Putative ATP-binding Domain Research Articles

Ovatodiolide inhibits SARS-CoV-2 replication and ameliorates pulmonary fibrosis through suppression of the TGF-β/TβRs signaling pathway

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to pose threats to public health. The clinical manifestations of lung pathology in COVID-19 patients include sustained inflammation and pulmonary fibrosis. The macrocyclic diterpenoid ovatodiolide (OVA) has been reported to have anti-inflammatory, anti-cancer, anti-allergic, and analgesic activities. Here, we investigated the pharmacological mechanism of OVA in suppressing SARS-CoV-2 infection and pulmonary fibrosis in vitro and in vivo. Our results revealed that OVA was an effective SARS-CoV-2 3CLpro inhibitor and showed remarkable inhibitory activity against SARS-CoV-2 infection. On the other hand, OVA ameliorated pulmonary fibrosis in bleomycin (BLM)-induced mice, reducing inflammatory cell infiltration and collagen deposition in the lung. OVA decreased the levels of pulmonary hydroxyproline and myeloperoxidase, as well as lung and serum TNF-ɑ, IL-1β, IL-6, and TGF-β in BLM-induced pulmonary fibrotic mice. Meanwhile, OVA reduced the migration and fibroblast-to-myofibroblast conversion of TGF-β1-induced fibrotic human lung fibroblasts. Consistently, OVA downregulated TGF-β/TβRs signaling. In computational analysis, OVA resembles the chemical structures of the kinase inhibitors TβRI and TβRII and was shown to interact with the key pharmacophores and putative ATP-binding domains of TβRI and TβRII, showing the potential of OVA as an inhibitor of TβRI and TβRII kinase. In conclusion, the dual function of OVA highlights its potential for not only fighting SARS-CoV-2 infection but also managing injury-induced pulmonary fibrosis.

PSMC1 variant causes a novel neurological syndrome.

Proteasome 26S, the eukaryotic proteasome, serves as the machinery for cellular protein degradation. It is composed of the 20S core particle and one or two 19S regulatory particles, composed of a base and a lid. To date, several human diseases have been associated with mutations within the 26S proteasome subunits; only one of them affects a base subunit. We now delineate an autosomal recessive syndrome of failure to thrive, severe developmental delay and intellectual disability, spastic tetraplegia with central hypotonia, chorea, hearing loss, micropenis and undescended testes, as well as mild elevation of liver enzymes. None of the affected individuals achieved verbal communication or ambulation. Ventriculomegaly was evident on MRI. Homozygosity mapping combined with exome sequencing revealed a disease‐associated p.I328T PSMC1 variant. Protein modeling demonstrated that the PSMC1 variant is located at the highly conserved putative ATP binding and hydrolysis domain, and is suggested to interrupt a hydrophobic core within the protein. Fruit flies in which we silenced the Drosophila ortholog Rpt2 specifically in the eye exhibited an apparent phenotype that was highly rescued by the human wild‐type PSMC1, yet only partly by the mutant PSMC1, proving the functional effect of the p.I328T disease‐causing variant.

Identification of cbiO Gene Critical for Biofilm Formation by MRSA CFSa36 Strain Isolated from Pediatric Patient with Cystic Fibrosis.

The colonization of Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), has a detrimental effect on the respiratory care of pediatric patients with cystic fibrosis (CF). In addition to being resistant to multiple antibiotics, S. aureus also has the ability to form biofilms, which makes the infection more difficult to treat and eradicate. In this study, we examined the ability of S. aureus strains isolated from pediatric patients with CF to form biofilms. We screened a transposon mutant library of MRSA and identified a putative cobalt transporter ATP binding domain (cbiO) that is required for biofilm formation. We discovered that deleting cbiO creating a cbiO null mutant in CFSa36 (an MRSA strain isolated from a patient with cystic fibrosis) significantly hinders the ability of CFSa36 to form biofilm. The complementation of cbiO restored the ability of the cbiO deletion mutant to generate biofilm. Interestingly, we revealed that incorporating extra copper ions to the chemically defined medium (CDM) complemented the function of cbiO for biofilm formation in a dose-dependent manner, while the addition of extra iron ions in CDM enhanced the effect of cbiO null mutation on biofilm formation. In addition, neither the addition of certain extra amounts of copper ions nor iron ions in CDM had an impact on bacterial growth. Taken together, our findings suggest that cbiO mediates biofilm formation by affecting the transportation of copper ions in the MRSA CFSa36 strain. This study provides new insights into the molecular basis of biofilm formation by S. aureus.

Structural and functional mapping of Rtg2p determinants involved in retrograde signaling and aging of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae mitochondrial dysfunction induces retrograde signaling, a pathway of communication from mitochondria to the nucleus that promotes a metabolic remodeling to ensure sufficient biosynthetic precursors for replication. Rtg2p is a positive modulator of this pathway that is also required for cellular longevity. This protein belongs to the ASKHA superfamily, and contains a putative N-terminal ATP-binding domain, but there is no detailed structural and functional map of the residues in this domain that accounts for their contribution to retrograde signaling and aging. Here we use Decomposition of Residue Correlation Networks and site-directed mutagenesis to identify Rtg2p structural determinants of retrograde signaling and longevity. We found that most of the residues involved in retrograde signaling surround the ATP-binding loops, and that Rtg2p N-terminus is divided in three regions whose mutants have different aging phenotypes. We also identified E137, D158 and S163 as possible residues involved in stabilization of ATP at the active site. The mutants shown here may be used to map other Rtg2p activities that crosstalk to other pathways of the cell related to genomic stability and aging.

Antigenic group II chaperonin inMethanobrevibacter oralismay cross-react with human chaperonin CCT

Methanobrevibacter oralis is an archaeal species frequently isolated from sites of severe periodontitis. However, its pathogenic roles remain unclear. Here, we aimed to isolate group II chaperonin from M. oralis and examine its antigenicity. The genes encoding two chaperonin subunits (Cpn-1 and Cpn-2) were cloned from M. oralis using polymerase chain reaction and genome walking procedures. Recombinant proteins Cpn-1 and Cpn-2 were generated, and the reactivities of sera from patients with periodontitis were examined by Western immunoblotting. The open reading frames of Cpn-1 and Cpn-2 genes consisted of 1641 and 1614 base pairs, respectively. Putative ATP-binding domains conserved among the chaperonin family were observed in both genes. The deduced amino acid sequences of the two genes showed 28.8-40.0% identity to each of the subunits of human CCT (CCT1-8). Thirty and 29 of 36 patients' sera reacted with the recombinant Cpn-1 and recombinant Cpn-2, respectively. Western immunoblotting using antiserum against human CCT subunits indicated that anti-CCT3 and anti-CCT8 antibodies recognized recombinant Cpn-1. In addition, anti-CCT1, CCT3, CCT6, and CCT8 antibodies recognized an antigen of approximately 60 kDa in M. oralis. The results suggested that the chaperonin subunits of M. oralis were antigenic molecules that were recognized by periodontitis patients and that may cross-react with human chaperonin CCT.

Small-Molecule Activators of Insulin-Degrading Enzyme Discovered through High-Throughput Compound Screening

BackgroundHypocatabolism of the amyloid β-protein (Aβ) by insulin-degrading enzyme (IDE) is implicated in the pathogenesis of Alzheimer disease (AD), making pharmacological activation of IDE an attractive therapeutic strategy. However, it has not been established whether the proteolytic activity of IDE can be enhanced by drug-like compounds.Methodology/Principal FindingsBased on the finding that ATP and other nucleotide polyphosphates modulate IDE activity at physiological concentrations, we conducted parallel high-throughput screening campaigns in the absence or presence of ATP and identified two compounds—designated Ia1 and Ia2—that significantly stimulate IDE proteolytic activity. Both compounds were found to interfere with the crosslinking of a photoaffinity ATP analogue to IDE, suggesting that they interact with a bona fide ATP-binding domain within IDE. Unexpectedly, we observed highly synergistic activation effects when the activity of Ia1 or Ia2 was tested in the presence of ATP, a finding that has implications for the mechanisms underlying ATP-mediated activation of IDE. Notably, Ia1 and Ia2 activated the degradation of Aβ by ∼700% and ∼400%, respectively, albeit only when Aβ was presented in a mixture also containing shorter substrates.Conclusions/SignificanceThis study describes the first examples of synthetic small-molecule activators of IDE, showing that pharmacological activation of this important protease with drug-like compounds is achievable. These novel activators help to establish the putative ATP-binding domain as a key modulator of IDE proteolytic activity and offer new insights into the modulatory action of ATP. Several larger lessons abstracted from this screen will help inform the design of future screening campaigns and facilitate the eventual development of IDE activators with therapeutic utility.

Differential Modulation of Type-1, Type-2 and Type-3 Inositol (1,4,5)-Trisphosphate Receptors by ATP

Inositol (1,4,5)-trisphosphate receptors (InsP3R) are the predominant route of Ca2+ release in non-excitable cells and they play a vital role in regulating intracellular Ca2+ signals. There are three isoforms (InsP3R-1, InsP3R-2 and InsP3R-3) of InsP3R expressed in mammalian cells. This sequence diversity along with varied tissue distributions suggests that there are isoform-specific regulatory mechanisms. One such regulatory mechanism is the modulation of Ca2+ release from InsP3R by cytosolic ATP. ATP positively regulates all three InsP3R isoforms, but with distinct functional characteristics. We found that ATP was required for maximal InsP3-induced Ca2+ release from InsP3R-1 and InsP3R-3 while InsP3R-2 attained maximal activity in the absence of ATP. Furthermore, InsP3R-2 was more sensitive to ATP modulation than either InsP3R-1 or InsP3R-3. All three isoforms contain putative ATP binding domains, but the contributions of these sites to ATP modulation of InsP3R are poorly understood. InsP3R-1 contains two predicted ATP binding domains (ATPA, and ATPB) while InsP3R-2 and InsP3R-3 each express a single ATPB site. We examined the contributions of these ATP binding sites to the subtype-specific effects of ATP on InsP3R isoforms. ER Ca2+ measurements from permeabilized DT40 cells and single channel recordings of InsP3R were used to measure the effects of ATP on wild-type and mutated InsP3R. We found that ablation of the ATPB site in InsP3R-2 eliminated the enhancing effects of ATP on this isoform. Surprisingly, the positive effects of ATP were retained in InsP3R-1 and InsP3R-3 devoid of their respective ATP binding sites. ATP, therefore, differentially regulates the three InsP3R isoforms and likely regulates InsP3R-1 and InsP3R-3 via novel ATP binding sites. The implications of this differential regulation on Ca2+ signals would likely be determined by the relative ratios of the three isoforms expressed in a given cell.

Identification of Functional Domains in the RAD51L2 (RAD51C) Protein and Its Requirement for Gene Conversion

The RAD51 protein plays a key part in the process of homologous recombination through its catalysis of homologous DNA pairing and strand exchange. Additionally five novel mammalian RAD51-like proteins have been identified in mammalian cells, but their roles in homologous recombination are much less well established. These RAD51-like proteins form two different complexes, but only the RAD51L2 (RAD51C) protein is a part of both complexes. By using site-directed mutagenesis of RAD51L2, we show that non-conservative mutation of the putative ATP-binding domain severely reduces its function, whereas a conservative mutation shows partial loss of function. We find that the protein is localized to the nucleus by tagging RAD51L2 with the green fluorescent protein and provisionally identify a C-terminal domain that acts as a nuclear localization signal. Further, a RAD51L2-deficient cell line was found to have significantly reduced homology-directed repair of a DNA double-strand break by gene conversion. This recombination defect could be partially restored by ectopic expression of the human RAD51L2 protein. Therefore we have identified protein domains that are important for the correct functioning of RAD51L2 and have shown that there is a specific requirement for RAD51L2 in gene conversion in mammalian cells.

Mutational analysis of the Menkes copper P-type ATPase (ATP7A)

The Menkes protein (ATP7A; MNK) is a ubiquitous human copper-translocating P-type ATPase and it has a key role in regulating copper homeostasis. Previously we characterised fundamental steps in the catalytic cycle of the Menkes protein. In this study we analysed the role of several conserved regions of the Menkes protein, particularly within the putative cytosolic ATP-binding domain. The results of catalytic studies have indicated an important role of 1086 His in catalysis. Our findings provide a biochemical explanation for the most common Wilson disease-causing mutation (H1069Q in the homologous Wilson copper-translocating P-type ATPase). Furthermore, we have identified a unique role of 1230 Asp , within the DxxK motif, in coupling ATP binding and acylphosphorylation with copper translocation. Finally, we found that the Menkes protein mutants with significantly reduced catalytic activity can still undergo copper-regulated exocytosis, suggesting that only the complete loss of catalytic activity prevents copper-regulated trafficking of the Menkes protein.

Functional analysis of mutant and wild-type Drosophila origin recognition complex.

The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. We have reconstituted a functional recombinant Drosophila ORC and compared activities of the wild-type and several mutant ORC variants. Drosophila ORC is an ATPase, and our studies show that the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA(+) nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. We also provide evidence that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features among Drosophila and budding yeast ORC complexes.

XRCC2 Is a Nuclear RAD51-like Protein Required for Damage-dependent RAD51 Focus Formation without the Need for ATP Binding

The human XRCC2 gene was recently identified by its ability to complement a hamster cell line, irs1, which is sensitive to DNA-damaging agents and shows genetic instability. The XRCC2 protein is highly conserved in mammalian species and has structural features, including a putative ATP-binding domain (P-loop), consistent with membership of the RecA/RAD51 family of recombination-repair proteins. We show that a hybrid XRCC2-green fluorescent protein, which was found to be functional by complementation, localizes to the nucleus. We have established a functional link between XRCC2 and RAD51 by looking at damage-dependent RAD51 focus formation in the irs1 cell line. Little or no formation of RAD51 foci occurred in irs1. This effect was specific to the loss of XRCC2 because transfection of the gene into irs1 restored normal levels of focus formation. Surprisingly, XRCC2 genes carrying site-specific mutations in P-loop residues were found to be able to complement the XRCC2-deficient irs1 line for a number of different end points. We conclude that XRCC2 is important in the early stages of homologous recombination in mammalian cells to facilitate RAD51-dependent recombination repair but that it does not make use of ATP binding to promote this function.

The molecular biology of the group VIA Ca 2+-independent phospholipase A 2

The group VIA PLA2 is a member of the PLA2 superfamily. This enzyme, which is cytosolic and Ca2+-independent, has been designated iPLA2beta to distinguish it from another recently cloned Ca2+-independent PLA2. Features of iPLA2beta molecular structure offer some insight into possible cellular functions of the enzyme. At least two catalytically active iPLA2beta isoforms and additionalsplicing variants are derived from a single gene that consists of at least 17 exons located on human chromosome 22q13.1. Potential tumor suppressor genes also reside at or near this locus. Structural analyses reveal that iPLA2beta contains unique structural features that include a serine lipase consensus motif (GXSXG), a putative ATP-binding domain, an ankyrin-repeat domain, a caspase-3 cleavage motif DVTD138Y/N, a bipartite nuclear localization signal sequence, and a proline-rich region in the human long isoform. iPLA2beta is widely expressed among mammalian tissues, with highest expression in testis and brain. iPLA2beta prefers to hydrolyze fatty acid at the sn-2 fatty acid substituent but also exhibits phospholipase A1, lysophospholipase, PAF acetylhydrolase, and transacylase activities. iPLA2beta may participate in signaling, apoptosis, membrane phospholipid remodeling, membrane homeostasis, arachidonate release, and exocytotic membrane fusion. Structural features and the existence of multiple splicing variants of iPLA2beta suggest that iPLA2beta may be subject to complex regulatory mechanisms that differ among cell types. Further study of its regulation and interaction with other proteins may yield insight into how its structural features are related to its function.

Calcium transport across the sarcoplasmic reticulum: structure and function of Ca2+-ATPase and the ryanodine receptor.

Contraction of striated muscle results from a rise in cytoplasmic calcium concentration in a process termed excitation/contraction coupling. Most of this calcium moves back and forth across the sarcoplasmic-reticulum membrane in cycles of contraction and relaxation. The channel responsible for release from the sarcoplasmic reticulum is the ryanodine receptor, whereas Ca2+-ATPase effects reuptake in an ATP-dependent manner. The structures of these two molecules have been studied by cryoelectron microscopy, with helical crystals in the case of Ca2+-ATPase and as isolated tetramers in the case of ryanodine receptor. Structures of Ca2+-ATPase at 8-A resolution reveal the packing of transmembrane helices and have allowed fitting of a putative ATP-binding domain among the cytoplasmic densities. Comparison of ATPases in different conformations gives hints about the conformational changes that accompany the reaction cycle. Structures of ryanodine receptor at 30-A resolution reveal a multitude of isolated domains in the cytoplasmic portion, as well as a distinct transmembrane assembly. Binding sites for various protein ligands have been determined and conformational changes induced by ATP, calcium and ryanodine have been characterized. Both molecules appear to use large conformational changes to couple interactions in their cytoplasmic domains with calcium transport through their membrane domains, and future studies at higher resolution will focus on the mechanisms for this coupling.

Cloning and Chromosomal Mapping of the Human DNA Polymerase θ (POLQ), the Eighth Human DNA Polymerase

We have cloned the cDNA for the eighth human DNA polymerase, DNA polymerase θ. The human cDNA encodes a putative DNA polymerase of 1762 amino acids with a calculated molecular mass of 198 kDa. The derived protein sequence is homologous to the Drosophila melanogaster mus308 protein product, a putative DNA polymerase–helicase involved in repair of interstrand crosslinks. The C-terminal region contains the canonical DNA polymerase motifs A, B, and C found in the family A type of DNA polymerases, which includes Escherichia coli polymerase I. The N-terminal region contains a putative ATP binding domain but not motifs for a helicase. The gene was mapped by radiation hybrid analysis to chromosome 3q within an interval flanked by proximal marker D3S1303 and distal marker D3S3576 and, based on proximity to a gene that has been mapped cytogenetically, within band 3q13.31.

Glucose-induced autophagy of peroxisomes in Pichia pastoris requires a unique E1-like protein.

Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Delta had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(DeltaATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25-30 kDa protein. This conjugate was not observed in cells expressing Gsa7(DeltaATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.

Structure of pyridoxal kinase from sheep brain and role of the tryptophanyl residues.

The primary structure of sheep brain pyridoxal kinase has been determined by direct chemical and physical methods. The enzyme contains 312 amino acid residues with an acetylated methionine at the N-terminus, yielding a molecular mass of 34,861 Da. The functional role played by the two tryptophanyl residues in positions 52 and 244 of the polypeptide chain has been investigated by fluorescence spectroscopy. The tryptophanyl residues are not completely exposed to the rapidly relaxing solvent and they are poorly accessible to collisional quenchers. Chemical modification with NBS abolishes the catalytic activity of the kinase. The amino acid sequence of the sheep brain enzyme shows high similarity (86.2% identity) with the human pyridoxal kinase recently reported [Hanna, Turner, and Kirkness, (1997), J. Biol. Chem. 272, 10756-10760]. Comparison of the mammalian proteins with bacterial and yeast putative pyridoxal kinases retrieved from the Swiss-Prot data bank shows a low degree of overall similarity. In particular, the putative ATP-binding domain is conserved, whereas the region that appears to be crucial in the binding of the pyridoxal substrate is not. Thus, the assignment of the bacterial and yeast cDNA-deduced proteins as pyridoxal kinases should be taken with caution.

Molecular cloning and expression of the KIF3A gene in the frog brain and testis.

KIF3A is a member of the kinesin superfamily proteins (KIFs), but its gene has been cloned only in mouse and sea urchin. We have cloned a homolog of KIF3A from the frog, Rana rugosa (rrKIF3A). The sequence encoded a 699 amino acid protein that shares 93% similarity with mouse KIF3A (mKIF3A) and 69% with sea urchin kinesin-related protein (SpKRP85). The putative ATP-binding domain was completely identical to that of mKIF3A and SpKRP85. The level of rrKIF3A mRNA appeared to be high in the brain and testis of adult frogs, but low in the heart, lung and kidney. The results suggest that the rrKIF3A gene is expressed in the brain and testis more than other tissues of adult frogs examined, and that KIF3A is widely distributed in eukaryotic organisms.

Activation of the c-Jun N-terminal kinase pathway by a novel protein kinase related to human germinal center kinase.

The c-Jun N-terminal kinase (JNK), or stress-activated protein kinase plays a crucial role in cellular responses stimulated by environmental stress and proinflammatory cytokines. However, the mechanisms that lead to the activation of the JNK pathway have not been elucidated. We have isolated a cDNA encoding a novel protein kinase that has significant sequence similarities to human germinal center kinase (GCK) and human hematopoietic progenitor kinase 1. The novel GCK-like kinase (GLK) has a nucleotide sequence that encodes an ORF of 885 amino acids with 11 kinase subdomains. Endogenous GLK could be activated by UV radiation and proinflammatory cytokine tumor necrosis factor alpha. When transiently expressed in 293 cells, GLK specifically activated the JNK, but not the p42/44(MAPK)/extracellular signal-regulated kinase or p38 kinase signaling pathways. Interestingly, deletion of amino acids 353-835 in the putative C-terminal regulatory region, or mutation of Lys-35 in the putative ATP-binding domain, markedly reduced the ability of GLK to activate JNK. This result indicates that both kinase activity and the C-terminal region of GLK are required for maximal activation of JNK. Furthermore, GLK-induced JNK activation could be inhibited by a dominant-negative mutant of mitogen-activated protein kinase kinase kinase 1 (MEKK1) or mitogen-activated protein kinase kinase 4/SAPK/ERK kinase 1 (SEK1), suggesting that GLK may function upstream of MEKK1 in the JNK signaling pathway.

The third member of the Tetrahymena CCT subunit gene family, TpCCT alpha, encodes a component of the hetero-oligomeric chaperonin complex.

The sequence of a third member of the Tetrahymena pyriformis chaperonin CCT ('chaperonin containing TCP1') subunit gene family is presented. This gene, designated TpCCT alpha, is the orthologue of the mouse chaperonin gene TCP1/CCT alpha. To characterize the CCT complex in this ciliate, we have produced polyclonal antibodies against synthetic peptides based on C-terminal sequences deduced from the primary sequences of the TpCCT alpha, TpCCT gamma and TpCCT eta subunits. We have also used polyclonal antibodies produced against recombinant yeast CCT alpha and CCT beta subunits. Using these antibodies, we show that Tetrahymena cells contain a hetero-oligomeric CCT chaperonin comprising at least seven distinct subunits. Three of these were assigned to specific TpCCT genes, whereas a fourth was recognized by the polyclonal antibody against yeast CCT beta, suggesting that this gene is also present in the ciliate. The CCT complex also contains other unidentified proteins that were recognized by the polyclonal antibody UM-1, raised against the putative ATP binding domain of the chaperonin proteins. TpCCT alpha gene expression was shown in exponentially growing cells and cells regenerating their cilia for different periods to have a similar pattern to the previously identified genes TpCCT gamma and TpCCT eta, and also to tubulin genes.

Structural Motif and Characteristics of the Extracellular Domain of P2XReceptors

Seven sequences of the proteins recently cloned from rat tissues and identified as P2Xreceptors are examined. Their putative transmembrane and extracellular domains and the most probable locations of disulphide bridges are discussed. Some surface accessible regions are identified and the structure of the putative ATP binding domain is discussed in the light of known selectivities and physiological characteristics of individual P2Xsubunits. The summarised information and the theoretical considerations regarding the protein structure of P2Xreceptors have been used to select amino acid sequences for raising antibodies against the P2X1receptor subunit. Initial results suggest that, in rat brainstem, P2X1receptor subunits are found on a distinct subpopulation of neuronal perikarya in distributions consistent with distributions of known α,β-methylene-ATP autoradiography and physiological effects of purinoceptor activation.