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At least 235 records · Page 13Linked to original sources
PubMed1998

ATP binding induces large conformational changes in the apical and equatorial domains of the eukaryotic chaperonin containing TCP-1 complex.

The chaperonin-containing TCP-1 complex (CCT) is a heteromeric particle composed of eight different subunits arranged in two back-to-back 8-fold pseudo-symmetric rings. The structural and functional implications of nucleotide binding to the CCT complex was addressed by electron microscopy and image processing. Whereas ADP binding to CCT does not reveal major conformational differences when compared with nucleotide-free CCT, ATP binding induces large conformational changes in the apical and equatorial domains, shifting the latter domains up to 40 degrees (with respect to the inter-ring plane) compared with 10 degrees for nucleotide-free CCT or ADP-CCT. This equatorial ATP-induced shift has no counterpart in GroEL, its prokaryotic homologue, which suggests differences in the folding mechanism for CCT.

Adenosine Triphosphate↗
PubMed1998

The chaperone cofactor Hop/p60 interacts with the cytosolic chaperonin-containing TCP-1 and affects its nucleotide exchange and protein folding activities.

The folding of protein structures often requires the presence of molecular chaperones and/or chaperonin complexes. We here investigated the inhibitory effects of the chaperone cofactors Hop/p60 and Hap46. By coimmunoprecipitation, we observed a direct interaction of the eukaryotic chaperonin-containing TCP-1 (CCT) purified from rabbit reticulocyte lysate with Hop/p60. By contrast, Hap46 was not coprecipitated. Binding of Hop/p60 to CCT is dependent on the presence of ATP or ADP and occurs through carboxyl-terminal sequences of Hop/p60. Hop/p60 significantly stimulates nucleotide exchange on CCT but not its ATPase activity, while Hap46 has no effects. We used denatured firefly luciferase as a model protein and found decreased binding to CCT in the presence of Hop/p60 and ATP. This coincides with the inhibitory effect of Hop/p60 on luciferase reactivation in an assay using purified CCT in combination with hsc70 and hsp40. We also observed that an antibody directed against one of the subunits of CCT efficiently inhibits refolding in a system which depends on crude reticulocyte lysate.

Adenosine Diphosphate↗
PubMed1995

Characterization of four new tcp-1-related cct genes from the nematode Caenorhabditis elegans.

In this report we present the sequences of four new cct chaperonin genes from the nematode Caenorhabditis elegans. The four genes, cct-2, cct-4, cct-5, and cct-6 are orthologs of the mouse chaperonin genes Cctb, Cctd, Ccte, and Cctz, sharing 66%, 63%, 68%, and 67% deduced amino acid sequence identity, respectively. The C. elegans multigene family includes these four genes as well as cct-1 (tcp-1), and displays 23-35% pairwise predicted amino acid sequence identity between members, and 31-35% identity to the closely related archaebacterial chaperonin TF55. The five C. elegans cct genes are expressed in all life stages (egg, four larval stages, and adult). Members of the multigene family occur as a loosely associated group of three genes on chromosome II, and two widely separated genes on chromosome III. The predicted secondary structures of all five C. elegans CCT deduced protein sequences are nearly identical. Moreover, all chaperonins examined had comparable predicted secondary structures. Algorithmic predictions of the secondary structures of GroEL, Hsp60, and Rubisco subunit-binding protein (RuBP) are almost identical, and are very similar to the known GroEL secondary structure. The CCT/TF55 family predicted secondary structures are essentially identical to each other and are also related to GroEL, Hsp60, and RuBP. The most notable difference between the CCT/TF55 and the GroEL/Hsp60/RuBP families is in the presumed polypeptide binding domain.

Amino Acid Sequence↗
PubMed1996

Peptide mass fingerprinting of chaperonin-containing TCP-1 (CCT) and copurifying proteins.

The chaperonin-containing TCP-1 (CCT), found in the eukaryotic cytosol, is currently the focus of extensive research, CCT isolated from mouse testis lysate sediments at 20S in a sucrose gradient and accounts for about 70% of the total protein in this fraction. We intend to identify all the other proteins that copurify with CCT and to compile a reference profile for future studies. Their identification can be accelerated by a combination of protease digestion, matrix-assisted laser desorption-mass spectrometry, and database matching known as peptide mass fingerprinting. We applied this strategy to 32 polypeptides resolved by 2-dimensional gel electrophoresis, and 23 known proteins and 6 novel proteins were identified. We analyzed isoelectric variants of the CCT subunits and differences in the peptide mass spectra of two CCT theta isoforms indicated a novel posttranslational modification of this subunit.

Amino Acid Sequence↗
PubMed2002

Assembly of the SMRT-histone deacetylase 3 repression complex requires the TCP-1 ring complex.

The acetylation of histone tails is a primary determinant of gene activity. Histone deacetylase 3 (HDAC3) requires the nuclear receptor corepressor SMRT for HDAC enzyme activity. Here we report that HDAC3 interacts with SMRT only after priming by cellular chaperones including the TCP-1 ring complex (TRiC), which is required for proper folding of HDAC3 in an ATP-dependent process. SMRT displaces TRiC from HDAC3, yielding an active HDAC enzyme. The SMRT-HDAC3 repression complex thus joins the VHL-elongin BC tumor suppression complex and the cyclin E-Cdk2 cell cycle regulation complex as critical cellular machines requiring TRiC for proper assembly and function. The strict control of HDAC3 activity underscores the cellular imperative that histone deacetylation occur only in targeted regions of the genome.

Adenosine Triphosphate↗
PubMed2001

Nested allosteric interactions in the cytoplasmic chaperonin containing TCP-1.

Initial rates of ATP hydrolysis by the chaperonin containing TCP-1 (CCT) from bovine testis were measured as a function of ATP concentration. Two allosteric transitions are observed: one at relatively low concentrations of ATP (<100 microM) and the second at higher concentrations of ATP. The data suggest that CCT has positive intra-ring cooperativity and negative inter-ring cooperativity in ATP hydrolysis, with respect to ATP, as previously observed in the case of GroEL. It is shown that the relatively weak positive intra-ring cooperativity found in the case of CCT may be due to heterogeneity in its subunit composition. Our results suggest that nested allosteric behavior may be common to chaperone double-ring systems.

Adenosine Triphosphate↗
PubMed1995

The chaperonin containing t-complex polypeptide 1 (TCP-1). Multisubunit machinery assisting in protein folding and assembly in the eukaryotic cytosol.

Many proteins in the cell require assistance from molecular chaperones at stages in their life cycles in order to attain correctly folded states and functional conformations during protein synthesis or during recovery from denatured states. A recently discovered molecular chaperone, which is abundant in the eukaryotic cytosol and is called the chaperonin containing TCP-1 (CCT), has been shown to assist the folding of some proteins in cytosol. This chaperone is a member of the chaperonin family which includes GroEL, 60-kDa heat shock protein (Hsp60), Rubisco subunit binding protein (RBP) and thermophilic factor 55 (TF55), but is distinct from the other members in several respects. Presently the most intriguing feature is the hetero-oligomeric nature of the CCT; at least eight subunit species which are encoded by independent and highly diverged genes are known. These genes are calculated to have diverged around the starting point of the eukaryotic lineage and they are maintained in all eukaryotes investigated, suggesting a specific function for each subunit species. The amino acid sequences of these subunits share approximately 30% identity and have some highly conserved motifs probably responsible for ATPase function, suggesting this function is common to all subunits. Thus, each subunit is thought to have both specific and common functions. These observations, in conjunction with biochemical and genetic analysis, suggest that CCT functions as a very complex machinery for protein folding in the eukaryotic cell and that its chaperone activity may be essential for the folding and assembly of various newly synthesized polypeptides. This complex behaviour of CCT may have evolved to cope with the folding and assembly of certain highly evolved proteins in eukaryotic cells.

Amino Acid Sequence↗
PubMed1998

The chaperonin containing TCP-1 (CCT) displays a single-ring mediated disassembly and reassembly cycle.

The chaperonin-containing TCP-1 (CCT) assists in the folding of actins and tubulins in eukaryotic cells. CCT is composed of 8 subunit species encoded by separate genes. CCT purifies as a single hetero-oligomeric protein complex of 950 kDa through multiple chromatographic and antibody affinity procedures. The CCT 16-mer contains 7 polypeptide species in equimolar amounts (CCTalpha, beta, gamma, delta, epsilon, zeta, eta), together with another subunit (CCTtheta) which is around half-molar. Here we show, by in vitro translation of CCT subunit mRNAs in rabbit reticulocyte lysate, that none of the CCT subunit proteins are themselves folded by CCT. However, the newly translated CCT subunits can incorporate into the endogenous CCT complex present in the lysate via a mechanism involving a nucleotide-dependent disassembly reaction to produce single-rings and then a reassembly reaction whereby free CCT subunits assemble onto these single-rings. This cycling behaviour is an inherent property of the CCT chaperonin complex and provides a powerful method for introducing single amino acid residue changes into this 8578 residue protein complex.

Adenosine Diphosphate↗
PubMed1997

Bioactive bone cement: comparison of AW-GC filler with hydroxyapatite and beta-TCP fillers on mechanical and biological properties.

Three types of bioactive bone cement (designated AWC, HAC, and TCPC), each consisting of bisphenol-alpha-glycidyl methacrylate (Bis-GMA)-based resin and a bioactive filler of apatite and wollastonite containing glass-ceramic (AW-GC), sintered hydroxyapatite (HA), or beta-tricalcium phosphate (beta-TCP) powder were made in order to evaluate the influence of the bioactive filler on the mechanical and biological properties of bone cement. The proportion of filler added to the cements was 70% w/w. The compressive, bending, and tensile strengths and the fracture toughness of AWC were higher than HAC and TCPC under wet conditions. The cements were evaluated in vivo by packing them into the intramedullary canals of rat tibiae. An affinity index that equalled the length of bone in direct apposition to the cement was calculated for each cement and expressed as a percentage of the total length of the cement surface. Histological examination of rat tibiae up to 8 weeks after implantation revealed that AWC had higher bioactivity than HAC and TCPC. New bone had formed along the AWC surface within 2 weeks, and at 4 weeks newly formed bone surrounded the cement surface almost completely. In HAC- and TCPC-implanted tibiae, immature bone had formed directly toward but not along the cement surface at 2 weeks. Observation of cement-bone interfaces showed that AWC had bonded to the bone via a so-called "Ca-P-rich layer"; the cement-bone interface remained stable, and the width of the CA-P-rich layer became thicker with time. On the other hand, in HAC- and TCPC-implanted tibiae, the cement surface fillers were surrounded by new bone and were absorbed gradually to become bone matrix. The cement-bone interfaces went inside the cement with time. Our results indicate that stronger interstitial bonding between the inorganic filler and the organic matrix resin in AWC lead to higher mechanical properties; results also indicate that the more stable cement-bone interface and higher bioactivity of AWC are due to early and uniform apatite formation on the cement surface.

Animals↗
PubMed2005

Degradation of poly-L/DL-lactide versus TCP composite pins: a three-year animal study.

Biodegradable polylactide implants allow secure fixation of osteochondral fractures. This quasirandomized parallel-group animal study investigates whether a composite implant of poly-L/DL-lactide (PLDLLA) with additional 10% beta-tricalcium phosphate produces an osteoconductive effect, whether the admixture positively influences implant degradation (assessed by comparing time to implant degradation), and whether the pin sites of degraded implants are replaced with bone tissue. On 36 medial femoral condyles of sheep, osteotomies were fixed with either three PLDLLA pins or three composite pins. At 3, 18, and 36 months, the pin sites were measured histologically and the state of degradation assessed according to Pistner's classification. All fractures healed without clinically relevant complications and without displacement. Both pin types led to asymptomatic pin-site enlargement at 18 months, which disappeared by 36 months. At 18 months, 14 of 18 PLDLLA pins were no longer evident, whereas 16 of 18 composite pins showed only peripheral degradation. By 36 months, all implants from both groups were completely degraded and replaced with scar (2/18) or bone tissue (16/18). At 36 months there was no evidence for significant improvement of either degradation performance or osseointegration through use of the 10% TCP composite mixture.

Animals↗
PubMed1997

A search for cholera toxin (CT), toxin coregulated pilus (TCP), the regulatory element ToxR and other virulence factors in non-01/non-0139 Vibrio cholerae.

Twenty-four selected non-O1/non-O139 Vibrio cholerae strains were examined for the presence of virulence associated genes like ctxA, tcpA, toxR and the repetitive sequence (RS element). Seventeen of these were isolated from diarrhoeal stool samples while the remaining seven were of local environmental origin. Nine and four respectively of these strains were positive for ctxA and tcpA by Multiplex PCR analysis. The majority (16 out of 18 tested) of the strains (including the four tcpA + strains) contained toxR sequences as determined by another PCR assay. The presence of RS element was demonstrable in ctxA+ strains only. Interestingly, three of these non-O1/non-O139 strains were shown to contain all the three virulence associated genes (ctxA, tcpA and toxR) as well as the RS element. Two of these belonged to serogroups 037 (V2) and 064 (CG15) while the third one (V315-1) was untypable. These three strains also produced cholera toxin, expressed toxin coregulated pilus (TCP) and/or TcpA related antigens when grown under appropriate culture conditions. Southern hybridization analysis of their chromosomal DNA fragments using DNA probes representing ctxA, zot, ace and RS element revealed that the strains V2 and CG15 contained, at least, two complete copies of the CTX genetic element, while the strain V315-1 had three or more copies of the same. Presence of the RS element in these strains led to tandem duplication of the CTX genetic element in the chromosome of V2 and V315-1, but not in CG15 where the copies were likely to be present at different loci. These results also indicate the presence of additional copies of incomplete "core region' with zot and ace genes, but not ctxA, in strains V2 and CG15. The significance of these results in terms of the pathogenic and epidemic potential of V. cholerae strains is discussed.

Animals↗
PubMed2001

Analysis of a 2,4,6-trichlorophenol-dehalogenating enrichment culture and isolation of the dehalogenating member Desulfitobacterium frappieri strain TCP-A.

An anaerobic, 2,4,6-trichlorophenol ortho-dehalogenating mixed culture was enriched from sediment of the river Saale (Germany). Two isolated dechlorinating colonies (MK1 and MK2) consisted of rods of different lengths and thicknesses, indicating heterogeneity. Following subcultivation with thiosulfate as alternative electron acceptor and cocultivation with Clostridium celerecrescensT, the 2,4,6-trichlorophenol-dehalogenating bacterium Desulfitobacterium frappieri strain TCP-A was isolated and characterized regarding its taxonomic properties and the spectrum of chlorophenols that it dehalogenated. Four other bacterial strains were coenriched and identified as organisms with closest phylogenetic relatedness to the Clostridium type strains C. indolis, C. glycolicum, C. hydroxybenzoicum and C. sporosphaeroides (16S rDNA sequence identities of 99.5, 99.2, 94.4, and 93.5%, respectively). Amplified ribosomal DNA restriction analysis of the original dehalogenating cultures MK1 and MK2 (when not exposed to thiosulfate) confirmed the microbial heterogeneity and revealed the presence of two additional species related to the type strains of C. celerecrescens and Clostridium propionicum. Only one copy of the 16S rRNA genes of Desulfitobacterium frappieri in each of the clone libraries of MK1 and MK2 (containing 136 and 56 clones, respectively) was found by dot-blot hybridization, suggesting a relatively low number of the dehalogenating bacterium within the enrichment culture.

Anaerobiosis↗
PubMed1987

Quantitative autoradiographic localization of NMDA receptors in rat brain using [3H]CPP: comparison with [3H]TCP binding sites.

The regional distribution of N-methyl-D-aspartate (NMDA) receptors in rat brain using the selective NMDA receptor ligand [3H]3-[+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) has been quantitated by in vitro autoradiography. [3H]CCP binding was highest in the CA1 region of the hippocampus. Relatively high levels of binding were also observed in the cerebral cortex, while moderate binding was obtained in the thalamus, striatum and granule cell layer of the cerebellum. Concurrent studies examining the phencyclidine receptor ligand [3H]1-[1-(2-thienyl)cyclohexyl]-piperidine (TCP), revealed a similar pattern of binding that correlated well with the localization of [3H]CPP-labeled NMDA receptors (r = 0.88, P less than 0.01).

Animals↗
PubMed1995

Antibody characterisation of two distinct conformations of the chaperonin-containing TCP-1 from mouse testis.

We describe a panel of antibodies specific to individual subunits of the chaperonin-containing TCP-1 (CCT) and one antibody that reacts with all the subunits of CCT. Immunoblot analysis of CCT purified from mouse testis suggests that the testis-specific subunit, S6, may be related to CCT zeta and that a co-purifying 63 kDa protein may be a novel subunit of CCT. Using these antibodies in the analysis of CCT subjected to nondenaturing IEF we observed the resolution of two distinct conformations of CCT, which differ in their susceptibility to proteolysis and in the number of associated polypeptides.

Animals↗
PubMed1995

WIN 63480, a hydrophilic TCP-site ligand, has reduced agonist-independent NMDA ion channel access compared to MK-801 and phencyclidine.

NMDA channel blockers are potentially advantageous therapeutic agents for the treatment of ischemia and head trauma, which greatly elevate extracellular glutamate, because they should most effectively inhibit high levels of receptor activation. A novel high affinity TCP site ligand, WIN 63480, does not produce MK-801- or PCP-like behavioral activation at anti-ischemic doses. While WIN 63480, MK-801 and PCP were all observed to be effective blockers of open NMDA channels, WIN 63480 had much less access to closed NMDA channels. This difference may be due to the fact that WIN 63480 is hydrophilic (logD = -4.1) while MK-801 and PCP are lipophilic (logD = +1.8). In vivo, closed channel access may result in a non-competitive profile of antagonism for MK-801 and PCP compared to a more uncompetitive profile for WIN 63480. Release of glutamate, and depolarization, are likely to produce a high level of NMDA receptor activation in ischemic areas compared to normal tissue. Consequently, at anti-ischemic doses, WIN 63480 may produce less inhibition of physiological NMDA-mediated processes in neural systems involved in behavioral regulation than MK-801 or PCP, leading to an improved side effect profile.

Animals↗
PubMed1987

Glycine potentiates N-methyl-D-aspartate-induced [3H]TCP binding to rat cortical membranes.

In extensively washed preparations of rat cortical membranes, N-methyl-D-aspartate (NMDA) increases the specific binding of [3H]TCP by over 4-fold in a concentration dependent manner (EC50 = 3.1 microM). Glycine (1 microM) potentiates the maximal effect of NMDA by a factor of 1.7. The effect of glycine is concentration dependent (EC50 = 380 nM) and strychnine insensitive. These data are discussed with reference to the recently reported effects of glycine on the NMDA operated cation channel and the relationship between the PCP and NMDA receptors.

Animals↗
PubMed1989

Ligand-binding characteristics of [3H]QNB, [3H]prazosin, [3H]rauwolscine, [3H]TCP and [3H]nitrendipine to cerebral cortical and hippocampal membranes of senescence accelerated mouse.

The senescence accelerated mouse (SAM) is known as a murine model of aging and memory dysfunction. In the cerebral cortical membranes of male 9-month-old SAM mice, the Bmax values of [3H]rauwolscine and [3H]nitrendipine binding, and the values of both Kd and Bmax of [3H]TCP binding in the accelerated aging strain SAM-P/8, were significantly increased compared with the values in the control strain SAM-R/1. In hippocampal membranes, however, the Bmax values of [3H]quinuclidinyl benzilate and [3H]nitrendipine binding were significantly decreased in SAM-P/8 compared with those in SAM-R/1. These results suggest that muscarinic acetylcholine receptors, alpha 2-adrenoceptors, N-methyl-D-aspartate receptor channels and L-type Ca2+ channels are changed in cerebral cortex and hippocampus in SAM-P/8 at 9 months.

Aging↗