Trehalose and glycerol stabilize and renature yeast inorganic pyrophosphatase inactivated by very high temperatures

A number of naturally occurring small organic molecules, primarily involved in maintaining osmotic pressure in the cell, display chaperone-like activity, stabilizing the native conformation of proteins, and protecting them from various kinds of stress. Most of them are sugars, polyols, amino acids or methylamines. Similar to molecular chaperones, most of these compounds have no substrate specificity, but some specifically stabilize certain proteins. In the present work, the capacity of trehalose and glycerol, two well-known osmolytes, to stabilize and renature inorganic pyrophosphatase is demonstrated. Both trehalose and glycerol significantly protect pyrophosphatase against thermoinactivation achieved by incubating the enzyme at temperatures up to 95 degrees C, and allow the enzyme already inactivated in the presence of these osmolytes to renature upon incubation at low temperatures. To the best of our knowledge, there are no data on the effects of these compounds on renaturation of thermoinactivated proteins. The correlation between the recovery of enzyme activity and structural changes indicated by fluorescence spectroscopy contribute to better understanding of the protein stabilization mechanism.     

Quality control of integral membrane proteins

Integral membrane proteins (IMPs) are essential components of the plasma and organellar membranes of the eukaryotic cell. Non-native IMPs, which can arise as a result of mutations, errors during biosynthesis or cellular stress, can disrupt these membranes and potentially lead to cell death. To protect against this outcome, the cell possesses quality control (QC) systems that detect and dispose of non-native IMPs from cellular membranes. Recent studies suggest that recognition of non-native IMPs by the QC machinery is correlated with the thermodynamic stability of these proteins. Consistent with this, small molecules known as chemical and pharmacological chaperones have been identified that stabilize non-native IMPs and enable them to evade QC. These findings have far-reaching implications for treating human diseases caused by defective IMPs.     

Influence of molecular and chemical chaperones on protein folding

Protein folding inside the cell involves the Participation of accessory components known as molecular chaperones. In addition to their active participation in the folding process, molecular chaperones serve as a type of ‘quality control system’, recognizing, retaining and targeting misfolded proteins for their eventual degradation. It is now known that a number of human diseases arise as a consequence of specific point mutations or deletions within genes encoding essential proteins. In many cases these mutations/deletions are not so sever as to totally destroy the biological activity of the particular gene product. Rather, the mutations often result in only subtle folding abnormalities which lead to the newly synthesized protein being retained at the endoplasmic reticulum by the actions of the cellylar quality control system. In this short review article we discuss our recent studies showing that the protein folding defect associated with the most common mutation in patients with cystic fibriosis can be overcome by a novel strategy. As shown in the paper by Brown et al in this issue (Brown et al 1996), a number of different low molecular weight compounds, all known to stabilize proteins in their native conformation, are effective in rescuing the processing defect of the mutant cystic fibrosis transmembrane conductance regulator protein. We then discuss how these same compounds, which we now call chemical chaperones, also may prove to be effective in correcting a number of other protein folding abnormalities which constitute the underlying basis of a large number of different human diseases.     

Roles of molecular chaperones in protein misfolding diseases

Human misfolding diseases result from the failure of proteins to reach their active state or from the accumulation of aberrantly folded proteins. The mechanisms by which molecular chaperones influence the development of these diseases is beginning to be understood. Mutations that compromise the activity of chaperones lead to several rare syndromes. In contrast, the more frequent amyloid-related neurodegenerative diseases are caused by a gain of toxic function of misfolded proteins. Toxicity in these disorders may result from an imbalance between normal chaperone capacity and production of dangerous protein species. Increased chaperone expression can suppress the neurotoxicity of these molecules, suggesting possible therapeutic strategies.     

Heat shock proteins: Molecules with assorted functions

Heat shock proteins (Hsps) or molecular chaperones, are highly conserved protein families present in all studied organisms. Following cellular stress, the intracellular concentration of Hsps generally increases several folds. Hsps undergo ATP-driven conformational changes to stabilize unfolded proteins or unfold them for translocation across membranes or mark them for degradation. They are broadly classified in several families according to their molecular weights and functional properties. Extensive studies during the past few decades suggest that Hsps play a vital role in both normal cellular homeostasis and stress response. Hsps have been reported to interact with numerous substrates and are involved in many biological functions such as cellular communication, immune response, protein transport, apoptosis, cell cycle regulation, gametogenesis and aging. The present review attempts to provide a brief overview of various Hsps and summarizes their involvement in diverse biological activities.     

Inhibitors of CLK Protein Kinases Suppress Cell Growth and Induce Apoptosis by Modulating Pre-mRNA Splicing

Accumulating evidence has demonstrated the importance of alternative splicing in various physiological processes, including the development of different diseases. CDC-like kinases (CLKs) and serine-arginine protein kinases (SRPKs) are components of the splicing machinery that are crucial for exon selection. The discovery of small molecule inhibitors against these kinases is of significant value, not only to delineate the molecular mechanisms of splicing, but also to identify potential therapeutic opportunities. Here we describe a series of small molecules that inhibit CLKs and SRPKs and thereby modulate pre-mRNA splicing. Treatment with these small molecules (Cpd-1, Cpd-2, or Cpd-3) significantly reduced the levels of endogenous phosphorylated SR proteins and caused enlargement of nuclear speckles in MDA-MB-468 cells. Additionally, the compounds resulted in splicing alterations of RPS6KB1 (S6K), and subsequent depletion of S6K protein. Interestingly, the activity of compounds selective for CLKs was well correlated with the activity for modulating S6K splicing as well as growth inhibition of cancer cells. A comprehensive mRNA sequencing approach revealed that the inhibitors induced splicing alterations and protein depletion for multiple genes, including those involved in growth and survival pathways such as S6K, EGFR, EIF3D, and PARP. Fluorescence pulse-chase labeling analyses demonstrated that isoforms with premature termination codons generated after treatment with the CLK inhibitors were degraded much faster than canonical mRNAs. Taken together, these results suggest that CLK inhibitors exhibit growth suppression and apoptosis induction through splicing alterations in genes involved in growth and survival. These small molecule inhibitors may be valuable tools for elucidating the molecular machinery of splicing and for the potential development of a novel class of antitumor agents.     

Why molecular chaperones buffer mutational damage: a case study with a yeast Hsp40/70 system

The malfunctioning of molecular chaperones may result in uncovering genetic variation. The molecular basis of this phenomenon remains largely unknown. Chaperones rescue proteins unfolded by environmental stresses and therefore they might also help to stabilize mutated proteins and thus mask damages. To test this hypothesis, we carried out a genomewide mutagenesis followed by a screen for mutations that were synthetically harmful when the RAC-Ssb1/2 cytosolic chaperones were inactive. Mutants with such a phenotype were found and mapped to single nucleotide substitutions. However, neither the genes identified nor the nature of genetic lesions implied that folding of the mutated proteins was being supported by the chaperones. In a second screen, we identified temperature-sensitive (ts) mutants, a phenotype indicative of structural instability of proteins. We tested these for an association with sensitivity to loss of chaperone activity but found no such correlation as might have been expected if the chaperones assisted the folding of mutant proteins. Thus, molecular chaperones can mask the negative effects of mutations but the mechanism of such buffering need not be direct. A plausible role of chaperones is to stabilize genetic networks, thus making them more tolerant to malfunctioning of their constituents.     

Intracellular Refolding Assay

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding¹ and have a crucial role in promoting cell survival upon stress insults like heat shock², nutrient starvation and exposure to chemicals/poisons³. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells⁴ as well as neurodegeneration⁵. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy⁷ and neurodegenerative disorders⁹. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones^10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene ¹¹ (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45°C for 30 minutes. Upon recovery at 37°C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).     

药物分子伴侣: 蛋白质折叠运输缺陷的新疗法

大量遗传性疾病的发生是由于基因突变引起蛋白质错误折叠而不能运输到作用位点,从而导致功能缺陷．近年来兴起的药物分子伴侣是恢复蛋白质折叠运输缺陷的新疗法,这类化合物一般为目的蛋白的底物类似物、受体配基或酶抑制剂等化学小分子,具细胞通透性,能在内质网中特异性识别并结合突变蛋白,校正并稳定其正确构象,协助其运输到正确位点,直接恢复突变蛋白功能,可治疗各种南蛋白质折叠运输缺陷导致的内分泌及代谢疾病．目前已报道的由药物分子伴侣恢复功能的突变蛋白主要为质膜蛋白及细胞器蛋白,如ATP结合盒转运蛋白、G-蛋白耦联受体及溶酶体酶等．大量的细胞及动物实验结果显示了药物分子伴侣的临床应用前景广阔,目前已有一例临床实验获得了成功．     

The role of stress proteins in prostate cancer

The development of therapeutic resistance, after hormone or chemotherapy for example, is the underlying basis for most cancer deaths. Exposure to anticancer therapies induces expression of many stress related proteins, including small heat shock proteins (HSPs). HSPs interact with various client proteins to assist in their folding and enhance the cellular recovery from stress, thus restoring protein homeostasis and promoting cell survival. The vents of cell stress and cell death are linked, as the induction of molecular chaperones appears to function at key regulatory points in the control of apoptosis. On the basis of these observations and on the role of molecular chaperones in the regulation of steroid receptors, kinases, caspases, and other protein remodelling events involved in chromosome replication and changes in cell structure, it is not surprising that molecular chaperones have been implicated in the control of cell growth and in resistance to various anticancer treatments that induce apoptosis. Recently, several molecular chaperones such as Clusterin and HSP27 have been reported to be involved in development and progression of hormone-refractory prostate cancer. In this review, we address some of the molecular and cellular events initiated by treatment induced stress, and discuss the potential role of chaperone proteins as targets for prostate cancer treatment.     

Heat shock proteins and aging in Drosophila melanogaster

Heat shock proteins (Hsps) are conserved molecular chaperones that are upregulated following exposure to environmental stress and during aging. The mechanisms underlying the aging process are only beginning to be understood. The beneficial effects of Hsps on aging revealed in mild stress and overexpression experiments suggest that these proteins are part of an important cell protection system rather than being unspecific molecular chaperones. Among the Hsps families, small Hsps have the greatest influence on aging and the modulation of their expression during aging in Drosophila suggest that they are involved in pathways of longevity determination.     

Effect of Chemical Chaperones on Glucose-Induced Lysozyme Modifications

Nonenzymatic glycation of biomacromolecules occurs due to the diabetes mellitus and ageing. A number of small molecules, known as chemical chaperones, stabilize protein conformation against thermal and chemically induced denaturation. These compounds are including: polyamines (e.g. spermine and spermidine), amino acids (e.g. lysine) and polyols (e.g. glycerol). In this study the effect of spermidine (Spd), spermine (Spm), and glycerol on glycation, structure and function of lysozyme (LZ), as an extra-cellular protein, by different techniques is investigated. LZ is incubated with or without glucose (50 or 100 mM) in the absence or presence of Spd/Spm/glycerol at 37 °C up to 16 weeks. All the observed changes of glycated-LZ in comparison with the native protein, including: increased fluorescence emission, alteration in the secondary and tertiary structure, and reduced electrophoretic mobility- indicate its structural changes that are accompanied with its reduced activity. Glucose in the presence or absence of Spd induces the protein dimerization, but glucose plus Spm induces its trimmerization. In contrast, glycerol inhibits the LZ glycation and prevents the large changes on its structure and function. Glucose binds lysine residues, decreases the protein positive charges and induces some alterations in its structure and activity. Polyamines also directly bind to LZ, increase its positive charges and hence induce more glycation; more conformational changes, oligomerization and its inactivation in the presence of glucose, but glycerol affect the protein environment and preserve protein from these harmful effects.     

Yeast protein glycation in vivo by methylglyoxal. Molecular modification of glycolytic enzymes and heat shock proteins

Protein glycation by methylglyoxal is a nonenzymatic post-translational modification whereby arginine and lysine side chains form a chemically heterogeneous group of advanced glycation end-products. Methylglyoxal-derived advanced glycation end-products are involved in pathologies such as diabetes and neurodegenerative diseases of the amyloid type. As methylglyoxal is produced nonenzymatically from dihydroxyacetone phosphate and d-glyceraldehyde 3-phosphate during glycolysis, its formation occurs in all living cells. Understanding methylglyoxal glycation in model systems will provide important clues regarding glycation prevention in higher organisms in the context of widespread human diseases. Using Saccharomyces cerevisiae cells with different glycation phenotypes and MALDI-TOF peptide mass fingerprints, we identified enolase 2 as the primary methylglyoxal glycation target in yeast. Two other glycolytic enzymes are also glycated, aldolase and phosphoglycerate mutase. Despite enolase's activity loss, in a glycation-dependent way, glycolytic flux and glycerol production remained unchanged. None of these enzymes has any effect on glycolytic flux, as evaluated by sensitivity analysis, showing that yeast glycolysis is a very robust metabolic pathway. Three heat shock proteins are also glycated, Hsp71/72 and Hsp26. For all glycated proteins, the nature and molecular location of some advanced glycation end-products were determined by MALDI-TOF. Yeast cells experienced selective pressure towards efficient use of d-glucose, with high methylglyoxal formation as a side effect. Glycation is a fact of life for these cells, and some glycolytic enzymes could be deployed to contain methylglyoxal that evades its enzymatic catabolism. Heat shock proteins may be involved in proteolytic processing (Hsp71/72) or protein salvaging (Hsp26).     

Structure of acid beta-glucosidase with pharmacological chaperone provides insight into Gaucher disease

Gaucher disease results from mutations in the lysosomal enzyme acid beta-glucosidase (GCase). Although enzyme replacement therapy has improved the health of some affected individuals, such as those with the prevalent N370S mutation, oral treatment with pharmacological chaperones may be therapeutic in a wider range of tissue compartments by restoring sufficient activity of endogenous mutant GCase. Here we demonstrate that isofagomine (IFG, 1) binds to the GCase active site, and both increases GCase activity in cell lysates and restores lysosomal trafficking in cells containing N370S mutant GCase. We also compare the crystal structures of IFG-bound GCase at low pH with those of glycerol-bound GCase at low pH and apo-GCase at neutral pH. Our data indicate that IFG induces active GCase, which is secured by interactions with Asn370. The design of small molecules that stabilize substrate-bound conformations of mutant proteins may be a general therapeutic strategy for diseases caused by protein misfolding and mistrafficking.     

Chaperones and protein folding

Chaperones are centrally involved in the control of protein structure, function, localization and transport. A flurry of scientific activity continues to examine the molecular nature of chaperone-substrate recognition and the role of auxiliary chaperones (cohort proteins) and small molecules that expedite these processes. Chaperones have been implicated in processes as diverse as protein secretion, nuclear transport, thermotolerance, the steroid receptor signal transduction pathway, T-cell receptor and major histocompatibility complex class I and II multimeric assembly and bacterial virulence.     

Small heat shock proteins--role in apoptosis, cancerogenesis and diseases associated with protein aggregation

Small heat shock proteins (sHsps) belong to molecular chaperones, which protect prokaryotic and eukaryotic cells against deleterious effects, of stress. sHsps prevent stress induced, irreversible aggregation of damaged proteins and facilitate renaturation of bound substrates cooperating with other molecular chaperones. This review summarizes recent studies focused mainly on the involvement of sHsps in diseases related to protein aggregation. sHsps are often a component of protein aggregates forming during progress of neurodegenerative disorders. Mutation in sHsps genes have been identified, which are responsible for development of cataract, desmin related myopathy and neuropathies. sHsps protect cells against oxidative stress resulting from ischemia/reperfusion during heart or brain stroke. Several studies indicate that sHsp participate in regulation of apoptosis and are involved in cancerogenesis. Uncovering the sHsps role in diseases enable to develop new therapeutic strategies.     

Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.     

Rescue of vasopressin V2 receptor mutants by chemical chaperones: specificity and mechanism

Because missense mutations in genetic diseases of membrane proteins often result in endoplasmic reticulum (ER) retention of functional proteins, drug-induced rescue of their cell surface expression and understanding the underlying mechanism are of clinical value. To study this, we tested chemical chaperones and sarco(endo)plasmic reticulum Ca2+ ATPase pump inhibitors on Madin-Darby canine kidney cells expressing nine ER-retained vasopressin type-2 receptor (V2R) mutants involved in nephrogenic diabetes insipidus. Of these nine, only V2R-V206D showed improved maturation and plasma membrane rescue with glycerol, dimethyl sulfoxide (DMSO), thapsigargin/curcumin, and ionomycin but not with other osmolytes or growth at 27 degrees C. This revealed that rescue is mutant specific and that this mutant is prone to rescue by multiple compounds. Rescue did not involve changed expression of molecular chaperones calnexin, heat-shock protein (HSP) 70, or HSP90. V2R antagonist SR121463B treatment revealed that V2R-V206D and V2R-S167T were rescued and matured to a greater extent, suggesting that the rescuing activity of a pharmacological versus chemical chaperone is broader and stronger. Calcium measurements showed that rescue of V2R-V206D by thapsigargin, curcumin, and ionomycin was because of increased cytosolic calcium level, rather than decreased endoplasmic reticulum calcium level. The molecular mechanism underlying rescue by DMSO, glycerol, and SR121463B is different, because with these compounds intracellular calcium levels were unaffected.