Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chaperonins containing t-complex polypeptide-1 (CCT) are cytosolic molecular chaperone particles implicated especially in the biogenesis of cytoskeletal proteins by promoting the correct folding of the major ubiquitous cytoskeletal components, tubulin and actin. We have purified cytosolic chaperonins from the ND7/23 cell line, determined their subunit composition and examined changes in the intracellular locations of their components during differentiation of ND7/23 cells to a neuronal phenotype by using immunocytochemistry and immunoblots. Chaperonins containing the CCT alpha (TCP1) subunit enter neuritic processes and are particularly noticeable at the leading edge of growth cone-like structures where they co-localise with actin. Chaperonins containing three other components (CCT beta, epsilon and gamma), however, remain predominantly restricted to perikaryal cytoplasm. These findings suggest a heterogeneous population of chaperonin particles within single differentiated ND7/23 cells and this may reflect specialisation of chaperonin function in different cytoplasmic compartments of a neurone. Further, since ribosomes do not enter neurites while CCT alpha-containing chaperonins do, the latter may play roles, subsequent to translation, which influence cytoskeletal elaboration during neuritogenesis.
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PMID:Cytoplasmic chaperonin complexes enter neurites developing in vitro and differ in subunit composition within single cells. 761 68

A new member of the tubulin superfamily, gamma-tubulin, is localized at microtubule-organizing centers (MTOCs) in a variety of organisms. Chlamydomonas cDNA coding for the full-length sequence of gamma-tubulin was expressed in insect ovarian Sf9 cells using the baculovirus expression system. Approximately half of the induced 52 kDa gamma-tubulin was recovered in the supernatant after centrifugation of Sf9 cell lysates at 18,000 g for 15 minutes. When the cell supernatant was analyzed by FPLC on a Superdex 200 sizing column, Chlamydomonas gamma-tubulin separated into two major peaks. The lagging peak contained a monomeric form of gamma-tubulin with a sedimentation coefficient of 2.5 S, which interacted with the Superdex column in a salt-dependent manner. The leading peak, with an apparent molecular mass of 900 kDa, corresponded to a molecular chaperonin complex, and TCP1 chaperonin released folded gamma-tubulin polypeptide from the complex in the presence of MgATP. The released gamma-tubulin monomers were capable of binding to microtubules in vitro and biochemical quantities of active monomers were further purified using a combination of size-exclusion and ion-exchange column chromatography. The endogenous Sf9 cell gamma-tubulin migrated faster than Chlamydomonas gamma-tubulin with an apparent molecular mass of 49 kDa on gels. Analyses on gel filtration and sucrose density gradient centrifugation showed that, while overexpressed Chlamydomonas gamma-tubulin was present in a monomeric form, endogenous gamma-tubulin from Sf9 and HeLa cells exists as a dimer. These results may suggest the possibility that gamma-tubulin could form a heterodimer with hitherto unknown molecule(s).
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PMID:Identification of intrinsic dimer and overexpressed monomeric forms of gamma-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas gamma-tubulin sequence. 762 95

The Escherichia coli chaperonin GroEL and its regulator GroES are thought to mediate adenosine triphosphate-dependent protein folding as an asymmetrical complex, with substrate protein bound within the GroEL cylinder. In contrast, a symmetrical complex formed between one GroEL and two GroES oligomers, with substrate protein binding to the outer surface of GroEL, was recently proposed to be the functional chaperonin unit. Electron microscopic and biochemical analyses have now shown that unphysiologically high magnesium concentrations and increased pH are required to assemble symmetrical complexes, the formation of which precludes the association of unfolded polypeptide. Thus, the functional significance of GroEL:(GroES)2 particles remains to be demonstrated.
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PMID:Functional significance of symmetrical versus asymmetrical GroEL-GroES chaperonin complexes. 763

The chaperonins GroEL and GroES of Escherichia coli facilitate protein folding in an adenosine triphosphate (ATP)-dependent reaction cycle. The kinetic parameters for the formation and dissociation of GroEL-GroES complexes were analyzed by surface plasmon resonance. Association of GroES and subsequent ATP hydrolysis in the interacting GroEL toroid resulted in the formation of a stable GroEL:ADP:GroES complex. The complex dissociated as a result of ATP hydrolysis in the opposite GroEL toroid, without formation of a symmetrical GroEL:(GroES)2 intermediate. Dissociation was accelerated by the addition of unfolded polypeptide. Thus, the functional chaperonin unit is an asymmetrical GroEL:GroES complex, and substrate protein plays an active role in modulating the chaperonin reaction cycle.
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PMID:Asymmetrical interaction of GroEL and GroES in the ATPase cycle of assisted protein folding. 763 1

The heat shock protein GroEL from Escherichia coli is a tetradecameric oligomer that facilitates the refolding of nonnative polypeptides in an ATP-hydrolysis dependent reaction. A mutant in GroEL was prepared in which lysine 3 was substituted with glutamate, which destabilizes the oligomeric structure of GroEL (Horovitz, A., Bochkareva, E.S., and Girshovich, A.S. (1993) J. Biol. Chem. 268, 9957-9959). The highly expressed and purified GroELK3E was judged to be monomeric by sedimentation equilibrium, yielding a molecular weight of 54,500, despite a weak tendency of the mutant to reversibly form higher order aggregates above 4 mg ml-1. The monomeric variant appears to be folded based on the far UV circular dichroism spectrum, which shows significant alpha-helical content, but with slight differences in conformation relative to wild-type GroEL. The increase in exposed hydrophobic surface of the monomer was probed with the dye 4,4'-bis-1-anilino-3-naphthalenesulfonate (bis-ANS). The fluorescence of bis-ANS increases approximately 150-fold in the presence of the mutant, and about 4 mol of bis-ANS bind per mol of monomer, with a binding constant of 1.6 microM. Adenosine nucleotide binding to monomeric GroELK3E resulted in considerable quenching of bis-ANS fluorescence, correlating with significant structural changes as seen in the far UV circular dichroism, and permitted the measurement of binding isotherms for ATP and ADP. Hyperbolic ATP binding isotherms yield a dissociation constant of 82 microM, about 4-fold weaker than the K0.5 for ATP seen in steady-state kinetics assays of the wild-type GroEL ATPase.A similar difference was seen for ADP binding. These results suggest that the mutation disrupts the native tetradecameric quaternary structure through conformational changes that may also weaken nucleotide binding. The monomeric mutant exhibited no chaperone activity as evidenced by a filure to inhibit or facilitate the refolding of chemically denatured enolase, an inability to refold denatured rhodanese above spontaneous levels, and a lack of binding to alpha-casein, a competitor in many chaperonin-promoted refolding reactions. Thus, the formation of assembly incompetent monomers by the lysine 3 to glutamate mutation results in a dramatic decrease in the affinity for nonnative polypeptide chains and suggests that the oligomeric nature of GroEL is crucial for its molecular chaperone function.
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PMID:A monomeric variant of GroEL binds nucleotides but is inactive as a molecular chaperone. 765 15

Unfolded rhodanese in a complex with the chaperonin GroEL was subjected to limited proteolysis. Sequence analysis indentified a GroEL-bound fragment of approximately 11,000 M(r) and a well defined fragment of approximately 7,000 M(r) from the two homologous domains of rhodanese. The shorter segment contains one hydrophobic and one amphiphilic alpha-helix mapping to the domain interface while the other fragment contains the homologous regions and an additional hydrophobic helix. Our results suggest a mechanism for the GroEL-mediated folding of rhodanese in which the domain-forming regions of the polypeptide are kept apart and are then released, perhaps sequentially, resulting in correct folding.
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PMID:Binding of defined regions of a polypeptide to GroEL and its implications for chaperonin-mediated protein folding. 766 27

A 65-kDa protein and a 10-kDa protein are two of the more strongly immunoreactive components of Mycobacterium tuberculosis, the causative agent of tuberculosis. The 65-kDa antigen has homology with members of the GroEL or chaperonin-60 (Cpn60) family of heat shock proteins. The 10-kDa antigen has homology with the GroES or chaperonin-10 family of heat shock proteins. These two proteins are encoded by separate genes in M. tuberculosis. The studies reported here reveal that M. tuberculosis contains a second Cpn60 homolog located 98 bp downstream of the 10-kDa antigen gene. The second Cpn60 homolog (Cpn60-1) displays 61% amino acid sequence identity with the 65-kDa antigen (Cpn60-2) and 53% and 41% identity with the Escherichia coli GroEL protein and the human P60 protein, respectively. Primer-extension analysis revealed that transcription starts 29 bp upstream of the translation start of the Cpn60-1 homolog and protein purification studies indicate that the cpn60-1 gene is expressed as an approximately 60-kDa polypeptide.
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PMID:Mycobacterium tuberculosis expresses two chaperonin-60 homologs. 768 82

The interaction between the pyruvate decarboxylase (E1) component and a di-domain (lipoyl domain plus peripheral subunit-binding domain) from the dihydrolipoyl acetyltransferase (E2) component of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex was investigated. Only 1 mol of di-domain (binding domain) was bound to 1 mol of heterotetrameric E1 (alpha 2 beta 2) and the binding was without effect on the kinetic activity of E1. Similarly, the di-domain bound to separate E1 beta subunits at a maximal polypeptide chain ratio of 1:2, but no detectable interaction was found with the E1 alpha subunit. However, addition of the monomeric E1 alpha subunit to an E1 beta-di-domain complex generated a fully functional E1 (alpha 2 beta 2)-di-domain complex, indicating that the E1 beta subunit plays the critical part in binding the E1 component to the di-domain and suggesting that no chaperonin is needed in vitro to promote the assembly of the three separate proteins. Mixing the E1 and dihydrolipoyl dehydrogenase (E3) components in the presence of di-domain revealed that E1 and E3 cannot bind simultaneously to the same molecule of di-domain, a new feature of the assembly pathway and an important factor in determining the ultimate structure of the assembled enzyme complex.
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PMID:Interaction of component enzymes with the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: stoichiometry and specificity in self-assembly. 770 67

We have used expression of human medium chain acyl-CoA dehydrogenase (MCAD) in Escherichia coli as a model system for dissecting the molecular effects of two mutations detected in patients with MCAD deficiency. We demonstrate that the R28C mutation predominantly affects polypeptide folding. The amounts of active R28C mutant enzyme produced could be modulated between undetectable to 100% of the wild-type control by manipulating the level of available chaperonins and the growth temperature. For the prevalent K304E mutation, however, the amounts of active mutant enzyme could be modulated only in a range from undetectable to approximately 50% of the wild-type, and the assembled mutant enzyme displayed a decreased thermal stability. Two artificially constructed mutants (K304Q and K304E/D346K) yielded clearly higher amounts of active MCAD enzyme than the K304E mutant but were also responsive to chaperonin co-overexpression and growth at low temperature. The thermal stability profile of the K304E/D346K double mutant was shifted to even lower temperatures than that of the K304E mutant, whereas that of the K304Q mutant was closely similar to the wild-type. Taken together, the results show that the K304E mutation affects (i) polypeptide folding due to elimination of the positively charged lysine and (ii) oligomer assembly and stability due to replacement of lysine 304 with the negatively charged glutamic acid.
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PMID:Effects of two mutations detected in medium chain acyl-CoA dehydrogenase (MCAD)-deficient patients on folding, oligomer assembly, and stability of MCAD enzyme. 773 Mar 33

Chloroplasts contain a 21-kDa co-chaperonin polypeptide (cpn21) formed by two GroES-like domains fused together in tandem. Expression of a double-domain spinach cpn21 in Escherichia coli groES mutant strains supports growth of bacteriophages lambda and T5, and will also suppress a temperature-sensitive growth phenotype of a groES619 strain. Each domain of cpn21 expressed separately can function independently to support bacteriophage lambda growth, and the N-terminal domain will additionally suppress the temperature-sensitive growth phenotype. These results indicate that chloroplast cpn21 has two functional domains, either of which can interact with GroEL in vivo to facilitate bacteriophage morphogenesis. Purified spinach cpn21 has a ring-like toroidal structure and forms a stable complex with E. coli GroEL in the presence of ADP and is functionally interchangeable with bacterial GroES in the chaperonin-facilitated refolding of denatured ribulose-1,5-bisphosphate carboxylase. Cpn21 also inhibits the ATPase activity of GroEL. Cpn21 binds with similar efficiency to both the alpha and beta subunits of spinach cpn60 in the presence of adenine nucleotides, with ATP being more effective than ADP. The tandemly fused domains of cpn21 evolved early and are present in a wide range of photosynthetic eukaryotes examined, indicating a high degree of conservation of this structure in chloroplasts.
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PMID:Spinach chloroplast cpn21 co-chaperonin possesses two functional domains fused together in a toroidal structure and exhibits nucleotide-dependent binding to plastid chaperonin 60. 773 7


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