Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.13.12.7 (firefly luciferase)
 2,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have reconstituted an ATP-dependent protein folding machinery using purified yeast cytosolic proteins. The S. cerevisiae Hsp70 Ssa1p and the DnaJ homolog Ydj1p refolded denatured firefly luciferase. In E. coli, efficient refolding of luciferase requires the Hsp70 DnaK and two modulators, DnaJ and GrpE, that synergistically stimulate its ATPase activity. Exchanging DnaJ homologs between the S. cerevisiae and E. coli systems revealed that their ability to stimulate Hsp70 ATPase activity was conserved. In contrast, GrpE further stimulated only DnaK's ATPase activity. Efficient refolding of luciferase by Ssa1p and DnaJ, but not by DnaK and Ydj1p, suggests that a compatible Hsp70/DnaJ homolog pair can act as a protein folding machinery.
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PMID:Conserved ATPase and luciferase refolding activities between bacteria and yeast Hsp70 chaperones and modulators. 763 93

The effects of the human DnaJ homolog, Hsp40, on the ATPase and chaperone functions of the constitutively expressed Hsp70 homolog, Hsc70, were analyzed. Hsp40 stimulates the hydrolysis of ATP by Hsc70, causing a approximately 7-fold increase in its steady-state ATPase activity. In contrast to the prokaryotic Hsp70 system, ATP-hydrolysis and not the release of bound ADP is the rate-limiting step in the overall ATPase cycle of mammalian Hsc70. The ability to activate the Hsc70 ATPase is partially preserved in a deletion mutant containing the J-domain and the G/F region of Hsp40 but not in a deletion mutant that contains the J-domain alone. As a result of its ATPase stimulating activity, addition of Hsp40 allows Hsc70 to bind peptide in the presence of ATP, whereas in the absence of Hsp40, peptide is efficiently released upon ATP binding to Hsc70. The functional cooperation of Hsp40 with Hsc70 is essential to ensure the ATP hydrolysis-dependent binding of aggregation-sensitive denatured polypeptides, such as thermally denatured firefly luciferase and chemically denatured rhodanese. Binding of these proteins results in the formation of ternary complexes of Hsc70, Hsp40, and substrates. Hsc70 and Hsp40 cooperate with further factors in protein renaturation, as demonstrated by the finding that luciferase, thermally denatured in the presence of Hsc70, Hsp40, and ATP, refolds upon addition of rabbit reticulocyte cytosol. Our results indicate that Hsp40 has a critical regulatory function in the Hsc70 ATPase cycle that is required for the efficient loading of peptide substrate onto Hsc70.
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PMID:Regulation of the heat-shock protein 70 reaction cycle by the mammalian DnaJ homolog, Hsp40. 870 58

Mdj1p, a DnaJ homolog in the mitochondria of Saccharomyces cerevisiae, is involved in the folding of proteins in the mitochondrial matrix. In this capacity, Mdj1p cooperates with mitochondrial Hsp70 (mt-Hsp70). Here, we analyzed the role of Mdj1p as a chaperone for newly synthesized proteins encoded by mitochondrial DNA and for nucleus-encoded proteins as they enter the mitochondrial matrix. A series of conditional mutants of mdj1 was constructed. Mutations in the various functional domains led to a partial loss of Mdj1p function. The mutant Mdj1 proteins were defective in protecting the tester protein firefly luciferase against heat-induced aggregation in isolated mitochondria. The mitochondrially encoded var1 protein showed enhanced aggregation after synthesis in mdj1 mutant mitochondria. Mdj1p and mt-Hsp70 were found in a complex with nascent polypeptide chains on mitochondrial ribosomes. Mdj1p was not found to interact with translocation intermediates of imported proteins spanning the two membranes and exposing short segments into the matrix, in accordance with the lack of requirement of Mdj1p in the mt-Hsp70-mediated protein import into mitochondria. On the other hand, precursor proteins in transit which had further entered the matrix were found in a complex with Mdj1p. Our results suggest that Mdj1p together with mt-Hsp70 plays an important role as a chaperone for mitochondrially synthesized polypeptide chains emerging from the ribosome and for translocating proteins at a late import step.
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PMID:Role of the mitochondrial DnaJ homolog Mdj1p as a chaperone for mitochondrially synthesized and imported proteins. 894 61

Hsp70 molecular chaperones facilitate protein folding and translocation by binding to hydrophobic regions of nascent or unfolded proteins, thereby preventing their aggregation. N-Ethylmaleimide (NEM) inhibits the ATPase and protein translocation-stimulating activities of the yeast Hsp70 Ssa1p by modifying its three cysteine residues, which are located in its ATPase domain. NEM alters the conformation of Ssa1p and disrupts the coupling between its nucleotide- and polypeptide-binding domains. Ssa1p and the yeast DnaJ homolog Ydj1p constitute a protein folding machinery of the yeast cytosol. Using firefly luciferase as a model protein to study chaperone-dependent protein refolding, we have found that NEM also inhibits the protein folding activity of Ssa1p. Interestingly, the NEM-modified protein (NEM-Ssa1p) is a potent inhibitor of protein folding. NEM-Ssa1p can prevent the aggregation of luciferase and stimulate the ATPase activity of Ssa1p suggesting that it acts as an inhibitor by binding to nonnative forms of luciferase and by competing with them for the polypeptide binding site of Ssa1p. NEM-Ssa1p inhibits Ssa1p/Ydj1p-dependent protein refolding at different stages indicating that the chaperones bind and release nonnative forms of luciferase multiple times before folding is completed.
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PMID:N-Ethylmaleimide-modified Hsp70 inhibits protein folding. 1046 52