Bifunctional Anti-Huntingtin Proteasome-Directed Intrabodies Mediate Efficient Degradation of Mutant Huntingtin Exon 1 Protein Fragments
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Mendeley | Further Information

{"title"=>"Bifunctional anti-huntingtin proteasome-directed intrabodies mediate efficient degradation of mutant huntingtin exon 1 protein fragments", "type"=>"journal", "authors"=>[{"first_name"=>"David C.", "last_name"=>"Butler", "scopus_author_id"=>"10045281000"}, {"first_name"=>"Anne", "last_name"=>"Messer", "scopus_author_id"=>"7006121897"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"22216210", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "doi"=>"10.1371/journal.pone.0029199", "issn"=>"19326203", "scopus"=>"2-s2.0-84055182535", "pui"=>"363132822", "sgr"=>"84055182535"}, "id"=>"6ad0a57e-69b2-36c8-8c07-a3cd1b3ee8ff", "abstract"=>"Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG)(n) repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q) tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt), formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs), expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1) significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC) to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q) by ~80-90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation.", "link"=>"http://www.mendeley.com/research/bifunctional-antihuntingtin-proteasomedirected-intrabodies-mediate-efficient-degradation-mutant-hunt", "reader_count"=>30, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Researcher"=>7, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>9, "Student > Postgraduate"=>1, "Student > Master"=>4, "Other"=>2, "Student > Bachelor"=>5}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Researcher"=>7, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>9, "Student > Postgraduate"=>1, "Student > Master"=>4, "Other"=>2, "Student > Bachelor"=>5}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Biochemistry, Genetics and Molecular Biology"=>8, "Medicine and Dentistry"=>6, "Agricultural and Biological Sciences"=>10, "Neuroscience"=>3, "Physics and Astronomy"=>1, "Psychology"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>6}, "Neuroscience"=>{"Neuroscience"=>3}, "Physics and Astronomy"=>{"Physics and Astronomy"=>1}, "Psychology"=>{"Psychology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>10}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>8}}, "reader_count_by_country"=>{"United States"=>1}, "group_count"=>2}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/699280"], "description"=>"<p>Viability and GFP geometric mean fluorescent intensity (MFI) were assayed by flow cytometric analysis 48 h after transfection. For scFv-C4-PEST vs.scFv-C4: <b>A</b>. The percentage of propidium iodide (PI) positive nonviable cells is reduced. <b>B</b>. Total httex1-25Q-GFP and httex1-72Q-GFP reduction was confirmed. (means ± SEM; *** p<0.001 within groups.)</p>", "links"=>[], "tags"=>["further", "enhances", "viability", "st14a", "co-transfection", "experiments", "compared"], "article_id"=>369666, "categories"=>["Biotechnology", "Neuroscience", "Genetics", "Immunology"], "users"=>["David C. Butler", "Anne Messer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029199.g002", "stats"=>{"downloads"=>4, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_scFv_C4_PEST_further_enhances_viability_in_ST14A_co_transfection_experiments_compared_to_scFv_C4_/369666", "title"=>"scFv-C4-PEST further enhances viability in ST14A co-transfection experiments compared to scFv-C4.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 10:30:16"}
  • {"files"=>["https://ndownloader.figshare.com/files/699697"], "description"=>"<p>ST14A cells co-transfected with 1∶2 or 1∶5 transfection ratio of intrabody-PEST to httex1-72Q-GFP plasmids. <b>A</b>. Live cell imaging. Httex1-72Q is diffuse at 1∶2, and being begins to form aggregates at a 1∶5 transfection ratio of intrabody to httex1-72Q-GFP in scFv-C4 transfected cells. Fusion scFv-C4-PEST clears mhtt at 1∶2, and keeps mhtt diffuse at 1∶5. <b>B, C</b>. Western blots of EM48 immunoreactivity measuring the soluble (<b>B</b>; SDS-PAGE) and insoluble (<b>C</b>; AGERA) material to confirm live cell imaging.</p>", "links"=>[], "tags"=>["degradation", "htt", "occurs", "dose-dependent"], "article_id"=>370083, "categories"=>["Biotechnology", "Neuroscience", "Genetics", "Immunology"], "users"=>["David C. Butler", "Anne Messer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029199.g006", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Targeted_degradation_of_htt_occurs_in_a_dose_dependent_manner_/370083", "title"=>"Targeted degradation of htt occurs in a dose-dependent manner.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 10:32:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/699595"], "description"=>"<p>ST14A cells co-transfected with httex1-72Q-GFP and either empty vector, scFv-6E, or scFv-6E-PEST plasmids. Cells imaged and processed at 48 h. <b>A</b>. Live cell imaging. scFv-6E-PEST does not appear to alter the aggregation of htt compared to empty vector or scFv-6E. <b>B</b>. Western blotting. Monomeric htt is similar between groups. HA-tagged intrabody levels are similar between groups. <b>C</b>. Detergent-insoluble material resolved by AGERA is similar between empty vector and 6E-PEST groups.</p>", "links"=>[], "tags"=>["pest", "motif", "anti", "htt", "fibril-specific", "scfv-6e", "intrabody", "degrade", "fragments", "st14a"], "article_id"=>369966, "categories"=>["Biotechnology", "Neuroscience", "Genetics", "Immunology"], "users"=>["David C. Butler", "Anne Messer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029199.g005", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Fusion_of_PEST_motif_to_anti_htt_fibril_specific_scFv_6E_intrabody_does_not_degrade_htt_fragments_in_ST14A_cells_/369966", "title"=>"Fusion of PEST motif to anti htt fibril-specific scFv-6E intrabody does not degrade htt fragments in ST14A cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 10:31:45"}
  • {"files"=>["https://ndownloader.figshare.com/files/699813"], "description"=>"<p>Physico-chemical determinants of soluble intrabody expression. Solubility is improved by an overall negative charge at pH 7.4 and reduced GRAVY score. The GRAVY score is the average hydropathy score for all the amino acids in the protein, with a more negative score indicating increased hydrophilicity. The PEST-FIND program <a href=\"http://emboss.bioinformatics.nl/cgi-bin/emboss/pestfind\" target=\"_blank\">http://emboss.bioinformatics.nl/cgi-bin/emboss/pestfind</a> objectively produces a score ranging from about −50 to +50. A score above +5 denotes a PEST region <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029199#pone.0029199-Rechsteiner1\" target=\"_blank\">[33]</a>.</p>", "links"=>[], "tags"=>["properties", "scfv-c4"], "article_id"=>370201, "categories"=>["Biotechnology", "Neuroscience", "Genetics", "Immunology"], "users"=>["David C. Butler", "Anne Messer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029199.t001", "stats"=>{"downloads"=>2, "page_views"=>27, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Physico_chemical_properties_of_scFv_C4_intrabodies_/370201", "title"=>"Physico-chemical properties of scFv-C4 intrabodies.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-20 10:32:58"}
  • {"files"=>["https://ndownloader.figshare.com/files/699484"], "description"=>"<p>Using dual transfections as above, scFv-HA levels were compared among scFv-C4, scFv-C4-PEST, and scFv-C4-PEST-SCR, in the presence and absence of httex1-72Q-GFP. Levels of scFv-C4-PEST were reduced by ∼25% when compared with scFv-C4 or the scrambled (inactive) PEST. The presence or absence of antigen had no significant effect on intrabody-PEST levels. (n = 4; mean ± SEM; *p<0.05. for C4 vs. C4-PEST/C4-PEST-SCR.)</p>", "links"=>[], "tags"=>["fusion", "steady-state", "scfv-c4", "intrabody", "levels"], "article_id"=>369865, "categories"=>["Biotechnology", "Neuroscience", "Genetics", "Immunology"], "users"=>["David C. Butler", "Anne Messer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029199.g004", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PEST_fusion_reduces_steady_state_scFv_C4_intrabody_levels_modestly_and_independent_of_antigen_/369865", "title"=>"PEST fusion reduces steady-state scFv-C4 intrabody levels modestly, and independent of antigen.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 10:31:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/699163"], "description"=>"<p>ST14A cells were co-transfected with GFP-tagged httex1-25Q (httex1-25Q-GFP) or (httex1-72Q-GFP), plus either empty vector, scFv-C4, or scFv-C4-PEST plasmids. <b>A</b>. Representative live cell imaging depicting reduction of httex1-72Q-GFP and httex1-25Q-GFP fluorescence in the scFv-C4-PEST co-transfected groups. Phase contrast confirms uniform cell integrity. 48 h; bar = 20 µm. <b>B</b>. SDS-PAGE Western blot and densitometry probed for htt (EM48), quantified vs. actin. Monomeric soluble mhttex1 protein fragments were quantitatively reduced in scFv-C4-PEST vs. scFv-C4 co-transfected cells. (Mean ± SEM; *p<0.05 comparing httex1-25Q-GFP co-transfections; *p<0.05 comparing httex1-72Q-GFP co-transfections) Note that <b>B</b> shows only soluble httex1-72Q fragment levels, which are low unless intrabody is present. <b>C</b>. Agarose Gel Electrophoresis for Resolving Aggregates (AGERA) shows the decrease of detergent-insoluble material in httex1-72Q-GFP to scFv-C4-PEST co-transfected cells compared to other groups.</p>", "links"=>[], "tags"=>["enhances", "degradation", "gfp-tagged", "httex1", "soluble", "insoluble"], "article_id"=>369550, "categories"=>["Biotechnology", "Neuroscience", "Genetics", "Immunology"], "users"=>["David C. Butler", "Anne Messer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029199.g001", "stats"=>{"downloads"=>1, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_scFv_C4_PEST_enhances_degradation_of_GFP_tagged_httex1_soluble_and_insoluble_protein_fragments_/369550", "title"=>"scFv-C4-PEST enhances degradation of GFP-tagged httex1 soluble and insoluble protein fragments.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 10:29:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/699372"], "description"=>"<p><b>A</b>. Representative live cell images of dual transfections. Diffuse httex1-72Q-GFP fluorescence is similar between scFv-C4-PEST-SCR and scFv-C4 cells, and much higher than scFv-C4-PEST. <b>B</b>. Biochemical fractionation of HA-tagged fluorobodies from ST14A cells. Detergent-soluble and -insoluble cell lysates were prepared as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029199#s4\" target=\"_blank\">Materials and Methods</a>. Empty vector and scFv-C4-PEST transfected cells express minimal levels of monomeric htt, while scFv-C4-PEST-SCR and scFv-C4 transfected cells have similar levels of monmeric htt <b>C</b>. AGERA. There is an increase of detergent-insoluble material in scFv-C4-PEST-SCR transfected cells compared to scFv-C4-PEST. <b>D</b>. Western blotting of total protein. Monomeric mhtt levels do not differ between scFv-C4 and scFv-C4-PEST-SCR co-transfected cells. C4-PEST reduces soluble mhtt by ∼90%, and empty vector control mhtt is insoluble <b>E</b>. Proteasome inhibition reduced clearance of insoluble httex1-72Q by scFv-C4-PEST. ST14A cells were co-transfected with httex1-72Q-GFP and scFv-C4-PEST. 36 h after transfection, cells were treated with 10 µM epoxomicin (+), a potent and selective proteasome inhibitor <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029199#pone.0029199-Meng1\" target=\"_blank\">[34]</a>; or DMSO vehicle control. Insoluble mhtt was assessed at 48 h using AGERA. Proteasome inhibition resulted in reduced clearance of htt exon1 protein fragments by scFv-C4-PEST.</p>", "links"=>[], "tags"=>["pest", "inhibition", "proteasome", "eliminates", "enhanced", "degradation"], "article_id"=>369759, "categories"=>["Biotechnology", "Neuroscience", "Genetics", "Immunology"], "users"=>["David C. Butler", "Anne Messer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0029199.g003", "stats"=>{"downloads"=>2, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Scrambling_the_PEST_sequence_and_specific_inhibition_of_the_proteasome_eliminates_the_enhanced_degradation_of_httex1_72Q_GFP_/369759", "title"=>"Scrambling the PEST sequence and specific inhibition of the proteasome eliminates the enhanced degradation of httex1-72Q-GFP.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 10:30:42"}

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Relative Metric

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