Attila Glatz

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.

Heat shock response in photosynthetic organisms: membrane and lipid connections.

The ability of photosynthetic organisms to adapt to increases in environmental temperatures is becoming more important with climate change. Heat stress is known to induce heat-shock proteins (HSPs) many of which act as chaperones. Traditionally, it has been thought that protein denaturation acts as a trigger for HSP induction. However, increasing evidence has shown that many stress events cause HSP induction without commensurate protein denaturation. This has led to the membrane sensor hypothesis where the membranes physical and structural properties play an initiating role in the heat shock response. In this review, we discuss heat-induced modulation of the membranes physical state and changes to these properties which can be brought about by interaction with HSPs. Heat stress also leads to changes in lipid-based signaling cascades and alterations in calcium transport and availability. Such observations emphasize the importance of membranes and their lipids in the heat shock response and provide a new perspective for guiding further studies into the mechanisms that mediate cellular and organismal responses to heat stress.

The Synechocystis model of stress: From molecular chaperones to membranes

Abstract A highly conserved and exquisitely regulated cellular response to most stress conditions, such as cold, heat and visible or UV-light, is common to all organisms. Cells that have been pre-exposed to a sublethal dose of these stimuli can acquire a transient resistance against the killing effect of a subsequent stress. Specific membrane lipids and stress proteins, many of them molecular chaperones, play a fundamental role in these acclimation processes. According to the presently discussed model, cellular membranes are not simply the primary sites of stress damage, they are also able to sense extreme environmental changes and to activate stress-defence genes remotely as a consequence of a rapid modification in their physical state and microdomain organisation. Underlying the interdependence of the redox sensory pathway and membrane fluidity, sudden changes in membrane order can be signaled to gene expression via the light requiring redox sensory pathway. The photosynthetic Synechocystis PCC 6803 has several features that render it a particularly suitable model for such studies. While the general assembly and lipid composition of its thylakoid is similar to that of higher plants, it can easily be transformed by foreign DNA. Nonetheless, its whole genome has been determined and has become available via the internet.

Membrane-Lipid Therapy in Operation: The HSP Co-Inducer BGP-15 Activates Stress Signal Transduction Pathways by Remodeling Plasma Membrane Rafts

Aging and pathophysiological conditions are linked to membrane changes which modulate membrane-controlled molecular switches, causing dysregulated heat shock protein (HSP) expression. HSP co-inducer hydroxylamines such as BGP-15 provide advanced therapeutic candidates for many diseases since they preferentially affect stressed cells and are unlikely have major side effects. In the present study in vitro molecular dynamic simulation, experiments with lipid monolayers and in vivo ultrasensitive fluorescence microscopy showed that BGP-15 alters the organization of cholesterol-rich membrane domains. Imaging of nanoscopic long-lived platforms using the raft marker glycosylphosphatidylinositol-anchored monomeric green fluorescent protein diffusing in the live Chinese hamster ovary (CHO) cell plasma membrane demonstrated that BGP-15 prevents the transient structural disintegration of rafts induced by fever-type heat stress. Moreover, BGP-15 was able to remodel cholesterol-enriched lipid platforms reminiscent of those observed earlier following non-lethal heat priming or membrane stress, and were shown to be obligate for the generation and transmission of stress signals. BGP-15 activation of HSP expression in B16-F10 mouse melanoma cells involves the Rac1 signaling cascade in accordance with the previous observation that cholesterol affects the targeting of Rac1 to membranes. Finally, in a human embryonic kidney cell line we demonstrate that BGP-15 is able to inhibit the rapid heat shock factor 1 (HSF1) acetylation monitored during the early phase of heat stress, thereby promoting a prolonged duration of HSF1 binding to heat shock elements. Taken together, our results indicate that BGP-15 has the potential to become a new class of pharmaceuticals for use in ‘membrane-lipid therapy’ to combat many various protein-misfolding diseases associated with aging.

Only one dnaK homolog, dnaK2, is active transcriptionally and is essential in Synechocystis.

We examined the expression and the function of the DnaK chaperone family in the photoautotrophic cyanobacterium, Synechocystis PCC 6803. Surprisingly, only one of the three dnaK genes was transcribed either under normal or heat shock conditions. Their predicted cochaperones (four dnaJs and one grpE) proved to be uninducible under our experimental conditions. Attempts to inactivate the active dnaK2 has failed, indicating that the gene is essential. The partial mutant displayed lower inducibility of chaperones (especially GroEL and HSP17) both at mRNA and protein levels upon heat shock. The mutant showed temperature sensitive phenotype, but was able to acquire thermotolerance.

Changes in Membrane Fluid State and Heat Shock Response Cause Attenuation of Virulence

So far attenuation of pathogens has been mainly obtained by chemical or heat treatment of microbial pathogens. Recently, live attenuated strains have been produced by genetic modification. We have previously demonstrated that in several prokaryotes as well as in yeasts and mammalian cells the heat shock response is controlled by the membrane physical state (MPS). We have also shown that in Salmonella enterica serovar Typhimurium LT2 (Salmonella Typhimurium) overexpression of a Delta(12)-desaturase gene alters the MPS, inducing a sharp impairment of transcription of major heat shock genes and failure of the pathogen to grow inside macrophage (MPhi) (A. Porta et al., J. Bacteriol. 192:1988-1998, 2010). Here, we show that overexpression of a homologous Delta(9)-desaturase sequence in the highly virulent G217B strain of the human fungal pathogen Histoplasma capsulatum causes loss of its ability to survive and persist within murine MPhi along with the impairment of the heat shock response. When the attenuated strain of H. capsulatum was injected in a mouse model of infection, it did not cause disease. Further, treated mice were protected when challenged with the virulent fungal parental strain. Attenuation of virulence in MPhi of two evolutionarily distant pathogens was obtained by genetic modification of the MPS, suggesting that this is a new method that may be used to produce attenuation or loss of virulence in both other intracellular prokaryotic and eukaryotic pathogens. This new procedure to generate attenuated forms of pathogens may be used eventually to produce a novel class of vaccines based on the genetic manipulation of a pathogens membrane fluid state and stress response.

Involvement of small heat shock proteins, trehalose, and lipids in the thermal stress management in Schizosaccharomyces pombe.

Changes in the levels of three structurally and functionally different important thermoprotectant molecules, namely small heat shock proteins (sHsps), trehalose, and lipids, have been investigated upon heat shock in Schizosaccharomyces pombe. Both α-crystallin-type sHsps (Hsp15.8 and Hsp16) were induced after prolonged high-temperature treatment but with different kinetic profiles. The shsp null mutants display a weak, but significant, heat sensitivity indicating their importance in the thermal stress management. The heat induction of sHsps is different in wild type and in highly heat-sensitive trehalose-deficient (tps1Δ) cells; however, trehalose level did not show significant alteration in shsp mutants. The altered timing of trehalose accumulation and induction of sHsps suggest that the disaccharide might provide protection at the early stage of the heat stress while elevated amount of sHsps are required at the later phase. The cellular lipid compositions of two different temperature-adapted wild-type S. pombe cells are also altered according to the rule of homeoviscous adaptation, indicating their crucial role in adapting to the environmental temperature changes. Both Hsp15.8 and Hsp16 are able to bind to different lipids isolated from S. pombe, whose interaction might provide a powerful protection against heat-induced damages of the membranes. Our data suggest that all the three investigated thermoprotectant macromolecules play a pivotal role during the thermal stress management in the fission yeast.

Metabolic crosstalk between membrane and storage lipids facilitates heat stress management in Schizosaccharomyces pombe

Cell membranes actively participate in stress sensing and signalling. Here we present the first in-depth lipidomic analysis to characterize alterations in the fission yeast Schizosaccharomyces pombe in response to mild heat stress (HS). The lipidome was assessed by a simple one-step methanolic extraction. Genetic manipulations that altered triglyceride (TG) content in the absence or presence of HS gave rise to distinct lipidomic fingerprints for S. pombe. Cells unable to produce TG demonstrated long-lasting growth arrest and enhanced signalling lipid generation. Our results reveal that metabolic crosstalk between membrane and storage lipids facilitates homeostatic maintenance of the membrane physical/chemical state that resists negative effects on cell growth and viability in response to HS. We propose a novel stress adaptation mechanism in which heat-induced TG synthesis contributes to membrane rigidization by accommodating unsaturated fatty acids of structural lipids, enabling their replacement by newly synthesized saturated fatty acids.

Thermal Acclimation and Heat Stress Response of Synechocystis PCC6803: The Possible Role of Thylakoid Physical State, Lipid Saturation and Molecular Chaperones

There is unequivocal evidence that in higher plant cells exposed to heat stress the photosynthetic apparatus is irreversibly damaged prior to impairment of other cellular functions (Berry and Bjorkman 1980). The general assembly of the photosynthetic membranes in cyanobacteria is similar to that of higher plants, whereas the genetic organization is simpler and can be manipulated more easily. Correspondingly, our attention has been focused on the molecular mechanism of adaptation and resistance to high temperatures of a cyanobacterial strain, Synechocystis PCC6803 (Vigh et al. 1990, Lehel et al. 1992, 1993a, 1993b).

Is the Membrane the Primary Target in the Biological Perception of Temperature? Effect of Membrane Physical State on the Expression of Stress-Defence Genes

Biological organisms are capable of perceiving environmental changes caused by a variety of agents. But whereas sensors of light (rhodopsins in animals or phytochromes in plants) are well-characterized, no knowledge is available concerning the thermal sensors. The only clue hitherto known about the thermal perception is that temperature decrease would result in a reduction of molecular motion in membranes, which is attained mainly via specific desaturation of fatty acids of membrane lipids [1]. It is assumed, that the response of systems to temperature stress via physiological, morphological and biochemical adjustments aimed at offset or compensate for the temperature-induced disturbances. Amongst the many compensatory responses, it seems that the major adjustment is the reorganization of the physical state of the cellular membranes, a phenomenon called “homeoviscous adaptation”.