Ibolya Horváth

HSP72 protects against obesity-induced insulin resistance

Patients with type 2 diabetes have reduced gene expression of heat shock protein (HSP) 72, which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signaling proteins such as c-jun amino terminal kinase (JNK), inhibitor of κB kinase, and tumor necrosis factor-α, can induce insulin resistance, but HSP 72 can block the induction of these molecules in vitro. Accordingly, we examined whether activation of HSP72 can protect against the development of insulin resistance. First, we show that obese, insulin resistant humans have reduced HSP72 protein expression and increased JNK phosphorylation in skeletal muscle. We next used heat shock therapy, transgenic overexpression, and pharmacologic means to overexpress HSP72 either specifically in skeletal muscle or globally in mice. Herein, we show that regardless of the means used to achieve an elevation in HSP72 protein, protection against diet- or obesity-induced hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance was observed. This protection was tightly associated with the prevention of JNK phosphorylation. These findings identify an essential role for HSP72 in blocking inflammation and preventing insulin resistance in the context of genetic obesity or high-fat feeding.

Small heat-shock proteins regulate membrane lipid polymorphism

Thermal stress in living cells produces multiple changes that ultimately affect membrane structure and function. We report that two members of the family of small heat-shock proteins (sHsp) (α-crystallin and Synechocystis HSP17) have stabilizing effects on model membranes formed of synthetic and cyanobacterial lipids. In anionic membranes of dimyristoylphosphatidylglycerol and dimyristoylphosphatidylserine, both HSP17 and α-crystallin strongly stabilize the liquid-crystalline state. Evidence from infrared spectroscopy indicates that lipid/sHsp interactions are mediated by the polar headgroup region and that the proteins strongly affect the hydrophobic core. In membranes composed of the nonbilayer lipid dielaidoylphosphatidylethanolamine, both HSP17 and α-crystallin inhibit the formation of inverted hexagonal structure and stabilize the bilayer liquid-crystalline state, suggesting that sHsps can modulate membrane lipid polymorphism. In membranes composed of monogalactosyldiacylglycerol and phosphatidylglycerol (both enriched with unsaturated fatty acids) isolated from Synechocystis thylakoids, HSP17 and α-crystallin increase the molecular order in the fluid-like state. The data show that the nature of sHsp/membrane interactions depends on the lipid composition and extent of lipid unsaturation, and that sHsps can regulate membrane fluidity. We infer from these results that the association between sHsps and membranes may constitute a general mechanism that preserves membrane integrity during thermal fluctuations.

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.

Membranes: a meeting point for lipids, proteins and therapies

•  Introduction •  Membrane lipid composition •  Membrane lipid structure •  Membrane lipid organization ‐  Why so many different lipids? ‐  Lipid mixing and demixing ‐  Lateral pressure ‐  Surface electrostatics •  Role of lipids in cell functions •  Lipid influence in transmembrane protein function ‐  Prokaryotic potassium channel (KcsA) ‐  Mechanosensitive channels ‐  Voltage‐gated potassium channel (KvAP) ‐  Nicotinic acetylcholine receptor (nAcChR) ‐  G protein‐coupled receptors ‐  Other examples •  Non‐permanent proteins in membranes ‐  Proteins that interact reversibly with the bilayers ‐  Proteins that interact irreversibly with the bilayers ‐  Proteins that interact weakly with the membrane ‐  Proteins that interact strongly with the membrane ‐  G proteins and their interactions with membranes ‐  Small monomeric G proteins: the Ras and Ras‐like family ‐  Protein kinase C •  Membrane microdomains and lipid mediators in the control of heat‐shock protein response ‐  Stress sensing and signalling: the membrane sensor theory ‐  Hsp signalling in cancer and diabetes ‐  The role of membrane microdomains ‐  Lipid mediators of the stress response •  A subpopulation of Hsps can interact with and translocate through membranes ‐  Hsp90 in eukaryotic membranes ‐  Hsp70 in cell membranes ‐  Hsp27‐membrane interactions ‐  Secreted Hsps ‐  Representative cases where Hsps interact with membranes or release from the cells •  Concluding remarks

The hyperfluidization of mammalian cell membranes acts as a signal to initiate the heat shock protein response

The concentrations of two structurally distinct membrane fluidizers, the local anesthetic benzyl alcohol (BA) and heptanol (HE), were used at concentrations so that their addition to K562 cells caused identical increases in the level of plasma membrane fluidity as tested by 1,6‐diphenyl‐1,3,5‐hexatriene (DPH) anisotropy. The level of membrane fluidization induced by the chemical agents on isolated membranes at such concentrations corresponded to the membrane fluidity increase seen during a thermal shift up to 42 °C. The formation of isofluid membrane states in response to the administration of BA or HE resulted in almost identical downshifts in the temperature thresholds of the heat shock response, accompanied by increases in the expression of genes for stress proteins such as heat shock protein (HSP)‐70 at the physiological temperature. Similarly to thermal stress, the exposure of the cells to these membrane fluidizers elicited nearly identical increases of cytosolic Ca2+ concentration in both Ca2+‐containing and Ca2+‐free media and also closely similar extents of increase in mitochondrial hyperpolarization. We obtained no evidence that the activation of heat shock protein expression by membrane fluidizers is induced by a protein‐unfolding signal. We suggest, that the increase of fluidity in specific membrane domains, together with subsequent alterations in key cellular events are converted into signal(s) leading to activation of heat shock genes.

Imaging of mobile long-lived nanoplatforms in the live cell plasma membrane.

The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, which are discussed in the context of “lipid rafts” or “membrane rafts.” Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition, and heterogeneity. We present here a method that allows for the first time the direct imaging of nanoscopic long-lived platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker proteins or lipids on a time scale of seconds. A special photobleaching protocol was used to reduce the surface density of labeled mobile platforms down to the level of well isolated diffraction-limited spots without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single molecule brightness analysis. For demonstration, we used the consensus raft marker glycosylphosphatidylinositol-anchored monomeric GFP and the fluorescent lipid analog BODIPY-GM1, which preferentially partitions into liquid-ordered phases. For both markers, we found cholesterol-dependent homo-association in the plasma membrane of living CHO and Jurkat T cells in the resting state, thereby demonstrating the existence of small, mobile, long-lived platforms containing these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures, the glycosylphosphatidylinositol-anchored monomeric GFP homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.

The temperature-dependent expression of the desaturase gene desA in Synechocystis PCC6803

We examined the temperature‐dependent regulation of the expression of the desA gene, which encodes Δ12 desaturase of Synechocystis PCC6803. The level of desA transcript increased 10‐fold within 1 h upon a decrease in temperature from 36°C to 22°C. This suggests that the low‐temperature‐induced desaturation of membrane lipid fatty acids is regulated at the level of the expression of the desaturase genes. The accumulation of the desA transcript depended on the extent of temperature change over a certain threshold level, but not on the absolute temperature.

Hyperfluidization-coupled membrane microdomain reorganization is linked to activation of the heat shock response in a murine melanoma cell line

Targeting of the Hsp function in tumor cells is currently being assessed as potential anticancer therapy. An improved understanding of the molecular signals that trigger or attenuate the stress protein response is essential for advances to be made in this field. The present study provides evidence that the membrane fluidizer benzyl alcohol (BA), a documented nondenaturant, acts as a chaperone inducer in B16(F10) melanoma cells. It is demonstrated that this effect relies basically on heat shock transcription factor 1 (HSF1) activation. Under the conditions tested, the BA-induced Hsp response involves the up-regulation of a subset of hsp genes. It is shown that the same level of membrane fluidization (estimated in the core membrane region) attained with the closely analogous phenethyl alcohol (PhA) does not generate a stress protein signal. BA, at a concentration that activates heat shock genes, exerts a profound effect on the melting of raft-like cholesterol-sphingomyelin domains in vitro, whereas PhA, at a concentration equipotent with BA in membrane fluidization, has no such effect. Furthermore, through the in vivo labeling of melanoma cells with a fluorescein labeled probe that inserts into the cholesterol-rich membrane domains [fluorescein ester of polyethylene glycol-derivatized cholesterol (fPEG-Chol)], we found that, similarly to heat stress per se, BA, but not PhA, initiates profound alterations in the plasma membrane microdomain structure. We suggest that, apart from membrane hyperfluidization in the deep hydrophobic region, a distinct reorganization of cholesterol-rich microdomains may also be required for the generation and transmission of stress signals to activate hsp genes.

TPPP/p25 promotes tubulin assemblies and blocks mitotic spindle formation.

Recently, we isolated from bovine brain a protein, TPPP/p25 and identified as p25, a brain-specific protein that induced aberrant tubulin assemblies. The primary sequence of this protein differs from that of other proteins identified so far; however, it shows high homology with p25-like hypothetical proteins sought via blast. Here, we characterized the binding of TPPP/p25 to tubulin by means of surface plasmon resonance; the kinetic parameters are as follows: kon, 2.4 × 104 M–1·s–1; koff, 5.4 × 10–3 s–1; and Kd, 2.3 × 10–7 M. This protein at substoichometric concentration promotes the polymerization of tubulin into double-walled tubules and polymorphic aggregates or bundles paclitaxel-stabilized microtubules as judged by quantitative data of electron and atomic force microscopies. Injection of bovine TPPP/p25 into cleavage Drosophila embryos expressing tubulin–GFP fusion protein reveals that TPPP/p25 inhibits mitotic spindle assembly and nuclear envelope breakdown without affecting other cellular events like centrosome replication and separation, microtubule nucleation by the centrosomes, and nuclear growth. GTP counteracts TPPP/p25 both in vitro and in vivo.

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.