Biomedical subjects

C Georgopoulos

Publications and source records attributed to C Georgopoulos.

At least 55 records · Page 3Linked to original sources

PubMed1995

The conserved G/F motif of the DnaJ chaperone is necessary for the activation of the substrate binding properties of the DnaK chaperone.

The universally conserved DnaK and DnaJ molecular chaperone proteins bind in a coordinate manner to protein substrates to prevent aggregation, to disaggregate proteins, or to regulate proper protein function. To further examine their synergistic mechanism of action, we constructed and characterized two DnaJ deletion proteins. One has an 11-amino-acid internal deletion that spans amino acid residues 77-87 (DnaJ delta 77-87) and the other amino acids 77-107 (DnaJ delta 77-107). The DnaJ delta 77-87 mutant protein, was normal in all respects analyzed. The DnaJ delta 77-107 mutant protein has its entire G/F (Gly/Phe) motif deleted. This motif is found in most, but not all DnaJ family members. In vivo, DnaJ delta 77-107 supported bacteriophage lambda growth, albeit at reduced levels, demonstrating that at least some protein function was retained. However, DnaJ delta 77-107 did not exhibit other wild type properties, such as proper down-regulation of the heat-shock response, and had an overall poisoning effect of cell growth. The purified DnaJ delta 77-107 protein was shown to physically interact and stimulate DnaK's ATPase activity at wild type levels, unlike the previously characterized DnaJ259 point mutant (DnaJH33Q). Moreover, both DnaJ delta 77-107 and DnaJ259 bound to substrate proteins, such as sigma 32, at similar affinities as DnaJ+. However, DnaJ delta 77-107 was found to be largely defective in activating the ATP-dependent substrate binding mode of DnaK. In vivo, the ability of the mutant DnaJ proteins to down-regulate the heat-shock response was correlated only with their in vitro ability to activate DnaK to bind sigma 32, in an ATP-dependent manner, and not with their ability to bind sigma 32. We conclude, that although the G/F motif of DnaJ does not directly participate in the stimulation of DnaK's ATPase activity, nevertheless, it is involved in an important manner in modulating DnaK's substrate binding activity.

Base Sequence↗

PubMed1995

The Escherichia coli heat shock response and bacteriophage lambda development.

The Escherichia coli/bacteriophage lambda genetic interaction system has been used to uncover the existence of various biological machines. The starting point of all these studies was the isolation and characterization of E. coli mutants that blocked lambda growth, and the corresponding lambda compensatory mutations. In this manner, the lambda N-promoted transcriptional anti-termination machine was discovered composed of the NusA/NusB/NusE/NusG host proteins. In addition, the DnaK and GroEL chaperone machines were discovered composed of DnaK/DnaJ/GrpE and GroES/GroEL heat shock proteins. The individual members of the DnaK and GroEL chaperone machines have been conserved throughout evolution in both function and structure. Their biological roles include a direct involvement in lambda DNA replication and morphogenesis, the protection of proteins from aggregation, the disaggregation of various protein aggregates, the manipulation of protein structure and function, as well as the autoregulation of the heat shock response. The evolution of lambda to extensively rely on the status of the heat shock response of E. coli is likely linked to its lytic versus lysogenic choice of lifestyle. The bacteriophage T4 gp31 protein has been purified and shown to substitute for many of GroES' co-chaperonin activities.

Bacteriophage lambda↗

PubMed1994

NMR structure determination of the Escherichia coli DnaJ molecular chaperone: secondary structure and backbone fold of the N-terminal region (residues 2-108) containing the highly conserved J domain.

DnaJ from Escherichia coli is a 376-amino acid protein that functions in conjunction with DnaK and GrpE as a chaperone machine. The N-terminal fragment of residues 2-108, DnaJ-(2-108), retains many of the activities of the full-length protein and contains a structural motif, the J domain of residues 2-72, which is highly conserved in a superfamily of proteins. In this paper, NMR spectroscopy was used to determine the secondary structure and the three-dimensional polypeptide backbone fold of DnaJ-(2-108). By using 13C/15N doubly labeled DnaJ-(2-108), nearly complete sequence-specific assignments were obtained for 1H, 15N, 13C alpha, and 13C beta, and about 40% of the peripheral aliphatic carbon resonances were also assigned. Four alpha-helices in polypeptide segments of residues 6-11, 18-31, 41-55, and 61-68 in the J domain were identified by sequential and medium-range nuclear Overhauser effects. For the J domain, the three-dimensional structure was calculated with the program DIANA from an input of 536 nuclear Overhauser effect upper-distance constraints and 52 spin-spin coupling constants. The polypeptide backbone fold is characterized by the formation of an antiparallel bundle of two long helices, residues 18-31 and 41-55, which is stabilized by a hydrophobic core of side chains that are highly conserved in homologous J domain sequences. The Gly/Phe-rich region from residues 77 to 108 is flexibly disordered in solution.

Amino Acid Sequence↗

PubMed1994

A mitochondrial homolog of bacterial GrpE interacts with mitochondrial hsp70 and is essential for viability.

Mitochondrial hsp70 (mhsp70) is located in the matrix and an essential component of the mitochondrial protein import system. To study the function of mhsp70 and to identify possible partner proteins we constructed a yeast strain in which all mhsp70 molecules carry a C-terminal hexa-histidine tag. The tagged mhsp70 appears to be functional in vivo. When an ATP depleted mitochondrial extract was incubated with a nickel-derivatized affinity resin, the resin bound not only mhsp70, but also a 23 kDa protein. This protein was dissociated from mhsp70 by ATP. ADP and GTP were much less effective in promoting dissociation whereas CTP and TTP were inactive. We cloned the gene encoding the 23 kDa protein. This gene, termed GRPE, encodes a 228 residue protein, whose sequence closely resembles that of the bacterial GrpE protein. Microsequencing the purified 23 kDa protein established it as the product of the yeast GRPE gene. Yeast GrpEp is made as a precursor that is cleaved upon import into isolated mitochondria. GrpEp is essential for viability. We suggest that this protein interacts with mhsp70 in a manner analogous to that of GrpE with DnaK of E.coli.

Adenosine Triphosphate↗

PubMed1994

The Escherichia coli dsbC (xprA) gene encodes a periplasmic protein involved in disulfide bond formation.

We have identified and functionally characterized a new Escherichia coli gene, dsbC, whose product is involved in disulfide bond formation in the periplasmic space. It corresponds to a previously sequenced open reading frame mapping upstream of recJ with no previously assigned function. Null mutations in dsbC were obtained using a screen for dithiothreitol (DTT)-sensitive mutants and were shown to result in the accumulation of reduced forms of a variety of disulfide bond-containing periplasmic proteins. This defect could be rescued by the addition of either oxidized DTT or cystine or by multicopy expression of dsbA, a known periplasmic disulfide oxidase. The DsbC protein is synthesized as a precursor form of 25.5 kDa which is processed to a 23.3 kDa mature species located in the periplasmic space. The DsbC protein was overexpressed, purified to homogeneity and shown to catalyse the reduction of insulin in a DTT-dependent manner at levels comparable with those of purified DsbA. The replacement of either cysteine residue of the predicted active site, F-(X4)-C-G-Y-C, completely inactivates DsbC protein function. We have further shown that in vivo overexpression of DsbC can functionally substitute for a loss of DsbA function. Taken together, all of our results demonstrate that DsbC acts in vivo as a disulfide oxidase.

Amino Acid Sequence↗

PubMed1994

Bacteriophage T4 encodes a co-chaperonin that can substitute for Escherichia coli GroES in protein folding.

Several bacteriophages use the Escherichia coli GroES and GroEL chaperonins for folding and assembly of their morphogenetic structures. Bacteriophage T4 is unusual in that it encodes a specialized protein (Gp31) that is thought to interact with the host GroEL and to be absolutely required for the correct assembly of the major capsid protein (Gp23) in vivo. Here we show that despite the absence of amino-acid sequence similarity between Gp31 and GroES, Gp31 can functionally substitute for the GroES co-chaperonin in the morphogenesis of bacteriophages lambda and T5, the in vivo and in vitro chaperonin-dependent assembly of ribulose bisphosphate carboxylase (Rubisco), as well as overall bacterial growth at the non-permissive temperature. Like GroES, the bacteriophage Gp31 protein forms a stable complex with the E. coli GroEL protein in the presence of Mg-ATP and inhibits the ATPase activity of GroEL in vitro.

Bacterial Proteins↗

PubMed1994

The NH2-terminal 108 amino acids of the Escherichia coli DnaJ protein stimulate the ATPase activity of DnaK and are sufficient for lambda replication.

The Escherichia coli heat shock proteins DnaK and DnaJ function cooperatively as molecular chaperones. Central to their biochemical functions is the ability of DnaJ to interact with DnaK and to stimulate its ATPase activity. Here, we report the genetic isolation of dnaJ12, which has a nonsense mutation at codon 109, yet was able to support lambda growth at 30 degrees C. The 12-kDa DnaJ12 protein was purified to homogeneity and shown to be active in an in vitro lambda-DNA replication system and to be capable of stimulating DnaK's ATPase activity, specifically at the step of ATP hydrolysis. The previously well studied and characterized dnaJ259 mutation was also cloned and sequenced, revealing a single His-->Gln amino acid change at codon 33. The purified DnaJ259 protein was inactive in an in vitro lambda-DNA replication system and was unable to stimulate DnaK's ATPase activity. Consistent with this, an NH2-terminal deletion of the first 34 amino acids or an Asp insertion at residue 35 of DnaJ resulted in a protein that completely lacked DnaJ activity. Collectively, these results demonstrate that the highly conserved NH2-terminal region of DnaJ, the so-called J region, is necessary and sufficient for stimulating both DnaK's ATPase activity and lambda-DNA replication. These results may be applicable to other eukaryotic proteins that contain this conserved J domain as proteins that interact and stimulate the hydrolysis of ATP by their cognate HSP70 proteins.

Adenosine Triphosphatases↗

PubMed1994

Heat shock proteins as carrier molecules: in vivo helper effect mediated by Escherichia coli GroEL and DnaK proteins requires cross-linking with antigen.

In the past few years we have shown that mycobacterial heat shock proteins (hsp) of 65 and 70 kD exert a very strong helper effect in mice and monkeys when conjugated to peptides and oligosaccharides and given in the absence of adjuvants. In the present study we show that this adjuvant-free helper effect (i) is not due to lipopolysaccharide (LPS), since it was observed in LPS-resistant mice (C3H/HeJ) immunized with hsp-based constructs containing the malaria peptide (NANP)40, and (ii) is characteristic of hsp, since it was not observed with conjugates containing the mycobacterial p38 antigen, which is not a stress protein. Interestingly, the hsp GroEL and DnaK of Escherichia coli, which share a high degree of homology with the mycobacterial 65-kD and 70-kD hsp, respectively, exhibit a strong in vivo helper effect when conjugated to the (NANP)40 peptide, and the conjugates given in the absence of adjuvants. This in vivo helper behaviour of the GroEL and DnaK proteins corresponds well to that observed with the mycobacterial 65-kD and 70-kD hsp, respectively, since the hsp65- and GroEL-based constructs require previous priming of the animals with live bacille Calmette-Guérin (BCG), which is not needed for the hsp70- and DnaK-based constructs. Finally, using both mycobacterial and E. coli hsp we show that their in vivo helper effect in the absence of adjuvants requires cross-linking to the synthetic peptide. Taken together, our results suggest that the adjuvant-free helper effect observed with mycobacterial and E. coli hsp may be a generalized phenomenon, exhibited by hsp from diverse microorganisms. These findings may find applications in the design of vaccine constructs.

Adjuvants, Immunologic↗

PubMed1994

Two classes of extragenic suppressor mutations identify functionally distinct regions of the GroEL chaperone of Escherichia coli.

The GroES and GroEL proteins of Escherichia coli function together as the GroE molecular chaperone machine to (i) prevent denaturation and aggregation and (ii) assist the folding and oligomerization of other proteins without being part of the final structure. Previous genetic and biochemical analyses have determined that this activity requires interactions of the GroES 7-mer with the GroEL 14-mer. Recently, we have identified a region of the GroES protein that interacts with the GroEL protein. To identify those residues of the GroEL protein that interact with GroES, we have exploited the thermosensitive phenotype of strains bearing mutations at one or the other of two GroEL-interacting residues of GroES. We have isolated, cloned, and sequenced six suppressor mutations in groEL, three independent isolates for each groES mutant. Changes of only three different amino acid substitutions in GroEL protein were found among these six groEL suppressor mutations. On the basis of a number of in vivo analyses of the chaperone activity of various combinations of groES mutant alleles and groEL suppressor alleles, we propose that an amino-proximal region of the GroEL protein which includes amino acid residues 174 and 190 interacts with GroES and that a carboxyl-proximal region which includes residue 375 interacts with substrate proteins.

Bacterial Proteins↗

PubMed1994

Isolation and characterization of point mutations in the Escherichia coli grpE heat shock gene.

The Escherichia coli grpE gene (along with dnaK, dnaJ, groEL, and groES) was originally identified as one of the host factors required for phage lambda growth. The classical grpE280 mutation was the only grpE mutation that resulted from the initial screen and shown to specifically block the initiation of lambda DNA replication. Here we report the isolation of several new grpE missense mutations, again using phage lambda resistance as a selection. All mutants fall into two groups based on their temperature-dependent phenotype for lambda growth. Members of the first group (I), including grpE17 and grpE280, which was obtained again, are resistant to lambda growth at both 30 and 42 degrees C. Members of the second group (II), including grpE25, grpE66, grpE103, grpE13a, grpE57b, and grpE61, are sensitive to lambda growth at 30 degrees C but resistant at 42 degrees C. All mutations are recessive, since an E. coli grpE null mutant strain carrying these mutant alleles on low-copy-number plasmids are sensitive to infection by the lambda grpE+ transducing phage. Both group I and group II mutants are temperature sensitive for E. coli growth above 42 degrees C. The nucleotide changes were identified by sequencing analyses and shown to be dispersed throughout the latter 75% of the grpE coding region. Most of the amino acid changes occur at conserved residues, as judged by sequence comparisons between E. coli and other bacterial and yeast GrpE homologs. The isolation of these new mutations is the first step toward a structure-function analysis of the GrpE protein.

Amino Acid Sequence↗

PubMed1993

Both the Escherichia coli chaperone systems, GroEL/GroES and DnaK/DnaJ/GrpE, can reactivate heat-treated RNA polymerase. Different mechanisms for the same activity.

In this work we show that the GroEL (Hsp60 equivalent) chaperone protein can protected purified Escherichia coli RNA polymerase (RNAP) holoenzyme from heat inactivation better than the DnaK (Hsp70 equivalent) chaperone can. In this protection reaction, the GroES protein is not essential, but its presence reduces the amount of GroEL required. GroEL and GroES can also reactivate heat-inactivated RNAP in the presence of ATP. The mutant GroEL673 protein, with or without GroES, is incapable of reactivating heat-inactivated RNAP. GroEL673 can only protect RNAP, and this protecting ability is not stimulated by GroES. The mechanism by which the DnaJ and GrpE heat shock proteins contribute to DnaK's ability to reactivate heat-inactivated RNAP GroEL673 has also been investigated. We found that the DnaJ protein substantially reduces the levels of DnaK protein needed in this reactivation assay. However, the observed lag in reactivation is diminished only in the additional presence of the GrpE protein. Hence, DnaJ and GrpE are involved in both steps of this reactivation reaction (recognition of substrate and release of chaperone from the substrate-chaperone complex) while, in the case of the GroEL-dependent reaction, GroES is involved only during the release of chaperone from the substrate-chaperone complex.

Bacterial Proteins↗

PubMed1993

Autoregulation of the Escherichia coli heat shock response by the DnaK and DnaJ heat shock proteins.

All organisms respond to various forms of stress, including heat shock. The heat shock response has been universally conserved from bacteria to humans. In Escherichia coli the heat shock response is under the positive transcriptional control of the sigma 32 polypeptide and involves transient acceleration in the rate of synthesis of a few dozen genes. Three of the heat shock genes--dnaK, dnaJ, and grpE--are special because mutations in any one of these lead to constitutive levels of heat shock gene expression, implying that their products negatively autoregulate their own synthesis. The DnaK, DnaJ, and GrpE proteins have been known to function in various biological situations, including bacteriophage lambda replication. Here, we report the formation of an ATP hydrolysis-dependent complex of DnaJ, sigma 32, and DnaK proteins in vitro. This DnaJ-sigma 32-DnaK complex has been seen under different conditions, including glycerol gradient sedimentation and co-immunoprecipitation. The DnaK and DnaJ proteins in the presence of ATP can interfere with the efficient binding of sigma 32 to the RNA polymerase core, and are capable of disrupting a preexisting sigma 32-RNA polymerase complex. Our results suggest a possible mechanism for the autoregulation of the heat shock response.

Adenosine Triphosphate↗

PubMed1993

Function of the GrpE heat shock protein in bidirectional unwinding and replication from the origin of phage lambda.

The initiation of DNA replication by phage lambda depends on a specialized nucleoprotein structure that provides for the precise localization and activity of the Escherichia coli DnaB helicase at the lambda replication origin. Previous work has shown that the DnaJ and DnaK heat shock proteins function in the initiation pathway by releasing the DnaB helicase from the initiation complex to carry out localized unwinding of origin DNA. This DnaJ.DnaK pathway results in mainly unidirectional DNA unwinding and replication, whereas replication in vivo is mainly bidirectional. Based on recent replication work indicating an important role for the GrpE heat shock protein, we have used electron microscopy to study the action of GrpE in the DNA unwinding and replication reactions. We have found that GrpE acts with DnaJ and DnaK to facilitate the unwinding reaction at low concentrations of DnaK. In the presence of GrpE, bidirectional unwinding occurs in approximately half of the unwound DNA molecules. In addition, GrpE significantly increases the frequency of replication proceeding leftward from the origin. We suggest that reactions including GrpE result in more complete disassembly of the preinitiation nucleoprotein structure, thus allowing replication to proceed in both directions from the origin.

Bacterial Proteins↗

PubMed1993

Isolation and characterization of ClpX, a new ATP-dependent specificity component of the Clp protease of Escherichia coli.

We have used 14C-labeled bacteriophage lambda O-DNA replication protein as a probe to identify and purify Escherichia coli proteases capable of its degradation. In this manner, five different proteases (termed Lop) have been identified capable of degrading lambda O protein to acid-soluble fragments in an ATP-dependent fashion. One of these activities was purified to homogeneity and shown to be composed of two different polypeptides. The 23,000-Da component (LopP) was identified as the previously characterized ClpP protein, known to complex with ClpA to form the ClpAP, an ATP-dependent protease, capable of degrading casein. The second 46,000-Da component was identified as ClpX (LopC), coded by a gene located in the same operon, but promoter distal to that coding for ClpP (Gottesman, S., Clark, W. P., de Crecy-Lagard, V., and Maurizi, M. R. (1993) J. Biol. Chem. 268, 22618-22626). This identification was based on the determination of the sequence of the first 24 amino acid residues of the purified ClpX protein and its identity with that predicted by the DNA sequence. The ClpXP protease is substrate specific, since it degrades casein (known to be degraded by ClpAP), lambda P, or DnaK proteins slowly or not at all. These results suggest that ClpX protein directs ClpP protease to specific substrates. It is estimated that 50% of all lambda O-specific protease activity present in crude E. coli extracts is due to the ClpXP protease. We propose that transient inhibition of lambda O degradation observed in vivo during the later stages of lambda-DNA replication in vivo is responsible for the switch from bidirectional to unidirectional replication. One round unidirectional replication will lead to strand separation resulting in a switch from early (theta) to late (sigma) mode of lambda-DNA replication.

ATP-Dependent Proteases↗

PubMed1993

Identification and characterization of the Escherichia coli gene dsbB, whose product is involved in the formation of disulfide bonds in vivo.

We have identified and characterized the Escherichia coli gene dsbB, whose product is required for disulfide bond formation of periplasmic proteins, by using two different approaches: (i) screening of a multicopy plasmid library for clones which protect E. coli from the lethal effects of dithiothreitol (DTT), and (ii) screening of insertion libraries of E. coli for DTT-sensitive mutants. Mapping and characterization of mutations conferring a DTT-sensitive phenotype also identified the dsbA, trxA, and trxB genes, whose products are involved in different oxidation-reduction pathways. Null mutations in dsbB conferred pleiotropic phenotypes such as sensitivity to benzylpenicillin and inability to support plaque formation of filamentous phages, and they were shown to severely affect disulfide bond oxidation of secreted proteins such as OmpA and beta-lactamase. These phenotypes resemble the phenotype of bacteria carrying either a null mutation in the dsbA gene or the double mutation dsbA dsbB. Sequencing and expression of the dsbB gene revealed that it encodes a 20-kDa protein predicted to possess an "exchangeable" disulfide bond in -Cys-Val-Leu-Cys-. The dsbB gene maps at 26.5 min on the genetic map of the E. coli chromosome, and its transcription is directed from two promoters, neither of which resembles the canonical E sigma 70-recognized promoter.

Amino Acid Sequence↗

PubMed1993

Characterization of a functionally important mobile domain of GroES.

Although genetic and biochemical evidence has established that GroES is required for the full function of the molecular chaperone, GroEL, little is known about the molecular details of their interaction. GroES enhances the cooperativity of ATP binding and hydrolysis by GroEL (refs 4, 5) and is necessary for release and folding of several GroEL substrates. Here we report that native GroES has a highly mobile and accessible polypeptide loop whose mobility and accessibility are lost upon formation of the GroES/GroEL complex. In addition, lesions present in eight independently isolated mutant groES alleles map in the mobile loop. Studies with synthetic peptides suggest that the loop binds in a hairpin conformation at a site on GroEL that is distinct from the substrate-binding site. Flexibility may be required in the mobile loops on the GroES seven-mer to allow them to bind simultaneously to sites on seven GroEL subunits, which may themselves be able to adopt different arrangements, and thus to modulate allosterically GroEL/substrate affinity.

Amino Acid Sequence↗

PubMed1993

Initiation of lambda DNA replication. The Escherichia coli small heat shock proteins, DnaJ and GrpE, increase DnaK's affinity for the lambda P protein.

It is known that the initiation of bacteriophage lambda replication requires the orderly assembly of the lambda O.lambda P.DnaB helicase protein preprimosomal complex at the ori lambda DNA site. The DnaK, DnaJ, and GrpE heat shock proteins act together to destabilize the lambda P.DnaB complex, thus freeing DnaB and allowing it to unwind lambda DNA near the ori lambda site. The first step of this disassembly reaction is the binding of DnaK to the lambda P protein. In this report, we examined the influence of the DnaJ and GrpE proteins on the stability of the lambda P.DnaK complex. We present evidence for the existence of the following protein-protein complexes: lambda P.DnaK, lambda P.DnaJ, DnaJ.DnaK, DnaK.GrpE, and lambda P.DnaK.GrpE. Our results suggest that the presence of GrpE alone destabilizes the lambda P.DnaK complex, whereas the presence of DnaJ alone stabilizes the lambda P.DnaK complex. Using immunoprecipitation, we show that in the presence of GrpE, DnaK exhibits a higher affinity for the lambda P.DnaJ complex than it does alone. Using cross-linking with glutaraldehyde, we show that oligomeric forms of DnaK exhibit a higher affinity for lambda P than monomeric DnaK. However, in the presence of GrpE, monomeric DnaK can efficiently bind lambda P protein. These findings help explain our previous results, namely that in the GrpE-dependent lambda DNA replication system, the DnaK protein requirement can be reduced up to 10-fold.

Bacterial Proteins↗

PubMed1993

Heat shock proteins in multiple sclerosis and other autoimmune diseases.

Because of the abundance of the highly conserved heat shock proteins (hsp) in microbial pathogens and in mammalian cells, hsps have been considered candidates as target antigens in autoimmune disorders such as multiple sclerosis (MS). Consequently, this workshop examined the current understanding of the biology of hsps and discussed the evidence that they may contribute to autoimmune disease processes. This article reports the outcome of the discussion.

Animals↗