title: Biochemical and Proteomic Analysis of Ubiquitination of Hsc70 and Hsp70 by the E3 Ligase CHIP.
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.
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, United States of America; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, United States of America.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Soss
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, United States of America; Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee, United States of America.
authors.forename: Kristie L
authors.initials: KL
authors.lastname: Rose
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, United States of America; Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee, United States of America.
authors.forename: Salisha
authors.initials: S
authors.lastname: Hill
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, United States of America; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, United States of America.
authors.forename: Sophie
authors.initials: S
authors.lastname: Jouan
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, United States of America; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, United States of America; Department of Chemistry, Vanderbilt University, Nashville, Tennessee, United States of America.
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Chazin
mesh_terms: D050883:HSC70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D050883:HSC70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D011994:Recombinant Proteins; D025801:Ubiquitin; C097308:UBE2D1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C467728:STUB1 protein, human; D044767:Ubiquitin-Protein Ligases