Hop (Hsp70-Hsp90 Organizing Protein) is a co-chaperone of two major molecular chaperones, Hsp70 and Hsp90, and acts by transferring substrates from Hsp70 to Hsp90. Although under normal conditions Hop is predominantly localized within the cytosol, Hop has been detected in the nucleus under certain conditions including cell cycle arrest. A putative nuclear localization signal (NLS) has been identified within Hop, which overlaps with the TPR2A domain (previously shown to be critical for Hop-Hsp90 interactions). Hop is phosphorylated in vitro by two cell cycle kinases, namely, casein kinase II (CKII) at S189 and cdc2-kinase at T198; both residues are found upstream of the putative NLS and TPR2A domain. Mimicking phosphorylation at either phosphorylation site appeared to affect the subcellular localization of Hop. The aim of this study was to characterize Hop with respect to its phosphorylation status in vivo, as well as its subcellular localization pattern under heat stress and determine how these properties affected its interaction with Hsp90 as a co-chaperone. Dephosphorylation of proteins under normal and heat shock conditions changed the isoform composition of Hop, providing strong evidence that Hop was phosphorylated in vivo. Surface plasmon resonance (SPR) and glutatione-S-transferase (GST) co-precipitation studies showed that a cdc2-kinase phosphorylated mimic of Hop disrupted Hop-Hsp90 binding. A full length Hop-EGFP construct, as well as substitution mutants of the predicted NLS residues within the Hop-EGFP construct, were transfected into baby hamster kidney (BHK)-21 cells in order to establish the subcellular localization of Hop under heat stress and to test whether predicted residues were critical for nuclear localization of Hop. Under normal conditions, both Hop-EGFP and the NLS mutants were predominantly cytosolic, but when the cells were subjected to heat stress, Hop and its NLS-mutants were localized to both the cytosol and the nucleus. SPR and GST co-precipitation studies showed that substitution of the residues within the major arm of the putative NLS abrogated Hop-Hsp90 interactions. The data obtained from this study, showed for the first time, that Hop was phosphorylated in vivo and suggested that phosphorylation of Hop by cdc2-kinase could inhibit Hop-Hsp90 interactions. Moreover, these results suggested that the subcellular localization of Hop was dependent on stress levels of the cell, particularly heat stress. We propose that the nuclear localization of Hop may be primarily regulated by stress and secondarily by cell cycle arrest. The major arm of the putative NLS did not affect the localization of Hop directly, but was shown to be critical for Hop-Hsp90 binding in vitro. The results of this study suggested that binding of Hop to Hsp90 sequestered Hop within the cytosol and that Hsp90 acted as a cytosolic retention factor for Hop. Both phosphorylation of Hop, and its subcellular localization, appeared to be intimately related to its interaction with Hsp90 as a co-chaperone.

Self-renewal of mouse embryonic stem (mES) cells is dependent upon the presence of leukemia inhibitory factor (LIF). LIF induces tyrosine phosphorylation and nuclear translocation of STAT3 (signal transducer and activator of transcription 3) which is thought to promote self-renewal by inducing key target genes. The molecular chaperone heat shock protein 90 (Hsp90) is involved in signal transduction pathways and regulates STAT3 activity in different cell types. However, the role of Hsp90 in regulating STAT3 activity in mES cells has not previously been investigated. The aim of this study was to investigate if Hsp90 interacts with STAT3 in mES cells and to determine if this interaction is important for the maintenance of self-renewal. It was found that when mES cells were cultured for 24.0 hours in the absence of LIF, the expression levels of total STAT3, tyrosine-phosphorylated STAT3 (pYSTAT3), and the pluripotency marker, Nanog, were down regulated. However, the expression level of Hsp90 was found to be slightly up-regulated over the same period. Significantly, it was found that the amount of STAT3 in differentiating mES cells available for binding to Hsp90 was decreased upon down-regulation of STAT3 by LIF withdrawal. Therefore, STAT3-Hsp90 interactions in mES cells were dependent on the presence of LIF, which suggested that the reduction in STAT3-Hsp90 interaction may have resulted from the low levels of STAT3. Despite a dramatic reduction in the expression levels of pYSTAT3 upon 24.0 hours of culture of mES cells in the presence of the STAT3 tyrosine phosphorylation inhibitor, cucurbitanin I, there was no obvious reduction in the levels of total STAT3, Oct-3/4 or Nanog. These results suggested that the levels of unphosphorylated STAT3 rather than pYSTAT3, maybe more important in the maintenance of mES cells self-renewal.

Stress-inducible protein 1 (Sti1) is a 60 kDa eukaryotic protein that is important under stress and non-stress conditions. Human Sti1 is also known as the Hsp70/Hsp90 organising protein (Hop) that coordinates the functional cooperation of heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) during the folding of various transcription factors and kinases, including certain oncogenic proteins and prion proteins. Limited studies have been conducted on the STI1 gene structure. Thus, the aim of this study was to develop a comprehensive description of human STI1 (hSTI1), mouse STI1 (mSTI1), and yeast STI1 (ySTI1) genes, using a bioinformatic approach. Genes encoded near the STI1 loci were identified for the three organisms using National Centre for Biotechnology Information (NCBI) MapViewer and the Saccharomyces Genome Database. Exon/intron boundaries were predicted using Hidden Markov model gene prediction software (HMMGene) and Genscan, and by alignment of the mRNA sequence with the genomic DNA sequence. Transcription factor binding sites (TFBS) were predicted by scanning the region 1000 base pairs (bp) upstream of the STI1 orthologues’ transcription start site (TSS) with Alibaba, Transcription element search software (TESS) and Transcription factor search (TFSearch). The promoter region was defined by comparing the number, type and position of TFBS across the orthologous STI1 genes. Additional putative TFBS were identified for ySTI1 by searching with software that aligns nucleic acid conserved elements (AlignACE) for over-represented motifs in the region upstream of the TSS of genes thought to be co-regulated with ySTI1. This study showed that hSTI1 and mSTI1 occur in a region of synteny with a number of genes of related function. Both hSTI1 and mSTI1 comprised 14 putative exons, while ySTI1 was encoded on a single exon. Human and mouse STI1 shared a perfectly conserved 55 bp region spanning their predicted TSS, although their TATA boxes were not conserved. A putative CpG island was identified in the region from -500 to +100 bp relative to the hSTI1 and mSTI1 TSS. This region overlapped with a region of high TFBS density, suggesting that the core promoter region was located in the region approximately 100 to 200 bp upstream of the TSS. Several conserved clusters of TFBS were also identified upstream of this promoter region, including binding sites for stimulatory protein 1 (Sp1), heat shock factor (HSF), nuclear factor kappa B (NF-kappaB), and the cAMP/enhancer binding protein (C/EBP). Microarray data suggested that ySTI1 was co-regulated with several heat shock proteins and substrates of the Hsp70/Hsp90 heterocomplex, and several putative regulatory elements were identified in the upstream region of these co-regulated genes, including a motif for HSF binding. The results of this research suggest several avenues of future experimental work, including the confirmation of the proposed core promoter, upstream regulatory elements, and CpG island, and the investigation into the co-regulation of mammalian STI1 with its surrounding genes. These results could also be used to inform STI1 gene knockout experiments in mice, to assess the biological importance of mammalian STI1.

Normal mRNA turnover is essential for genetic regulation within cells. The E. coli RNA degradosome, a large multi-component protein complex which originates through specific protein interactions, has been referred to as the “RNA decay machine” and is responsible for mRNA turnover. The degradosome functions to process RNA and its key components have been identified. The scaffold protein is RNase E and it tethers the degradosome to the cytoplasmic membrane. Polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlB helicase) and the glycolytic enzyme enolase associate with RNase E to form the degradosome. Polyphosphate kinase associates with the degradosome in substoichiometric amounts, as do the molecular chaperones DnaK and GroEL. The role of DnaK as well as that of enolase in the RNA degradosome is unknown. Very limited research has been conducted on the components of the RNA degradosome under conditions of stress. The aim of this study was to understand the role played by enolase in the assembly of the degradosome under conditions of stress, as well as investigating the protein levels of molecular chaperones under these conditions. The RNA degradosome was successfully purified through its scaffold protein using nickel-affinity chromatography. In vivo studies were performed to investigate the protein levels of DnaK and GroEL present in the degradosome under conditions of heat stress, and whether GroEL could functionally replace DnaK in the degradosome. To investigate the recruitment of enolase to the degradosome under heat stress, a subcellular fractionation was performed to determine the localization of enolase upon heat shock in vivo. The elevated temperature resulted in an increased concentration of enolase in the membrane fraction. To determine whether there is an interaction between enolase and DnaK, enolase activity assays were conducted in vitro. The effect of DnaK on enolase activity was measured upon quantifying DnaK and adding it to the enolase assays. For the first time it was observed that the activity of enolase increased with the addition of substoichiometric amounts of DnaK. This indicates that DnaK may be interacting with the RNA degradosome via enolase.

Plasmodium falciparum is the pathogen that is responsible for the most virulent, severe and dangerous form of human malaria infection, accounting for nearly a million deaths every year. To survive and develop in the unusual environment of the red blood cells, the parasite causes structural remodelling of the host cell and biochemical changes through the export of virulence factors. Among the exportome are the molecular chaperones of the heat shock protein family, of which Hsp40s and Hsp70s are prominent. PF A0660w, a type II P. falciparum Hsp40, has been shown to be exported in complex with PfHsp70-x into the infected erythrocyte, suggesting possible functional interactions. However, the chaperone properties of PF A0660w and its interactions with proteins of parasite and human origin are yet to be investigated. Using a codon optimised coding region, PF A0660w was successfully expressed in E. coli M 15 [pREP4] cells. However, the expressed protein was largely deposited as insoluble pellet, and analysis of the pellets revealed a high percentage of PF A0660w, characteristic of inclusion body formation. PF A0660w was purified from inclusion bodies using additive enhanced solubilisation and refolding buffers followed by nickel affinity chromatography. SDS-PAGE and western analysis revealed that the purified protein was of high purity. Size exclusion chromatography showed that the protein existed as a monomer in solution and the secondary structure analysis using Fourier transformed infrared spectroscopy (FTIR) confirmed the success of the refolding approach. Its monomeric state suggests that PF A0660w may be functionally different from other Hsp40 that form dimers and that for PF A0660w, dimer formation may not be needed to maintain the stability of the protein in solution, but may occur in response to functional necessities during its interaction with partner Hsp70. PFA0660w was able to significantly stimulate the ATPase activity ofPfl-Isp70-x but not Pfl-Isp70-1 or human Hsp70 (HsHsp70), suggesting a specific functional interaction. Also, PF A0660w produced a dose dependent suppression of rhodanese aggregation and cooperated with Pfl-Isp70-1, PfHsp70-x and HsHsp70 to cause enhanced aggregation suppression. Its ability to independently suppress aggregation may help to maintain substrates in an unfolded conformation for eventual transfer to partner Hsp70s during refolding processes. Also, the in vivo characterisation using a PF A0660w peptide specific antibody confirmed that PF A0660w was exported into the cytosol of infected erythrocytes. Its lack of induction upon heat shock suggests that PF A0660w may not be involved in the response of the parasite to heat stress. Overall, this study has provided the first heterologous over-expression, purification and biochemical evidence for the possible functional role of PF A0660w, and has thereby provided the needed background for further exploration of this protein as a potential target for drug discovery.

Neste trabalho realizamos um programa de seqüenciamento em larga escala de cDNAs obtidos de bibliotecas construídas a partir de mRNA de células de B. emersonii submetidas ao choque térmico e ao estresse por cádmio. Obtivemos 6350 seqüências expressas (ESTs) de alta qualidade, que representam 2326 seqüências únicas putativas (unigenes) do fungo. Destes unigenes putativos, 1282 genes foram classificados em pelo menos uma das categorias do Consórcio Gene Ontology (GO). A análise do transcriptoma parcial de B. emersonii determinado até o momento permitiu a identificação de 78 unigenes codificando chaperones moleculares de todas as famílias conhecidas. Para avaliarmos a expressão global dos genes em resposta a estresses ambientais, como o choque térmico e o cádmio, realizamos ensaios de microarranjos de DNA nestas condições de estresse. Observamos que em resposta ao choque térmico, B. emersonii induz a expressão de genes que codificam proteínas relacionadas com o enovelamento de proteínas e com a proteólise, o que seria esperado em condições de temperaturas elevadas, assim como genes que codificam proteínas com propriedades antioxidantes, além de proteínas envolvidas no metabolismo de nucleotídeos e no metabolismo de carboidratos. Em resposta ao estresse por cádmio, verificou-se a indução de genes que codificam principalmente proteínas com propriedades antioxidantes, proteínas envolvidas no metabolismo de aminoácidos, proteínas relacionadas com o transporte celular e proteínas envolvidas no enovelamento de proteínas e proteólise. Uma das conseqüências do estresse por cádmio é o aumento do estresse oxidativo e proteínas antioxidantes têm um papel fundamental na resposta a este tipo de estresse. Dentre os genes observados durante o seqüenciamento das ESTs de B. emersonii, observamos dez genes codificando proteínas distintas da família Hsp70. Nove genes hsp70 são expressos em pelo menos um dos estágios do desenvolvimento do fungo e sete apresentam uma indução significativa após o choque térmico. Estes dados sugerem que estes genes desempenham um papel importante durante o desenvolvimento e em resposta ao estresse térmico em B. emersonii. Outro dado interessante obtido neste trabalho foi o enriquecimento de ESTs que continham íntrons em sua seqüência nas bibliotecas de estresse. Portanto, o choque térmico e o estresse por cádmio em B. emersonii diminuem a eficiência de processamento dos íntrons permitindo sua caracterização. O cDNA da proteína Hsp17 foi o que apresentou o maior número de ESTs seqüenciadas nas bibliotecas de estresse. Experimentos de Northern blot indicaram que o gene hsp17 possui um nível de expressão muito baixo durante o ciclo de vida de B. emersonii, no entanto, como esperado sua expressão aumenta drasticamente quando as células de esporulação ou germinação são submetidas a choque térmico. Os níveis da proteína Hsp17 acompanham os níveis do seu mRNA, indicando que o controle da expressão do gene hsp17 deve ocorrer em nível de transcrição. / In this work we realized a large scale, sequencing program of cDNAs libraries obtained from mRNA of B. emersonii cells submitted to heat shock and cadmium stress. A total of 6350 high quality expressed sequence tags (ESTs) were obtained, representing 2326 unique putative genes (unigenes) of this fungus. From these putative unigenes, 1282 genes were classified at least in one of the three Gene Ontology Consortium (GO) categories. The analysis of the partial transcriptome of B. emersonii, determined until now, allowed the identification of 78 unigenes encoding molecular chaperones of all known protein families. To evaluate the global expression of the genes in response to environmental stresses, such as heat shock and cadmium, DNA microarray assays were performed. We observed that in response to heat shock B. emersonii induces the expreession of genes encoding proteins related to protein folding and proteolysis, as expected under high temperature conditions, as well as genes encoding proteins with antioxidant properties and proteins involved in nucleotide and carbohydrate metabolism. In response to cadmium stress, we mainly verified the induction of genes for proteins with antioxidant properties, proteins involved in amino acid metabolism, proteins related to cellular transport and proteins related to protein folding and proteolysis. One of the consequences of the exposure to cadmium is the increase of oxidative stress, and antioxidant proteins have a fundamental role in the response to this kind of injury. Amongst the genes observed during the B. emersonii EST sequencing program, ten genes encoding distinct proteins from the Hsp70 family were observed. Nine of them are expressed at least in one stage of the fungus development and seven genes presented a significant induction during heat shock treatment. These data suggest that the hsp70 genes perform an important role during development and in response to heat stress in B. emersonii. Another interesting result from this work was the enrichment of ESTs containing introns in the stress libraries. Thus, heat shock and cadmium stress decrease the efficiency of intron processing in B. emersonii, allowing for intron characterization. The cDNA for the Hsp17 protein presented the highest number of ESTs sequenced from the stress libraries. Northern blot experiments indicated that the hsp17 gene is expressed at very low levels throughout the life cycle of B. emersonii, however, as expected its expression increases drastically when sporulation or germination cells are submitted to heat shock. Hsp17 protein levels accompany its mRNA levels, indicating that the control of expression of the hsp17 gene occurs at a transcriptional level.

No description available.

No description available.

No description available.

Protein folding is the process in which polypeptides in their non-native states attain the unique folds of their native states. Adverse environmental conditions and genetic predisposition challenge the folding process and accelerate the production of proteotoxic misfolded proteins. Misfolded proteins are selectively recognized and removed from the cell by processes of protein quality control (PQC). In PQC molecular chaperones of the Heat shock protein 70 kDa (Hsp70) family play important roles by recognizing and facilitating the removal of misfolded proteins. Hsp70 function is dependent on cofactors that regulate the intrinsic ATPase activity of the chaperone. In this thesis I have used yeast genetic, cell biological and biochemical experiments to gain insight into the regulation of Hsp70 function in PQC by nucleotide-exchange factors (NEFs). Study I shows that the NEF Fes1 is a key factor essential for cytosolic PQC. A reverse genetics approach demonstrated that Fes1 NEF activity is required for the degradation of misfolded proteins associated with Hsp70 by the ubiquitin-proteasome system. Specifically, Fes1 association with Hsp70-substrate complexes promotes interaction of the substrate with downstream ubiquitin E3 ligase Ubr1. The consequences of genetic removal of FES1 (fes1Δ) are the failure to degrade misfolded proteins, the accumulation of protein aggregates and constitutive induction of the heat-shock response. Taken the experimental data together, Fes1 targets misfolded proteins for degradation by releasing them from Hsp70. Study II describes an unusual example of alternative splicing of FES1 transcripts that leads to the expression of the two alternative splice isoforms Fes1S and Fes1L. Both isoforms are functional NEFs but localize to different compartments. Fes1S is localized to the cytosol and is required for the efficient degradation of Hsp70-associated misfolded proteins. In contrast, Fes1L is targeted to the nucleus and represents the first identified nuclear NEF in yeast. The identification of distinctly localized Fes1 isoforms have implications for the understanding of the mechanisms underlying nucleo-cytoplasmic PQC. Study III reports on the mechanism that Fes1 employs to regulate Hsp70 function. Specifically Fes1 carries an N-terminal domain (NTD) that is conserved throughout the fungal kingdom. The NTD is flexible, modular and is required for the cellular function of Fes1. Importantly, the NTD forms ATP-sensitive complexes with Hsp70 suggesting that it competes substrates of the chaperone during Fes1-Hsp70 interactions. Study IV reports on methodological development for the efficient assembly of bacterial protein-expression plasmids using yeast homologous recombination cloning and the novel vector pSUMO-YHRC. The findings support the notion that Fes1 plays a key role in determining the fate of Hsp70-associated misfolded substrates and thereby target them for proteasomal degradation. From a broader perspective, the findings provide information essential to develop models that describe how Hsp70 function is regulated by different NEFs to participate in protein folding and degradation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>