Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Heat shock proteins (HSPs) of the Hsp70 and GroEL families associate with a variety of cell proteins in vivo. However, the formation of such complexes has not been systematically studied. A 31-kDa fusion protein (CRAG), which contains 12 residues of cro repressor, truncated protein A, and 14 residues of beta-galactosidase, when expressed in Escherichia coli, was found in complexes with DnaK, GrpE, protease La, and GroEL. When an E. coli extract not containing CRAG was applied to an affinity column containing CRAG, DnaK, GroEL, and GrpE were selectively bound. These HSPs did not bind to a normal protein A column. DnaK, GrpE, and the fraction of GroEL could be eluted from the CRAG column with ATP but not with a nonhydrolyzable ATP analog. The ATP-dependent release of DnaK and GroEL also required Mg2+, but GrpE dissociated with ATP alone. The binding and release of DnaK and GroEL were independent events, but the binding of GrpE required DnaK. Inactivation of DnaJ, GrpE, and GroES did not affect the association or dissociation of DnaK or GroEL from CRAG. The DnaK and GrpE proteins could be eluted with 10(-6) M ATP, but 10(-4) M was required for GroEL release. This approach allows a one-step purification of these proteins from E. coli and also the isolation of the DnaK and GroEL homologs from yeast mitochondria. Competition experiments with oligopeptide fragments of CRAG showed that DnaK and GroEL interact with different sites on CRAG and that the cro-derived domain of CRAG contains the DnaK-binding site.
...
PMID:Formation in vitro of complexes between an abnormal fusion protein and the heat shock proteins from Escherichia coli and yeast mitochondria. 193 19

Induction of heat shock proteins in Escherichia coli is caused by a transient increase in the cellular level of sigma 32 (the rpoH gene product), a protein required for transcription of heat shock genes. Both increased synthesis and stabilization of sigma 32 contribute to the increase in sigma 32. We previously showed that heat-induced translation of sigma 32-beta-galactosidase fusion protein encoded by an rpoH-lacZ gene fusion was mediated by an mRNA secondary structure formed between two 5'-proximal segments (A and B) of rpoH coding sequence spanning some 200 nt. We now report that a portion of the sigma 32 polypeptide that corresponds to further downstream (designated region C) is involved in the DnaK-mediated negative control resulting in the shutoff of heat-induced synthesis and degradation of fusion protein. Gene fusions carrying the 5' half (433 nt) or more of the rpoH coding sequence exhibited normal shutoff of synthesis, and the fusion proteins produced were very unstable, like authentic sigma 32; both the shutoff of synthesis and the instability of protein were markedly affected by the dnaK and dnaJ mutations. In contrast, gene fusions carrying < or = 364 nt (lacking region C) and a fusion carrying most of the rpoH sequence but with a frameshift mutation specifically affecting region C exhibited little or no shutoff and produced stable proteins. These results indicate that a distinct segment of sigma 32 plays a critical role in the negative feedback control of sigma 32. The control may be exerted during or after completion of sigma 32 synthesis mediated by interaction between nascent or mature sigma 32 and DnaK/DnaJ proteins.
...
PMID:A distinct segment of the sigma 32 polypeptide is involved in DnaK-mediated negative control of the heat shock response in Escherichia coli. 793 41

PreS2-S'-beta-galactosidase, a three-domain fusion protein that aggregates extensively in the cytoplasm of Escherichia coli, was used to systematically investigate the effects of heat-shock protein (hsp) overproduction on protein misfolding and inclusion body formation. While the co-overexpression of the DnaK and DnaJ molecular chaperones led to a 3-6 fold increase in the recovery of enzymatically active preS2-S'-beta-galactosidase over a wide range of growth temperatures (30-42 degrees C), an increase in the concentration of the GroEL and GroES chaperonins had a significant effect at 30 degrees C only. Co-immunoprecipitation experiments confirmed that preS2-S'-beta-galactosidase formed a stable complex with DnaK, but not with GroEL, at 42 degrees C. When the intracellular concentration of chromosomal heat-shock proteins was increased by overproduction of the heat-shock transcription factor sigma 32, or by addition of 3% ethanol (v/v) to the growth medium, a 2-3 fold higher recovery of active enzyme was observed at 30 and 42 degrees C, but not at 37 degrees C. The overexpression of all heat-shock proteins or specific chaperone operons did not significantly affect the synthesis rates or stability of preS2-S'-beta-galactosidase and did not lead to the disaggregation of preformed inclusion bodies. Rather, the improvements in the recovery of soluble and active fusion protein resulted primarily from facilitated folding and assembly. Our findings suggest that titration of the DnaK-DnaJ early folding factors leads to the formation of preS2-S'-beta-galactosidase inclusion bodies.
...
PMID:Protein misfolding and inclusion body formation in recombinant Escherichia coli cells overexpressing Heat-shock proteins. 862 59

In Escherichia coli and mitochondria, the molecular chaperone DnaJ is required not only for protein folding but also for selective degradation of certain abnormal polypeptides. Here we demonstrate that in the yeast cytosol, the homologous chaperone Ydj1 is also required for ubiquitin-dependent degradation of certain abnormal proteins. The temperature-sensitive ydj1-151 mutant showed a large defect in the overall breakdown of short-lived cell proteins and abnormal polypeptides containing amino acid analogs, especially at 38 degrees C. By contrast, the degradation of long-lived cell proteins, which is independent of ubiquitin, was not altered nor was cell growth affected. The inactivation of Ydj1 markedly reduced the rapid, ubiquitin-dependent breakdown of certain beta-galactosidase (beta-gal) fusion polypeptides. Although degradation of N-end rule substrates (arginine-beta-gal and leucine-beta-gal) and the B-type cyclin Clb5-beta-gal occurred normally, degradation of the abnormal polypeptide ubiquitin-proline-beta-gal (Ub-P-beta-gal) and that of the short-lived normal protein Gcn4 were inhibited. As a consequence of reduced degradation of Ub-P-beta-gal, the beta-gal activity was four to five times higher in temperature-sensitive ydj1-151 mutant cells than in wild-type cells; thus, the folding and assembly of this enzyme do not require Ydj1 function. In wild-type cells, but not in ydj1-151 mutant cells, this chaperone is associated with the short-lived substrate Ub-P-beta-gal and not with stable beta-gal constructs. Furthermore, in the ydj1-151 mutant, the ubiquitination of Ub-P-beta-gal was blocked and the total level of ubiquitinated protein in the cell was reduced. Thus, Ydj1 is essential for the ubiquitin-dependent degradation of certain proteins. This chaperone may facilitate the recognition of unfolded proteins or serve as a cofactor for certain ubiquitin-ligating enzymes.
...
PMID:Involvement of the molecular chaperone Ydj1 in the ubiquitin-dependent degradation of short-lived and abnormal proteins in Saccharomyces cerevisiae. 875 35

We have systematically investigated the influence of mutations in the sigma(32) heat-shock transcription factor and the DnaK-DnaJ-GrpE and GroEL-GroES molecular chaperone machines on the folding of preS2-beta-galactosidase. This 120 kDa fusion protein between the hepatitis B surface antigen preS2 sequence and beta-galactosidase was synthesized in a highly soluble and enzymatically active form in wild-type Escherichia coli cells cultured at temperatures between 30 degrees C and 42 degrees C, but aggregated extensively in an rpoH165 (Am) mutant. Proper folding was partially restored upon co-overexpression of the dnaKJ operon, but not when the groE operon or dnaK alone were overproduced. The enzymatic activities in dnaK103, dnaJ259 and grpE280 mutants were 40-60% lower relative to a dnaK756 mutant or isogenic wild-type cells at 30 degrees C and 37 degrees C. At 42 degrees C, only 10-40% of the wild-type activity was present in each of the early-folding-factor mutants. Although the synthesis levels of preS2-beta-galactosidase were reduced in the dnaK103, dnaJ259 and grpE280 genetic backgrounds, aggregation was primarily responsible for the loss of activity when the cells were grown at 37 degrees C or 42 degrees C. By contrast, the groEL140, groES30 and groES619 mutations, which induced the aggregation of homodimeric ribulose bisphosphate carboxylase (Rubisco), did not affect the solubility of preS2-beta-galactosidase at temperatures up to 42 degrees C. Our results are discussed in terms of the current understanding of the E. coli protein-folding cascade. The potential usefulness of heat-shock protein mutants for the production of soluble proteins in an inclusion-body form is addressed.
...
PMID:Protein folding in the cytoplasm of Escherichia coli: requirements for the DnaK-DnaJ-GrpE and GroEL-GroES molecular chaperone machines. 889 87

The dnaK gene of an ammonia-oxidizing bacterium, Nitrosomonas europaea, was cloned and sequenced. It was found that the dnaK gene product was highly homologous to previously analyzed dnaK gene products from other organisms at the amino acid level. Two partial open reading frames located upstream and downstream of the dnaK gene were also found and identified as grpE and dnaJ genes, respectively, by the predicted amino acid homology of their gene products to other bacterial GrpE and DnaJ proteins. Transcription of the dnaK gene was strongly induced by a heat shock from 30 to 37 degrees C. An analysis of the expression of the dnaK gene fused to the lacZ translational reporter gene also showed eightfold increase in beta-galactosidase activity after the heat shock induction. Heat-inducible transcription start sites of the dnaK gene, revealed by primer extension analysis, were located 16 and 17 nucleotides upstream from the translational start codon of the dnaK gene, and the predicted promoter sequence showed a homology to the consensus sequence of sigma 32-dependent heat shock promoters of gram-negative bacteria. The upstream region of the dnaK gene did not contain the inverted repeat structure that was involved in the regulation of the heat shock gene of several gram-negative and gram-positive bacteria. Therefore, we conclude that the heat shock regulatory mechanism of the N. europaea dnaK gene may be similar to the sigma 32-dependent mechanism observed in other gram-negative bacteria.
...
PMID:Cloning, nucleotide sequence, and regulatory analysis of the Nitrosomonas europaea dnaK gene. 914 12

The proper folding of aggregation-prone recombinant proteins in Escherichia coli can be facilitated by co-overexpressing specific molecular chaperones or by culturing the cells in the presence of ethanol or other agents that upregulate the synthesis of all heat-shock proteins (hsps). We have investigated the effect of combining direct chaperone overproduction with ethanol supplementation on the cytoplasmic folding of two aggregation-prone model proteins, preS2-S'-beta-galactosidase and human SPARC. In 25-ml shake flask cultures grown at 30 degrees C, addition of 3% (v/v) ethanol to the growth medium prior to inoculation improved the chaperone-mediated increase in the yields of active preS2-S'-beta-galactosidase 1.5- to 2-fold. When cultures overexpressing the dnaKJ operon were grown in the presence of ethanol, the levels of enzymatic activity were 5-fold higher relative to control cells and preS2-S'-beta-galactosidase aggregation was almost entirely abolished. Combining DnaK-DnaJ overexpression and growth of the cells at temperatures lower than 30 degrees C did not result in a comparable increase in activity. Although the individual effects of ethanol supplementation and dnaKJ overproduction were more limited when the culture volume was raised, a synergistic improvement in preS2-S'-beta-galactosidase activity was observed when the two approaches were used in concert. In contrast, ethanol supplementation promoted the aggregation of human SPARC, a protein exhibiting a chaperone dependency similar to that of preS2-S'-beta-galactosidase. Our results show that ethanol can exert complex and divergent effects on inclusion body formation and that the beneficial effect of the solvent on recombinant protein folding cannot simply be explained by an increase in the intracellular concentration of molecular chaperones.
...
PMID:Divergent effects of chaperone overexpression and ethanol supplementation on inclusion body formation in recombinant Escherichia coli. 942 34

Escherichia coli null dnaJ and dnaKdnaJ mutants were defective in the biosynthesis and secretion of several enzymes. The synthesis of beta-galactosidase induced in delta dnaJ and delta dnaKdnaJ mutants was abolished at 42 degrees C and significantly decreased at 30 and 37 degrees C. The activity of alkaline phosphatase in the periplasm in both mutant strains at high temperature was lower than in the wild-type strain. The synthesis of b-type cytochromes was defective in two deletion mutants while the synthesis of nitrate reductase-A at 42 degrees C was influenced by dnaK mutation only. The lack of DnaK and DnaJ does not impair the activity of catechol 2,3-dioxygenase irrespective of growth temperature.
...
PMID:Biosynthesis and secretion of several enzymes in Escherichia coli dnaK and dnaJ mutants. 1075 20

DnaK-DnaJ-GrpE and GroEL-GroES are the best-characterized molecular chaperone systems in the cytoplasm of Escherichia coli. A number of additional proteins, including ClpA, ClpB, HtpG and IbpA/B, act as molecular chaperones in vitro, but their function in cellular protein folding remains unclear. Here, we examine how these chaperones influence the folding of newly synthesized recombinant proteins under heat-shock conditions. We show that the absence of either CIpB or HtpG at 42 degrees C leads to increased aggregation of preS2-beta-galactosidase, a fusion protein whose folding depends on DnaK-DnaJ-GrpE, but not GroEL-GroES. However, only the deltaclpB mutation is deleterious to the folding of homodimeric Rubisco and cMBP, two proteins requiring the GroEL-GroES chaperonins to reach a proper conformation. Null mutations in clpA or the ibpAB operon do not affect the folding of these model substrates. Overexpression of ClpB, HtpG, IbpA/B or ClpA does not suppress inclusion body formation by the aggregation-prone protein preS2-S'-beta-galactosidase in wild-type cells or alleviate recombinant protein misfolding in dnaJ259, grpE280 or groES30 mutants. By contrast, higher levels of DnaK-DnaJ, but not GroEL-GroES, restore efficient folding in deltaclpB cells. These results indicate that ClpB, and to a lesser extent HtpG, participate in de novo protein folding in mildly stressed E. coli cells, presumably by expanding the ability of the DnaK-DnaJ-GrpE team to interact with newly synthesized polypeptides.
...
PMID:ClpB and HtpG facilitate de novo protein folding in stressed Escherichia coli cells. 1093 Dec 86

ClpB/Hsp104 collaborates with the Hsp70 system to promote the solubilization and reactivation of proteins that misfold and aggregate following heat shock. In Escherichia coli and other eubacteria, two ClpB isoforms (ClpB95 and ClpB80) that differ by the presence or absence of a highly mobile 149-residues long N-terminus domain are synthesized from the same transcript. Whether and how the N-domain contributes to ClpB chaperone activity remains controversial. Here, we show that, whereas fusion of a 20-residues long hexahistidine extension to the N-terminus of ClpB95 interferes with its in vivo and in vitro activity, the same tag has no detectable effect on ClpB80 function. In addition, ClpB95 is more effective than ClpB80 at restoring the folding of the model protein preS2-beta-galactosidase as stress severity increases, and is superior to ClpB80 in improving the high temperature growth and low temperature recovery of dnaK756 DeltaclpB cells. Our results are consistent with a model in which the N-domain of ClpB95 maximizes substrate processing under conditions where the cellular supply of free DnaK-DnaJ is limiting.
...
PMID:The N-terminal domain of Escherichia coli ClpB enhances chaperone function. 1605 Dec 21


1

---