TorsinA and heat shock proteins act as molecular chaperones: suppression of alpha-synuclein aggregation

TorsinA, a protein with homology to yeast heat shock protein104, has previously been demonstrated to colocalize with alpha-synuclein in Lewy bodies, the pathological hallmark of Parkinson's disease. Heat shock proteins are a family of chaperones that are both constitutively expressed and induced by stressors, and that serve essential functions for protein refolding and/or degradation. Here, we demonstrate that, like torsinA, specific molecular chaperone heat shock proteins colocalize with alpha-synuclein in Lewy bodies. In addition, using a cellular model of alpha-synuclein aggregation, we demonstrate that torsinA and specific heat shock protein molecular chaperones colocalize with alpha-synuclein immunopositive inclusions. Further, overexpression of torsinA and specific heat shock proteins suppress alpha-synuclein aggregation in this cellular model, whereas mutant torsinA has no effect. These data suggest that torsinA has chaperone-like activity and that the disease-associated GAG deletion mutant has a loss-of-function phenotype. Moreover, these data support a role for chaperone proteins, including torsinA and heat shock proteins, in cellular responses to neurodegenerative inclusions.     

Calnexin and BiP act as sequential molecular chaperones during thyroglobulin folding in the endoplasmic reticulum

Before secretion, newly synthesized thyroglobulin (Tg) folds via a series of intermediates: disulfide-linked aggregates and unfolded monomers-->folded monomers-->dimers. Immediately after synthesis, very little Tg associated with calnexin (a membrane-bound molecular chaperone in the ER), while a larger fraction bound BiP (a lumenal ER chaperone); dissociation from these chaperones showed superficially similar kinetics. Calnexin might bind selectively to carbohydrates within glycoproteins, or to hydrophobic surfaces of secretory proteins while they form proper disulfide bonds (Wada, I., W.-J. Ou, M.-C. Liu, and G. Scheele, J. Biol. Chem. 1994. 269:7464-7472). Because Tg has multiple disulfides, as well as glycans, we tested a brief exposure of live thyrocytes to dithiothreitol, which resulted in quantitative aggregation of nascent Tg, as analyzed by SDS-PAGE of cells lysed without further reduction. Cells lysed in the presence of dithiothreitol under non-denaturing conditions caused Tg aggregates to run as reduced monomers. For cells lysed either way, after in vivo reduction, Tg coprecipitated with calnexin. After washout of dithiothreitol, nascent Tg aggregates dissolved intracellularly and were secreted ultimately. 1 h after washout, > or = 92% of labeled Tg was found to dissociate from calnexin, while the fraction of labeled Tg bound to BiP rose from 0 to approximately 40%, demonstrating a "precursor-product" relationship. Whereas intralumenal reduction was essential for efficient Tg coprecipitation with calnexin, Tg glycosylation was not required. These data are among the first to demonstrate sequential chaperone function involved in conformational maturation of nascent secretory proteins within the ER.     

Photoresponsive nanogels formed by the self-assembly of spiropyrane-bearing pullulan that act as artificial molecular chaperones

Novel photoresponsive nanogels were prepared by the self-assembly of spiropyrane-bearing pullulan (SpP). The solution properties of the nanogels could be controlled by photostimulation via isomerization between hydrophobic spiropyrane and hydrophilic merocyanine. The molecular chaperone-like activity of the nanogels in protein refolding was investigated. The activity of citrate synthase significantly increased when the amphiphilicity of SpP nanogels was switched by photostimulation.     

Quadruplex formation as a molecular switch to turn on intrinsically fluorescent nucleotide analogs

Quadruplexes are involved in the regulation of gene expression and are part of telomeres at the ends of chromosomes. In addition, they are useful in therapeutic and biotechnological applications, including nucleic acid diagnostics. In the presence of K⁺ ions, two 15-mer sequences d(GGTTGGTGTGGTTGG) (thrombin binding aptamer) and d(GGGTGGGTGGGTGGG) (G3T) fold into antiparallel and parallel quadruplexes, respectively. In the present study, we measured the fluorescence intensity of one or more 2-aminopurine or 6-methylisoxanthopterin base analogs incorporated at loop-positions of quadruplex forming sequences to develop a detection method for DNA sequences in solution. Before quadruplex formation, the fluorescence is efficiently quenched in all cases. Remarkably, G3T quadruplex formation results in emission of fluorescence equal to that of a free base in all three positions. In the case of thrombin binding aptamer, the emission intensity depends on the location of the fluorescent nucleotides. Circular dichroism studies demonstrate that the modifications do not change the overall secondary structure, whereas thermal unfolding experiments revealed that fluorescent analogs significantly destabilize the quadruplexes. Overall, these studies suggest that quadruplexes containing fluorescent nucleotide analogs are useful tools in the development of novel DNA detection methodologies.     

Novel trisubstituted acridines as human telomeric quadruplex binding ligands

A novel series of trisubstituted acridines were synthesized with the aim of mimicking the effects of BRACO19. These compounds were synthesized by modifying the molecular structure of BRACO19 at positions 3 and 6 with heteroacyclic moieties. All of the derivatives presented in the study exhibited stabilizing effects on the human telomeric DNA quadruplex. UV–vis spectroscopy, circular dichroism, linear dichroism and viscosimetry were used in order to study the nature of the DNA binding in more detail. The results show that all of the novel derivatives were able to fold the single-stranded DNA sequences into antiparallel G-quadruplex structures, with derivative 15 exhibiting the highest stabilizing capability. Cell cycle analysis revealed that a primary trend of the “braco”-like derivatives was to arrest the cells in the S- and G₂M-phases of the cell cycle within the first 72 h, with derivative 13 and BRACO19 proving particularly effective in suppressing cell proliferation. All studies derivatives were less toxic to human fibroblast cell line in comparison with HT 29 cancer cell line.     

Fluorescence-based melting assays for studying quadruplex ligands

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomeres and telomerase are relevant targets in oncology, and telomere ligands and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analysed the FRET method used to measure the stabilization and selectivity of quadruplex ligands towards the human telomeric G-quadruplex. The stabilization value depends on the nature of the fluorescent tags, the incubation buffer, and the method chosen for T(m) calculation, complicating a direct comparison of the results obtained by different laboratories.     

Role of molecular chaperones in inclusion body formation

Protein misfolding and aggregation are linked to several degenerative diseases and are responsible for the formation of bacterial inclusion bodies. Roles of molecular chaperones in promoting protein deposition have been speculated but not proven in vivo. We have investigated the involvement of individual chaperones in inclusion body formation by producing the misfolding-prone but partially soluble VP1LAC protein in chaperone null bacterial strains. Unexpectedly, the absence of a functional GroEL significantly reduced aggregation and favoured the incidence of the soluble protein form, from 4 to 35% of the total VP1LAC protein. On the other hand, no regular inclusion bodies were then formed but more abundant small aggregates up to 0.05 microm(3). Contrarily, in a DnaK(-) background, the amount of inclusion body protein was 2.5-fold higher than in the wild-type strain and the average volume of the inclusion bodies increased from 0.25 to 0.38 microm(3). Also in the absence of DnaK, the minor fraction of soluble protein appears as highly proteolytically stable, suggesting an inverse connection between proteolysis and aggregation managed by this chaperone. In summary, GroEL and DnaK appear as major antagonist controllers of inclusion body formation by promoting and preventing, respectively, the aggregation of misfolded polypeptides. GroEL might have, in addition, a key role in driving the protein transit from the soluble to the insoluble cell fraction and also in the opposite direction. Although chaperones ClpB, ClpA, IbpA and IbpB also participate in these processes, the impact of the respective null mutations on bacterial inclusion body formation is much more moderate.     

Heat shock proteins as molecular chaperones

Exposure to different conditions or agents that destabilize cell homeostasis often alters protein folding. Depending on stress intensity irreversible protein aggregation and cell death can occur. Cells have developed a conserved defense mechanism aimed at reducing the deleterious effects induced by protein folding alteration. This mechanism is characterized by the expression of a small number of genes encoding specific proteins, named Hsps. Several of these proteins act as molecular chaperones through their ability to refold polypeptides with an altered conformation. Moreover, constitutive Hsps homologues have been characterized that participate in the folding of newly made polypeptides, in the assembly of protein complexes in the endoplasmic reticulum, in the translocation of polypeptides through membranes or in masking mutations that alter protein folding. Neurodegeneratives and cancereous diseases are discussed as examples where high levels of Hsp expression can be either beneficial or deleterious to the cells.     

Liposome membrane can act like molecular and metal chaperones for oxidized and fragmented superoxide dismutase

A mechanism for liposome-recruited activity of oxidized and fragmented superoxide dismutase (Fr.-SOD) [Tuan LQ, Umakoshi H, Shimanouchi T, Kuboi R. Liposome-recruited activity of oxidized and fragmented superoxide dismutase. Langmuir 2008;24:350–4] was further investigated, focusing on the secondary structure of Fr.-SOD. Liposome membrane was found to assist the conformational change of Fr.-SOD and reactivate the enzymatic activity, like molecular and metal chaperones. The loss of SOD activity and its secondary structure was observed during 6 h oxidation in 2 mM hydrogen peroxide. The contents of the α-helix and β-sheet structures in the oxidized and fragmented SOD (2 μM) were increased only in the presence of 10 μM Cu²⁺ and Zn²⁺ together, or in the presence of 2 mM POPC liposomes. The mixture of all of these elements (fragmented SOD and POPC liposomes with Cu²⁺ and Zn²⁺) gave not only the increase of the α-helix and β-sheet contents but also the mediation of the high SOD-like activity.     

Disordered plant LEA proteins as molecular chaperones

Plants often respond to abiotic stresses by the increased expression of LEA (late embryogenesis abundant) proteins, so called because they also accompany seed formation. Whereas the cellular function of LEA proteins in mitigating the damage caused by stress is clear, the molecular mechanisms of their action are rather enigmatic. Several models have been developed, based on their putative activities as ion sinks, stabilizers of membrane structure, buffers of hydrate water, antioxidants and/or chaperones. Due to their known structural flexibility, this latter idea has received little experimental attention thus far. Recently, however, it has been suggested that intrinsically disordered proteins (IDPs) may exert chaperone activity by an “entropy transfer” mechanism. In our subsequent study published in the May issue of Plant Physiology, we provided evidence that two group 2 LEA proteins, ERD (early response to dehydration) 10 and 14, are potent molecular chaperones. This observation may have far-reaching implications, as it may explain how LEA proteins of ill-defined structures protect plant cells during dehydration, and it may also lead to the general experimental validation of the entropy transfer model of disordered chaperones.Key words: abiotic stress, dehydration stress, stress tolerance, late embryogenesis abundant protein, chaperone, disordered protein, unstructured protein     

Structure of bradavidin-C-terminal residues act as intrinsic ligands

Bradavidin is a homotetrameric biotin-binding protein from Bradyrhizobium japonicum, a nitrogen fixing and root nodule-forming symbiotic bacterium of the soybean. Wild-type (wt) bradavidin has 138 amino acid residues, whereas the C-terminally truncated core-bradavidin has only 118 residues. We have solved the X-ray structure of wt bradavidin and found that the C-terminal amino acids of each subunit were uniquely bound to the biotin-binding pocket of an adjacent subunit. The biotin-binding pocket occupying peptide (SEKLSNTK) was named "Brad-tag" and it serves as an intrinsic stabilizing ligand in wt bradavidin. The binding of Brad-tag to core-bradavidin was analysed by isothermal titration calorimetry and a binding affinity of ～25 μM was measured. In order to study the potential of Brad-tag, a green fluorescent protein tagged with Brad-tag was prepared and successfully concentrated from a bacterial cell lysate using core-bradavidin-functionalized Sepharose resin.     

Chromosomal toxin-antitoxin systems may act as antiaddiction modules

Toxin-antitoxin (TA) systems are widespread among bacterial chromosomes and mobile genetic elements. Although in plasmids TA systems have a clear role in their vertical inheritance by selectively killing plasmid-free daughter cells (postsegregational killing or addiction phenomenon), the physiological role of chromosomally encoded ones remains under debate. The assumption that chromosomally encoded TA systems are part of stress response networks and/or programmed cell death machinery has been called into question recently by the observation that none of the five canonical chromosomally encoded TA systems in the Escherichia coli chromosome seem to confer any selective advantage under stressful conditions (V. Tsilibaris, G. Maenhaut-Michel, N. Mine, and L. Van Melderen, J. Bacteriol. 189:6101-6108, 2007). Their prevalence in bacterial chromosomes indicates that they might have been acquired through horizontal gene transfer. Once integrated in chromosomes, they might in turn interfere with their homologues encoded by mobile genetic elements. In this work, we show that the chromosomally encoded Erwinia chrysanthemi ccd (control of cell death) (ccd(Ech)) system indeed protects the cell against postsegregational killing mediated by its F-plasmid ccd (ccd(F)) homologue. Moreover, competition experiments have shown that this system confers a fitness advantage under postsegregational conditions mediated by the ccd(F) system. We propose that ccd(Ech) acts as an antiaddiction module and, more generally, that the integration of TA systems in bacterial chromosomes could drive the evolution of plasmid-encoded ones and select toxins that are no longer recognized by the antiaddiction module.     

Enkephalins may act as sensory transmitters in earthworms

Summary Part of the sensory cells of the earthworm (Lumbricus terrestris) epidermis stain immunocytochemically with enkephalin antisera of different region specificities. The immunocytochemical results suggest the existence of peptides identical with or closely resembling met- and leu-enkephalin in these cells. The processes of enkephalin-immunoreactive cells become collected to form sensory nerves before entering the ventral ganglionic chain where they project as enkephalin-immunoreactive sensory bundles. Injections of the opiate receptor antagonist naloxone in earthworms inhibit their touch-induced withdrawal reflex. Recovery occurs within 2 hours. Moreover, anaesthesia of earthworms in dilute ethanol brings about abolishment of the withdrawal reflex as well as disappearance of enkephalin immunoreactivity from the cell bodies, but not from the sensory hairs. Together, these data suggest that opioid peptides, possibly enkephalins, act as sensory transmitters or modulators in earthworms.     

Cell surface receptors for molecular chaperones

Heat shock proteins are intracellular molecular chaperones. However, extracellular heat shock proteins have recently been shown to mediate a range of powerful effects in inflammatory cells, neuronal cells and immune cells. These effects are transmitted by a number of cell surface receptors including LRP/CD91, CD40, Toll-like receptors, Scavenger receptors and c-type Lectins. However, although extracellular heat shock proteins are products of at least five different gene superfamilies, similar receptor types often trigger their effects. We have assessed heat shock protein binding to the different receptor types with particular regard to its role in tumor immunology. Heat shock protein 70 released from dying tumor cells or injected as part of a vaccine induces a remarkable range of immune effects. This molecular chaperone induces powerful pro-inflammatory signaling cascades leading to the activation of antigen presenting cells. In addition, heat shock protein 70 is able to transport antigenic peptides as cargo from the tumor cell cytoplasm across the membranes of antigen presenting cells and deliver them to major histocompatability class I molecules, a process known as "cross-presentation". The resulting major histocompatability class I-peptide complexes are then displayed on the cell surface by antigen presenting cells, leading to activation of cytotoxic T lymphocytes and tumor cell killing. Understanding how heat shock protein-receptor binding orchestrates individual components of tumor immunity will permit enhanced design of molecular chaperone based immunotherapy.     

Amino acid sequence determinants and molecular chaperones in amyloid fibril formation

Amyloid consists of cross-β-sheet fibrils and is associated with about 25 human diseases, including several neurodegenerative diseases, systemic and localized amyloidoses and type II diabetes mellitus. Amyloid-forming proteins differ in structures and sequences, and it is to a large extent unknown what makes them convert from their native conformations into amyloid. In this review, current understanding of amino acid sequence determinants and the effects of molecular chaperones on amyloid formation are discussed. Studies of the nonpolar, transmembrane surfactant protein C (SP-C) have revealed amino acid sequence features that determine its amyloid fibril formation, features that are also found in the amyloid β-peptide in Alzheimer’s disease and the prion protein. Moreover, a proprotein chaperone domain (CTC_Brichos) that prevents amyloid-like aggregation during proSP-C biosynthesis can prevent fibril formation also of other amyloidogenic proteins.     

The role of heat-shock proteins as molecular chaperones.

Recent studies have revealed that protein folding and assembly events in vivo require the participation of accessory components, now being referred to as 'molecular chaperones'. A number of chaperones have been identified as members of the heat-shock (or stress) protein family. This review discusses the roles of two classes of chaperones, the heat-shock protein 70 and groEL/ES families, in facilitating protein maturation, and describes how such events are perturbed in the cell subjected to metabolic stress.     

Epigenetic mechanisms in the dentate gyrus act as a molecular switch in hippocampus-associated memory formation

We make strong memories of significant events in our lives which may serve to increase our resilience and adaptation capacity to deal with future challenges. It is well established that the neurotransmitter glutamate and the ERK MAPK intracellular signaling pathway play a principal role in memory formation. In addition, stress-associated hormones like glucocorticoids released during such events are known to strengthen formation of memories. But, how do these hormones work? Do they interact with the ERK MAPK pathway or otherwise? What are the more distal, epigenomic effects? We discovered in rats and mice that confrontation with a psychological challenge (e.g. forced swimming, Morris water maze) would lead, through NMDA-ERK signaling, to MSK1 and Elk-1 activation in dentate gyrus neurons (a part of the hippocampus involved in encoding of memories) resulting in histone H3 S10-phosphorylation and K14-acetylation, H4 hyper-acetylation, gene induction and formation of memories of the event. Moreover, glucocorticoid hormones via the glucocorticoid receptor (GR) greatly facilitated the epigenomic mechanisms and cognitive performance. Therefore, we propose that formation of enduring memories of significant events requires an interaction of GRs with the NMDA/ERK/MSK1/Elk-1 signaling pathways to allow an optimal epigenomic activation pattern in dentate gyrus neurons to accommodate their altered neurophysiological function.     

Overexpressed proteins may act as mops removing their ligands from the host cells: A case study of calf PNP

Calf purine nucleoside phosphorylase (PNP) was overexpressed in Escherichia coli. The basic kinetic parameters of recombinant PNP were found to be similar to the values published previously for non-recombinant PNP from calf spleen. However, upon titration of the recombinant enzyme with the tight-binding multisubstrate analogue inhibitor DFPP-DG, endothermic as well as exothermic signals were obtained. This was not the case for PNP isolated from calf spleen for which only the endothermic process was observed. Further calorimetric titrations of the recombinant and non-recombinant enzyme with its potent and moderate ligands, and studied involving partial inactivation of the enzyme, lead to the conclusion that a part of the recombinant enzyme forms a complex with its product, hypoxanthine, although hypoxanthine was not present at any purification stage except for its natural occurrence in E. coli cells. Binding of hypoxanthine is accompanied with a large negative change of the free enthalpy, and therefore the replacement of this compound by DFPP-DG yields positive heat signal. Our data obtained with calf PNP indicate that similar processes - moping of ligands from the host cells - may take place in the case of other proteins with high overexpression yield.