Synechocystis HSP17 is an amphitropic protein that stabilizes heat-stressed membranes and binds denatured proteins for subsequent chaperone-mediated refolding

The small heat shock proteins (sHSPs) are ubiquitous stress proteins proposed to act as molecular chaperones to prevent irreversible protein denaturation. We characterized the chaperone activity of Synechocystis HSP17 and found that it has not only protein-protective activity, but also a previously unrecognized ability to stabilize lipid membranes. Like other sHSPs, recombinant Synechocystis HSP17 formed stable complexes with denatured malate dehydrogenase and served as a reservoir for the unfolded substrate, transferring it to the DnaK/DnaJ/GrpE and GroEL/ES chaperone network for subsequent refolding. Large unilamellar vesicles made of synthetic and cyanobacterial lipids were found to modulate this refolding process. Investigation of HSP17-lipid interactions revealed a preference for the liquid crystalline phase and resulted in an elevated physical order in model lipid membranes. Direct evidence for the participation of HSP17 in the control of thylakoid membrane physical state in vivo was gained by examining an hsp17− deletion mutant compared with the isogenic wild-type hsp17+ revertant Synechocystis cells. We suggest that, together with GroEL, HSP17 behaves as an amphitropic protein and plays a dual role. Depending on its membrane or cytosolic location, it may function as a “membrane stabilizing factor” as well as a member of a multichaperone protein-folding network. Membrane association of sHSPs could antagonize the heat-induced hyperfluidization of specific membrane domains and thereby serve to preserve structural and functional integrity of biomembranes.

The Wheat Peptidyl Prolyl cis-trans-Isomerase FKBP77 Is Heat Induced and Developmentally Regulated1

We isolated a cDNA encoding a 568-amino acid, heat-stress-induced peptidyl prolyl isomerase belonging to the FK506-binding-protein (FKBP) family. The open reading frame encodes for a peptidyl prolyl isomerase that possesses three FKBP-12-like domains, a putative tetratricopeptide motif, and a calmodulin-binding domain. Specific antibodies showed that the open reading frame encodes a heat-induced 77-kD protein, the wheat FKBP77 (wFKBP77), which exhibits 84% identity with the wFKBP73 and 42% identity with the human FKBP59. Because of the high similarity in sequence to wFKBP73, wFKBP77 was designated as the heat-induced isoform. The wFKBP77 mRNA steady-state level was 14-fold higher at 37°C than at 25°C. The wFKBP77 transcript abundance was the highest in mature embryos that had imbibed and 2-d-old green shoots exposed to 37°C, and decreased to 6% in 6-d-old green shoots. The transcript level returned to the level detected at 25°C after recovery of the embryos for 90 min at 25°C. We compared wFKBP73 and wFKBP77 with the heat-shock proteins having cognate and heat-stress-induced counterparts.

High-Molecular-Weight FK506-Binding Proteins Are Components of Heat-Shock Protein 90 Heterocomplexes in Wheat Germ Lysate1

In animal cell lysates the multiprotein heat-shock protein 90 (hsp90)-based chaperone complexes consist of hsp70, hsp40, and p60. These complexes act to convert steroid hormone receptors to their steroid-binding state by assembling them into heterocomplexes with hsp90, p23, and one of several immunophilins. Wheat germ lysate also contains a hsp90-based chaperone system that can assemble the glucocorticoid receptor into a functional heterocomplex with hsp90. However, only two components of the heterocomplex-assembly system, hsp90 and hsp70, have thus far been identified. Recently, purified mammalian p23 preadsorbed with JJ3 antibody-protein A-Sepharose pellets was used to isolate a mammalian p23-wheat hsp90 heterocomplex from wheat germ lysate (J.K. Owens-Grillo, L.F. Stancato, K. Hoffmann, W.B. Pratt, and P. Krishna [1996] Biochemistry 35: 15249–15255). This heterocomplex was found to contain an immunophilin(s) of the FK506-binding class, as judged by binding of the radiolabeled immunosuppressant drug [3H]FK506 to the immune pellets in a specific manner. In the present study we identified the immunophilin components of this heterocomplex as FKBP73 and FKBP77, the two recently described high-molecular-weight FKBPs of wheat. In addition, we present evidence that the two FKBPs bind hsp90 via tetratricopeptide repeat domains. Our results demonstrate that binding of immunophilins to hsp90 via tetratricopeptide repeat domains is a conserved protein interaction in plants. Conservation of this protein-to-protein interaction in both plant and animal cells suggests that it is important for the biological action of the high-molecular-weight immunophilins.

Polypeptides of the Maize Amyloplast Stroma1 : Stromal Localization of Starch-Biosynthetic Enzymes and Identification of an 81-Kilodalton Amyloplast Stromal Heat-Shock Cognate

In the developing endosperm of monocotyledonous plants, starch granules are synthesized and deposited within the amyloplast. A soluble stromal fraction was isolated from amyloplasts of immature maize (Zea mays L.) endosperm and analyzed for enzyme activities and polypeptide content. Specific activities of starch synthase and starch-branching enzyme (SBE), but not the cytosolic marker alcohol dehydrogenase, were strongly enhanced in soluble amyloplast stromal fractions relative to soluble extracts obtained from homogenized kernels or endosperms. Immunoblot analysis demonstrated that starch synthase I, SBEIIb, and sugary1, the putative starch-debranching enzyme, were each highly enriched in the amyloplast stroma, providing direct evidence for the localization of starch-biosynthetic enzymes within this compartment. Analysis of maize mutants shows the deficiency of the 85-kD SBEIIb polypeptide in the stroma of amylose extender cultivars and that the dull mutant lacks a >220-kD stromal polypeptide. The stromal fraction is distinguished by differential enrichment of a characteristic group of previously undocumented polypeptides. N-terminal sequence analysis revealed that an abundant 81-kD stromal polypeptide is a member of the Hsp70 family of stress-related proteins. Moreover, the 81-kD stromal polypeptide is strongly recognized by antibodies specific for an Hsp70 of the chloroplast stroma. These findings are discussed in light of implications for the correct folding and assembly of soluble, partially soluble, and granule-bound starch-biosynthetic enzymes during import into the amyloplast.

Heat-Shock-Induced Changes in Intracellular Ca2+ Level in Tobacco Seedlings in Relation to Thermotolerance1

Exposure of plants to elevated temperatures results in a complex set of changes in gene expression that induce thermotolerance and improve cellular survival to subsequent stress. Pretreatment of young tobacco (Nicotiana plumbaginifolia) seedlings with Ca2+ or ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid enhanced or diminished subsequent thermotolerance, respectively, compared with untreated seedlings, suggesting a possible involvement of cytosolic Ca2+ in heat-shock (HS) signal transduction. Using tobacco seedlings transformed with the Ca2+-sensitive, luminescent protein aequorin, we observed that HS temperatures induced prolonged but transient increases in cytoplasmic but not chloroplastic Ca2+. A single HS initiated a refractory period in which additional HS signals failed to increase cytosolic Ca2+. However, throughout this refractory period, seedlings responded to mechanical stimulation or cold shock with cytosolic Ca2+ increases similar to untreated controls. These observations suggest that there may be specific pools of cytosolic Ca2+ mobilized by heat treatments or that the refractory period results from a temporary block in HS perception or transduction. Use of inhibitors suggests that HS mobilizes cytosolic Ca2+ from both intracellular and extracellular sources.

Sequence-specific DNA-binding dominated by dehydration.

Fluorescence spectroscopy and isothermal titration calorimetry were used to study the thermodynamics of binding of the glucocorticoid receptor DNA-binding domain to four different, but similar, DNA-binding sites. The binding sites are two naturally occurring sites that differ in the composition of one base pair, i.e., an A-T to G-C mutation, and two sites containing chemical intermediates of these base pairs. The calorimetrically determined heat capacity change (Delta C(p)o(obs)) for glucocorticoid receptor DNA-binding domain binding agrees with that calculated for dehydration of solvent-accessible surface areas. A dominating effect of dehydration or solvent reorganization on the thermodynamics is also consistent with an observed linear relationship between observed enthalpy change (Delta Ho(obs)) and observed entropy change (Delta So(obs)) with a slope close to the experimental temperature. Comparisons with structural data allow us to rationalize individual differences between Delta Ho(obs) (and Delta So(obs)) for the four complexes. For instance, we find that the removal of a methyl group at the DNA-protein interface is enthalpically favorable but entropically unfavorable, which is consistent with a replacement by an ordered water molecule.

Muscle-specific expression of Drosophila hsp70 in response to aging and oxidative stress.

Induction of Drosophila hsp70 protein was detected during aging in flight muscle and leg muscle in the absence of heat shock, using an hsp70-specific monoclonal antibody, and in transgenic flies containing hsp70-beta-galactosidase fusion protein reporter constructs. While hsp70 and reporter proteins were induced during aging, hsp70 message levels were not, indicating that aging-specific induction is primarily posttranscriptional. In contrast, hsp22 and hsp23 were found to be induced during aging at the RNA level and with a broader tissue distribution. The same muscle-specific hsp70 reporter expression pattern was observed in young flies mutant for catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6). In catalase (cat) hypomorphic lines where flies survived to older ages, the time course of hsp70 reporter expression during aging was accelerated, and the initial and ultimate levels of expression were increased. The hsp70 reporter was also induced in young flies mutant for copper/zinc superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1). Taken together, the results suggest that aging-specific hsp70 expression may be a result of oxidative damage.

Effect of periodontal treatment on the serum antibody levels to heat shock proteins

We have shown previously that both humoral and cellular immune responses to heat shock protein 60 (HSP60) are elevated in chronic periodontitis patients compared with non-diseased subjects. The aim of the present study was to determine whether periodontal treatment could influence the level of serum antibodies to human HSP60 and Porphyromonas gingivalis GroEL, a bacterial homologue of human HSP60. Sera were obtained from 21 patients with moderate to advanced chronic periodontitis at the baseline examination and again after completion of treatment. Antibody levels were determined using an enzyme-linked immunosorbent assay. The mean anti-P. gingivalis GroEL antibody levels were down-regulated significantly by periodontal treatment when recombinant P. gingivalis GroEL was used as an antigen, whereas antibody levels to P. gingivalis GroEL-specific peptide were significantly elevated following successful periodontal therapy. The mean level of anti-human HSP60 antibody remained unchanged although individual levels of antibody either increased or decreased after periodontal treatment, suggesting that synthesis of these antibodies might be regulated independently during the course of periodontal infection. Although their regulatory mechanisms in chronic infection are not understood, further study would provide insight not only into the role of these antibodies in the pathogenesis of periodontitis but also into the possible link between periodontitis and systemic diseases such as coronary heart disease.

Mathematical modelling and optimisation of a water heat pump

The purpose of the work described here has been to seek methods of narrowing the present gap between currently realised heat pump performance and the theoretical limit. The single most important pre-requisite to this objective is the identification and quantitative assessment of the various non-idealities and degradative phenomena responsible for the present shortfall. The use of availability analysis has been introduced as a diagnostic tool, and applied to a few very simple, highly idealised Rankine cycle optimisation problems. From this work, it has been demonstrated that the scope for improvement through optimisation is small in comparison with the extensive potential for improvement by reducing the compressor's losses. A fully instrumented heat pump was assembled and extensively tested. This furnished performance data, and led to an improved understanding of the systems behaviour. From a very simple analysis of the resulting compressor performance data, confirmation of the compressor's low efficiency was obtained. In addition, in order to obtain experimental data concerning specific details of the heat pump's operation, several novel experiments were performed. The experimental work was concluded with a set of tests which attempted to obtain definitive performance data for a small set of discrete operating conditions. These tests included an investigation of the effect of two compressor modifications. The resulting performance data was analysed by a sophisticated calculation which used that measurements to quantify each dagradative phenomenon occurring in that compressor, and so indicate where the greatest potential for improvement lies. Finally, in the light of everything that was learnt, specific technical suggestions have been made, to reduce the losses associated with both the refrigerant circuit and the compressor.

Modelling of transient heat transfer in annealing furnaces

This is a study of heat transfer in a lift-off furnace which is employed in the batch annealing of a stack of coils of steel strip. The objective of the project is to investigate the various factors which govern the furnace design and the heat transfer resistances, so as to reduce the time of the annealing cycle, and hence minimize the operating costs. The work involved mathematical modelling of patterns of gas flow and modes of heat transfer. These models are: Heat conduction and its conjectures in the steel coils;Convective heat transfer in the plates separating the coils in the stack and in other parts of the furnace; and Radiative and convective heat transfer in the furnace by using the long furnace model. An important part of the project is the development of numerical methods and computations to solve the transient models. A limited number of temperature measurements was available from experiments on a test coil in an industrial furnace. The mathematical model agreed well with these data. The model has been used to show the following characteristics of annealing furnaces, and to suggest further developments which would lead to significant savings: - The location of the limiting temperature in a coil is nearer to the hollow core than to the outer periphery. - Thermal expansion of the steel tends to open the coils, reduces their thermal conductivity in the radial direction, and hence prolongs the annealing cycle. Increasing the tension in the coils and/or heating from the core would overcome this heat transfer resistance. - The shape and dimensions of the convective channels in the plates have significant effect on heat convection in the stack. An optimal design of a channel is shown to be of a width-to-height ratio equal to 9. - Increasing the cooling rate, by using a fluidized bed instead of the normal shell and tube exchanger, would shorten the cooling time by about 15%, but increase the temperature differential in the stack. - For a specific charge weight, a stack of different-sized coils will have a shorter annealing cycle than one of equally-sized coils, provided that production constraints allow the stacking order to be optimal. - Recycle of hot flue gases to the firing zone of the furnace would produce a. decrease in the thermal efficiency up to 30% but decreases the heating time by about 26%.

A heat metering system for energy management on an industrial site

Faced with a future of rising energy costs there is a need for industry to manage energy more carefully in order to meet its economic objectives. A problem besetting the growth of energy conservation in the UK is that a large proportion of energy consumption is used in a low intensive manner in organisations where they would be responsibility for energy efficiency is spread over a large number of personnel who each see only small energy costs. In relation to this problem in the non-energy intensive industrial sector, an application of an energy management technique known as monitoring and targeting (M & T) has been installed at the Whetstone site of the General Electric Company Limited in an attempt to prove it as a means for motivating line management and personnel to save energy. The objective energy saving for which the M & T was devised is very specific. During early energy conservation work at the site there had been a change from continuous to intermittent heating but the maintenance of the strategy was receiving a poor level of commitment from line management and performance was some 5% - 10% less than expected. The M & T is concerned therefore with heat for space heating for which a heat metering system was required. Metering of the site high pressure hot water system posed technical difficulties and expenditure was also limited. This led to a ‘tin-house' design being installed for a price less than the commercial equivalent. The timespan of work to achieve an operational heat metering system was 3 years which meant that energy saving results from the scheme were not observed during the study. If successful the replication potential is the larger non energy intensive sites from which some 30 PT savings could be expected in the UK.

The development of heat recovery equipment from conception to commercialisation

A comprehensive survey of industrial sites and heat recovery products revealed gaps between equipment that was required and that which was available. Two heat recovery products were developed to fill those gaps: a gas-to-gas modular heat recovery unit; a gas-to-liquid exhaust gas heat exchanger. The former provided an entire heat recovery system in one unit. It was specifically designed to overcome the problems associated with existing component system of large design commitment, extensive installation and incompatibility between parts. The unit was intended to recover heat from multiple waste gas sources and, in particular, from baking ovens. A survey of the baking industry defined typical waste gas temperatures and flow rates, around which the unit was designed. The second unit was designed to recover heat from the exhaust gases of small diesel engines. The developed unit differed from existing designs by having a negligible effect on engine performance. In marketing terms these products are conceptual opposites. The first, a 'product-push' product generated from site and product surveys, required marketing following design. The second, a 'market-pull' product, resulted from a specific user need; this had a captive market and did not require marketing. Here marketing was replaced by commercial aspects including the protection of ideas, contracting, tendering and insurance requirements. These two product development routes are compared and contrasted. As a general conclusion this work suggests that it can be beneficial for small companies (as was the sponsor of this project) to undertake projects of the market-pull type. Generally they have a higher probability of success and are less capital intensive than their product-push counterparts. Development revealed shortcomings in three other fields: British Standards governing heat exchangers; financial assessment of energy saving schemes; degree day procedure of calculating energy savings. Methods are proposed to overcome these shortcomings.

The political shaping of energy technology:combined heat and power in Britain

There has been negligible adoption of combined heat and power (CHP) for district heating (DH) in Britain, despite continued advocacy. This thesis constructs an account of the treatment of the option, and devises a framework for explanation. Analysis of technological development and adoption, it is argued, should be similar to that of other social processes, and be subject to the same requirements and criticisms. They will, however, show features peculiar to the institutions developing and selecting technologies, their relation to different social groups, and the forms of knowledge in and about technology. Conventional approaches - organisation and interorganisation theories, and analyses of policy-making - give useful insights but have common limitations. Elements of an analytical framework situating detailed issues and outcomes in a structured historical context are derived from convergent radical critiques. Thus activity on CHP/DH is essentially shaped by the development and relations of energy sector institutions: central and local government, nationalised industries and particularly the electricity industry. Analysis of them is related to the specific character of the British state. A few CHP and DH installations were tried before 1940. During postwar reconstruction, extensive plans for several cities were abandoned or curtailed. In the 1960s and 70s, many small non-CHP DH schemes were installed on housing estates. From the mid-70s, the national potential of CHP/DH has been reappraised, with widespread support and favourable evaluations, but little practical progress. Significant CHP/DH adoption is shown to have been systematically excluded ultimately by the structure of energy provision; centralised production interests dominate and co-ordination is weak. Marginal economics and political commitment have allowed limited development in exceptional circumstances. Periods of upheaval provided greater opportunity and incentive for CHP/DH but restructuring eventually obstructed it. Explanation of these outcomes is shown to require analysis at several levels, from broad context to detailed action.

Solvability and asymptotics of the heat equation with mixed variable lateral conditions and applications in the opening of the exocytotic fusion pore in cells

We investigate a mixed problem with variable lateral conditions for the heat equation that arises in modelling exocytosis, i.e. the opening of a cell boundary in specific biological species for the release of certain molecules to the exterior of the cell. The Dirichlet condition is imposed on a surface patch of the boundary and this patch is occupying a larger part of the boundary as time increases modelling where the cell is opening (the fusion pore), and on the remaining part, a zero Neumann condition is imposed (no molecules can cross this boundary). Uniform concentration is assumed at the initial time. We introduce a weak formulation of this problem and show that there is a unique weak solution. Moreover, we give an asymptotic expansion for the behaviour of the solution near the opening point and for small values in time. We also give an integral equation for the numerical construction of the leading term in this expansion.

Repeated evolution of heat responsiveness among Brassicaceae COPIA transposable elements

Eukaryotic genomes contain repetitive DNA sequences. This includes simple repeats and more complex transposable elements (TEs). Many TEs reach high copy numbers in the host genome, owing to their amplification abilities by specific mechanisms. There is growing evidence that TEs contribute to gene transcriptional regulation. However, excess of TE activity may lead to reduced genome stability. Therefore, TEs are suppressed by the transcriptional gene silencing machinery via specific chromatin modifications. In contrary, effectiveness of the epigenetic silencing mechanisms imposes risk for TE survival in the host genome. Therefore, TEs may have evolved specific strategies for bypassing epigenetic control and allowing the emergence of new TE copies. Recent studies suggested that the epigenetic silencing can be, at least transiently, attenuated by heat stress in A. thaliana. Heat stress induced strong transcriptional activation of COPIA78 family LTR-retrotransposons named ONSEN, and even their transposition in mutants deficient in siRNA-biogenesis. ONSEN transcriptional activation was facilitated by the presence of heat responsive elements (HREs) within the long terminal repeats, which serve as a binding platform for the HEAT SHOCK FACTORs (HSFs). This thesis focused on the evolution of ONSEN heat responsiveness in Brassicaceae. By using whole-transcriptome sequencing approach, multiple Arabidopsis lyrata ONSENs with conserved heat response were found and together with ONSENs from other Brassicaceae were used to reconstruct the evolution of ONSEN HREs. This indicated ancestral situation with two, in palindrome organized, HSF binding motifs. In the genera Arabidopsis and Ballantinia, a local duplication of this locus increased number of HSF binding motifs to four, forming a high-efficiency HRE. In addition, whole transcriptome analysis revealed novel heat-responsive TE families COPIA20, COPIA37 and HATE. Notably, HATE represents so far unknown COPIA family which occurs in several Brassicaceae species but is absent in A. thaliana. Putative HREs were identified within the LTRs of COPIA20, COPIA37 and HATE of A. lyrata, and could be preliminarily validated by transcriptional analysis upon heat induction in subsequent survey of Brassicaeae species. Subsequent phylogenetic analysis indicated a repeated evolution of heat responsiveness within Brassicaceae COPIA LTR-retrotransposons. This indicates that acquisition of heat responsiveness may represent a successful strategy for survival of TEs within the host genome.