Biochemical and Proteomic Analysis of Ubiquitination of Hsc70 and Hsp70 by the E3 Ligase CHIP
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{"title"=>"Biochemical and proteomic analysis of ubiquitination of Hsc70 and Hsp70 by the E3 ligase CHIP", "type"=>"journal", "authors"=>[{"first_name"=>"Sarah E.", "last_name"=>"Soss", "scopus_author_id"=>"16317181300"}, {"first_name"=>"Kristie L.", "last_name"=>"Rose", "scopus_author_id"=>"23029040100"}, {"first_name"=>"Salisha", "last_name"=>"Hill", "scopus_author_id"=>"7402766249"}, {"first_name"=>"Sophie", "last_name"=>"Jouan", "scopus_author_id"=>"56663126600"}, {"first_name"=>"Walter J.", "last_name"=>"Chazin", "scopus_author_id"=>"7006727022"}], "year"=>2015, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "pmid"=>"26010904", "scopus"=>"2-s2.0-84930225523", "doi"=>"10.1371/journal.pone.0128240", "sgr"=>"84930225523", "pui"=>"604521487"}, "id"=>"339408df-704a-35a8-972b-2ae732fa0f52", "abstract"=>"The E3 ubiquitin ligase CHIP is involved in protein triage, serving as a co-chaperone for refolding as well as catalyzing ubiquitination of substrates. CHIP functions with both the stress induced Hsp70 and constitutive Hsc70 chaperones, and also plays a role in maintaining their balance in the cell. When the chaperones carry no client proteins, CHIP catalyzes their polyubiquitination and subsequent proteasomal degradation. Although Hsp70 and Hsc70 are highly homologous in sequence and similar in structure, CHIP mediated ubiquitination promotes degradation of Hsp70 with a higher efficiency than for Hsc70. Here we report a detailed and systematic investigation to characterize if there are significant differences in the CHIP in vitro ubiquitination of human Hsp70 and Hsc70. Proteomic analysis by mass spectrometry revealed that only 12 of 39 detectable lysine residues were ubiquitinated by UbcH5a in Hsp70 and only 16 of 45 in Hsc70. The only conserved lysine identified as ubiquitinated in one but not the other heat shock protein was K159 in Hsc70. Ubiquitination assays with K-R ubiquitin mutants showed that multiple Ub chain types are formed and that the distribution is different for Hsp70 versus Hsc70. CHIP ubiquitination with the E2 enzyme Ube2W is predominantly directed to the N-terminal amine of the substrate; however, some internal lysine modifications were also detected. Together, our results provide a detailed view of the differences in CHIP ubiquitination of these two very similar proteins, and show a clear example where substantial differences in ubiquitination can be generated by a single E3 ligase in response to not only different E2 enzymes but subtle differences in the substrate.", "link"=>"http://www.mendeley.com/research/biochemical-proteomic-analysis-ubiquitination-hsc70-hsp70-e3-ligase-chip", "reader_count"=>14, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Researcher"=>3, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>7, "Student > Master"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Researcher"=>3, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>7, "Student > Master"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>4, "Agricultural and Biological Sciences"=>6, "Medicine and Dentistry"=>1, "Chemistry"=>2}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Chemistry"=>{"Chemistry"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>6}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>4}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"Netherlands"=>1, "India"=>1}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2081774"], "description"=>"<p>A) In vitro ubiquitination reactions with CHIP and UbcH5a were incubated up to 90 minutes as labeled and the products were separated by SDS-PAGE. The singly and doubly-ubiquitinated bands were excised for in-gel digestion and LC-MS/MS analysis. B) Example MS/MS spectrum of Hsc70 LSK(GG)EDIER peptide identifying that Ub was attached to K512. The K* indicates the lysine residue is modified by the addition of a di-glycine peptide. Observed b- and y-type product ions, resulting from amide bond cleavage following collision-induced dissociation, are annotated above the corresponding product ion peaks in the spectrum. C) Example MS/MS spectrum of Hsp70 peptide LSK(GG)EEIER for the equivalent site.</p>", "links"=>[], "tags"=>["Hsc 70. CHIP ubiquitination", "HSP 70", "Hsc 70", "substrate", "Ub chain types", "Hsc 70. Ubiquitination assays", "heat shock protein", "constitutive Hsc 70 chaperones", "E 2 enzyme Ube 2W", "E 3 Ligase CHIP", "Hsc 70.", "Hsc 70. Proteomic analysis", "E 3 ligase", "E 2 enzymes", "lysine", "E 3 ubiquitin ligase CHIP"], "article_id"=>1425651, "categories"=>["Biological Sciences"], "users"=>["Sarah E. Soss", "Kristie L. Rose", "Salisha Hill", "Sophie Jouan", "Walter J. Chazin"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0128240.g001", "stats"=>{"downloads"=>0, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Identification_of_ubiquitinated_lysines_for_Hsc70_Ub_and_Hsp70_Ub_/1425651", "title"=>"Identification of ubiquitinated lysines for Hsc70-Ub and Hsp70-Ub.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-05-26 14:53:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/2081776"], "description"=>"<p>Concurrent ubiquitination and degradation reactions were incubated for 2 hours at 37°C before separating the products on the gel. Key bands are highlighted and quantification of both the Coomassie stained and immunoblot analyses confirm that only Hsp70 ubiquitinated by UbcH5a is significantly degraded under these conditions.</p>", "links"=>[], "tags"=>["Hsc 70. CHIP ubiquitination", "HSP 70", "Hsc 70", "substrate", "Ub chain types", "Hsc 70. Ubiquitination assays", "heat shock protein", "constitutive Hsc 70 chaperones", "E 2 enzyme Ube 2W", "E 3 Ligase CHIP", "Hsc 70.", "Hsc 70. Proteomic analysis", "E 3 ligase", "E 2 enzymes", "lysine", "E 3 ubiquitin ligase CHIP"], "article_id"=>1425653, "categories"=>["Biological Sciences"], "users"=>["Sarah E. Soss", "Kristie L. Rose", "Salisha Hill", "Sophie Jouan", "Walter J. Chazin"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0128240.g002", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Hsp70_is_more_rapidly_degraded_than_Hsc70_by_26S_in_vitro_/1425653", "title"=>"Hsp70 is more rapidly degraded than Hsc70 by 26S in vitro.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-05-26 14:53:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/2081777"], "description"=>"<p>Homology models based on the structure of DnaK (PDB ID 2KHO) were created using SWISS-MODEL, and are here used to map the sequence coverage and ubiquitination sites found by mass spectrometry proteomics. Observed regions are colored in red, with unobserved regions in grey. All lysine side-chains are explicitly shown with confirmed ubiquitination sites highlighted in yellow.</p>", "links"=>[], "tags"=>["Hsc 70. CHIP ubiquitination", "HSP 70", "Hsc 70", "substrate", "Ub chain types", "Hsc 70. Ubiquitination assays", "heat shock protein", "constitutive Hsc 70 chaperones", "E 2 enzyme Ube 2W", "E 3 Ligase CHIP", "Hsc 70.", "Hsc 70. Proteomic analysis", "E 3 ligase", "E 2 enzymes", "lysine", "E 3 ubiquitin ligase CHIP"], "article_id"=>1425654, "categories"=>["Biological Sciences"], "users"=>["Sarah E. Soss", "Kristie L. Rose", "Salisha Hill", "Sophie Jouan", "Walter J. Chazin"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0128240.g003", "stats"=>{"downloads"=>3, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CHIP_ubiquitination_of_Hsc70_A_and_Hsp70_B_is_wide_spread_but_largely_focused_on_the_C_terminal_half_of_the_protein_/1425654", "title"=>"CHIP ubiquitination of Hsc70 (A) and Hsp70 (B) is wide-spread but largely focused on the C-terminal half of the protein.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-05-26 14:53:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/2081779"], "description"=>"<p>All Ub species were incubated for 30 minutes with UbcH5a or Ube2W+ UbcH13/Uev1a as the E2 as labeled. Both the Coomassie stain, left, and immunoblot, right, are shown for each set of reactions. A) CHIP autoubiquitination, B) Hsc70 ubiquitination by CHIP, and C) Hsp70 ubiquitination by CHIP. The * indicates a weak signal in the western blot that is more clearly seen by Coomassie stain. Note that K11R Ub retains a fusion tag that results in a larger protein and thus larger ladder spacing.</p>", "links"=>[], "tags"=>["Hsc 70. CHIP ubiquitination", "HSP 70", "Hsc 70", "substrate", "Ub chain types", "Hsc 70. Ubiquitination assays", "heat shock protein", "constitutive Hsc 70 chaperones", "E 2 enzyme Ube 2W", "E 3 Ligase CHIP", "Hsc 70.", "Hsc 70. Proteomic analysis", "E 3 ligase", "E 2 enzymes", "lysine", "E 3 ubiquitin ligase CHIP"], "article_id"=>1425656, "categories"=>["Biological Sciences"], "users"=>["Sarah E. Soss", "Kristie L. Rose", "Salisha Hill", "Sophie Jouan", "Walter J. Chazin"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0128240.g004", "stats"=>{"downloads"=>3, "page_views"=>132, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Ubiquitin_mutants_alter_the_rate_of_reaction_in_vitro_/1425656", "title"=>"Ubiquitin mutants alter the rate of reaction in vitro.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-05-26 14:53:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/2081782"], "description"=>"<p>In vitro ubiquitination reactions with the fusion protein and various Ub mutants were incubated as above for 30 min (A). To isolate N-terminal ubiquitination, 0.2 μM H3C protease (to cleave His-GST from Hsp70) and 50 mM L-Cys (to quench E1 activity) were then added to a portion of the reaction and allowed to incubate for 30 min at room temperature prior to separation by SDS-PAGE (B). The cleaved reaction products in B were also investigated by western blots for C) Hsp70 and D) GST. The majority of the ubiquitination products in all reactions containing Ube2W (lanes 4–15) follow His-GST and indicate ubiquitination of the N-terminus.</p>", "links"=>[], "tags"=>["Hsc 70. CHIP ubiquitination", "HSP 70", "Hsc 70", "substrate", "Ub chain types", "Hsc 70. Ubiquitination assays", "heat shock protein", "constitutive Hsc 70 chaperones", "E 2 enzyme Ube 2W", "E 3 Ligase CHIP", "Hsc 70.", "Hsc 70. Proteomic analysis", "E 3 ligase", "E 2 enzymes", "lysine", "E 3 ubiquitin ligase CHIP"], "article_id"=>1425659, "categories"=>["Biological Sciences"], "users"=>["Sarah E. Soss", "Kristie L. Rose", "Salisha Hill", "Sophie Jouan", "Walter J. Chazin"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0128240.g005", "stats"=>{"downloads"=>2, "page_views"=>39, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Ubiquitin_is_predominantly_attached_to_the_N_terminus_of_His_GST_Hsp70_by_Ube2W_/1425659", "title"=>"Ubiquitin is predominantly attached to the N-terminus of His-GST-Hsp70 by Ube2W.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-05-26 14:53:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/2081783"], "description"=>"<p>Summary of results from mass spectrometry proteomics analysis of +1 or +2 Ub samples from <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128240#pone.0128240.g001\" target=\"_blank\">Fig 1A</a>.</p>", "links"=>[], "tags"=>["Hsc 70. CHIP ubiquitination", "HSP 70", "Hsc 70", "substrate", "Ub chain types", "Hsc 70. Ubiquitination assays", "heat shock protein", "constitutive Hsc 70 chaperones", "E 2 enzyme Ube 2W", "E 3 Ligase CHIP", "Hsc 70.", "Hsc 70. Proteomic analysis", "E 3 ligase", "E 2 enzymes", "lysine", "E 3 ubiquitin ligase CHIP"], "article_id"=>1425660, "categories"=>["Biological Sciences"], "users"=>["Sarah E. Soss", "Kristie L. Rose", "Salisha Hill", "Sophie Jouan", "Walter J. Chazin"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0128240.t001", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Summary_of_results_from_mass_spectrometry_proteomics_analysis_of_1_or_2_Ub_samples_from_Fig_1A_/1425660", "title"=>"Summary of results from mass spectrometry proteomics analysis of +1 or +2 Ub samples from Fig 1A.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2015-05-26 14:53:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/2081784", "https://ndownloader.figshare.com/files/2081785", "https://ndownloader.figshare.com/files/2081787", "https://ndownloader.figshare.com/files/2081788", "https://ndownloader.figshare.com/files/2081789", "https://ndownloader.figshare.com/files/2081790"], "description"=>"<div><p>The E3 ubiquitin ligase CHIP is involved in protein triage, serving as a co-chaperone for refolding as well as catalyzing ubiquitination of substrates. CHIP functions with both the stress induced Hsp70 and constitutive Hsc70 chaperones, and also plays a role in maintaining their balance in the cell. When the chaperones carry no client proteins, CHIP catalyzes their polyubiquitination and subsequent proteasomal degradation. Although Hsp70 and Hsc70 are highly homologous in sequence and similar in structure, CHIP mediated ubiquitination promotes degradation of Hsp70 with a higher efficiency than for Hsc70. Here we report a detailed and systematic investigation to characterize if there are significant differences in the CHIP in vitro ubiquitination of human Hsp70 and Hsc70. Proteomic analysis by mass spectrometry revealed that only 12 of 39 detectable lysine residues were ubiquitinated by UbcH5a in Hsp70 and only 16 of 45 in Hsc70. The only conserved lysine identified as ubiquitinated in one but not the other heat shock protein was K159 in Hsc70. Ubiquitination assays with K-R ubiquitin mutants showed that multiple Ub chain types are formed and that the distribution is different for Hsp70 versus Hsc70. CHIP ubiquitination with the E2 enzyme Ube2W is predominantly directed to the N-terminal amine of the substrate; however, some internal lysine modifications were also detected. Together, our results provide a detailed view of the differences in CHIP ubiquitination of these two very similar proteins, and show a clear example where substantial differences in ubiquitination can be generated by a single E3 ligase in response to not only different E2 enzymes but subtle differences in the substrate.</p></div>", "links"=>[], "tags"=>["Hsc 70. CHIP ubiquitination", "HSP 70", "Hsc 70", "substrate", "Ub chain types", "Hsc 70. Ubiquitination assays", "heat shock protein", "constitutive Hsc 70 chaperones", "E 2 enzyme Ube 2W", "E 3 Ligase CHIP", "Hsc 70.", "Hsc 70. Proteomic analysis", "E 3 ligase", "E 2 enzymes", "lysine", "E 3 ubiquitin ligase CHIP"], "article_id"=>1425661, "categories"=>["Biological Sciences"], "users"=>["Sarah E. Soss", "Kristie L. Rose", "Salisha Hill", "Sophie Jouan", "Walter J. Chazin"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0128240.s001", "https://dx.doi.org/10.1371/journal.pone.0128240.s002", "https://dx.doi.org/10.1371/journal.pone.0128240.s003", "https://dx.doi.org/10.1371/journal.pone.0128240.s004", "https://dx.doi.org/10.1371/journal.pone.0128240.s005", "https://dx.doi.org/10.1371/journal.pone.0128240.s006"], "stats"=>{"downloads"=>23, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Biochemical_and_Proteomic_Analysis_of_Ubiquitination_of_Hsc70_and_Hsp70_by_the_E3_Ligase_CHIP_/1425661", "title"=>"Biochemical and Proteomic Analysis of Ubiquitination of Hsc70 and Hsp70 by the E3 Ligase CHIP", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-05-26 14:53:23"}

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{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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