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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The infectious prion protein, PrP(Sc), a predominantly beta-sheet aggregate, is derived from PrP(C), the largely alpha-helical cellular isoform of PrP. Conformational conversion of PrP(C) into PrP(Sc) has been suggested to involve a chaperone-like factor. Here we report that the bacterial chaperonin GroEL, a close homolog of eukaryotic Hsp60, can catalyze the aggregation of chemically denatured and of folded, recombinant PrP in a model reaction in vitro. Aggregates form upon ATP-dependent release of PrP from chaperonin and have certain properties of PrP(Sc), including a high beta-sheet content, the ability to bind the dye Congo red, detergent-insolubility and increased protease-resistance. A conserved sequence segment of PrP (residues 90-121), critical for PrP(Sc) generation in vivo, is also required for chaperonin-mediated aggregate formation in vitro. Initial binding of refolded, alpha-helical PrP to chaperonin is mediated by the unstructured N-terminal segment of PrP (residues 23-121) and is followed by a rearrangement of the globular PrP core-domain. These results show that chaperonins of the Hsp60 class can, in principle, mediate PrP aggregation de novo, i.e. independently of a pre-existent PrP(Sc) template.
J Mol Biol 2001 Nov 02
PMID:Chaperonin-mediated de novo generation of prion protein aggregates. 1169 9

Chaperonins are a subclass of molecular chaperones that assist both the folding of newly synthesized proteins and the maintenance of proteins in a folded state during periods of stress. The best studied members of this family are the type I chaperonins, occurring in bacteria and evolutionarily derived organelles. Type II chaperonins occur in archaea and the eukaryotic cytosol. An intriguing question pertains to the mechanism by which chaperonins themselves are folded and assembled into functional oligomers. The available evidence for the assembly/disassembly of type I and II chaperonins points to a process that is highly cooperative and suggests a prominent role for nucleotides. Interestingly, the intracellular assembly of type I chaperonins appears to be a chaperone-dependent process itself and requires functional preformed chaperonin complexes.
Mol Biotechnol 2001 Oct
PMID:Assembly of chaperonin complexes. 1172 84

Retroprocessed pseudogenes, calmodulin II (psi1, psi2, and psi3 CALMII), psi alpha-tubulin, pi-glutathione S-transferase (psi pi-GST) from rat, lactic acid dehydrogenase (psi LDH) from mouse, and heat shock protein 60 chaperonin (psi HSP60) from Chinese hamster, were examined for their presence in these species by polymerase chain reaction (PCR). Pseudogenes of these murine rodents were detected by PCR only in those species in which the genes were originally identified, suggesting that the selected pseudogene of one species arose too recently to be detected in the genomes of the other rodent species. The calculated ages of the rodent pseudogenes ranged from 1.7 Myr (psi alpha-tubulin) to 7.5 Myr (psi3 CALMII) when employing a homologous functional gene of the taxon as a reference in the relative rate test with the mouse or rat as the outgroup. Given the high rate of divergence of the genes of rodents relative to other species, selection of an outgroup with similar mutation rates seems warranted. To justify further the conclusion that the selected pseudogenes were indeed retroprocessed after these three taxa diverged, the presence of the pseudogenes in the genome of different rat species was examined. The existence of psi3 CALMII and psi alpha-tubulin pseudogenes of Rattus norvegicus among species belonging to Rattus sensu stricto is evidence for the common ancestry of this group.
J Mol Evol 2002 Jan
PMID:Age and detection of retroprocessed pseudogenes in murine rodents. 1173 3

Many proteins display complex folding kinetics, which represent multiple parallel folding pathways emanating from multiple unfolded forms and converging to the unique native form. The small protein thioredoxin from Escherichia coli is one such protein. The effect of the chaperonin GroEL on modulating the complex energy landscape that separates the unfolded ensemble from the native state of thioredoxin has been studied. It is shown that while the fluorescence change accompanying folding occurs in five kinetic phases in the absence of GroEL, only the two slowest kinetic phases are discernible in the presence of saturating concentrations of GroEL. This result is shown to be consistent with only one out of several available folding routes being operational in the presence of GroEL. It is shown that native protein, which forms via fast as well as slow routes in the absence of GroEL, forms only via a slow route in its presence. The effect of GroEL on the folding of thioredoxin is shown to be the consequence of it binding differentially to the many folding-competent forms. While some of these forms can continue folding when bound to GroEL, others cannot. All molecules are then drawn into the operational folding route by the law of mass action. This observation indicates a new role for GroEL, which is to bias the energy landscape of a folding polypeptide towards fewer available pathways. It is suggested that such channeling might be a mechanism to avoid possible aggregation-prone routes available to a refolding polypeptide in vivo.
J Mol Biol 2001 Dec 14
PMID:GroEL channels the folding of thioredoxin along one kinetic route. 1174 32

Group II chaperonins of archaea and eukaryotes are distinct from group I chaperonins of bacteria. Whereas group I chaperonins require the co-chaperonin Cpn-10 or GroES for protein folding, no co-chaperonin has been known for group II. The protein folding mechanism of group II chaperonins is not yet clear. To understand this mechanism, we examined protein refolding by the recombinant alpha or beta-subunit chaperonin homo-oligomer (alpha16mer and beta16mer) from a hyperthermoplilic archaeum, Thermococcus strain KS-1, using a model substrate, green fluorescent protein (GFP). The alpha16mer and beta16mer captured the non-native GFP and promoted its refolding without any co-chaperonin in an ATP dependent manner. A non-hydrolyzable ATP analog, AMP-PNP, induced the GFP refolding mediated by beta16mer but not by the alpha16mer. A mutant alpha-subunit chaperonin homo-oligomer (trap-alpha) could capture the non-native protein but lacked the ability to refold it. Although trap-alpha suppressed ATP-dependent refolding of GFP mediated by alpha16mer or beta16mer, it did not affect the AMP-PNP-dependent refolding. This indicated that the GFP refolding mediated by beta16mer with AMP-PNP was not accessible to the trap-alpha. Gel filtration chromatography and a protease protection experiment revealed that this refolded GFP, in the presence of AMP-PNP, was associated with beta16mer. After the completion of GFP refolding mediated by beta16mer with AMP-PNP, addition of ATP induced an additional refolding of GFP. Furthermore, the beta16mer preincubated with AMP-PNP showed the ability to capture the non-native GFP. These suggest that AMP-PNP induced one of two chaperonin rings (cis-ring) to close and induced protein refolding in this ring, and that the other ring (trans-ring) could capture the unfolded GFP which was refolded by adding ATP. The present data indicate that, in the group II chaperonin of Thermococcus strain KS-1, the protein folding proceeds in its cis-ring in an ATP-dependent fashion without any co-chaperonin.
J Mol Biol 2002 Jan 04
PMID:Archaeal group II chaperonin mediates protein folding in the cis-cavity without a detachable GroES-like co-chaperonin. 1177 67

Most proteins in eukaryotic cells are degraded by 26-S proteasomes, usually after being conjugated to ubiquitin. In the absence of ATP, 26-S proteasomes fall apart into their two sub-complexes, 20-S proteasomes and PA700, which reassemble upon addition of ATP. Conceivably, 26-S proteasomes dissociate and reassemble during initiation of protein degradation in a ternary complex with the substrate, as in the dissociation-reassembly cycles found for ribosomes and the chaperonin GroEL/GroES. Here we followed disassembly and assembly of 26-S proteasomes in cell extracts as the exchange of PA700 subunits between mouse and human 26-S proteasomes. Compared to the rate of proteolysis in the same extract, the disassembly-reassembly cycle was much too slow to present an obligatory step in a degradation cycle. It has been suggested that subunit S5a (Mcb1, Rpn10), which binds poly-ubiquitin substrates, shuttles between a free state and the 26-S proteasome, bringing substrate to the complex. However, S5a was not found in the free state in HeLa cells. Besides, all subunits in PA700, including S5a, exchanged at similar low rates. It therefore seems that 26-S proteasomes function as stable entities during degradation of proteins.
J Mol Biol 2002 Jan 25
PMID:26 S proteasomes function as stable entities. 1181 35

We examined the biogenesis of the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL) in vitro and in vivo. pVHL formed a complex with the cytosolic chaperonin containing TCP-1 (CCT or TRiC) en route to assembly with elongin B/C and the subsequent formation of the VCB-Cul2 ubiquitin ligase. Blocking the interaction of pVHL with elongin B/C resulted in accumulation of pVHL within the CCT complex. pVHL present in purified VHL-CCT complexes, when added to rabbit reticulocyte lysate, proceeded to form VCB and VCB-Cul2. Thus, CCT likely functions, at least in part, by retaining VHL chains pending the availability of elongin B/C for final folding and/or assembly. Tumor-associated mutations within exon II of the VHL syndrome had diverse effects upon the stability and/or function of pVHL-containing complexes. First, a pVHL mutant lacking the entire region encoded by exon II did not bind to CCT and yet could still assemble into complexes with elongin B/C and elongin B/C-Cul2. Second, a number of tumor-derived missense mutations in exon II did not decrease CCT binding, and most had no detectable effect upon VCB-Cul2 assembly. Many exon II mutants, however, were found to be defective in the binding to and subsequent ubiquitination of hypoxia-inducible factor 1alpha (HIF-1alpha), a substrate of the VCB-Cul2 ubiquitin ligase. We conclude that the selection pressure to mutate VHL exon II during tumorigenesis does not relate to loss of CCT binding but may reflect quantitative or qualitative defects in HIF binding and/or in pVHL-dependent ubiquitin ligase activity.
Mol Cell Biol 2002 Mar
PMID:Diverse effects of mutations in exon II of the von Hippel-Lindau (VHL) tumor suppressor gene on the interaction of pVHL with the cytosolic chaperonin and pVHL-dependent ubiquitin ligase activity. 1186 71

The jakobids are free-living mitochondriate protists that share ultrastructural features with certain amitochondriate groups and possess the most bacterial-like mitochondrial genomes described thus far. Jakobids belong to a diverse group of mitochondriate and amitochondriate eukaryotes, the excavate taxa. The relationships among the various excavate taxa and their relationships to other putative deep-branching protist groups are largely unknown. With the hope of clarifying these issues, we have isolated the cytosolic chaperonin CCTalpha gene from the jakobid Reclinomonas americana (strains 50394 and 50283), the jakobid-like malawimonad Malawimonas jakobiformis, two heteroloboseans (Acrasis rosea and Naegleria gruberi), a euglenozoan (Trypanosoma brucei), and a parabasalid (Monocercomonas sp.). We also amplified the CCTdelta gene from M. jakobiformis. The Reclinomonas and Malawimonas sequences presented here are among the first nuclear protein-coding genes to be described from these organisms. Unlike other putative early diverging protist lineages, a high density of spliceosomal introns was found in the jakobid and malawimonad CCTs-similar to that observed in vertebrate protein-coding genes. An analysis of intron positions in CCT genes from protists, plants, animals, and fungi suggests that many of the intron-sparse or intron-lacking protist lineages may not be primitively so but have lost spliceosomal introns during their evolutionary history. In phylogenetic trees constructed from CCTalpha protein sequences, R. americana (but not M. jakobiformis) shows a weak but consistent affinity for the Heterolobosea and Euglenozoa.
Mol Biol Evol 2002 Apr
PMID:The chaperonin genes of jakobid and jakobid-like flagellates: implications for eukaryotic evolution. 1191 83

The thermostability of the recombinant alpha- and beta-subunit homo-oligomers (alpha16mer and beta16mer) and of natural chaperonins purified from cultured Thermococcus strain KS-1 cells was measured to understand the mechanism for the thermal acclimatization of T. KS-1. The beta-subunit content of the natural chaperonin from cells grown at 90 degrees C was higher than that at 80 degrees C. The optimum temperature for ATPase activity of the natural chaperonins was 80-90 degrees C, whereas that for alpha16mer and beta16mer was 60 degrees C and over 90 degrees C respectively. Judging from the ATPase activity, beta16mer was more thermostable than alpha16mer. The thermostabilities of the natural chaperonins were intermediate between alpha16mer and beta16mer, whereas the natural chaperonin with a higher beta-subunit content was more stable than that with a lower beta-subunit content. Native polyacrylamide gel electrophoresis (PAGE) revealed that the chaperonin oligomers thermally dissociated to their ATPase-inactive monomers. The thermal denaturation process monitored by circular dichroism showed that the free beta-subunit was more stable than the free alpha-subunit, and that the secondary structure of the chaperonin monomer in the oligomer was more stable than that in the free monomer. These results suggest that the structure of these subunits was stabilized in the oligomer, and that an increase in the beta-subunit content conferred higher thermostability to the natural hetero-oligomeric chaperonin.
Mol Microbiol 2002 May
PMID:Two kinds of archaeal group II chaperonin subunits with different thermostability in Thermococcus strain KS-1. 1199 56

alpha and beta-Tubulin fold in a series of chaperone-assisted steps. At least five protein cofactors are involved in the post-chaperonin tubulin folding pathway and required to maintain the supply of tubulin; some of them also participate in microtubule dynamics. The first tubulin chaperone identified in the tubulin folding pathway was cofactor A (CoA). Here we describe the three-dimensional structure of human CoA at 1.7 A resolution, determined by multiwavelength anomalous diffraction (MAD). The structure is a monomer with a rod-like shape and consists of a three-alpha-helix bundle, or coiled coil, with the second helix kinked by a proline break, offering a convex surface at one face of the protein. The helices are connected by short turns, one of them, between alpha2 and alpha3, including a 3(10)-helix. Peptide mapping analysis and competition experiments with peptides show that CoA interacts with beta-tubulin via the three alpha-helical regions but not with the rod-end loops. The main interaction occurs with the middle kinked alpha2 helix, at the convex face of the rod. Strong 3D structural homology is found with the Hsp70 chaperone cofactor BAG domain, suggesting that these proteins define a family of cofactors of simple compact architecture. Further structural homology is found with alpha-spectrin/alpha-actinin repeats, all are rods of identical length of ten helical turns. We propose to call these three-helix bundles alpha ten modules.
J Mol Biol 2002 May 10
PMID:Three-dimensional structure of human tubulin chaperone cofactor A. 1205 8


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