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.     

Transient kinetic analysis of ATP-induced allosteric transitions in the eukaryotic chaperonin containing TCP-1

The chaperonin CCT (chaperonin containing t-complex polypeptide 1 (TCP-1)) from bovine testis was mixed rapidly with different concentrations of ATP and the time-resolved change in fluorescence emission, upon excitation at 280 nm, was followed. Two kinetic phases were observed and assigned by (i) analyzing the dependence of the corresponding observed rate constants on ATP concentration; and (ii) by carrying out mixing experiments also with ADP, ATPgammaS and ATP without K(+). The values of the observed rate constants corresponding to both phases are found to be dependent on ATP concentration. The observed rate constant corresponding to the fast phase displays a bi-sigmoidal dependence on ATP concentration with Hill coefficients that are similar to those determined in steady-state ATPase experiments. This phase most likely reflects ATP binding-induced conformational changes. The rate constant of the conformational change in the presence of excess ATP is about 17s(-1) (at 25 degrees C) and is tenfold slower than the corresponding rate constant of GroEL. The observed rate constant corresponding to the second slower phase displays a hyperbolic dependence on ATP concentration. This phase is not observed in mixing experiments of CCT with ADP, ATPgammaS or ATP without K(+) and it, therefore, reflects a conformational change associated with ATP hydrolysis. Taken together, our results indicate that the kinetic mechanism of the allosteric transitions of CCT differs considerably from that of GroEL.     

Activities of the chaperonin containing TCP-1 (CCT): implications for cell cycle progression and cytoskeletal organisation.

The chaperonin containing TCP-1 (CCT) is required for the production of native actin and tubulin and numerous other proteins, several of which are involved in cell cycle progression. The mechanistic details of how CCT acts upon its folding substrates are intriguing: whilst actin and tubulin bind in a sequence-specific manner, it is possible that some proteins could use CCT as a more general binding interface. Therefore, how CCT accommodates the folding requirements of its substrates, some of which are produced in a cell cycle-specific manner, is of great interest. The reliance of folding substrates upon CCT for the adoption of their native structures results in CCT activity having far-reaching implications for a vast array of cellular processes. For example, the dependency of the major cytoskeletal proteins actin and tubulin upon CCT results in CCT activity being linked to any cellular process that depends on the integrity of the microfilament and microtubule-based cytoskeletal systems.     

Hemoglobin from the Antarctic fish Notothenia coriiceps neglecta. 1. Purification and characterisation

Antarctic fishes live at a constant temperature of -1.8 degrees C, in an oxygen-rich environment. In comparison with fishes that live in temperate or tropical waters, their blood contains less erythrocytes and hemoglobin. A study was initiated on the structure and function of Antarctic fish hemoglobin. The erythrocytes of the Antarctic benthic teleost Notothenia coriiceps neglecta, of the family Nototheniidae, have been shown to contain two hemoglobins, accounting for about 90% and 5% of the total content. These hemoglobins have been isolated, and obtained in crystalline form. They are tetramers and contain two pairs of globin chains. The globin chains of each hemoglobin have been purified and characterised. The two hemoglobins appear to have one of the two globin chains in common. The Root and Bohr effects have been investigated in erythrocytes, 'stripped' hemolysates and pure hemoglobins, indicating that the functional properties are finely regulated by pH and allosteric effectors.     

Differential effects of co-chaperonin homologs on cpn60 oligomers

In this study, we have investigated the relationship between chaperonin/co-chaperonin binding, ATP hydrolysis, and protein refolding in heterologous chaperonin systems from bacteria, chloroplast, and mitochondria. We characterized two types of chloroplast cpn60 oligomers, ch-cpn60 composed of α and β subunits (α₇β₇ ch-cpn60) and one composed of all β subunits (β₁₄ ch-cpn60). In terms of ATPase activity, the rate of ATP hydrolysis increased with protein concentration up to 60 μM, reflecting a concentration at which the oligomers are stable. At high concentrations of cpn60, all cpn10 homologs inhibited ATPase activity of α₇β₇ ch-cpn60. In contrast, ATPase of β₁₄ ch-cpn60 was inhibited only by mitochondrial cpn10, supporting previous reports showing that β₁₄ is functional only with mitochondrial cpn10 and not with other cpn10 homologs. Surprisingly, direct binding assays showed that both ch-cpn60 oligomer types bind to bacterial, mitochondrial, and chloroplast cpn10 homologs with an equal apparent affinity. Moreover, mitochondrial cpn60 binds chloroplast cpn20 with which it is not able to refold denatured proteins. Protein refolding experiments showed that in such instances, the bound protein is released in a conformation that is not able to refold. The presence of glycerol, or subsequent addition of mitochondrial cpn10, allows us to recover enzymatic activity of the substrate protein. Thus, in our systems, the formation of co-chaperonin/chaperonin complexes does not necessarily lead to protein folding. By using heterologous oligomer systems, we are able to separate the functions of binding and refolding in order to better understand the chaperonin mechanism.     

Solution structure of MT_nc,a novel metallothionein from the Antarctic fish Notothenia coriiceps

The structure of [113Cd(7)]-metallothionein (MT_nc) of the Antarctic fish Notothenia coriiceps, the first three-dimensional structure of a fish metallothionein, was determined by homonuclear 1H NMR experiments and heteronuclear [1H, 113Cd]-correlation spectroscopy. MT_nc is composed of an N-terminal beta domain with 9 cysteines and 3 metal ions and a carboxy-terminal alpha-domain with 11 cysteines and 4 metal ions. The position of the ninth Cys of the alpha domain of MT_nc is different from the corresponding Cys of mammalian MTs. As a result, the last CXCC motif in the mammalian MT sequence becomes CXXXCC in the fish MT. This difference leads to a structural change of the alpha domain and, in turn, to a different charge distribution with respect to that observed in mammalian metallothioneins.     

Stability and conformational dynamics of metallothioneins from the antarctic fish Notothenia coriiceps and mouse

The structural properties and the conformational dynamics of antarctic fish Notothenia coriiceps and mouse metallothioneins were studied by Fourier-transform infrared and fluorescence spectroscopy. Infrared data revealed that the secondary structure of the two metallothioneins is similar to that of other metallothioneins, most of which lack periodical secondary structure elements such as alpha-helices and beta-sheets. However, the infrared spectra of the N. coriiceps metallothionein indicated the presence of a band, which for its typical position in the spectrum and for its sensitivity to temperature was assigned to alpha-helices whose content resulted in 5% of the total secondary structure of the protein. The short alpha-helix found in N. coriiceps metallothionein showed an onset of denaturation at 30 degrees C and a T(m) at 48 degrees C. The data suggest that in N. coriiceps metallothionein a particular cysteine is involved in the alpha-helix and in the metal-thiolate complex. Moreover, infrared spectra revealed that both proteins investigated possess a structure largely accessible to the solvent. The time-resolved fluorescence data show that N. coriiceps metallothionein possesses a more flexible structure than mouse metallothionein. The spectroscopic data are discussed in terms of the biological function of the metallothioneins.     

Sequential ATP-induced allosteric transitions of the cytoplasmic chaperonin containing TCP-1 revealed by EM analysis

The eukaryotic cytoplasmic chaperonin containing TCP-1 (CCT) is a hetero-oligomeric complex that assists the folding of actins, tubulins and other proteins in an ATP-dependent manner. To understand the allosteric transitions that occur during the functional cycle of CCT, we imaged the chaperonin complex in the presence of different ATP concentrations. Labeling by monoclonal antibodies that bind specifically to the CCTalpha and CCTdelta subunits enabled alignment of all the CCT subunits of a given type in different particles. The analysis shows that the apo state of CCT has considerable apparent conformational heterogeneity that decreases with increasing ATP concentration. In contrast with the concerted allosteric switch of GroEL, ATP-induced conformational changes in CCT are found to spread around the ring in a sequential fashion that may facilitate domain-by-domain substrate folding. The approach described here can be used to unravel the allosteric mechanisms of other ring-shaped molecular machines.     

Electrophoretic analysis of Antarctic fish from South Georgia

White muscle samples of Antarctic fish ( Notothenia rossii, N., neglecta. N. gibbe-rifrons and Chaenocephalus (aceratus ) were subjected to electrophoretic tests. lsoelectric focusing of general muscle proteins provides a quick method of iden-tyfying species. Six muscle enzymes were resolved by horizontal starch gel clectrophoresis. These are presumed to be the products of ten independent gene loci. Coefficients of genetic distance were calculated and substantially agree with the conventional taxnomy. Levels of genetic variability are within the normal range for marine teleosts. Two polymorphic enzymes. GPI and PGM. might prove useful as 'genetic tags' for stock identification.     

Hemoglobin from the Antarctic fish Notothenia coriiceps neglecta. 2. Amino acid sequence of the alpha chain of Hb1

The complete amino acid sequence of the alpha chain of the main hemoglobin of the Antarctic fish Notothenia coriiceps neglecta (family Nototheniidae) has been determined. It consists of 142 residues; an acetylated seryl residue is at the amino terminal. The molecular mass is 15,519 Da. In comparison with alpha-chain sequences of non-Antarctic poikilothermic fish hemoglobins, the homology appears to be significantly lower than that existing among the latter species. A higher homology has been found with the alpha-chain sequence of the non-poikilothermic bluefin tuna.     

Cpn20: Siamese twins of the chaperonin world

The chloroplast cpn20 protein is a functional homolog of the cpn10 co-chaperonin, but its gene consists of two cpn10-like units joined head-to-tail by a short chain of amino acids. This double protein is unique to plastids and was shown to exist in plants as well plastid-containing parasites. In vitro assays showed that this cpn20 co-chaperonin is a functional homolog of cpn10. In terms of structure, existing data indicate that the oligomer is tetrameric, yet it interacts with a heptameric cpn60 partner. Thus, the functional oligomeric structure remains a mystery. In this review, we summarize what is known about this distinctive chaperonin and use a bioinformatics approach to examine the expression of cpn20 in Arabidopsis thaliana relative to other chaperonin genes in this species. In addition, we examine the primary structure of the two homologous domains for similarities and differences, in comparison with cpn10 from other species. Lastly, we hypothesize as to the oligomeric structure and raison d’être of this unusual co-chaperonin homolog. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Quantitative analysis of the impact of a human pathogenic mutation on the CCT5 chaperonin subunit using a proxy archaeal ortholog

The human chaperonin complex is a ~ 1 MDa nanomachine composed of two octameric rings formed from eight similar but non-identical subunits called CCT. Here, we are elucidating the mechanism of a heritable CCT5 subunit mutation that causes profound neuropathy in humans. In previous work, we introduced an equivalent mutation in an archaeal chaperonin that assembles into two octameric rings like in humans but in which all subunits are identical. We reported that the hexadecamer formed by the mutant subunit is unstable with impaired chaperoning functions. This study quantifies the loss of structural stability in the hexadecamer due to the pathogenic mutation, using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The disassembly of the wild type complex, which is tightly coupled with subunit denaturation, was decoupled by the mutation without affecting the stability of individual subunits. Our results verify the effectiveness of the homo-hexadecameric archaeal chaperonin as a proxy to assess the impact of subtle defects in heterologous systems with mutations in a single subunit.     

Specific immunity proteomic profile of the skin mucus of Antarctic fish Chionodraco hamatus and Notothenia coriiceps

The white-blooded Antarctic icefish is the only known vertebrate lacking oxygen-transporting haemoglobins. Fish skin mucus, as the first line of defence against pathogens, can reflect fish welfare. In this study, we analysed the skin mucus proteome profiles of the two Antarctic fish species, the white-blooded Antarctic icefish, Chionodraco hamatus, and the red-blooded Antarctic fish, Notothenia coriiceps, unfolding the different proteins by liquid chromatography coupled with tandem mass spectrometry isobaric tags for relative and absolute quantitation (iTRAQ) technology. Of the 4444 totally identified proteins, 227 differentially expressed proteins (DEPs) were found in the comparison between C. hamatus and N. coriiceps, of which 121 were upregulated and 106 were downregulated in the icefish. In the Kyoto Encyclopedia of Genes and Genomes pathway annotation, we found two pathways “Legionellosis” and “Complement and coagulation cascades” were significantly enriched, among of which innate immune candidate proteins such as C3, CASP1, ASC, F3 and C9 were significantly upregulated, suggesting their important roles in C. hamatus immune system. Additionally, the DEP protein–protein interaction network analysis and “Response to stress” GO category provided candidate biomarkers for deep understanding of the distinct immune response of the two Antarctic fish underlying the cold adaptation.     

Eco-morphological patterns of the sagitta of Antarctic fish

Morphology and morphometry of the sagittae otolith were studied in pelagic and mesopelagic fish. The shape, margins and rostrum of four groups of otoliths from several species were analyzed: group 1 (pelagic fish associated with the under ice cover N = 42), group 2 (pelagic fish associated with water offshore N = 9), group 3 (mesopelagic fish associated with extensive vertical migration N = 57) and group 4 (mesopelagic fish associated with short vertical migration N = 54). E (maximum width of the sagitta /maximum length of the sagitta %), R (rostrum length (RL)/maximum length of the sagitta %) and S (sulcus area (SS)/otolith area (OS) %) indexes were calculated for each species. Sagittae of pelagic groups (1 and 2) showed the smallest sagitta dimensions in relation to the total length of the fish, in this group the sagitta shape is variable. Sagittae of mesopelagic fish (groups 3 and 4) showed variable shape and edges. The shape in group 4 was polygonal and these species have more width than length. Statistical analysis showed significant differences in the E, R and S indexes. These results were compared with other 19 species, belonging to six families, taken from a publisher-edited literature. E, R and S-values could be used to characterize the sagittae of the Antarctic fish and could be considered as a useful tool for fish ecology studies.     

Chaperonin-containing TCP-1 complex directly binds to the cytoplasmic domain of the LOX-1 receptor

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a scavenger receptor that binds oxidized low-density lipoprotein (OxLDL) and has a role in atherosclerosis development. The N-terminus intracellular region (cytoplasmic domain) of LOX-1 mediates receptor internalization and trafficking, potentially through intracellular protein interactions. Using affinity isolation, we identified 6 of the 8 components of the chaperonin-containing TCP-1 (CCT) complex bound to LOX-1 cytoplasmic domain, which we verified by coimmunoprecipitation and immunostaining in human umbilical vein endothelial cells. We found that the interaction between CCT and LOX-1 is direct and ATP-dependent and that OxLDL suppressed this interaction. Understanding the association between LOX-1 and the CCT complex may facilitate the design of novel therapies for cardiovascular disease.     

Subunits of the chaperonin CCT interact with F-actin and influence cell shape and cytoskeletal assembly

The integrity of the cytoskeleton is closely linked to the oligomeric chaperonin containing TCP-1 (CCT) via the folding requirements of actin and tubulin, but the role of CCT in cytoskeletal organization remains unclear. We address this issue by analyzing the effects of targeting CCT subunits via siRNA and assessing their location/assembly state in cultured mammalian cells. Reducing levels of individual CCT subunits implicates CCT? in influencing cell shape and reduced levels of this subunit limit the cells' ability to recover from microfilament depolymerization. Conversely, cells displayed enhanced microtubule regrowth when CCT subunit levels were altered by siRNA. Some CCT subunits co-localize with F-actin, whilst all are predominantly monomeric in extracts enriched for the cytoskeleton. This provides compelling evidence that some CCT subunits as monomers can influence cytoskeletal organization/polymerization. Therefore the activity of CCT may well extend beyond the folding of newly synthesized polypeptides, representing a novel function for CCT subunits distinct from their role in the CCT oligomer.     

Identification of a novel helitron transposon in the genome of Antarctic fish

Rolling-circle (RC) eukaryotic transposons, known as helitrons, are found in a wide range of organisms, from protist to mammals. Autonomous helitrons have a distinctive open reading frame (ORF) encoding a polypeptide that contains typical domains for RC replication (RCR): the Rep (RCR initiator) and the DNA helicase domains. These elements are believed to have an important role in the host genome evolution, owing to their frequent capture of host genes, some of which can evolve into novel genes or become essential for helitron transposition. We conducted a molecular analysis of the suborder Notothenioidei, a group of Perciformes that currently dominate the Antarctic waters by virtue of their remarkable cold-adaptation ability. A novel helitron from the genome of the icefish species Chionodraco hamatus, belonging to the Channichthyidae, the most derived Notothenioids family, was isolated, characterized and designated as HeliNoto (8.9 kb). Its ORF was compared to homologous sequences from different species in a comprehensive phylogenetic analysis. For the first time the putative functional domains of a helitron were subjected to a well accurate structural analysis including chromosomal localization. Finally, the distribution of HeliNoto among Notothenioids was investigated.     

Posttranslational modification of brain tubulins from the Antarctic fish Notothenia coriiceps: reduced C-terminal glutamylation correlates with efficient microtubule assembly at low temperature

We have shown previously that the tubulins of Antarctic fish assemble into microtubules efficiently at low temperatures (-2 to +2 degrees C) due to adaptations intrinsic to the tubulin subunits. To determine whether changes in posttranslational glutamylation of the fish tubulins may contribute to cold adaptation of microtubule assembly, we have characterized C-terminal peptides from alpha- and beta-tubulin chains from brains of adult specimens of the Antarctic rockcod Notothenia coriiceps by MALDI-TOF mass spectrometry and by Edman degradation amino acid sequencing. Of the four fish beta-tubulin isotypes, nonglutamylated isoforms were more abundant than glutamylated isoforms. In addition, maximal glutamyl side-chain length was shorter than that observed for mammalian brain beta tubulins. For the nine fish alpha-tubulin isotypes, nonglutamylated isoforms were also generally more abundant than glutamylated isoforms. When glutamylated, however, the maximal side-chain lengths of the fish alpha tubulins were generally longer than those of adult rat brain alpha chains. Thus, Antarctic fish adult brain tubulins are glutamylated differently than mammalian brain tubulins, resulting in a more heterogeneous population of alpha isoforms and a reduction in the number of beta isoforms. By contrast, neonatal rat brain tubulin possesses low levels of glutamylation that are similar to that of the adult fish brain tubulins. We suggest that unique residue substitutions in the primary structures of Antarctic fish tubulin isotypes and quantitative changes in isoform glutamylation act synergistically to adapt microtubule assembly to low temperatures.     

Studies on the cytoplasmic protein tyrosine kinase activity of the Antarctic psychrotrophic bacterium Pseudomonas syringae

The Antarctic psychrotrophic bacterium Pseudomonas syringae contains a 66-kDa cytoplasmic protein which was found to by phosphorylated on a tyrosine residue [Ray, M.K. et al. (1994) FEMS Microbiol. Lett. 122, pp. 49-54]. To investigate the nature of the cytoplasmic protein tyrosine kinase and its role in the bacterial physiology, we carried out some biochemical studies of the enzyme in vitro in the presence of exogenous peptide substrates and expression studies in vivo at low and high temperature during various phases of growth. The results suggest that the protein tyrosine kinase associated with the cytoplasmic fraction of the bacterium has certain similarities and dissimilarities with the known eukaryotic tyrosine kinases. The protein tyrosine kinase could phosphorylate exogenous substrate corresponding to the N-terminal peptide of p34cdc2 kinase but could not do so on poly(Glu:Tyr). The enzyme could not be inhibited by genistein, staurosporine and dimethyl aminopurine, but could be inhibited by piceatannol which is a known competitive inhibitor of the peptide binding site of mammalian protein tyrosine kinases. The enzyme activity in the cytoplasm is uniquely inhibited by sodium orthovanadate (IC50 = 20 microM) which is a known protein tyrosine phosphatase inhibitor. The expression studies show that the enzyme is produced more at a higher temperature (22 degrees C) of growth than at lower temperature (4 degrees C) and during the stationary phase of growth of P. syringae.