Abstract
Proteins with Bcl2-associated anthanogene (BAG) domains act as nucleotide exchange factors (NEFs) for the molecular chaperone heat shock protein 70 (Hsp70). There are six BAG family NEFs in humans, and each is thought to link Hsp70 to a distinct cellular pathway. However, little is known about how the NEFs compete for binding to Hsp70 or how they might differentially shape its biochemical activities. Toward these questions, we measured the binding of human Hsp72 (HSPA1A) to BAG1, BAG2, BAG3, and the unrelated NEF Hsp105. These studies revealed a clear hierarchy of affinities: BAG3 > BAG1 > Hsp105 ≫ BAG2. All of the NEFs competed for binding to Hsp70, and their relative affinity values predicted their potency in nucleotide and peptide release assays. Finally, we combined the Hsp70-NEF pairs with cochaperones of the J protein family (DnaJA1, DnaJA2, DnaJB1, and DnaJB4) to generate 16 permutations. The activity of the combinations in ATPase and luciferase refolding assays were dependent on the identity and stoichiometry of both the J protein and NEF so that some combinations were potent chaperones, whereas others were inactive. Given the number and diversity of cochaperones in mammals, it is likely that combinatorial assembly could generate a large number of distinct permutations.
Keywords:
ATPases; Cochaperones; Hsp40; Isothermal Titration Calorimetry; Molecular Chaperone; Protein Complexes; Protein Folding; Protein Misfolding; Protein-Protein Interactions.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Adaptor Proteins, Signal Transducing / chemistry*
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Adaptor Proteins, Signal Transducing / genetics
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Adaptor Proteins, Signal Transducing / metabolism
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Apoptosis Regulatory Proteins
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DNA-Binding Proteins / chemistry*
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism
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HSP110 Heat-Shock Proteins / chemistry*
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HSP110 Heat-Shock Proteins / genetics
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HSP110 Heat-Shock Proteins / metabolism
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HSP40 Heat-Shock Proteins / chemistry*
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HSP40 Heat-Shock Proteins / genetics
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HSP40 Heat-Shock Proteins / metabolism
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HSP70 Heat-Shock Proteins / chemistry*
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HSP70 Heat-Shock Proteins / genetics
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HSP70 Heat-Shock Proteins / metabolism
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Humans
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Molecular Chaperones / chemistry*
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Molecular Chaperones / genetics
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Molecular Chaperones / metabolism
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Multiprotein Complexes / chemistry*
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Multiprotein Complexes / genetics
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Multiprotein Complexes / metabolism
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Protein Binding / physiology
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Protein Folding
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Transcription Factors / chemistry*
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Transcription Factors / genetics
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Transcription Factors / metabolism
Substances
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Adaptor Proteins, Signal Transducing
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Apoptosis Regulatory Proteins
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BAG2 protein, human
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BAG3 protein, human
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BCL2-associated athanogene 1 protein
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DNA-Binding Proteins
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HSP110 Heat-Shock Proteins
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HSP40 Heat-Shock Proteins
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HSP70 Heat-Shock Proteins
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HSPA1A protein, human
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HSPH1 protein, human
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Molecular Chaperones
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Multiprotein Complexes
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Recombinant Proteins
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Transcription Factors