Sarah Perrett

Intrinsic peroxidase-like activity of ferromagnetic nanoparticles

Nanoparticles containing magnetic materials, such as magnetite (Fe3O4), are particularly useful for imaging and separation techniques. As these nanoparticles are generally considered to be biologically and chemically inert, they are typically coated with metal catalysts, antibodies or enzymes to increase their functionality as separation agents. Here, we report that magnetite nanoparticles in fact possess an intrinsic enzyme mimetic activity similar to that found in natural peroxidases, which are widely used to oxidize organic substrates in the treatment of wastewater or as detection tools. Based on this finding, we have developed a novel immunoassay in which antibody-modified magnetite nanoparticles provide three functions: capture, separation and detection. The stability, ease of production and versatility of these nanoparticles makes them a powerful tool for a wide range of potential applications in medicine, biotechnology and environmental chemistry.

Chirality of Glutathione Surface Coating Affects the Cytotoxicity of Quantum Dots

Quantum dots (QDs) have been extensively investigated as fluorescent probes and are emerging as a new class of agents for biomedical imaging and diagnosis because of their broad absorption profiles, tunable emission wavelengths, and high photooxidation stability. QDs consist of an inorganic core surrounded by an organic shell. Normally, different types of biomolecules, such as amino acids, DNA, or peptides, are used for the organic shell to facilitate water solubility and biocompatibility of the QDs. However, because the core may contain toxic heavy metals (e.g., Cd, Hg, Pb, and Zn), the potential cytotoxicity of QDs has been a major impediment to their widespread application. It has therefore become critical to fully understand the interactions between QDs and living cells in order to develop nontoxic and biocompatible QDs for clinical use. Early studies have suggested that the release of core components, the generation of reactive oxygen species (ROS), and nonspecific binding to cellular membranes and intracellular proteins are the major mechanisms of the observed cytotoxic effects of QDs. Despite a significant surge in the number of investigations into the cytotoxicity of QDs, there is currently only limited knowledge about the cytological and physiological mediators of these effects. Interestingly, recent data have suggested that the induction of autophagy by certain sizes of QDs could play an important role in their toxic actions. Autophagy is a metabolic process involved in protein and organelle degradation and plays key roles in maintaining cellular homeostasis and contributing to cellular defense. It has been recognized as a third pathway of cell death, after apoptosis and necrosis, and is responsive to various physicopathological stimuli. Recent work has shown that small QDs (< 10 nm) rather than those with larger sizes (40–50 nm) induce autophagy in cultured cells. This size-dependent induction of autophagy has also been reported for other nanoparticles (NPs). However, all the above studies focused on the effects of type and size of the NPs, while other factors that may induce autophagy remain unexplored. Although many studies have demonstrated that surface modification of QDs with biomolecules endows them with various biological functionalities, the impact on living organisms of the chirality of the surface biomolecules has been largely neglected. Chirality is an important phenomenon in living systems and nearly all biological polymers must be homochiral to function. For example, all amino acids in proteins are “left-handed”, whereas all sugars in DNA and RNA are “right-handed”. Different chiral properties of biomolecules may determine their ability to interact with other biomolecules and thereby modulate a range of downstream processes. More recently, several attempts to develop chiral QDs with optical activities using different chiral stabilizers have been reported. Herein, the effects of QDs capped with different chiral forms of the tripeptide glutathione (GSH) on cytotoxicity and induction of autophagy were examined. Two different sizes of cadmium telluride (CdTe) QDs coated with either l-GSH (lGSH-QDs) or d-GSH (d-GSH-QDs) were found to show dose-dependent cytotoxicity and to significantly increase the levels of autophagic vacuoles. The activation of autophagy was chirality-dependent, with l-GSH-QDs being more effective than d-GSH-QDs. The ability of QDs to induce cell death was correlated with their ability to induce autophagy. This chirality-associated regulation of cellular metabolism and cytotoxicity highlights the important role of the conformation of the stabilizers, and has important implications for the design of novel QDs with enhanced optical properties and reduced or no toxicity. In this study, negatively charged water-soluble CdTe QDs were synthesized according to the Rogach–Weller method and coated with different chiral forms of GSH as stabilizers (Figure 1a). To clearly understand the chirality effect, two series of QDs were prepared. Group 1 comprised small-sized [*] Y. Li, Prof. Y. Zhao, Prof. G. Nie CAS Key Laboratory for Biological Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology 11 Beiyijie, Zhongguancun, Beijing 100190 (China) E-mail: niegj@nanoctr.cn

Catalysis of Amide Proton Exchange by the Molecular Chaperones GroEL and SecB

Hydrogen-deuterium exchange of 39 amide protons of Bacillus amyloliquefaciens ribonuclease (barnase) was analyzed by two-dimensional nuclear magnetic resonance in the presence of micromolar concentrations of the molecular chaperones GroEL and SecB. Both chaperones bound to native barnase under physiological conditions and catalyzed exchange of deeply buried amide protons with solvent. Such exchange required complete unfolding of barnase, which occurred in the complex with the chaperones. Subsequent collapse of unfolded barnase to the exchange-protected folding intermediate was markedly slowed in the presence of GroEL or SecB. Thus, both chaperones have the potential to correct misfolding in proteins by annealing.

Effect of Nanoparticles on Protein Folding and Fibrillogenesis

The large surface area and small size of nanoparticles provide properties and applications that are distinct from those of bulk materials. The ability of nanoparticles to influence protein folding and aggregation is interesting, not only because of the potential beneficial applications, but also the potential risks to human health and the environment. This makes it essential that we understand the effect of nanoparticles on fundamental biological process, like protein folding. Here, we review studies that have examined the effect of nanoparticles on protein folding and aggregation, providing insight both into the mechanisms of these processes and how they may be controlled.

The yeast prion protein Ure2 shows glutathione peroxidase activity in both native and fibrillar forms.

Ure2p is the precursor protein of the Saccharomyces cerevisiae prion [URE3]. Ure2p shows homology to glutathione transferases but lacks typical glutathione transferase activity. A recent study found that deletion of the Ure2 gene causes increased sensitivity to heavy metal ions and oxidants, whereas prion strains show normal sensitivity. To demonstrate that protection against oxidant toxicity is an inherent property of native and prion Ure2p requires biochemical characterization of the purified protein. Here we use steady-state kinetic methods to characterize the multisubstrate peroxidase activity of Ure2p using GSH with cumene hydroperoxide, hydrogen peroxide, or tert-butyl hydroperoxide as substrates. Glutathione-dependent peroxidase activity was proportional to the Ure2p concentration and showed optima at pH 8 and 40 °C. Michaelis-Menten behavior with convergent straight lines in double reciprocal plots was observed. This excludes a ping-pong mechanism and implies either a rapid-equilibrium random or a steady-state ordered sequential mechanism for Ure2p, consistent with its classification as a glutathione transferase. The mutant 90Ure2, which lacks the unstructured N-terminal prion domain, showed kinetic parameters identical to wild type. Fibrillar aggregates showed the same level of activity as native protein. Demonstration of peroxidase activity for Ure2 represents important progress in elucidation of its role in vivo. Further, establishment of an in vitro activity assay provides a valuable tool for the study of structure-function relationships of the Ure2 protein as both a prion and an enzyme.

Relationship between stability of folding intermediates and amyloid formation for the yeast prion Ure2p: a quantitative analysis of the effects of pH and buffer system.

The dimeric yeast protein Ure2 shows prion-like behaviour in vivo and forms amyloid fibrils in vitro. A dimeric intermediate is populated transiently during refolding and is apparently stabilized at lower pH, conditions suggested to favour Ure2 fibril formation. Here we present a quantitative analysis of the effect of pH on the thermodynamic stability of Ure2 in Tris and phosphate buffers over a 100-fold protein concentration range. We find that equilibrium denaturation is best described by a three-state model via a dimeric intermediate, even under conditions where the transition appears two-state by multiple structural probes. The free energy for complete unfolding and dissociation of Ure2 is up to 50 kcal mol(-1). Of this, at least 20 kcal mol(-1) is contributed by inter-subunit interactions. Hence the native dimer and dimeric intermediate are significantly more stable than either of their monomeric counterparts. The previously observed kinetic unfolding intermediate is suggested to represent the dissociated native-like monomer. The native state is stabilized with respect to the dimeric intermediate at higher pH and in Tris buffer, without significantly affecting the dissociation equilibrium. The effects of pH, buffer, protein concentration and temperature on the kinetics of amyloid formation were quantified by monitoring thioflavin T fluorescence. The lag time decreases with increasing protein concentration and fibril formation shows pseudo-first order kinetics, consistent with a nucleated assembly mechanism. In Tris buffer the lag time is increased, suggesting that stabilization of the native state disfavours amyloid nucleation.

CDK-Dependent Hsp70 Phosphorylation Controls G1 Cyclin Abundance and Cell-Cycle Progression

Summary In budding yeast, the essential functions of Hsp70 chaperones Ssa1–4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity.

Hsp40 Interacts Directly with the Native State of the Yeast Prion Protein Ure2 and Inhibits Formation of Amyloid-like Fibrils

Ure2 is the protein determinant of the [URE3] prion phenotype in Saccharomyces cerevisiae and consists of a flexible N-terminal prion-determining domain and a globular C-terminal glutathione transferase-like domain. Overexpression of the type I Hsp40 member Ydj1 in yeast cells has been found to result in the loss of [URE3]. However, the mechanism of prion curing by Ydj1 remains unclear. Here we tested the effect of overexpression of Hsp40 members Ydj1, Sis1, and Apj1 and also Hsp70 co-chaperones Cpr7, Cns1, Sti1, and Fes1 in vivo and found that only Ydj1 showed a strong curing effect on [URE3]. We also investigated the interaction of Ydj1 with Ure2 in vitro. We found that Ydj1 was able to suppress formation of amyloid-like fibrils of Ure2 by delaying the process of fibril formation, as monitored by thioflavin T binding and atomic force microscopy imaging. Controls using bovine serum albumin, Sis1, or the human Hsp40 homologues Hdj1 or Hdj2 showed no significant inhibitory effect. Ydj1 was only effective when added during the lag phase of fibril formation, suggesting that it interacts with Ure2 at an early stage in fibril formation and delays the nucleation process. Using surface plasmon resonance and size exclusion chromatography, we demonstrated a direct interaction between Ydj1 and both wild type and N-terminally truncated Ure2. In contrast, Hdj2, which did not suppress fibril formation, did not show this interaction. The results suggest that Ydj1 inhibits Ure2 fibril formation by binding to the native state of Ure2, thus delaying the onset of oligomerization.

Amyloid-like aggregates of neuronal tau induced by formaldehyde promote apoptosis of neuronal cells

BackgroundThe microtubule associated protein tau is the principle component of neurofibrillar tangles, which are a characteristic marker in the pathology of Alzheimers disease; similar lesions are also observed after chronic alcohol abuse. Formaldehyde is a common environmental contaminant and also a metabolite of methanol. Although many studies have been done on methanol and formaldehyde intoxication, none of these address the contribution of protein misfolding to the pathological mechanism, in particular the effect of formaldehyde on protein conformation and polymerization.ResultsWe found that unlike the typical globular protein BSA, the natively-unfolded structure of human neuronal tau was induced to misfold and aggregate in the presence of ~0.01% formaldehyde, leading to formation of amyloid-like deposits that appeared as densely staining granules by electron microscopy and atomic force microscopy, and bound the amyloid-specific dyes thioflavin T and Congo Red. The amyloid-like aggregates of tau were found to induce apoptosis in the neurotypic cell line SH-SY5Y and in rat hippocampal cells, as observed by Hoechst 33258 staining, assay of caspase-3 activity, and flow cytometry using Annexin V and Propidium Iodide staining. Further experiments showed that Congo Red specifically attenuated the caspase-3 activity induced by amyloid-like deposits of tau.ConclusionThe results suggest that low concentrations of formaldehyde can induce human tau protein to form neurotoxic aggregates, which could play a role in the induction of tauopathies.

Insights into the mechanism of prion propagation

Proteins with prion properties have been identified in both mammals and fungi. The tractability of yeast as a genetic model has contributed significantly to our understanding of prion formation and propagation. A number of molecular chaperones have been found to modulate the ability of yeast prion proteins to propagate. The results of recent genetic and in vitro studies have shed light on the mechanism of prion propagation, the physical and structural basis of different prion strains and the species barrier, as well as the function and mechanism of the chaperones that interact with the prion proteins. Whether aspects of the mechanisms of formation, maintenance and clearance of prions are conserved between fungi and mammals remains to be seen.