Chaperonin Research Articles

Microbial chaperonin 60 inhibits osteoclast differentiation by interfering with RANK/RANKL binding and overexpression of lipocalin2

Osteoclasts play a crucial role in bone destruction in rheumatoid arthritis (RA). This study aimed to investigate the inhibitory effects of chaperonin 60 (CPN60), identified in the surface proteins of Propionibacterium freudenreichii MJ2, on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation, and elucidate the underlying mechanisms. Treatment with CPN60 inhibited RANKL-induced osteoclast differentiation by decreasing the expression of osteoclast differentiation-related genes and proteins. CPN60 interfered with the binding of RANKL to RANK, as elucidated using surface plasmon resonance (SPR) and immunofluorescence. In silico molecular docking analysis further supported the interference of CPN60 with the binding of RANKL and RANK. CPN60 suppressed the expression of molecules linked to the calcium-dependent pathway in RANKL-induced osteoclast differentiation at both mRNA and protein levels. Microarray analysis showed elevated expression of lipocalin 2 (Lcn2), which was closely linked to the inhibition of osteoclast differentiation in CPN60-treated RAW 264.7 cells. Inhibition of Lcn2 decreased the inhibitory effect of CPN60 on osteoclast differentiation, indicating that increased expression of Lcn2 by CPN60 contributes to the inhibition of osteoclastogenesis. In addition, CPN60 treatment alleviated arthritis symptoms in collagen-induced arthritis mice by reducing the generation of collagen-specific antibodies and inhibiting osteoclast differentiation. In conclusion, CPN60 of P. freudenreichii MJ2 interfered with RANKL–RANK binding, reduced the expression of genes and proteins related to osteoclast differentiation and upregulated Lcn2 expression, thereby inhibiting RANKL-induced osteoclast differentiation, which might contribute to ameliorate collagen-induced arthritis.

The role of exogenous glutamine on germination, plant development and transcriptional expression of some stress-related genes in onion under salt stress

ABSTRACT The purpose of this research was to explore the role of glutamine (Gln) on plant germination and growth under salinity [0, 50, 100, 150, 200 mM sodium chloride (NaCl)], and on the activation of some stress-related genes [CuZn-Superoxide Dismutase (SOD), Mn-SOD, L-ascorbate oxidase (AO), DNA Polymerase Delta 1 (POLD)-1, Chaperon (CHAPE) and Heat Shock Protein (HSP)-21], genetic template stability and photosynthetic pigment activation. Under salt stress, 2 mM Gln application reduced the mean germination time (MGT) (4.51 days), increased the coefficient of velocity of germination (CVG) (75.85), germination index (GI) (1.46) and germination percentage (GP) (82%), indicating that it was the best application for germination under stress. Gln promoted the development of aboveground plant organs. 3 mM Gln increased the number of leaves from 5.3 to 13.7 and 4.3 to 6.6 under unstress and stress conditions, respectively. Under salt stress, Gln increased photosynthetic pigments and genomic template stability (GTS) (80%). At 2 mM Gln, total chlorophyll and carotenoid content increased from 1.59 mg · g-1 TA to 2.23 mg · g-1 TA and from 0.28 mg · g-1 TA to 0.37 mg · g-1 TA, respectively. The effect on gene expression levels varied with the concentration. The application of 2 mM Gln, which enhanced germination and vegetative parameters under stress, caused a raise in CuZn-SOD gene expression and a 43% decrease in the transcriptional expression of the HSP-21 gene as an indicator of the functionality of the repair mechanism. The improved growth of Gln-treated plants under stress suggests that Gln is involved in bridging the energy deficit of the plant by acting as an alternative fuel for metabolic activities under stress.

Ectopic CH60 mediates HAPLN1-induced cell survival signaling in multiple myeloma.

Multiple myeloma (MM), the second most common hematological malignancy, is generally considered incurable because of the development of drug resistance. We previously reported that hyaluronan and proteoglycan link protein 1 (HAPLN1) produced by stromal cells induces activation of NF-κB, a tumor-supportive transcription factor, and promotes drug resistance in MM cells. However, the identity of the cell surface receptor that detects HAPLN1 and thereby engenders pro-tumorigenic signaling in MM cells remains unknown. Here, we performed an unbiased cell surface biotinylation assay and identified chaperonin 60 (CH60) as the direct binding partner of HAPLN1 on MM cells. Cell surface CH60 specifically interacted with TLR4 to evoke HAPLN1-induced NF-κB signaling, transcription of anti-apoptotic genes, and drug resistance in MM cells. Collectively, our findings identify a cell surface CH60-TLR4 complex as a HAPLN1 receptor and a potential molecular target to overcome drug resistance in MM cells.

Transcriptomic and proteomic profiles of II YOU 838 (Oryza sativa) provide insights into heat stress tolerance in hybrid rice

Heat stress is an increasing threat to rice production worldwide. To investigate the mechanisms of heat tolerance in hybrid rice and their contributions to rice heterosis, we compared the transcriptome of the hybrid rice II YOU 838 (II8) with the transcriptomes of its parents Fu Hui 838 (F8) and II-32A (II3) after heat stress at 42 °C for 0 h, 24 h, 72 h and 120 h. We also performed a proteomic analysis in II8 after heat stress at 42 °C for 24 h. The transcriptome data revealed time-dependent gene expression patterns under the heat stress conditions, and the heat stress response of II8 was greatly different from those of its parents. Gene ontology analysis of the differentially expressed genes that were clustered using k-means clustering showed that most of the up-regulated genes were involved in responses to stimuli, cell communication, and metabolic and transcription factor activities, whereas the down-regulated genes were enriched in photosynthesis and signal transduction. Moreover, 35 unique differentially abundant proteins, including a basic helix-loop-helix transcription factor (bHLH96), calmodulin-binding transcription activator, heat shock protein (Hsp70), and chaperonin 60 (CPN60), were detected in the proteomic analysis of II8 under heat stress. The co-regulatory analysis revealed novel genes and pathways involved in heat tolerance, namely, ferredoxin-NADP reductase, peroxidases, mitogen-activated protein kinase kinase kinase, and heat shock factor (HSF)–Hsp network. Members of the Hsp and HSF families had over-dominant expression patterns in the hybrid compared with its parents, to help maintain the higher photosynthesis and antioxidant defense systems in the hybrid. Our study suggests that the complex HSF–Hsp regulatory network contribute to the heat tolerance of the hybrid rice.

Effect of cigarette smoke and treatment with relaxin on guinea pig skin

Cigarette smoking causes microvascular dysfunction and skin aging. Relaxin, primarily but not exclusively involved in reproduction, has connective tissue among its targets. Within a project on the interference of relaxin with the effects of smoke on guinea pigs, we examined the skin response to those stimuli. Adult guinea pigs were exposed to cigarette smoke daily for 8 weeks, and some of them were treated also with relaxin, 1 or 10 µg/die. Controls were treated with relaxin vehicle alone. The skin was analyzed by light and electron microscopy and histochemistry for mast cells and the collagen specific chaperonin Hsp47. The epidermis appeared unaffected by any treatment. In the superficial dermis, smoke led to a decrease in mast cell number and intensity of astra blue staining, suggestive of granule discharge. Relaxin caused further significant reduction in mast cell number. In the superficial and deep dermis, the staining intensity of Hsp47 positive cells, assumed as active fibroblasts, increased upon smoke. The staining intensity decreased gradually in the superficial dermis upon relaxin, reaching significance after treatment with 10 µg/die relaxin, while in the deep dermis it decreased significantly upon treatment with 1 µg/die relaxin and underwent further, significant increase with 10 µg/die relaxin. The results suggest that relaxin can enhance skin mast cell secretory response, possibly antagonizing nicotine induced vasoconstriction and, depending on dose and localization of responding cells, can counteract the profibrotic stimulus of smoke on dermal fibroblasts.

Limited Trypsinolysis of GroES: The Effect on the Interaction with GroEL and Assembly In Vitro

Limited Trypsinolysis of GroES: The Effect on the Interaction with GroEL and Assembly In Vitro

Prefoldins in Archaea.

Molecular chaperones promote the correct folding of proteins in aggregation-prone cellular environments by stabilizing nascent polypeptide chains and providing appropriate folding conditions. Prefoldins (PFDs) are molecular chaperones found in archaea and eukaryotes, generally characterized by a unique jellyfish-like hexameric structure consisting of a rigid beta-barrel backbone with protrudingflexible coiled-coils. Unlike eukaryotic PFDs that mainly interact with cytoskeletal components, archaeal PFDs can stabilize a wide range of substrates; such versatility reflects PFD's role as a key element in archaeal chaperone systems, which often lack general nascent-chain binding chaperone components such as Hsp70. While archaeal PFDs mainly exist as hexameric complexes, their structural diversity ranges from tetramers to filamentous oligomers. PFDs bind and stabilize nonnative proteins using varying numbers of coiled-coils, and subsequently transfer the substrate to a group II chaperonin (CPN) for refolding. The distinct structure and specific function of archaeal PFDs have been exploited for a broad range of applications in biotechnology; furthermore, a filament-forming variant of PFD has been used to fabricate nanoscale architectures of defined shapes, demonstrating archaeal PFDs' potential applicability in nanotechnology.

Structural and mechanistic characterization of an archaeal-like chaperonin from a thermophilic bacterium

The chaperonins (CPNs) are megadalton sized hollow complexes with two cavities that open and close to encapsulate non-native proteins. CPNs are assigned to two sequence-related groups that have distinct allosteric mechanisms. In Group I CPNs a detachable co-chaperone, GroES, closes the chambers whereas in Group II a built-in lid closes the chambers. Group I CPNs have a bacterial ancestry, whereas Group II CPNs are archaeal in origin. Here we describe open and closed crystal structures representing a new phylogenetic branch of CPNs. These Group III CPNs are divergent in sequence and structure from extant CPNs, but are closed by a built-in lid like Group II CPNs. A nucleotide-sensing loop, present in both Group I and Group II CPNs, is notably absent. We identified inter-ring pivot joints that articulate during ring closure. These Group III CPNs likely represent a relic from the ancestral CPN that formed distinct bacterial and archaeal branches.

Leishmania donovani chaperonin 10 regulates parasite internalization and intracellular survival in human macrophages

Protozoa of the genus Leishmania infect macrophages in their mammalian hosts causing a spectrum of diseases known as the leishmaniases. The search for leishmania effectors that support macrophage infection is a focus of significant interest. One such candidate is leishmania chaperonin 10 (CPN10) which is secreted in exosomes and may have immunosuppressive properties. Here, we report for the first time that leishmania CPN10 localizes to the cytosol of infected macrophages. Next, we generated two genetically modified strains of Leishmania donovani (Ld): one strain overexpressing CPN10 (CPN10+++) and the second, a CPN10 single allele knockdown (CPN10+/-), as the null mutant was lethal. When compared with the wild-type (WT) parental strain, CPN10+/- Ld showed higher infection rates and parasite loads in human macrophages after 24h of infection. Conversely, CPN10+++ Ld was associated with lower initial infection rates. This unexpected apparent gain-of-function for the knockdown could have been explained either by enhanced parasite internalization or by enhanced intracellular survival. Paradoxically, we found that CPN10+/- leishmania were more readily internalized than WT Ld, but also displayed significantly impaired intracellular survival. This suggests that leishmania CPN10 negatively regulates the rate of parasite uptake by macrophages while being required for intracellular survival. Finally, quantitative proteomics identified an array of leishmania proteins whose expression was positively regulated by CPN10. In contrast, many macrophage proteins involved in innate immunity were negatively regulated by CPN10. Taken together, these findings identify leishmania CPN10 as a novel effector with broad based effects on macrophage cell regulation and parasite survival.

Comparative proteomic analysis of kit-positive gastrointestinal stromal tumors based on differential risk classification

Objective To screen new molecular markers related to invasion and metastasis of gastrointestinal stromal tumors (GISTs) by analyzing differential protein expression profiles between high-risk and low-risk GISTs with c-kit mutation. Methods Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to detect the differentially expressed proteins in 5 cases of high-risk and 5 cases of low-risk GIST tissues with c-kit mutation respectively, and the differentially expressed protein markers were analyzed and identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Results By comparative analysis of protein expression profiles we found 41 differentially expressed protein spots. Using MALDI-TOF-MS technique for mass spectrometry analysis of differentially expressed protein spots we successfully identified 27 different protein molecules. Of these, 14 proteins including aldehyde dehydrogenase 1 family, member A2 (ALDH1A2), ubiquinol cytochrome c reductase core protein 1 (UQCRC1), chaperonin containing TCP1 subunit 2 (CCT-2), pyruvate dehydrogenase beta (PDHB), peroxiredoxin 3 (PRDX3), protease activator 28 beta (Pa28β), cytochrome c oxidase subunit 5A (COX5A), caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP), Tu translation elongation factor, mitochondrial (TuFM), glutathione S-transferase omega 1 (GSTO1), hypocretin receptor 1 (HCRTR1), NM23-H1 and an unknown protein were upregulated in high-risk GISTs. 13 proteins included apolipoprotein A1 (APOA1), heme binding protein 1 (HEBP1), chromosome 1 open reading frame 123 (C1orf123), heat shock protein beta-6 (HSPB6), SH3 domain-binding glutamic acid-rich-like protein (SH3BGRL), glutathione S-transferase Mu 2 (GSTM2), tropomyosin alpha-1 chain (TPM1), protein disulfide-isomerase A3 (PDIA3), dimethylarginine dimethylaminohydrolase 1 (DDAH1), tetratricopeptide repeat domain 1 (TTC1), heterogeneous nuclear ribonucleoprotein C (HNRNPC) and DJ-1 were downregulated in high-risk GISTs. Conclusion The 27 differentially expressed proteins had the potential to be the specific tumor biomarkers for predicting the prognosis and warning of the metastasis of GISTs. Key words: Gastrointestinal stromal tumors; Proteomics; Molecular markers; Neoplasm metastasis

Purification, crystallization, and preliminary X-ray crystallographic analysis of the Group III chaperonin from Carboxydothermus hydrogenoformans.

Chaperonins (CPNs) are megadalton sized ATP-dependent nanomachines that facilitate protein folding through complex cycles of complex allosteric articulation. They consist of two back-to-back stacked multisubunit rings. CPNs are usually classified into Group I and Group II. Here, we report the crystallization of both the AMPPNP (an ATP analogue) and ADP bound forms of a novel CPN, classified as belonging to a third Group, recently discovered in the extreme thermophile Carboxydothermus hydrogenoformans. Crystals of the two forms were grown by the vapor batch crystallization method at 295 K. Crystals of the Ch-CPN/AMPPNP complex diffracted to 3.0 Å resolution and belonged to the space group P422, with unit-cell parameters a = b = 186.166, c = 160.742 Å. Assuming the presence of four molecules in the asymmetric unit, the solvent content was estimated to be about 60.02%. Crystals of the Ch-CPN/ADP complex diffracted to 4.0 Å resolution and belonged to the space group P4212, with unit-cell parameters a = b = 209.780, c = 169.813Å. Assuming the presence of four molecules in the asymmetric unit, the solvent content was estimated to be about 70.19%.

Proteomics of differentially-expressed proteins between human primary and lymph node metastatic gastric adenocarcinoma

Objective To identify the differentially-expressed proteins of human gastric adenocarcinoma cell in primary or metastatic lymph node tissues by comparative proteomics technology, and to screen the specific metastatic-associated proteins so as to investigate the metastatic molecular mechanism of lymph node metastasis in gastric cancer. Methods 11differential proteins were acquired previously from primary and metastatic lymphnode tissues in gastric adenocarcinoma patients by 2D-DIGE. Some selected differential protein spots were identified by PMF based on MALDI-TOF-MS and database search. Immunohistochemical staining of HSP70 was used to evaluate the reliability of the proteomic analysis results. Results After analyzed on 11 differential proteins by in-gel trypsin digestion and MALDI-TOF-MS-based PMF analysis, a total of 5 differential proteins were identified by searching Mascot-database, HSP70's 8 isoform 2 variant, chaperonin, chaperonin, leucine aminopeptidase, predicted: hypothetical protein XP_515584. Among the differential proteins identified, the levels of HSP70, chaperonin, leucine aminopeptidase expression had a significant up-regulation in gastric primary cancer compared with metastatic lymph node. HSP70 expression rate increased with the metastasis of lymph node and the progress of gastric cancer, agreed with the proteomics results. Conclusions They are similar in differentially-expressed proteins in primary or metastatic lymph node tissues because of the uniformity in source and differention. There are few protein changes in cancer cells between them, taking part in the metastatic of gastric cancer. HSP70 takes part in the progress of gastric cancer and relates to the metastasis of lymph node and malignant degree. Key words: Stomach neoplasms; Adenocarcinoma; Lymphatic metastasis; Microdissection; Spectrometry, mass, matrix-assisted laser desorption-ionization; Proteomics

Skeletal muscle heat shock protein 60 increases after endurance training in mice and induces peroxisome proliferation-activated receptor-γ coactivator-1 α1 expression

Heat shock protein (Hsp60) is a mitochondrial chaperonin whose unconventional cellular localizations and functions are discovered day by day. In the present study, the levels of Hsp60 in fibres of the soleus muscle and its correlation to the expression of four isoforms of peroxisome proliferation-activated receptor-γ (PPAR-γ) coactivator-1α (PGC1α) were investigated in 72 young (7-weeks old) healthy male mice (BALB/c AnNHsd) at baseline and after completing a 6-week endurance training program. The mice were assigned to one of the two experimental groups: SED (sedentary) or TR (trained). Short-term overexpression of hsp60, achieved by in vitro plasmid transfection, was then performed to determine whether this chaperonin could have a role in the activation of the expression levels of PGC-1α isoforms. The levels of Hsp60 protein were fibre-type specific in the posterior muscles at baseline, and endurance training increased its content in type I muscle fibers. Concomitantly with the increased levels of Hsp60 released in the blood stream of trained mice, mitochondrial copy number and the expression of three isoforms of PGC-1α increased. Overexpressing hsp60 in cultured myoblasts induced only the expression of PGC-1 α1, letting us suppose a direct correlation between Hsp60 overexpression and PGC-1 α1 activation. Overall, these results suggest that during endurance training Hsp60 is upregulated and activates the mitochondrial biogenesis pathway, probably as a response to the oxidative stress induced by exercise. This study reveals a molecular response of skeletal muscle to a mechanical stress induced by training which involves the molecular chaperonin Hsp60 and the transcriptional co-activator PGC-1 α1. The role of these proteins in aerobic adaptation and pathological conditions as cancer cachexia warrants further investigations.

Nuclear localization and new isoforms detection give new insights on Hsp10 functions in normal and cigarette smoke-stressed lung cells

Heat-shock protein (Hsp)10 is the co-chaperone for Hsp60 inside mitochondria, but it also resides outside the organelle. Variations in its levels and intracellular dis- tribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) is a potent stressor for the respiratory system, but its effects on the expression, function, and cellular locali- zation of mitochondrial chaperonins are still largely unknown. We studied in vivo (airways biopsies) the localization of Hsp10 and Hsp60 in patients (smokers and non-smokers) affected by mild-moderate COPD, and charac- terized the effects of non-lethal doses of CS extract (CSE) on the expression of these molecules in two human cell lines: lung fibroblasts (HFL-1) and bronchial epithelial cells (16HBE). We applied various in vitro methods: IHC, subcellular fractionation analyses (SFA), western blotting (WB), ICC, transmission electron microscopy (TEM) immunogold, chromati protein extracts (CPE), as well as 2D-gel based proteomics analyses. Bioinformatics was used to gather structural in silico data. IHC showed that Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers. ICC, SFA, and WB showed that 16HBE and HFL-1 cells featured nuclear Hsp10, before and after CSE exposure; TEM immunogold further confirmed this observation. Proteomics data showed that CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts. Bioinformatics analyses indicated that Hsp10 can localize in extramitochondrial sites, such as the nucleus, even if Hsp10 lacks known DNA-binding motifs or nuclear import signals. Hsp10 nuclear levels increased after CSE stimulation in HFL-1, indicating cytosol to nucleus migration, and although Hsp10 did not bind DNA, it bound a DNA-associated protein as suggested by CPE/gel retardation experiments. Data reported here indicate that in human cells of the respiratory mucosa there are at least three different intracellular locales for Hsp10: mitochondrial, nuclear, and cyto- solic. Further experiments are en route for the definition of the mechanisms underlying the transfer of Hsp10 to the nucleus and other cellular/extracellular compartments. This work was supported by grants from University of Palermo (FFR 2012) to GLR.

The role of heat shock proteins in the inflammatory state of vernal keratoconjunctivitis

The aim of the study was to analyse the role of heat shock proteins (HSPs) in vernal keratoconjunctivitis (VKC), a recurrent allergic ocular inflammatory disease. We evaluated the expression of some HSPs (Hsp10, Hsp27, Hsp40, Hsp60, Hsp70, Hsp90) in the mucosal biopsies of VKC patients by immunohistochemistry, and in conjunctival cells cultures treated with inflammatory stimuli (IL-1β, histamine, IL-4, TNFβ, UVB irradiation) by western blotting. Immunohistochemical analysis revealed that Hsp10, Hsp27, Hsp40, Hsp70 and Hsp90 expression was significantly increased in VKC whereas the Hsp60 level was unaltered. In vitro induction by inflammatory stimuli in Chang epithelial conjunctival cells revealed that Hsp70 protein expression was significantly increased in epithelial cells line after 4-10 h from histamine and IL-4 stimulation. The same molecule was also overexpressed in conjunctival fibroblast cultures after TNFβ treatment. Hsp90 protein level was increased in the same cell cultures by IL-1β at 4-10-24 h. The Hsp40 protein expression was increased both in epithelial and fibroblast cultures induced by all inflammatory stimuli. Moreover, UVB irradiation significantly increased Hsp90 expression in primary fibroblast culture and Hsp27 in conjunctival epithelial cells after 10 hours. These results indicate that HSPs levels increase in VKC. In particular, Hsp40 expression is up-regulated by all the typical inflammatory stimuli involved in VKC pathogenesis. The specific role of each one of these chaperonins to further induce or counteract inflammation need to be further investigated.

Potential marker proteins for ozone-induced yield reduction in rice

Three proteins - a 60-kDa chaperonin (CPN-60), chloroplastic ATP synthase, and enolase 1 - were evaluated as potential markers of ozone-induced yield responses in six rice (Oryza sativa L.) cultivars (‘Kirara 397’, ‘Koshihikari’, ‘Nipponbare’, ‘Takanari’, ‘Kasalath’, ‘Suphanburi 90’) under ozone stress in laboratory-scale tests. The levels of all three proteins decreased after ozone exposure in cultivars identified as ozone-sensitive while they increased or remained constant after ozone exposure in tolerant cultivars, although ATP synthase tended to decrease. Furthermore, the protein level and grain yield in each cultivar exposed to ozone were significantly positively correlated for all three proteins. Thus, CPN-60 and enolase 1 are potential markers for chronic ozone stress in rice

Detection of antibodies against immunodominant proteins of Chlamydia trachomatis in the sera of patients with urogenital Chlamydia trachomatis infection

Objective To detect antibodies against chlamydial plasmid-encoded protein 3 (Pgp3),outer membrane complex protein B C-terminal peptide (OmcBc),CT841 protein and heat shock protein 60 (HSP60) in the sera of patients with urogenital Chlamydia trachomatis infection.Methods Recombinant plasmids encoding the aforementioned four proteins and an empty plasmid were transformed into Escherichia coli separately followed by 2-hour isopropyl-1-thio-β-galactopyranoside (IPTG) induction and cell lysis.The expressed proteins were purified with glutathione magnetic beads and then used to coat 96-well enzyme-linked immunosorbent assay (ELISA) plates precoated by glutathione.Serum samples were collected from 20 patients with and 20 clients without urogenital C.trachomatis infection attending the sexually transmitted disease (STD) clinic of Tianjin Medical University General Hospital.ELISA with the expressed protein-coated plates was adopted to detect antibodies against these proteins in the serum samples.Results Of the 20 serum samples from C.trachomatis-infected patients,14 (70％)had anti-Pgp3 antibody,9 (45％) anti-OmcBc antibody,8 (40％) anti-CT841 antibody,and 5 (25％) anti-HSP60 antibody.Meanwhile,no antibody was detected in any of the serum samples from uninfected clients except for one with anti-HSP60 antibody.Conclusions Of the four studied C.trachomatis proteins,Pgp3 appears to have the strongest antigenicity with the highest antibody-detection rate,while HSP60 exhibits the weakest antigenicity with the lowest antibody-detection rate. Key words: Chlamydia trachomatis; Chaperonin 60; Pgp3; OmcB; CT841

Clinical anatomic, immunomorphologic and molecular anatomic data suggest interplay of thyroidal molecules, autoantibodies and Hsp60 in Hashimoto’s disease

Hsp60 is, typically, a mitochondrial protein, but it also occurs in the cytosol, vesicles, and plasma membrane, and in the intercellular space and biological fluids, e.g., blood. Changes in the levels and distribution of Hsp60 are linked to several pathologies, including cancer and chronic inflammatory and autoimmune disorders. What is the histopathological pattern of Hsp60 in the thyroid of Hashimoto’s patients? Are there indications of a pathogenic role of Hsp60 that may make Hashimoto’s thyroiditis a chaperonopathy? Experiments reported here provide information regarding those questions. We found by various immunomorphological techniques increased levels of Hsp60 in the thyroid from HT patients, localized to thyrocytes of small and degenerated follicles and to oncocytes (Hurtle cells). Immunofluorescence showed the chaperonin both inside the cells and also in the plasma membrane, especially in oncocytes. We also found that Hsp60 levels in the blood of HT patients were increased compared to controls and correlated with those of autoantibodies against two distinctive thyroidal proteins, thyroglobulin (TG) and thyroid peroxidase (TPO) (r=0.379, p=0.0103; r=0.484, p=0.0008; respectively). Molecular analysis of these two proteins in comparison with Hsp60 demonstrated various regions of high structural similarity shared by them, which could very well be immunologically crossreactive epitopes. Thus, it is likely that the three proteins potentiate each other as immunogens to elicit autoantibodies and, as antigens, to cause antigen-antibody reactions at those sites in which Hsp60 is exposed, for example the surface of oncocytes. This would lead to inflammation and oncocyte lysis with destruction of thyroidal tissue. The cytometric bead assay revealed that recombinant Hsp60 did not induce increment of cytokine production by peripheral blood mononuclear cells from HT patients. Consequently, we propose that Hsp60 is implicated in the pathogenesis of Hashimoto’s thyroiditis as autoantigen, via a participation of autoantibodies that also recognize TG and TPO, whereas participation of inflammatory cytokines induced by the chaperonin is unlikely. Supported by IEMEST (FC and AJLM).

Novel in vitro and in vivo data on the cellular localization of Hsp10 in smokers affected by COPD and in lung-derived cell lines exposed to cigarette smoke extract as stressor

Cigarette smoke is a potent stressor for the respiratory system, contributing to pathogenesis, for instance in chronic obstructive pulmonary disease (COPD), but its effects on the expression, function, and cellular localization of mitochondrial chaperonins are still largely unknown. We studied in vivo (airways biopsies) the localization of Hsp10 and Hsp60 in patients (smokers and non-smokers) affected by mild-moderate COPD, and characterized the effects of non-lethal doses of cigarette smoke extract (CSE) on the expression of these molecules in two human cell lines: lung fibroblasts (HFL-1) and bronchial epithelial (16HBE). We applied various in vitro methods: immunohistochemistry (IHC), subcellular fractionation analyses (SFA), Western blotting (WB), immunocytochemistry (ICC), and transmission electron microscopy (TEM) immunogold, and used bioinformatics and databases searches to gather structural in silico data for interpreting and complementing the in vitro results. IHC showed that in smokers and non-smokers COPD patients Hsp10 was localized in both, the cytoplasm and the nucleus of epithelial and lamina propria cells, while Hsp60 was present only in the cytosol. ICC, SFA, and WB on both CSE-exposed cell lines confirmed the presence of nuclear Hsp10, with an increasing trend in parallel to CSE concentration. TEM immunogold further confirmed Hsp10 in the nucleus, in addition to its presence in the cytoplasm and mitochondria, on both cell lines. Bioinformatics and in silico structural analyses indicated that Hsp10 can localize in extramitochondrial sites, such as the nucleus, even if Hsp10 lacks known DNA-binding motifs or nuclear import signals in its primary sequence. Our data suggest a link between exposure to exogenous oxidative stress and cell response, involving Hsp10, which would play roles different from its canonical functions. It is known that Hsp10 can display an array of functions depending on its location: cytoplasm, mitochondria, or extracellular. Here, we show for the first time the presence of Hsp10 in the nucleus of epithelial and stromal human-lung cell lines, paralleling the observations in vivo in COPD patients, and indicating that intranuclear Hsp10 levels are affected by oxidative stress due to an exogenous stressor like cigarette-smoke. The questions now are by what mechanism Hsp10 becomes a resident of the nucleus and what are its functions there.

Simulation of the shape of chaperonins using the small-angle x-ray scattering curves and torus form factor

The inverse scattering problem has been solved for protein complexes whose surfaces can be described by a set of the simplest doubly connected surfaces in the uniform approximation (a scattering potential inside the molecule is a constant). Solutions of two proteins—wellknown GroEL bacterial chap� eronin and poorstudied bacteriophage chaperonin, which is a product of 146 gene (gp146)—were taken for the experiment. The shapes of protein complexes have been efficiently reconstructed from the experimental scattering curves. The shell method, the method of the rotation of amino acid sequences with the use of the form factor of an amino acid, and the method of seeking the model parameters of a protein complex with the preliminarily obtained form factor of the model have been used to reconstruct the shape of these particles.