown", "Todd E. Golde", "David R. Borchelt"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0050750.g002", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Polyglutamine_length_does_not_affect_cleavage_efficiency_/198613", "title"=>"Polyglutamine length does not affect cleavage efficiency.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-12-07 02:23:33"}
• {"files"=>["https://ndownloader.figshare.com/files/528408"], "description"=>"<p>A, HEK293 cells were transfected with N171-18Q, treated with 3-MA, and either untreated, or treated with DAPT or LY411,575 followed by immunoblot analysis for htt (lane 4 is non-transfected). In all treatment scenarios, no change was detected in htt proteolysis; cp-A/1 (arrowhead on left panel) was detected at equivalent levels regardless of treatment. B, The efficacy of the γ-secretase inhibitor was confirmed by immunoblotting for APP C-terminal fragments (10 kDa CTF; arrow on right panel). The images shown are representative of at least 3 repetitions of the experiment.</p>", "links"=>[], "tags"=>["inhibitors", "htt"], "article_id"=>198896, "categories"=>["Biological Sciences", "Biochemistry", "Neuroscience", "Genetics"], "users"=>["Andrew T. N. Tebbenkamp", "Keith W. Crosby", "Zoe B. Siemienski", "Hilda H. Brown", "Todd E. Golde", "David R. Borchelt"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0050750.g006", "stats"=>{"downloads"=>1, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Gamma_secretase_inhibitors_fail_to_block_htt_proteolysis_/198896", "title"=>"Gamma-secretase inhibitors fail to block htt proteolysis.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-12-07 02:28:16"}
• {"files"=>["https://ndownloader.figshare.com/files/528188"], "description"=>"<p>A, The sequence of htt is shown through residue 171, the substrate used in these experiments. The first cysteine in htt is at residue 105 (bold) and represents the most N-terminal cysteine that could be labeled with sulfhydrylated biotin (Biotin-SH). This cysteine must be present for any labeling to occur. Subsequent immunoprecipitation (IP) followed by detection with streptavidin or htt antibodies was used to visualize the biotinylation. B, Detection using streptavidin-HRP of transfected cell lysate (Tfx N171-18Q, +) reacted with biotinylation reagent (Biotin, +) shows cp-B/2 and cp-A/1-sized products (arrows). Asterisks (*) signify possible multimers of htt. C, Detection of htt with the antibody htt3-16 confirms the identities of cp-B/2 and cp-A/1 (bottom two arrows). IgG from the immunoprecipitation is observed in all lanes (top arrow). The images shown are representative of at least 3 repetitions of the experiment.</p>", "links"=>[], "tags"=>["cleavage", "occurs", "c-terminal", "cysteine"], "article_id"=>198678, "categories"=>["Biological Sciences", "Biochemistry", "Neuroscience", "Genetics"], "users"=>["Andrew T. N. Tebbenkamp", "Keith W. Crosby", "Zoe B. Siemienski", "Hilda H. Brown", "Todd E. Golde", "David R. Borchelt"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0050750.g003", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Htt_cleavage_occurs_C_terminal_to_cysteine_105_/198678", "title"=>"Htt cleavage occurs C-terminal to cysteine 105.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-12-07 02:24:38"}
• {"files"=>["https://ndownloader.figshare.com/files/528275"], "description"=>"<p>HEK293 cells which stably express a shRNA against calpain-1 (shRNAi +) do not block cleavage of htt following transfection with cDNA encoding N171-18Q (Tfx +). Knock-down of capn1 was confirmed by immunoblot with an antibody to activated calpain-1. Htt cp-A/1 was detected with the htt81-90 (1∶3000) antibody. The images shown are representative of at least 3 repetitions of the experiment.</p>", "links"=>[], "tags"=>["calpain-1", "htt"], "article_id"=>198771, "categories"=>["Biological Sciences", "Biochemistry", "Neuroscience", "Genetics"], "users"=>["Andrew T. N. Tebbenkamp", "Keith W. Crosby", "Zoe B. Siemienski", "Hilda H. Brown", "Todd E. Golde", "David R. Borchelt"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0050750.g004", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Knock_down_of_calpain_1_does_not_block_htt_cleavage_/198771", "title"=>"Knock-down of calpain-1 does not block htt cleavage.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-12-07 02:26:11"}
• {"files"=>["https://ndownloader.figshare.com/files/528345"], "description"=>"<p>Top: inhibitors (3-methyladenine, 3-MA and Chloroquine, Chlrq) or activators (Rapamycin, Rapa) of autophagy failed to block proteolysis, as detected by immunoblot with 2B4 antibody (1∶1000). Treatment with 3-MA caused an increase in cp-B/2 and cp-A/1. Bottom: Detection of LC3-I and LC3-II. LC3 antibody was used at 1∶1000. The images shown are representative of at least 3 repetitions of the experiment.</p>", "links"=>[], "tags"=>["activators", "inhibitors", "htt"], "article_id"=>198834, "categories"=>["Biological Sciences", "Biochemistry", "Neuroscience", "Genetics"], "users"=>["Andrew T. N. Tebbenkamp", "Keith W. Crosby", "Zoe B. Siemienski", "Hilda H. Brown", "Todd E. Golde", "David R. Borchelt"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0050750.g005", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Autophagy_activators_or_inhibitors_do_not_block_htt_proteolysis_/198834", "title"=>"Autophagy activators or inhibitors do not block htt proteolysis.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-12-07 02:27:14"}

PMC Usage Stats | Further Information

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Relative Metric

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F1000Prime