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Query: UNIPROT:P06889 (Mol)
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We have identified a gene encoding the 60 kDa heat shock protein (hsp60) from a Plasmodium falciparum blood stage cDNA library. The deduced protein sequence encodes for a polypeptide of 577 amino acids with a calculated molecular weight of 62158 Da. The primary structure of P. falciparum hsp60 contains a putative mitochondrial targeting peptide at its amino-terminus and a GGM motif at its carboxyl-terminus. The overall structure exhibits strong conservation (approximately 50%) to the hsp60 from human and other eukaryotes, but only low homology (< 30%) to a recently reported P. falciparum chaperonin 60 gene. The P. falciparum hsp60 gene is located on chromosome 10. During heat shock, the level of hsp60 transcript in blood stage parasites increases significantly and its accumulation correlates with the duration of the induction.
Mol Biochem Parasitol 1996 Jul
PMID:Cloning of a Plasmodium falciparum gene related to the human 60-kDa heat shock protein. 884 68

We developed a bacterial expression system for the human alpha and beta cDNAs of propionyl-CoA carboxylase (PCC). These cDNAs (less the putative mitochondrial matrix targeting presequences) were co-expressed in Escherichia coli on one plasmid vector with each cDNA having its own IPTG-inducible promoter. Only negligible amounts of active PCC were measured despite the presence of both alpha and beta subunits as indicated by Western blot analysis and the almost complete biotinylation of the alpha subunit. Co-expression of this plasmid with a second plasmid vector over-expressing the E. coli chaperonin proteins, groES and groEL, resulted in a several hundred-fold increase in PCC specific activity, to a level comparable with that found in crude human liver extracts. PCC was partially purified on monomeric avidin affinity resin and the presence of both alpha and beta subunits was demonstrated, thereby confirming the assembly of both subunits into an active enzyme. Deficiency of either alpha PCC or beta PCC results in propionic acidemia, an autosomal recessive disorder. We used this expression system to characterize one missense mutation previously described in five Japanese alleles, namely C1283T (Thr428lle) in beta PCC. This mutation, when expressed in E.coli under the same conditions as that of wild-type PCC, had null activity, despite the presence of assembled alpha PCC and beta PCC subunits. This bacterial expression system can be useful for analysis of either alpha PCC or beta PCC mutations. Our findings indicated that the groES and groEL chaperonin proteins were essential for folding and assembly of the human PCC heteromeric subunits.
Hum Mol Genet 1996 Mar
PMID:Chaperonin-mediated assembly of wild-type and mutant subunits of human propionyl-CoA carboxylase expressed in Escherichia coli. 885 56

We have determined the structure of the Ascaris major sperm protein (MSP) to 2.5 A resolution using X-ray crystallography. The MSP polypeptide chain has an immunoglobulin-like fold based on a seven-stranded beta sandwich. In two strands, cis-proline residues impart distinctive kinks, and overall the structure most closely resembles that of the N-terminal domain of the bacterial chaperonin, PapD. In the C2 crystal form which we have solved here, two MSP chains are tightly associated in the asymmetric unit and are related by a non-crystallographic 2-fold rotation axis. This arrangement almost certainly represents the MSP dimer that is present in solution. Additionally, the arrangement of two MSP dimers at one of the crystallographic 2-fold axes in the 215 A unit cell suggests a possible mode for the assembly of MSP into the filaments which promote cell movement. This dimer-dimer association is based on a beta sheet extension mechanism between adjoining MSP monomers which resembles the interaction between PapD and its protein substrate.
J Mol Biol 1996 Oct 25
PMID:2.5 A resolution crystal structure of the motile major sperm protein (MSP) of Ascaris suum. 891 7

We isolated a full-length cDNA clone encoding a Xenopus laevis 70-kDa heat shock cognate protein, hsc70.I. The protein coding region exhibits a high degree of identity with a number of mammalian hsc70 proteins, such as rat hsc71 (92%), whereas the identity to Xenopus hsp70 is only 80%. These data suggest that the inducible and constitutive forms of hsp70 diverged long before the emergence of amphibians. The Xenopus hsc70.I contains a number of conserved elements, including the ATP-binding domain, a nuclear localization signal and the carboxy-terminal EEVD motif, which has been implicated in several activities associated with chaperonin function. Northern blot analyses revealed that maternal hsc70.I mRNA is present in cleavage and early blastula stages of Xenopus development. After the onset of zygotic transcription at the midblastula stage, the levels of hsc70.I message increase through to the tadpole stages. Furthermore, in contrast to hsp70 mRNA, the relative levels of hsc70.I mRNA are not enhanced after heat shock in embryos and in the kidney epithelial cell line, A6. The levels of hsc70.I mRNA are high in adult spleen and testis, with moderate levels in eye, heart, liver and brain and comparatively low levels in hindlimb muscle.
Comp Biochem Physiol B Biochem Mol Biol 1996 Apr
PMID:Isolation and characterization of a cDNA encoding a Xenopus 70-kDa heat shock cognate protein, Hsc70.I. 892 37

The structure of a Salmonella enterica serovar typhi gene located within the fim gene cluster and encoding a putative periplasmic chaperone-like protein involved in the assembly of type 1 pili was determined. This gene, named fimC, has the ability to encode a 26-kDa polypeptide which is similar, at the sequence level, to the PapD periplasmic chaperonin mediating the assembly of P pili of Escherichia coli, as well as to other periplasmic chaperone-like proteins involved in the biogenesis of pili or capsule-like structures of various Gram-negative bacteria. A comprehensive search through the literature and sequence databases identified 31 (putative) bacterial proteins that can be included in this protein family on the basis of sequence similarity. Results of a multiple sequence comparison analysis showed that several residues, including most of those known to be critical in maintaining the three-dimensional structure of PapD, are either conserved or conservatively substituted in all these proteins, suggesting an overall similar folding for all of them. It was also evident that members of this family are clustered into different subfamilies according to structural and phyletic data.
J Mol Evol 1997 Mar
PMID:Relatedness and phylogeny within the family of periplasmic chaperones involved in the assembly of pili or capsule-like structures of gram-negative bacteria. 906 Mar 96

Mitochondrial malate dehydrogenase (mMDH) folds more rapidly in the presence of GroEL, GroES and ATP than it does unassisted. The increase in folding rate as a function of the concentration of GroEL-ES reaches a maximum at a stoichiometry which is approximately equimolar (mMDH subunits:GroEL oligomer) and with an apparent dissociation constant K' for the GroE acceptor state of at least 1 x 10(-8) M. However, even at chaperonin concentrations which are 4000 x K', i.e. at negligible concentrations of free mMDH, the observed folding rate of the substrate remains at its optimum, showing not only that folding occurs in the chaperonin-mMDH complex but also that this rate is uninhibited by any interactions with sites on GroEL. Despite the ability of mMDH to fold on the chaperonin, trapping experiments show that its dwell time on the complex is only 20 seconds. This correlates with both the rate of ATP turnover and the dwell time of GroES on the complex and is only approximately 5% of the time taken for the substrate to commit to the folded state. The results imply that ATP drives the chaperonin complex through a cycle of three functional states: (1) an acceptor complex in which the unfolded substrate is bound tightly; (2) an encapsulation state in which it is sequestered but direct protein-protein contact is lost so that folding can proceed unhindered; and (3) an ejector state which forces dissociation of the substrate whether folded or not.
J Mol Biol 1997 Mar 07
PMID:Binding, encapsulation and ejection: substrate dynamics during a chaperonin-assisted folding reaction. 910 59

Actin and tubulin polypeptide chains acquire their native conformation in the presence of the cytoplasmic chaperonin containing TCP-1 (CCT, also called TRiC) and, in the case of alpha- and beta-tubulin, additional protein cofactors. It has been previously demonstrated that nucleotide exchange and ATP hydrolysis act to switch CCT between conformations that interact either strongly or weakly with unfolded substrates [Melki, R., & Cowan, N.J. (1994) Mol. Cell. Biol. 14, 2895-2904]. The present study further documents the conformational changes and function of CCT. It is first shown, by the use of a range of labeled denatured substrate proteins and a radiolabeled total soluble HeLa cell extract, that CCT in the absence of nucleotides can bind any of a large number of proteins in vitro with high affinity. Second, by the use of denatured labeled beta-actin and beta-tubulin as model substrates for binding to CCT, we demonstrate that the CCT particle can contain two substrate protein chains simultaneously. Third, by electron microscopy, sedimentation velocity, and intrinsic fluorescence measurements, we document the conformational difference between CCT in its ATP- and ADP-bound forms, as well as the change that results from binding of substrate protein. A model summarizes substrate association with CCT and the role of the nucleotide in regulating the affinity of CCT for target proteins.
 ...
PMID:Cytoplasmic chaperonin containing TCP-1: structural and functional characterization. 915 22

Using stopped-flow fluorescence techniques, we have examined both the refolding and unfolding reactions of four structurally homologous dihydrofolate reductases (murine DHFR, wild-type E. coli DHFR, and two E. coli DHFR mutants) in the presence and absence of the molecular chaperonin GroEL. We show that GroEL binds the unfolded conformation of each DHFR with second order rate constants greater than 3 x 10(7) M(-1)s(-1) at 22 degrees C. Once bound to GroEL, the proteins refold with rate constants similar to those for folding in the absence of GroEL. The overall rate of formation of native enzyme is decreased by the stability of the complex between GroEL and the last folding intermediate. For wild-type E. coli DHFR, complex formation is transient while for the others, a stable complex is formed. The stable complexes are the same regardless of whether they are formed from the unfolded or folded DHFR. When complex formation is initiated from the native conformation, GroEL binds to a pre-existing non-native conformation, presumably a late folding intermediate, rather than to the native state, thus shifting the conformational equilibrium toward the non-native species by mass action. The model presented here for the interaction of these four proteins with GroEL quantitatively describes the difference between the formation of a transient complex and a stable complex as defined by the rate constants for release and rebinding to GroEL relative to the rate constant for the last folding step. Due to this kinetic partitioning, three different mechanisms can be proposed for the formation of stable complexes between GroEL and either murine DHFR or the two E. coli DHFR mutants. These data show that productive folding of GroEL-bound proteins can occur in the absence of nucleotides or the co-chaperonin GroES and suggest that transient complex formation may be the functional role of GroEL under normal conditions.
J Mol Biol 1997 May 02
PMID:GroEL-mediated folding of structurally homologous dihydrofolate reductases. 915 87

Folding of newly synthesized proteins in vivo is believed to be facilitated by the cooperative interaction of a defined group of proteins known as molecular chaperones. We investigated the direct interaction of chaperones with nascent polypeptides in the cytosol of mammalian cells by multiple methods. A new approach using a polyclonal antibody to puromycin allowed us to tag and capture a population of truncated nascent polypeptides with no bias as to the identity of the bound chaperones. In addition, antibodies that recognize the cytosolic chaperones hsp70, CCT (TRiC), hsp40, p48 (Hip), and hsp90 were compared on the basis of their ability to coprecipitate nascent polypeptides, both before and after chemical cross-linking. By all three approaches, hsp70 was found to be the predominant chaperone bound to nascent polypeptides. The interaction between hsp70 and nascent polypeptides is apparently dynamic under physiological conditions but can be stabilized by depletion of ATP or by cross-linking. The cytosolic chaperonin CCT was found to bind primarily to full-length, newly synthesized actin, and tubulin. We demonstrate and caution that nascent polypeptides have a propensity for binding many proteins nonspecifically in cell lysates. Although current models of protein folding in vivo have described additional components in contact with nascent polypeptides, our data indicate that the hsp70 and, perhaps, the hsp90 families are the predominant classes of molecular chaperones that interact with the general population of cytosolic nascent polypeptides.
Mol Biol Cell 1997 Aug
PMID:Complexes between nascent polypeptides and their molecular chaperones in the cytosol of mammalian cells. 928 25

GroE, the chaperonin system of Escherichia coli, prevents the aggregation of partially folded or misfolded proteins by complexing them in a form competent for subsequent folding to the native state. We examined the exchange of amide protons of cyclophilin A (CypA) interacting with GroEL, using NMR spectroscopy. We have applied labeling pulses in H2O to the deuterated GroEL-CypA-complex. When ATP and GroES were added after the labeling pulse, refolding of CypA could be accelerated to rates comparable to the amide proton exchange. This allowed the calculation of protection factors (PF) for the backbone amide protons in the GroEL-bound substrate protein. A set of highly protected protons in the native state (PF 10(5) to 10(7)) was observed to be much less protected (PF 10(2) to 10(4)) in complex with GroEL and, in contrast to the native structure, the protection factors were found to be quite uniform along the sequence suggesting that CypA with native-like structure undergoes multiple cycles of unfolding while bound to GroEL, which are faster than unfolding in free solution. Because of the small sequence dependence of the protection factors, unfolding must be global, and in this way the chaperone appears to resolve off-pathway intermediates and to support protein folding by annealing. Although in the complex with GroEL native-like states still predominate over globally unfolded states, this equilibrium is shifted 10(2) to 10(4)-fold toward the unfolded state when compared to CypA in free solution. Repeated global unfolding may be a key step in achieving a high yield of correctly folded proteins.
J Mol Biol 1997 Sep 05
PMID:Multiple cycles of global unfolding of GroEL-bound cyclophilin A evidenced by NMR. 929 28


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