The cytosolic chaperonin CCT associates to cytoplasmic microtubular structures during mammalian spermiogenesis and to heterochromatin in germline and somatic cells

Spermiogenesis, the haploid phase of spermatogenesis, is characterised by a dramatic cytodifferentiation of spermatids. The two major steps, nuclear shaping and cytoplasmic reorganisation of the organelles, rely on an extensive remodelling of the microtubule cytoskeleton. Folding of alpha- and beta-tubulin is mediated by the cytoplasmic chaperonin containing TCP-1 (CCT), highly expressed in testis. We studied CCT cellular distribution throughout spermatogenesis by immunofluorescence and immunoelectron microscopy. We unveil two main cytoplasmic localisations for CCT: at the centrosome and at the microtubules of the manchette, a structure unique to male germ cells. Both structures are essential for spermatid differentiation and may require CCT function. Although CCT is essentially cytoplasmic, a few reports suggest that a subset may have a nuclear localisation. We demonstrate that in the nucleus of germline and somatic cells, part of CCT associates to heterochromatin. In interphase cells, CCT seems generally confined to constitutive heterochromatin. Nevertheless, in condensing nucleus of future spermatozoon, it is also associated with chromatin undergoing compaction. Finally, in fully-condensed mitotic chromosomes, CCT is located all along the chromosomes. Our finding that CCT is associated with constitutive heterochromatin and to compacting chromatin raises the possibility that it may be implicated in maintenance and remodelling of heterochromatin.     

ATP-induced allostery in the eukaryotic chaperonin CCT is abolished by the mutation G345D in CCT4 that renders yeast temperature-sensitive for growth

Saccharomyces cerevisiae yeast cells containing the chaperonin CCT (chaperonin-containing t-complex polypeptide 1 (TCP-1)) with the G345D mutation in subunit CCT4 (anc2-1) are temperature-sensitive for growth and display defects in organization of actin structure, budding and cell shape. In this first structure-function analysis of CCT, we show that this mutation abolishes both intra- and inter-ring cooperativity in ATP binding by CCT. The finding that a single mutation in only one subunit in each CCT ring has such drastic effects highlights the importance of allostery for its in vivo function. These results, together with other kinetic data for wild-type CCT reported in this study, provide support for the sequential model for ATP-dependent allosteric transitions in CCT.     

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.     

Equivalent Mutations in the Eight Subunits of the Chaperonin CCT Produce Dramatically Different Cellular and Gene Expression Phenotypes

The eukaryotic cytoplasmic chaperonin-containing TCP-1 (CCT) is a complex formed by two back-to-back stacked hetero-octameric rings that assists the folding of actins, tubulins, and other proteins in an ATP-dependent manner. Here, we tested the significance of the hetero-oligomeric nature of CCT in its function by introducing, in each of the eight subunits in turn, an identical mutation at a position that is conserved in all the subunits and is involved in ATP hydrolysis, in order to establish the extent of ‘individuality’ of the various subunits. Our results show that these identical mutations lead to dramatically different phenotypes. For example, Saccharomyces cerevisiae yeast cells with the mutation in subunit CCT2 display heat sensitivity and cold sensitivity for growth, have an excess of actin patches, and are the only strain here generated that is pseudo-diploid. By contrast, cells with the mutation in subunit CCT7 are the only ones to accumulate juxtanuclear protein aggregates that may reflect an impaired stress response in this strain. System-level analysis of the strains using RNA microarrays reveals connections between CCT and several cellular networks, including ribosome biogenesis and TOR2, that help to explain the phenotypic variability observed.     

Yeast Phosducin-Like Protein 2 Acts as a Stimulatory Co-Factor for the Folding of Actin by the Chaperonin CCT via a Ternary Complex

The eukaryotic chaperonin-containing TCP-1 (CCT) folds the cytoskeletal protein actin. The folding mechanism of this 16-subunit, 1-MDa machine is poorly characterised due to the absence of quantitative in vitro assays. We identified phosducin-like protein 2, Plp2p (=PLP2), as an ATP-elutable binding partner of yeast CCT while establishing the CCT interactome. In a novel in vitro CCT-ACT1 folding assay that is functional under physiological conditions, PLP2 is a stimulatory co-factor. In a single ATP-driven cycle, PLP2-CCT-ACT1 complexes yield 30-fold more native actin than CCT-ACT1 complexes. PLP2 interacts directly with ACT1 through the C-terminus of its thioredoxin fold and the CCT-binding subdomain 4 of actin. The in vitro CCT-ACT1-PLP2 folding cycle of the preassembled complex takes 90 s at 30 °C, several times slower than the canonical chaperonin GroEL. The specific interactions between PLP2, CCT and ACT1 in the yeast-component in vitro system and the pronounced stimulatory effect of PLP2 on actin folding are consistent with in vivo genetic approaches demonstrating an essential and positive role for PLP2 in cellular processes involving actin in Saccharomyces cerevisiae. In mammalian systems, however, several members of the PLP family, including human PDCL3, the orthologue of PLP2, have been shown to be inhibitory toward CCT-mediated folding of actin in vivo and in vitro. Here, using a rabbit-reticulocyte-derived in vitro translation system, we found that inhibition of β-actin folding by PDCL3 can be relieved by exchanging its acidic C-terminal extension for that of PLP2. It seems that additional levels of regulatory control of CCT activity by this PLP have emerged in higher eukaryotes.     

Subunits of the chaperonin CCT are associated with Tetrahymena microtubule structures and are involved in cilia biogenesis

The cytosolic chaperonin CCT is a heterooligomeric complex of about 900 kDa that mediates the folding of cytoskeletal proteins. We observed by indirect immunofluorescence that the Tetrahymena TpCCTalpha, TpCCTdelta, TpCCTepsilon, and TpCCTeta-subunits colocalize with tubulin in cilia, basal bodies, oral apparatus, and contractile vacuole pores. TpCCT-subunits localization was affected during reciliation. These findings combined with atomic force microscopy measurements in reciliating cells indicate that these proteins play a role during cilia biogenesis related to microtubule nucleation, tubulin transport, and/or axoneme assembly. The TpCCT-subunits were also found to be associated with cortex and cytoplasmic microtubules suggesting that they can act as microtubule-associated proteins. The TpCCTdelta being the only subunit found associated with the macronuclear envelope indicates that it has functions outside of the 900 kDa complex. Tetrahymena cytoplasm contains granular/globular-structures of TpCCT-subunits in close association with microtubule arrays. Studies of reciliation and with cycloheximide suggest that these structures may be sites of translation and folding. Combined biochemical techniques revealed that reciliation affects the oligomeric state of TpCCT-subunits being tubulin preferentially associated with smaller CCT oligomeric species in early stages of reciliation. Collectively, these findings indicate that the oligomeric state of CCT-subunits reflects the translation capacity of the cell and microtubules integrity.     

Miles to go (mtgo) encodes FNDC3 proteins that interact with the chaperonin subunit CCT3 and are required for NMJ branching and growth in Drosophila

Analysis of mutants that affect formation and function of the Drosophila larval neuromuscular junction (NMJ) has provided valuable insight into genes required for neuronal branching and synaptic growth. We report that NMJ development in Drosophila requires both the Drosophila ortholog of FNDC3 genes; CG42389 (herein referred to as miles to go; mtgo), and CCT3, which encodes a chaperonin complex subunit. Loss of mtgo function causes late pupal lethality with most animals unable to escape the pupal case, while rare escapers exhibit an ataxic gait and reduced lifespan. NMJs in mtgo mutant larvae have dramatically reduced branching and growth and fewer synaptic boutons compared with control animals. Mutant larvae show normal locomotion but display an abnormal self-righting response and chemosensory deficits that suggest additional functions of mtgo within the nervous system. The pharate lethality in mtgo mutants can be rescued by both low-level pan- and neuronal-, but not muscle-specific expression of a mtgo transgene, supporting a neuronal-intrinsic requirement for mtgo in NMJ development. Mtgo encodes three similar proteins whose domain structure is most closely related to the vertebrate intracellular cytosolic membrane-anchored fibronectin type-III domain-containing protein 3 (FNDC3) protein family. Mtgo physically and genetically interacts with Drosophila CCT3, which encodes a subunit of the TRiC/CCT chaperonin complex required for maturation of actin, tubulin and other substrates. Drosophila larvae heterozygous for a mutation in CCT3 that reduces binding between CCT3 and MTGO also show abnormal NMJ development similar to that observed in mtgo null mutants. Hence, the intracellular FNDC3-ortholog MTGO and CCT3 can form a macromolecular complex, and are both required for NMJ development in Drosophila.     

Elevated blood Hsp60, its structural similarities and cross-reactivity with thyroid molecules,and its presence on the plasma membrane of oncocytes point to the chaperonin as an immunopathogenic factor in Hashimoto's thyroiditis

The role Hsp60 might play in various inflammatory and autoimmune diseases is under investigation, but little information exists pertaining to Hashimoto’s thyroiditis (HT). With the aim to fill this gap, in the present work, we directed our attention to Hsp60 participation in HT pathogenesis. We found Hsp60 levels increased in the blood of HT patients compared to controls. The chaperonin was immunolocalized in thyroid tissue specimens from patients with HT, both in thyrocytes and oncocytes (Hurthle cells) with higher levels compared to controls (goiter). In oncocytes, we found Hsp60 not only in the cytoplasm but also on the plasma membrane, as shown by double immunofluorescence performed on fine needle aspiration cytology. By bioinformatics, we found regions in the Hsp60 molecule with remarkable structural similarity with the thyroglobulin (TG) and thyroid peroxidase (TPO) molecules, which supports the notion that autoantibodies against TG and TPO are likely to recognize Hsp60 on the plasma membrane of oncocytes. This was also supported by data obtained by ELISA, showing that anti-TG and anti-TPO antibodies cross-react with human recombinant Hsp60. Antibody-antigen (Hsp60) reaction on the cell surface could very well mediate thyroid cell damage and destruction, perpetuating inflammation. Experiments with recombinant Hsp60 did not show stimulation of cytokine production by peripheral blood mononuclear cells from HT patients. All together, these results led us to hypothesize that Hsp60 may be an active player in HT pathogenesis via an antibody-mediated immune mechanism.     

Inactivation of the phosphoglucomutase gene pgm in Corynebacterium glutamicum affects cell shape and glycogen metabolism

In Corynebacterium glutamicum formation of glc-1-P (α-glucose-1-phosphate) from glc-6-P (glucose-6-phosphate) by α-Pgm (phosphoglucomutase) is supposed to be crucial for synthesis of glycogen and the cell wall precursors trehalose and rhamnose. Furthermore, Pgm is probably necessary for glycogen degradation and maltose utilization as glucan phosphorylases of both pathways form glc-1-P. We here show that C. glutamicum possesses at least two Pgm isoenzymes, the cg2800 (pgm) encoded enzyme contributing most to total Pgm activity. By inactivation of pgm we created C. glutamicum IMpgm showing only about 12% Pgm activity when compared to the parental strain. We characterized both strains during cultivation with either glucose or maltose as substrate and observed that (i) the glc-1-P content in the WT (wild-type) and the mutant remained constant independent of the carbon source used, (ii) the glycogen levels in the pgm mutant were lower during growth on glucose and higher during growth on maltose, and (iii) the morphology of the mutant was altered with maltose as a substrate. We conclude that C. glutamicum employs glycogen as carbon capacitor to perform glc-1-P homeostasis in the exponential growth phase and is therefore able to counteract limited Pgm activity for both anabolic and catabolic metabolic pathways.     

Interaction of CacyBP/SIP with NPM1 and its influence on NPM1 localization and function in oxidative stress

Calcyclin (S100A6) binding protein/Siah‐1 interacting protein (CacyBP/SIP) is mainly a cytoplasmic protein; however, some literature data suggested its presence in the nucleus. In this work we examined more precisely the nuclear localization and function of CacyBP/SIP. By applying mass spectrometry, we have identified several nuclear proteins, among them is nucleophosmin (NPM1), that may interact with CacyBP/SIP. Subsequent assays revealed that CacyBP/SIP forms complexes with NPM1 in the cell and that the interaction between these two proteins is direct. Interestingly, although CacyBP/SIP exhibits phosphatase activity, we have found that its overexpression favors phosphorylation of NPM1 on S125. In turn, the RNA immunoprecipitation assay indicated that the altered CacyBP/SIP level has an impact on the amount of 28S and 18S rRNA bound to NPM1. The overexpression of CacyBP/SIP resulted in a significant increase in the binding of 28S and 18S rRNA to NPM1, whereas silencing of CacyBP/SIP expression decreased 28S rRNA binding and had no effect on the binding of 18S rRNA. Further studies have shown that under oxidative stress, CacyBP/SIP overexpression alters NPM1 distribution in cell nuclei. In addition, staining for a nucleolar marker, fibrillarin, revealed that CacyBP/SIP is indispensable for maintaining the nucleolar structure. These results are in agreement with data obtained by western blot analysis, which show that upon oxidative stress the NPM1 level decreases but that CacyBP/SIP overexpression counteracts the effect of stress. Altogether, our results show for the first time that CacyBP/SIP binds to and affects the properties of a nuclear protein, NPM1, and that it is indispensable for preserving the structure of nucleoli under oxidative stress.     

Integrated analysis of DNA methylation and mRNA expression profiling reveals candidate genes associated with cisplatin resistance in non-small cell lung cancer

DNA methylation plays a critical role during the development of acquired chemoresistance. The aim of this study was to identify candidate DNA methylation drivers of cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC). The A549/DDP cell line was established by continuous exposure of A549 cells to increasing concentrations of DDP. Gene expression and methylation profiling were determined by high-throughput microarrays. Relationship of methylation status and DDP response was validated in primary tumor cell culture and the Cancer Genome Atlas (TCGA) samples. Cell proliferation, apoptosis, cell cycle, and response to DDP were determined in vitro and in vivo. A total of 372 genes showed hypermethylation and downregulation in A549/DDP cells, and these genes were involved in most fundamental biological processes. Ten candidate genes (S100P, GDA, WISP2, LOXL1, TIMP4, ICAM1, CLMP, HSP8, GAS1, BMP2) were selected, and exhibited varying degrees of association with DDP resistance. Low dose combination of 5-aza-2′-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) reversed drug resistance of A549/DDP cells in vitro and in vivo, along with demethylation and restoration of expression of candidate genes (GAS1, TIMP4, ICAM1 and WISP2). Forced expression of GAS1 in A549/DDP cells by gene transfection contributed to increased sensitivity to DDP, proliferation inhibition, cell cycle arrest, apoptosis enhancement, and in vivo growth retardation. Together, our study demonstrated that a panel of candidate genes downregulated by DNA methylation induced DDP resistance in NSCLC, and showed that epigenetic therapy resensitized cells to DDP.     

The reaction of alpha-crystallin with the cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) demonstrates close proximity of the C termini of alphaA and alphaB in the native assembly

The chaperone-like activity of human lens alpha-crystallin in inhibiting the aggregation of denatured proteins suggests a role for alpha-crystallin in cataract prevention. Although a variety of techniques have generated structural information relevant to its chaperone-like activity, the size and heterogeneity of alpha-crystallin have prevented determination of its crystal structure. Even though synthetic cross-linkers have provided considerable information about protein structures, they have not previously been used to study the proximity and orientation of subunits within human alpha-crystallin. Cross-linkers provide structural insight into proteins by binding the side chains of amino acids within close proximity. To identify the cross-linked residues, the modified protein is digested and the resulting peptides are analyzed by mass spectrometry. Analysis of products from the reaction of alpha-crystallin with 3,3'dithiobis(sulfosuccinimidyl propionate), DTSSP, identified several modifications to both alphaA and alphaB. The most structurally informative of these modifications was a cross-link between lysine 166 of alphaA and lysine 175 of alphaB. This cross-link provides experimental evidence supporting theoretical structural models that place the C termini of alphaA and alphaB within close proximity in the native aggregate.     

PEXEL-independent trafficking of Plasmodium falciparum SURFIN4.2 to the parasite-infected red blood cell and Maurer's clefts

SURFIN_4.2 is a parasite-infected red blood cell (iRBC) surface associated protein of Plasmodium falciparum. To analyze the region responsible for the intracellular trafficking of SURFIN_4.2 to the iRBC and Maurer's clefts, a panel of transgenic parasite lines expressing recombinant SURFIN_4.2 fused with green fluorescent protein was generated and evaluated for their localization. We found that the cytoplasmic region containing a tryptophan rich (WR) domain is not necessary for trafficking, whereas the transmembrane (TM) region was. Two PEXEL-like sequences were shown not to be responsible for the trafficking of SURFIN_4.2, demonstrating that the protein is trafficked in a PEXEL-independent manner. N-terminal replacement, deletion of the cysteine-rich domain or the variable region also did not prevent the protein from localizing at the iRBC or Maurer's clefts. A recombinant SURFIN_4.2 protein possessing 50 amino acids upstream of the TM region, TM region itself and a part of the cytoplasmic region was shown to be trafficked into the iRBC and Maurer's clefts, suggesting that there are no essential trafficking motifs in the SURFIN_4.2 extracellular region. A mini-SURFIN_4.2 protein containing WR domain was shown by Western blotting to be more abundantly detected in a Triton X-100-insoluble fraction, compared to the one without WR domain. We suggest that the cytoplasmic region containing the WR may be responsible for their difference in solubility.     

非ST段抬高型急性冠脉综合征患者血浆S100A1水平与GRACE评分及短期预后相关性的研究

目的:探讨非ST段抬高型急性冠脉综合征(NST-ACS)患者血浆S100A1水平与全球急性冠状动脉事件注册(GRACE)评分之间的关系,以及S100A1水平对NST-ACS患者30天预后的判定价值。方法:共有162例NST-ACS患者符合入选标准,收集基本临床资料,进行GRACE评分,同时收集次日清晨空腹采集肘静脉血,检测血浆S100A1浓度,与患者的GRACE评分进行比较。根据S100A1的水平进行分组随访,KM生存分析不同组患者30天预后进行评价。结果:不同GRACE分组患者间S100A1水平具有显著性差异(P0.05);相关性分析显示,NST-ACS患者S100A1与GRACE评分呈显著正相关(r=0.49,P0.01);KM生存分析显示,S100A1水平3.41 ng/mL的患者30天内心血管事件发生率显著升高(P0.05)。结论:S100A1可作为预测NST-ACE患者病情的发生发展的生化指标;在NST-ACS患者中运用S100A1有助于对患者早期危险分层及评估预后有一定的临床价值。     

The Uba2 and Ufd1 proteins of Saccharomyces cerevisiae interact with poly(A) polymerase and affect the polyadenylation activity of cell extracts

Poly(A) polymerase is responsible for the addition of the adenylate tail to the 3′ ends of mRNA. Using the two-hybrid system, we have identified two proteins which interact specifically with the Saccharomyces cerevisiae poly(A) polymerase, Pap1. Uba2 is a homolog of ubiquitin-activating (E1) enzymes and Ufd1 is a protein whose function is probably also linked to the ubiquitin-mediated protein degradation pathway. These two proteins interact with Pap1 and with each other, but not with eight other target proteins which were tested in the two-hybrid system. The last 115 amino acids of Uba2, which contains an 82-amino acid region not present in previously characterized E1 enzymes, is sufficient for the interaction with Pap1. Both Uba2 and Ufd1 can be co-immunoprecipitated from extracts with Pap1, confirming in vitro the interaction identified by two-hybrid analysis. Depletion of Uba2 from cells produces extracts which polyadenylate precursor RNA with increased efficiency compared to extracts from nondepleted cells, while depletion of Ufd1 yields extracts which are defective in processing. These two proteins are not components of polyadenylation factors, and instead may have a role in regulating poly(A) polymerase activity. Received: 6 January 1997 / Accepted: 27 February 1997     

Modulation of the junctional integrity by low or high concentrations of cytochalasin B and dihydrocytochalasin B in associated with distinct changes in F-actin and ZO-1

In a study of Necturus gallbladder epithelium Benzel et al. (Benzel et al., 1980) found that low (0.2–1.2 M) and higher concentrations (1.5 M and more) of cytochalasin B (CB) caused an increase and decrease in the transepithelial electrical resistance (TER), respectively. Moreover, there were slight changes in the height and complexicity of tight junction (TJ) strands, as visualized by freeze-fracture and freeze-etching. To elucidate the mechanisms of these findings, we first demonstrated that the effect is also present in monolayers of Madin-Darby Canine Kidney strain I (MDCK-I) cells. Thus, a low concentration (0.1 ng/ml) cytochalasin B (CB) strengthened the permeability barrier, as evidenced quantitatively by increases in TER on transepithelial electrical measurements. Furthermore, indirect immunofluorescence and confocal microscopy demonstrated that this effect was paralleled with an accumulation of F-actin and the tight junction marker protein, ZO-1, at the level of TJ. Equimolar concentrations of dihydrocytochalasin B (dhCB), on the other hand, did not lead to a tightening of the epithelium. Confirming previous studies, there was a general decrease in epithelial resistance after treatment with high concentrations (1 g/ml) of CB and dhCB, which was accompanied by distinct changes in the F-actin network and distribution of ZO-1. We speculate that the divergent effects of CB and dhCB on the F-actin and ZO-1 organization might be due to specific effects on the transport of monosaccharides across the plasma membrane, or that CB and dhCB in distinct ways involve the turnover of phosphatidylinositols in the membrane, thereby modulating junctional permeability and F-actin structure.     

Different intracellular distributions of heat-shock and arsenite-induced proteins in Drosophila Kc cells. Possible relation with the phosphorylation and translocation of a major cytoskeletal protein

The intracellular distribution of heat shock proteins (hsps) from Drosophila Kc cells is different in heat and in arsenite-treated cells. While the cytoplasmic localization of hsp 84 is confirmed in both treatments, the association of hsp 70 with the nucleus occurs only in heat-treated cells. This heat-dependent association of certain hsps with the nuclear pellet is confirmed by incubation of cells at various temperatures ranging from 23 to 39 °C. Furthermore their presence in this nuclear pellet can be correlated with the translocation and phosphorylation of a major cellular cytoskeletal protein of M_r 45,000. It is concluded that the previously reported nuclear association of hsps is not necessarily indicative of a nuclear function. It is further suggested that hsps might have a structural function within the cell.