Over-expression of Arabidopsis DnaJ (Hsp40) contributes to NaCl-stress tolerance

DnaJ (Hsp40), a heat shock protein, is a molecular chaperones responsive to various environmental stress. To analyze the protective role of DnaJ, we obtained sense transgenic Arabidopsis plants that constitutively expressed elevated levels of DnaJ. In this study, sense transgenic plants show large thinner, fade color and malformed leaves, as well as less floss of back leaves. Plants with enhanced levels of DnaJ in their transgenic sense lines exhibited tolerance to NaCl stress. Under 120 mM NaCl, root length was higher in transgenic sense plants than wild-type plants. In vitro expression system, DnaJ protein shows tolerance to high NaCl. These results suggest that over-expression of DnaJ can confer NaCl-stress tolerance

Small Heat Shock Proteins Potentiate Amyloid Dissolution by Protein Disaggregases from Yeast and Humans

The authors define how small heat-shock proteins synergize to regulate the assembly and disassembly of a beneficial prion, and then they exploit this knowledge to identify the human amyloid depolymerase

Translational control by the multi-KH domain protein Scp160

The control of mRNA translation mediated by RNA-binding proteins (RBPs) is a key player in modulating gene expression. In S. cerevisiae, the multi-KH domain protein Scp160 associates with a large number of mRNAs and is present on membrane-bound and, to a lesser extent, cytosolic polysomes. Its binding site on the ribosome is close to the mRNA exit tunnel and in vicinity to Asc1, which constitutes a binding platform for signaling molecules. The present study focused on the closer characterization of the Scp160-ribosome interaction and on the suggested function of Scp160 in modulating the translation of specific target mRNAs. Using affinity purifications, the partial RNA-dependence of the Scp160-ribosome association was confirmed. In contrast to published results, ribosome association was found to be only slightly reduced but not abolished in the absence of Asc1 or the last two KH domains. Furthermore, the putative elongation regulator Stm1 was identified as a co-purifier of Scp160. In subcellular fractionation experiments, RNA-binding mutants of Scp160 were present in the ribosome-free cytosolic fraction and therefore partially deficient in ribosome association and/or mRNP formation. However, no physiological conditions were found that equally induce a shift of wildtype Scp160 towards the cytosolic fraction. Within the scope of a translational profiling approach, microarray analyses of RNA isolated from sucrose density gradient fractions were performed and led to the identification of a set of mRNAs that shift their position within the gradients upon Scp160 depletion, indicating changes in their translation rates. Consistent with the membrane localization of Scp160, transcripts encoding secreted proteins were significantly enriched. Using immunoprecipitation and subsequent quantitative real-time PCR (qRT-PCR), the interaction of Scp160 with a subgroup of the identified targets was confirmed and it was shown that their binding is dependent on the conserved GXXG motifs in the two C-terminal KH domains of Scp160. Furthermore, data were obtained indicating that Scp160 can act as a translational activator on some of its target mRNAs, probably on the level of translation elongation. Finally, first evidence was provided that the translational misregulation of specific target transcripts may be involved in the polyploidization that is a hallmark of Scp160-deprived cells. In summary, these data substantiate the assumption that Scp160 is involved in translational regulation of a specific, functionally related subset of mRNAs. This finding is in good accordance with the emerging view that RBPs co-regulate multiple transcripts in order to allow faster adaptation to environmental changes

Recovery of Yeast Saccharomyces cerevisiae after Thermal Insult

All organisms have evolved mechanisms to acquire thermotolerance. A moderately high temperature activates heat shock genes and triggers thermotolerance towards otherwise lethal high temperature. The focus of this work is the recovery mechanisms ensuring survival of Saccharomyces cerevisiae yeast cells after thermal insult. Yeast cells, first preconditioned at 37˚C, can survive a short thermal insult at 48-50˚C and are able to refold heat-denatured proteins when allowed to recover at physiological temperature 24˚C. The cytoplasmic chaperone Hsp104 is required for the acquisition of thermotolerance and dissolving protein aggregates in the cytosol with the assistance of disaccharide trehalose. In the present study, Hsp104 and trehalose were shown to be required for conformational repair of heat-denatured secretory proteins in the endoplasmic reticulum. A reporter protein was first accumulated in the lumen of endoplasmic reticulum and heat-denatured by thermal insult, and then failed to be repaired to enzymatically active and secretion-competent conformation in the absence of Hsp104 or trehalose. The efficient transport of a glycoprotein CPY, accumulated in the endoplasmic reticulum, to the vacuole after thermal insult also needed the presence of Hsp104 and trehalose. However, proteins synthesized after thermal insult at physiological temperature were secreted with similar kinetics both in the absence and in the presence of Hsp104 or trehalose, demonstrating that the secretion machinery itself was functional. As both Hsp104 and trehalose are cytosolic, a cross-talk between cytosolic and luminal chaperone machineries across the endoplasmic reticulum membrane appears to take place. Global expression profiles, obtained with the DNA microarray technique, revealed that the gene expression was shut down during thermal insult and the majority of transcripts were destroyed. However, the transcripts of small cytosolic chaperones Hsp12 and Hsp26 survived. The first genes induced during recovery were related to refolding of denatured proteins and resumption of de novo protein synthesis. Transcription factors Spt3p and Med3p appeared to be essential for acquisition of full thermotolerance. The transcription factor Hac1p was found to be subject to delayed up-regulation at mRNA level and this up-regulation was diminished or delayed in the absence of Spt3p or Med3p. Consequently, production of the chaperone BiP/Kar2p, a target gene of Hac1p, was diminished and delayed in Δspt3 and Δmed3 deletion strains. The refolding of heat-denatured secretory protein CPY to a transport-competent conformation was retarded, and a heat-denatured reporter enzyme failed to be effectively reactivated in the cytoplasm of the deletion strains.Korkea lämpötila on merkittävimpiä stressitekijöistä, joille eliöt altistuvat. Hankittu lämmönsietokyky on ilmiö, jossa esialtistus lievemmälle lämpöstressille auttaa soluja selviytymään muuten tappavan korkeista lämpötiloista. Esialtistus lisää lämpösokkiproteiinien tuotantoa soluissa, samalla kun muiden proteiinien tuotantoa vähennetään. Tässä työssä tutkittiin leivinhiivasolujen (Saccharomyces cerevisiae) palautumista voimakkaan lämpöstressin jälkeen. Lämpöstressiin liittyvät perusmekanismit ovat varsin samanlaisia eri eliöissä, joten hiivasoluilla saadut tulokset ovat merkittäviä myös monisoluisten eliöiden stressinsietotutkimuksen kannalta. Korkea lämpötila tuhoaa proteiinien oikean laskostumisen ja siten niiden toimintakyvyn. Voimakkaan lämpöstressin jälkeen hiivasolut pystyvät uudelleen laskostamaan lämmön vaurioittamia proteiineja, kun solujen annetaan palautua normaalissa kasvulämpötilassa. Hsp104 on lämmönsietokyvyn muodostumiselle välttämätön solulimassa esiintyvä lämpösokkiproteiini, joka osallistuu lämpövaurioituneiden proteiinien korjaukseen solulimassa olevan trehaloosi-sokerin avulla. Tässä työssä tutkittiin Hsp104:n ja trehaloosin merkitystä solulimakalvoston sisällä vaurioituneiden proteiinien korjaukselle. Työssä käytettiin hiivakantoja, joista toimiva Hsp104-proteiini tai trehaloosin tuotantoon tarvittava entsyymi oli poistettu. Reportteriproteiinien avulla seurattiin solujen palautumista voimakkaan lämpöstressin jälkeen normaalissa lämpötilassa. Työssä havaittiin, että toimivan Hsp104:n tai trehaloosin puuttuessa solulimakalvoston sisälle kertyneiden ja siellä lämpövaurioituneiden proteiinien korjaus estyi. Tulokset osoittavat, että soluliman proteiinikorjauskoneiston ja solulimakalvoston sisäisen proteiinikorjauskoneiston välillä on toiminnallinen yhteys. Työssä selvitettiin DNA-mikrosirukokeilla muutoksia hiivan koko genomin ilmentymisessä lämpöstressin aikana ja jälkeen. Voimakkaan lämpöstressin aikana suurin osa lähetti-RNA:sta tuhottiin. Toipumisen aikana ensimmäiseksi indusoituneet geenit liittyivät proteiinien uudelleen laskostumiseen tai proteiinisynteesin käynnistämiseen. Aineistosta valittiin jatkotutkimuksiin 30 geeniä, joista transkriptiotekijät Spt3p ja Med3p osoittautuivat merkittäviksi täyden lämmönsietokyvyn muodostumiselle ja transkriptiotekijä Hac1p:n määrän säätelylle

UNDERSTANDING APOLIPOPROTEIN B’S ABILITY TO AGGREGATE THROUGH LIPID DROPLETS AND CHAPERONE HOLDASE

Endoplasmic Reticulum (ER) associated degradation (ERAD) is the general process in which misfolded secretory proteins are monitored and degraded to protect the cell from a buildup of nonfunctioning proteins. Apolipoprotein B (ApoB), an ERAD substrate is a large hydrophobic secretory protein associated with the transport of lipids and cholesterol by lipoproteins in the body. ApoB synthesis involves cotranslational translocation through the Sec61 translocon into the ER. If properly folded and lipidated, ApoB is then retrotranslocated through the same pore. Since ApoB contains many aggregation-prone hydrophobic β-sheets, what prevents ApoB aggregation before degradation by ERAD? Initial considerations suggested that cytosolic factors, such as lipid droplets or chaperone “holdases,” “foldases,” and “disaggregases” may help to maintain ApoB’s solubility post retrotranslocation. To test this hypothesis, I adapted our yeast galactose inducible ApoB expression system to be β-estradiol inducible and used it to investigate various chaperone candidates to determine if they affect ApoB stability. Upon large scale isolation of lipid droplets, ApoB was found not to interact with lipid droplets. Next, I investigated potential chaperones. I found that the small heat shock proteins, a family of ATP-independent chaperones, and the TRiC complex, an Hsp60 family member, do not affect ApoB stability. However, I determined that Hsp104, a AAA+ ATPase which helps to refold and reactivate aggregated proteins, is a pro-degradation factor for ApoB. ApoB degradation was slowed in the absence of this chaperone while overexpression caused faster degradation. I then investigated Rvb2, the yeast homolog of the human functional analog of Hsp104, to determine its effect on ApoB stability. Unexpectedly, Rvb2 did not restore ApoB degradation in the absence of Hsp104. Together, my data indicate that ApoB does require chaperone disaggregase function prior to ERAD

Analysis of heat shock-, sodium arsenite- and proteasome inhibitor-induced heat shock protein gene expression in Xenopus laevis

Previous studies have focused on the effect of individual stressors on hsp gene expression in eukaryotic organisms. In the present study, I examined the effect of concurrent low doses of sodium arsenite and mild heat shock temperatures on the expression of hsp30 and hsp70 genes in Xenopus laevis A6 kidney epithelial cells. Northern hybridization and western blot analysis revealed that exposure of A6 cells to 1-10 μM sodium arsenite at a mild heat shock temperature of 30˚C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 μM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26˚C with larger responses at 28 and 30 ˚C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 μM ) and heat shock (30 ˚C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge at 37 ˚C. Acquired thermotolerance was not observed with cells treated with the two mild stresses individually. It is likely that the enhanced accumulation of HSPs under these conditions permits the organism to cope with multiple environmental stresses encountered in their natural aquatic habitat. Previous studies have shown that inhibiting the activity of the proteasome also leads to the accumulation of damaged or unfolded proteins within the cell. In the second phase of this study, I report that inhibition of proteasome activity by the inhibitors carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) and lactacystin induced the accumulation of HSP30 and HSP70 as well as their respective mRNAs. The accumulation of HSP30 and HSP70 in A6 cells recovering from MG132 exposure was still relatively high 24 h after treatment and it decreased substantially after 48 h. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. HSP30 localization in A6 cells was primarily in the cytoplasm as revealed by immunocytochemistry. In some A6 cells treated with higher concentrations of MG132 and lactacystin, HSP30 was also found to localize in relatively large cytoplasmic foci. In some MG132-treated cells, HSP30 staining was substantially depleted in the cytoplasmic regions surrounding these foci. The activation of HSF1 may be involved in MG132-induced hsp gene expression in A6 cells since KNK437 inhibited the accumulation of HSP30 and HSP70. Lastly, MG132 treatment also conferred a state of thermotolerance in A6 cells such that they were able to survive a subsequent thermal challenge. Analysis of this phenomenon is important given the fact that impaired proteasomal activity has been suggested as an explanation for some of the late-onset neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease

Structural features and interactions of substrates complexed with molecular chaperones

Protein misfolding and aggregation perturbs cellular functions and is involved in aging and numerous medical disorders. In cells, the first line of defense is the association of deleterious aggregating proteins with small Heat shock proteins (sHsp). These oligomeric, ATP-independent chaperones sequester misfolded proteins into complexes and facilitate subsequent substrate solubilization and refolding by ATP-dependent chaperones. The cytosol of S. cerevisiae contains two sHsps: Hsp42 is constitutively active, while Hsp26 is activated at elevated temperatures. In my thesis, I wanted to elucidate how sHsps change the structure of aggregates, facilitating substrate reactivation. To this end, I studied the impact of Hsp26 and Hsp42 incorporation on the architecture of heat-induced aggregates by amide hydrogen exchange (HX). I established the experimental conditions for HX of heat-induced protein aggregates using thermolabile malate dehydrogenase (MDH) as model substrate. My data show that the formation of heat-induced Hsp26/MDH or Hsp42/MDH complexes has profound impact on the MDH structure. In the aggregated state formed in absence of sHsps, almost the entire MDH polypeptide becomes accessible to HX, reflecting global, large misfolding. In contrast, a more protected form of MDH is detected when complexed with Hsp26 or Hsp42. I observed that the mass spectra of many MDH peptides derived from sHsp/MDH complexes exist as a mixture of two populations after HX: a native-like and an aggregate-like population. Higher excess of sHsps promoted the native-like state. Single-molecule experiments confirmed the binding of sHsps to near native substrate folds. Furthermore, FRET experiments showed that sHsps increase the spacing between MDH molecules in sHsp/MDH complexes, preventing intermolecular contacts of misfolded MDH species. Finally, crosslinking approaches identified peripheral, surface-exposed MDH sites showing high HX as major sHsp binding sites. Summarized, these findings indicate that sHsps capture early unfolding intermediates of substrates and keep parts of the protein in a native-like state. This activity of sHsps might facilitate chaperone-dependent disaggregation. I then investigated how the two sHsps of yeast interact with their substrates. The N-terminal extensions (NTE) of both yeast sHsps were found to be the major substrate interaction sites. Compared to all known sHsps, the NTE of Hsp42 is unusually elongated and it was shown to be involved in the organized deposition of misfolded proteins at CytoQ (cytosolic quality control compartment). Hsp42 NTE harbors the two prototypes of intrinsically disordered domains (IDD): a prion-like and an unstructured subdomain. IDDs play important roles in the formation of membrane-free compartments due to their ability to self-associate and to coalesce into inclusions. In this study, the roles of both NTE subdomains in CytoQ formation and Hsp42 chaperone activity were investigated. We found that the prion-like domain of Hsp42 has a dual function: It binds misfolded substrate proteins and triggers CytoQ formation. The unstructured domain is dispensable for CytoQ formation, but it has a regulatory function, controlling Hsp42 localization and CytoQ numbers. Deletion of the unstructured domain increases Hsp42 substrate interaction and holdase activity, i.e. the prevention of tight contacts between misfolded species. Together, the presented data show that the prion-like domain of Hsp42 is essential for CytoQ formation, extending the role of prion-like domains in inclusion formation from RNA granules to protein aggregates and emphasizing their crucial contributions to protein phase transitions. In a second part of my thesis I studied how the Hsp70 chaperone system interacts with RepE, a dimeric replication initiation protein in E. coli. The disassembly of RepE seems mechanistically related to the disaggregation process. As a dimer RepE represses its own transcription, as a monomer it initiates the replication of the mini-F plasmid. Monomerization is mediated by the DnaK chaperone system. So far, it remained elusive, how components of the DnaK chaperone system interact with RepE and how they change its structure, leading to the disassembly of the RepE dimer. In this study the binding of DnaK and DnaJ to dimeric RepE wt and to RepE54, a constitutively monomeric variant, was studied by HX. HX analysis of RepE wt revealed a putative DnaK binding site and conformational changes induced by chaperones. Only dimeric RepE wt, but not monomeric RepE54, interacts with DnaJ. In contrast, both oligomeric states of RepE were able to bind DnaK – at least in absence of DNA. In presence of their respective DNA-binding elements, the binding of DnaK was prevented, most likely due to sterical hindrance as the DNA and the putative DnaK binding sites in RepE are in close proximity. The binding of DnaJ probably occurs in aa 96-116, and it destabilized parts of the DNA binding region in RepE, indicating conformational changes. Although interaction with DnaJ was shown to enhance the binding affinity of RepE to DNA, the DnaJ-induced conformational change might enable DnaK to access its binding site. Crosslinking experiments, however, showed that DnaJ binding is not sufficient to allow for interaction of DnaK with DNA-complexed RepE wt. Only concomitant presence of DnaJ and GrpE enabled DnaK to interact with DNA-bound RepE wt. HX revealed, that concerted binding of DnaJ and DnaK causes substantial conformational changes in RepE: Destabilization of the C-terminal region and stabilization in helix α4 near the dimer interface. The latter might be implicated in the monomerization of RepE wt. In summary, my results provide major contributions to elucidate the chaperone-mediated RepE monomerization process

Effect of combined sodium arsenite and cadmium chloride treatment on heat shock protein gene expression in Xenopus laevis A6 kidney epithelial cells

Sodium arsenite and cadmium chloride are two widespread environmental toxicants which have deleterious effects on living organisms. At the cellular level, sodium arsenite and cadmium chloride cause oxidative stress, dysregulation of gene expression, apoptosis, and the unfolding of protein. Furthermore, both chemical stressors individually have the ability to induce heat shock protein (HSP) accumulation. HSPs are molecular chaperones that aid in protein folding, translocation and in preventing stress-induced protein aggregation. Previously, our laboratory demonstrated that treatment of A6 kidney epithelial cells of the frog Xenopus laevis, with either cadmium chloride or sodium arsenite plus a concurrent mild heat shock resulted in an enhanced accumulation of HSPs that was greater than found with the sum of the individual stressors. To the best of our knowledge, no information is available to date on the effect that these two chemical stressors have in combination on HSP accumulation in aquatic organisms. The present study examined the effect of simultaneous sodium arsenite and cadmium chloride treatment on the pattern of HSP30 and HSP70 accumulation in Xenopus A6 cells. Immunoblot analysis revealed that the relative levels of HSP30 and HSP70 accumulation in A6 cells treated concurrently with sodium arsenite and cadmium chloride for 12 h were significantly higher than the sum of HSP30 or HSP70 accumulation from cells subjected to the treatments individually. For instance, the combined 10 µM sodium arsenite plus 100 µM cadmium chloride treatment resulted in a 3.5 fold increase in HSP30 accumulation and a 2.5 fold increase in HSP70 accumulation compared to the sum of the stressors individually. This finding suggested a synergistic action between the two stressors. Pretreatment of cells with KNK437, an HSF1 inhibitor, inhibited the combined sodium arsenite- and cadmium chloride-induced accumulation of HSP30 and HSP70 suggesting that this accumulation of HSPs may be regulated, at least in part, at the level of transcription. Immunocytochemical analysis employing the use of laser scanning confocal microscopy (LSCM) revealed that simultaneous treatment of cells with the two stressors induced HSP30 accumulation primarily in the cytoplasm in a punctate pattern with some dysregulation of F-actin structure. Increased ubiquitinated protein accumulation was observed with combined 10 µM sodium arsenite and 10, 50 or 100 µM cadmium chloride treatment compared to individual stressors suggesting an impairment of the ubiquitin-proteasome degradation system. Finally, while incubation of A6 cells with 1 µM sodium arsenite plus 10 µM cadmium chloride did not induce a detectable accumulation of HSPs, the addition of a 30 °C mild heat shock resulted in a strong accumulation of HSP30 and HSP70. This study has demonstrated that concurrent sodium arsenite and cadmium chloride treatment can enhance HSP accumulation. Since HSP accumulation is triggered by proteotoxic stress, these findings are relevant given the fact that aquatic amphibians in their natural habitat may be exposed to multiple chemical stressors simultaneously