The extreme C terminus of progesterone receptor contains a transcriptional repressor domain that functions through a putative corepressor.

Binding of a hormone agonist to a steroid receptor leads to the dissociation of heat shock proteins, dimerization, specific DNA binding, and target gene activation. Although the progesterone antagonist RU486 can induce most of these events, it fails to activate human progesterone receptor (hPR)-dependent transcription. We have previously demonstrated that a conformational change is a key event leading to receptor activation. The major conformational distinction between hormone- and antihormone-bound receptors occurs within the C-terminal portion of the molecule. Furthermore, hPR mutants lacking the C terminus become transcriptionally active in the presence of RU486. These results suggest that the C terminus contains a repressor domain that inhibits the transcriptional activity of the RU486-bound hPR. In this study, we have defined a 12 amino acid (12AA) region in the C terminus of hPR that is necessary and sufficient for the repressor function when fused to the C-terminal truncated hPR or to the GAL4 DNA-binding domain. Mutations in the 12AA domain (aa 917-928) generate an hPR that is active in the presence of RU486. Furthermore, overexpression of the 12AA peptide activates the RU486-bound wild-type hPR without affecting progesterone-dependent activation. These results suggest that association of the 12AA repressor region with a corepressor might inactivate hPR activity when it is bound to RU486. We propose that binding of a hormone agonist to the receptor changes its conformation in the ligand-binding domain so that association with coactivator is promoted and activation of target gene occurs.

A common evolutionary origin for mitochondria and hydrogenosomes.

Trichomonads are among the earliest eukaryotes to diverge from the main line of eukaryotic descent. Keeping with their ancient nature, these facultative anaerobic protists lack two "hallmark" organelles found in most eukaryotes: mitochondria and peroxisomes. Trichomonads do, however, contain an unusual organelle involved in carbohydrate metabolism called the hydrogenosome. Like mitochondria, hydrogenosomes are double-membrane bounded organelles that produce ATP using pyruvate as the primary substrate. Hydrogenosomes are, however, markedly different from mitochondria as they lack DNA, cytochromes and the citric acid cycle. Instead, they contain enzymes typically found in anaerobic bacteria and are capable of producing molecular hydrogen. We show here that hydrogenosomes contain heat shock proteins, Hsp70, Hsp60, and Hsp10, with signature sequences that are conserved only in mitochondrial and alpha-Gram-negative purple bacterial Hsps. Biochemical analysis of hydrogenosomal Hsp60 shows that the mature protein isolated from the organelle lacks a short, N-terminal sequence, similar to that observed for most nuclear-encoded mitochondrial matrix proteins. Moreover, phylogenetic analyses of hydrogenosomal Hsp70, Hsp60, and Hsp10 show that these proteins branch within a monophyletic group composed exclusively of mitochondrial homologues. These data establish that mitochondria and hydrogenosomes have a common eubacterial ancestor and imply that the earliest-branching eukaryotes contained the endosymbiont that gave rise to mitochondria in higher eukaryotes.

HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome.

We have isolated a new type of ATP-dependent protease from Escherichia coli. It is the product of the heat-shock locus hslVU that encodes two proteins: HslV, a 19-kDa protein similar to proteasome beta subunits, and HslU, a 50-kDa protein related to the ATPase ClpX. In the presence of ATP, the protease hydrolyzes rapidly the fluorogenic peptide Z-Gly-Gly-Leu-AMC and very slowly certain other chymotrypsin substrates. This activity increased 10-fold in E. coli expressing heat-shock proteins constitutively and 100-fold in cells expressing HslV and HslU from a high copy plasmid. Although HslV and HslU could be coimmunoprecipitated from cell extracts of both strains with an anti-HslV antibody, these two components were readily separated by various types of chromatography. ATP stimulated peptidase activity up to 150-fold, whereas other nucleoside triphosphates, a nonhydrolyzable ATP analog, ADP, or AMP had no effect. Peptidase activity was blocked by the anti-HslV antibody and by several types of inhibitors of the eukaryotic proteasome (a threonine protease) but not by inhibitors of other classes of proteases. Unlike eukaryotic proteasomes, the HslVU protease lacked tryptic-like and peptidyl-glutamyl-peptidase activities. Electron micrographs reveal ring-shaped particles similar to en face images of the 20S proteasome or the ClpAP protease. Thus, HslV and HslU appear to form a complex in which ATP hydrolysis by HslU is essential for peptide hydrolysis by the proteasome-like component HslV.

Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility.

In addition to the five 70-kDa heat shock proteins (HSP70) common to germ cells and somatic tissues of mammals, spermatogenic cells synthesize HSP70-2 during meiosis. To determine if this unique stress protein has a critical role in meiosis, we used gene-targeting techniques to disrupt Hsp70-2 in mice. Male mice homozygous for the mutant allele (Hsp70-2 -/-) did not synthesize HSP70-2, lacked postmeiotic spermatids and mature sperm, and were infertile. However, neither meiosis nor fertility was affected in female Hsp70-2 -/- mice. We previously found that HSP70-2 is associated with synaptonemal complexes in the nucleus of meiotic spermatocytes from mice and hamsters. While synaptonemal complexes assembled in Hsp70-2 -/- spermatocytes, structural abnormalities became apparent in these cells by late prophase, and development rarely progressed to the meiotic divisions. Furthermore, analysis of nuclei and genomic DNA indicated that the failure of meiosis in Hsp70-2 -/- mice was coincident with a dramatic increase in spermatocyte apoptosis. These results suggest that HSP70-2 participates in synaptonemal complex function during meiosis in male germ cells and is linked to mechanisms that inhibit apoptosis.

A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning.

In search of proteins which interact with activated steroid hormone receptors, we screened a human liver lambda gt11 expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence of 1322 bp that encodes a protein of 274 aa. This protein consists predominantly of hydrophilic amino acids and contains a putative bipartite nuclear localization signal. The in vitro translated receptor-associating protein runs in SDS/polyacrylamide gels with an apparent molecular mass of 46 kDa. By use of the bacterially expressed fusion protein with glutathione S-transferase we have found that interaction is not limited to the glucocorticoid receptor but included other nuclear receptors--most notably, the estrogen and thyroid receptors. Binding also occurs with the glucocorticoid receptor complexed with the antiglucocorticoid RU 38486, with the estrogen receptor complexed with the antiestrogen 4-hydroxytamoxifen or ICI 164,384, and even with receptors not complexed with ligand. Association with steroid hormone receptors depends on prior receptor activation--i.e., release from heat shock proteins. The sequence identified here appears to be a general partner protein for nuclear hormone receptors, with the gene being expressed in a variety of mammalian tissues.

The GroES homolog of Helicobacter pylori confers protective immunity against mucosal infection in mice.

Helicobacter pylori is an important etiologic agent of gastroduodenal disease. In common with other organisms, H. pylori bacteria express heat shock proteins that share homologies with the GroES-GroEL class of proteins from Escherichia coli. We have assessed the heat shock proteins of H. pylori as potential protective antigens in a murine model of gastric Helicobacter infection. Orogastric immunization of mice with recombinant H. pylori GroES- and GroEL-like proteins protected 80% (n = 20) and 70% (n = 10) of animals, respectively, from a challenge dose of 10(4) Helicobacter felis bacteria (compared to control mice, P = 0.0042 and P = 0.0904, respectively). All mice (n = 19) that were immunized with a dual antigen preparation, consisting of H. pylori GroES-like protein and the B subunit of H. pylori urease, were protected against infection. This represented a level of protection equivalent to that provided by a sonicated Helicobacter extract (P = 0.955). Antibodies directed against the recombinant H. pylori antigens were predominantly of the IgG1 class, suggesting that a type 2 T-helper cell response was involved in protection. This work reports a protein belonging to the GroES class of heat shock proteins that was shown to induce protective immunity. In conclusion, GroES-like and urease B-subunit proteins have been identified as potential components of a future H. pylori subunit vaccine.

Endophytic colonization of barley (Hordeum vulgare) roots by the nematophagous fungus Pochonia chlamydosporia reveals plant growth promotion and a general defense and stress transcriptomic response

Plant crop yields are negatively conditioned by a large set of biotic and abiotic factors. An alternative to mitigate these adverse effects is the use of fungal biological control agents and endophytes. The egg-parasitic fungus Pochonia chlamydosporia has been traditionally studied because of its potential as a biological control agent of plant-parasitic nematodes. This fungus can also act as an endophyte in monocot and dicot plants, and has been shown to promote plant growth in different agronomic crops. An Affymetrix 22K Barley GeneChip was used in this work to analyze the barley root transcriptomic response to P. chlamydosporia root colonization. Functional gene ontology (GO) and gene set enrichment analyses showed that genes involved in stress response were enriched in the barley transcriptome under endophytism. An 87.5 % of the probesets identified within the abiotic stress response group encoded heat shock proteins. Additionally, we found in our transcriptomic analysis an up-regulation of genes implicated in the biosynthesis of plant hormones, such as auxin, ethylene and jasmonic acid. Along with these, we detected induction of brassinosteroid insensitive 1-associated receptor kinase 1 (BR1) and other genes related to effector-triggered immunity (ETI) and pattern-triggered immunity (PTI). Our study supports at the molecular level the growth-promoting effect observed in plants endophytically colonized by P. chlamydosporia, which opens the door to further studies addressing the capacity of this fungus to mitigate the negative effects of biotic and abiotic factors on plant crops.

Surdez induzida pelo trauma acústico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Identification of differentially expressed genes in a Giardia lamblia WB C6 clone resistant to nitazoxanide and metronidazole.

OBJECTIVES The characterization of differential gene expression in Giardia lamblia WB C6 strain C4 resistant to metronidazole and nitazoxanide using microarray technology and quantitative real-time PCR. METHODS In a previous study, we created and characterized the G. lamblia WB C6 clone C4 resistant to nitazoxanide and metronidazole. In this study, using a microarray-based approach, we have identified open-reading frames (ORFs) that were differentially expressed in C4 when compared with its wild-type WB C6. Using quantitative real-time PCR, we have validated the expression patterns of some of those ORFs, focusing on chaperones such as heat-shock proteins in wild-type and C4 trophozoites. In order to induce an antigenic shift, trophozoites of both strains were subjected to a cycle of en- and excystation. Expression of selected genes and resistance to nitazoxanide and metronidazole were investigated after this cycle. RESULTS Forty of a total of 9115 ORFs were found to be up-regulated and 46 to be down-regulated in C4 when compared with wild-type. After a cycle of en- and excystation, resistance of C4 to nitazoxanide and metronidazole was lost. Resistance formation and en-/excystation were correlated with changes in expression of ORFs encoding for major surface antigens such as the variant surface protein TSA417 or AS7 ('antigenic shift'). Moreover, expression patterns of the cytosolic heat-shock protein HSP70 B2, HSP40, and of the previously identified nitazoxanide-binding proteins nitroreductase and protein disulphide isomerase PDI4 were correlated with resistance and loss of resistance after en-/excystation. C4 trophozoites had a higher thermotolerance level than wild-type trophozoites. After en-/excystation, this tolerance was lost. CONCLUSIONS These results suggest that resistance formation in Giardia to nitazoxanide and metronidazole is correlated with altered expression of genes involved in stress response such as heat-shock proteins.

Compression Loading Induced Cellular Stress Response of Intervertebral Disc Cells in Organ Culture

Introduction: Mechanical stress is often associated to interverterbal disc (IVD) degeneration and the effect of mechanical loading on IVD has been studied and reviewed.1,2 Previously, expression of heat shock proteins, HSP70 and HSP27 has been found in pathological discs.3 However, there is no direct evidence on whether IVD cells respond to the mechanical loading by expression of HSPs. The objective of this study is to investigate the stress response of IVD cells during compressive loading in an organ culture. Materials and Methods: Fresh adult bovine caudal discs were cultured with compressive loading applied at physiological range. Effect of loading type (static and dynamic) and repeated loading (2 hours per day for 2 days) were studied. Nucleus pulposus (NP) and annulus fibrosus (AF) of the IVD were retrieved at different time points: right after loading and right after resting. Positive control discs were heat shocked (43°C). Cell activity was assessed and expression of stress response genes (HSP70 and HSF1) and matrix remodeling genes (ACAN, COL2, COL1, ADAMTS4, MMP3 and MMP13) were studied. Results: Cell activity was maintained in all groups. Both NP and AF expressed high level of HSP70 in heat shock groups, confirming their expression in response to stress. In NP, expression of HSP70 was up-regulated after static loading and dynamic loading with higher fold change was observed after static loading. During repeated loading, HSP70 appeared to be upregulated right after loading and decreased after resting. Such trend was not observed in AF and HSF1 levels. Expressions of matrix remodeling genes did not change significantly with loading except ADAMTS4 decreased in AF during static loading. Conclusion: This study demonstrated that NP cells upregulate expression of HSP70 in response to loading induced stress without changing cell activity and matrix remodeling significantly. Acknowledgments: This project was funded by AO Spine (AOSPN) (grant number: SRN_2011_14) and a fellowship exchange award by AO Spine Scientific Research Network (SRN).

Interaction of the Hsp90 cochaperone cyclophilin 40 with Hsc70

The high-affinity ligand-binding form of unactivated steroid receptors exists as a multicomponent complex that includes heat shock protein (Hsp)90; one of the immunophilins cyclophilin 40 (CyP40), FKBP51, or FKBP52; and an additional p23 protein component. Assembly of this heterocomplex is mediated by Hsp70 in association with accessory chaperones Hsp40, Hip, and Hop. A conserved structural element incorporating a tetratricopeptide repeat (TPR) domain mediates the interaction of the immunophilins with Hsp90 by accommodating the C-terminal EEVD peptide of the chaperone through a network of electrostatic and hydrophobic interactions. TPR cochaperones recognize the EEVD structural motif common to both Hsp90 and Hsp70 through a highly conserved clamp domain. In the present study, we investigated in vitro the molecular interactions between CyP40 and FKBP52 and other stress-related components involved in steroid receptor assembly, namely Hsp70 and Hop. Using a binding protein-retention assay with CyP40 fused to glutathione S-transferase immobilized on glutathione-agarose, we have identified the constitutively expressed form of Hsp70, heat shock cognate (Hsc)70, as an additional target for CyP40. Deletion mapping studies showed the binding determinants to be similar to those for CyP40-Hsp90 interaction. Furthermore, a mutational analysis of CyP40 clamp domain residues confirmed the importance of this motif in CyP40-Hsc70 interaction. Additional residues thought to mediate binding specificity through hydrophobic interactions were also important for Hsc70 recognition. CyP40 was shown to have a preference for Hsp90 over Hsc70. Surprisingly, FKBP52 was unable to compete with CyP40 for Hsc70 binding, suggesting that FKBP52 discriminates between the TPR cochaperone-binding sites in Hsp90 and Hsp70. Hop, which contains multiple units of the TPR motif, was shown to be a direct competitor with CyP40 for Hsc70 binding. Similar to Hop, CyP40 was shown not to influence the adenosine triphosphatase activity of Hsc70. Our results suggest that CyP40 may have a modulating role in Hsc70 as well as Hsp90 cellular function.

Echinacea intake induces an immune response through altered expression of leucocyte hsp70, increased white cell counts and improved erythrocyte antioxidant defences

Objective: To study the effect of Echinacea tablets on the expression of leucocyte heat shock protein 70 (hsp70), erythrocyte haemolysis, plasma antioxidant status, serum chemistry, haematological values and plasma alkylamide concentrations. Method: Eleven healthy individuals (26-61 years of age) were evaluated at baseline (day 1) and on day 15 after consuming two commercially blended Echinacea tablets daily for 14 days. Results: Echinacea supplementation enhanced the fold increase in leucocyte hsp70 expression after a mild heat shock (P=0.029). White cell counts (WCC) were also increased (P=0.043). We also observed a preventative effect against free radical induced erythrocyte haemolysis (P=0.006) indicative of an antioxidant effect. Conclusion: The pilot study suggests that Echinacea may invoke an immune response through altered expression of hsp70 and increased WCC.

Isolation, characterization and discovery of new alleles of sHsp genes in durum wheat (Triticum durum Desf.)

Gli organismi vegetali mostrano una notevole capacità di adattamento alle condizioni di stress e lo studio delle componenti molecolari alla base dell'adattamento in colture cerealicole di interesse alimentare, come il frumento, è di particolare interesse per lo studio di varietà che consentano una buona produzione con basso input anche in condizioni ambientali non ottimali. L'esposizione delle colture cerealicole a stress termico durante determinate fasi del ciclo vitale influisce negativamente sulla resa e sulla qualità, a questo fine è necessario chiarire le basi genetiche e molecolari della termotolleranza per identificare geni e alleli vantaggiosi da impiegare in programmi di incrocio volti al miglioramento genetico. Numerosi studi dimostrano il coinvolgimento delle sHSP a localizzazione cloroplastica (in frumento sHSP26) nel meccanismo di acquisizione della termotolleranza e la loro interazione con diverse componenti del fotosistema II (PSII) che determinerebbe un’azione protettiva in condizioni di stress termico e altri tipi di stress. Lo scopo del progetto è quello di caratterizzare in frumento duro nuove varianti alleliche correlate alla tolleranza a stress termico mediate l'utilizzo del TILLING (Target Induced Local Lesion In Genome), un approccio di genetica inversa che prevede la mutagenesi e l'identificazione delle mutazioni indotte in siti di interesse. Durante la tesi sono state isolate e caratterizzate 3 sequenze geniche complete per smallHsp26 denominate TdHsp26-A1; TdHsp26-A2; TdHsp26-B1 e un putativo pseudogene denominato TdHsp26-A3. I geni isolati sono stati usati come target in analisi di TILLING in due popolazioni di frumento duro mutagenizzate con EMS (EtilMetanoSulfonato). Nel nostro studio sono stati impiegati due differenti approcci di TILLING: un approccio di TILLING classico mediante screening con High Resolution Melting (HRM) e un approccio innovativo che sfrutta un database di TILLING recentemente sviluppato. La popolazione di mutanti cv. Kronos è stata analizzata per la presenza di mutazioni in tutti e tre i geni individuati mediante ricerca online nel database di TILLING, il quale sfrutta la tecnica dell’exome capture sulla popolazione di TILLING seguito da sequenziamento ad alta processività. Attraverso questa tecnica sono state individuate, nella popolazione mutagenizzata di frumento duro cv. Kronos, 36 linee recanti mutazioni missenso. Contemporaneamente lo screening con HRM, effettuato su 960 genotipi della libreria di TILLING di frumento duro cv. Cham1 ha consentito di individuare mutazioni in una regione di 211bp di interesse funzionale del gene TdHsp26-B1, tra le quali 3 linee mutanti recanti mutazioni missenso in omozigosi. Alcune mutazioni missenso individuate sui due geni TdHsp26-A1 e TdHsp26-B1 sono state confermate in vivo nelle piante delle rispettive linee mutanti generando marcatori codominanti KASP (Kompetitive Allele Specific PCR) con cui è stato possibile verificare anche il grado di zigosità di tali mutazioni. Al fine di ridurre il numero di mutazioni non desiderate nelle linee risultate più interessanti, è stato eseguito il re-incrocio dei mutanti con i relativi parentali wild type ed inoltre sono stati generati alcuni doppi mutanti che consentiranno di comprendere meglio i meccanismi molecolari presieduti da questa classe genica. Gli individui F1 degli incroci sono stati poi genotipizzati con i medesimi marcatori KASP specifici per la mutazione di interesse per verificare la buona riuscita dell’incrocio. Questo approccio ha permesso di individuare ed implementare risorse genetiche utili ad intraprendere studi funzionali relativi al ruolo di smallHSP plastidiche implicate nella acquisizione di termotolleranza in frumento duro e di generare marcatori potenzialmente utili in futuri programmi di breeding.

Proteomic analysis of the anti-inflammatory action of minocycline

Minocycline possesses anti-inflammatory properties independently of its antibiotic activity although the underlying molecular mechanisms are unclear. Lipopolysaccharide (LPS)-induced cytokines and pro-inflammatory protein expression are reduced by minocycline in cultured macrophages. Here, we tested a range of clinically important tetracycline compounds (oxytetracycline, doxycycline, minocycline and tigecycline) and showed that they all inhibited LPS-induced nitric oxide production. We made the novel finding that tigecycline inhibited LPS-induced nitric oxide production to a greater extent than the other tetracycline compounds tested. To identify potential targets for minocycline, we assessed alterations in the macrophage proteome induced by LPS in the presence or absence of a minocycline pre-treatment using 2-DE and nanoLC-MS. We found a number of proteins, mainly involved in cellular metabolism (ATP synthase ß-subunit and aldose reductase) or stress response (heat shock proteins), which were altered in expression in response to LPS, some of which were restored, at least in part, by minocycline. This is the first study to document proteomic changes induced by minocycline. The observation that minocycline inhibits some, but not all, of the LPS-induced proteomic changes shows that minocycline specifically affects some signalling pathways and does not completely inhibit macrophage activation.

The expression of Hsf1 in the rat myometrium during pregnancy and labour

Heat shock factor 1 (Hsf1) is a protein known to be involved in both stress and developmental processes through the regulation of heat shock proteins. However, to date, no studies have been performed on examining its expression in the myometrium during pregnancy. During pregnancy, the uterus undergoes many structural and functional changes, and it also endures both mechanical and hormonal stresses. Therefore, the purpose of this thesis was to characterize the expression of Hsf1, and its associated factors in the uterus during pregnancy. Immunoblot analysis determined that Hsf1 protein expression was high early in gestation (day (d) 6) and then decreased significantly from mid gestation onwards (specifically when compared to d15, d17 and d22, p<0.05, n=5). Immunofluorescence analysis, demonstrated that Hsf1 was readily detectable in the myometrium but did not markedly change over gestation. Hsf1 was also localized mainly in the cytoplasm of myometrial cells, with some granular staining in the nucleus. Many related proteins of Hsf1 were also detectable in the myometrium, during pregnancy, such as PARP-1 and Hsf2. These results indicate that Hsf1 could play an important role early in gestation either to aid in myometrial cell proliferation or to upregulate expression of key genes necessary for subsequent myometrial differentiation.