Dataset Information

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.

SUBMITTER: Soss SE

PROVIDER: S-EPMC4444009 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Publications

Biochemical and Proteomic Analysis of Ubiquitination of Hsc70 and Hsp70 by the E3 Ligase CHIP.

Soss Sarah E SE Rose Kristie L KL Hill Salisha S Jouan Sophie S Chazin Walter J WJ

PloS one 20150526 5

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 ...[more]

PMID: 26010904

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Project description:CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum-anchored cytochrome P450 enzyme engaged in the biotransformation of endo- and xenobiotics, including >50% of clinically relevant drugs. Alterations of CYP3A4 protein turnover can influence clinically relevant drug metabolism and bioavailability and drug-drug interactions. This CYP3A4 turnover involves endoplasmic reticulum-associated degradation via the ubiquitin (Ub)-dependent 26 S proteasomal system that relies on two highly complementary E2 Ub-conjugating-E3 Ub-ligase (UBC7-gp78 and UbcH5a-C terminus of Hsc70-interacting protein (CHIP)-Hsc70-Hsp40) complexes, as well as protein kinases (PK) A and C. We have documented that CYP3A4 Ser/Thr phosphorylation (Ser(P)/Thr(P)) by PKA and/or PKC accelerates/enhances its Lys ubiquitination by either of these E2-E3 systems. Intriguingly, CYP3A4 Ser(P)/Thr(P) and ubiquitinated Lys residues reside within the cytosol-accessible surface loop and/or conformationally assembled acidic Asp/Glu clusters, leading us to propose that such post-translational Ser/Thr protein phosphorylation primes CYP3A4 for ubiquitination. Herein, this possibility was examined through various complementary approaches, including site-directed mutagenesis, chemical cross-linking, peptide mapping, and LC-MS/MS analyses. Our findings reveal that such CYP3A4 Asp/Glu/Ser(P)/Thr(P) surface clusters are indeed important for its intermolecular electrostatic interactions with each of these E2-E3 subcomponents. By imparting additional negative charge to these Asp/Glu clusters, such Ser/Thr phosphorylation would generate P450 phosphodegrons for molecular recognition by the E2-E3 complexes, thereby controlling the timing of CYP3A4 ubiquitination and endoplasmic reticulum-associated degradation. Although the importance of phosphodegrons in the CHIP targeting of its substrates is known, to our knowledge this is the first example of phosphodegron involvement in gp78-substrate recruitment, an important step in CYP3A4 proteasomal degradation.

Dataset Information

Biochemical and Proteomic Analysis of Ubiquitination of Hsc70 and Hsp70 by the E3 Ligase CHIP.

Publications

Biochemical and Proteomic Analysis of Ubiquitination of Hsc70 and Hsp70 by the E3 Ligase CHIP.

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