Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies

Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into β-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington''s disease (HD), Alzheimer''s disease (AD), Parkinson''s disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.     

Differential expression of molecular chaperones in brain of patients with Down syndrome

Heat shock proteins (HSPs) in their molecular capacity as chaperones have been reported to regulate the apoptotic pathway and also play a critical role in protein conformational diseases such as Alzheimer's disease (AD). As all Down syndrome (DS) brains display AD-like neuropathology, neuronal loss in DS was shown to be mediated by apoptosis. We decided to investigate the expression patterns of HSPs in seven brain regions of adults with DS using two-dimensional polyacrylamide gel electrophoresis (2-DE). Following 2-DE, approximately 120 protein spots were successfully identified by matrix-assisted laser desorption/ionization--mass spectrometry (MALDI-MS) followed by quantification of the identified proteins. We unambiguously identified and quantified nine different chaperone proteins. Accordingly, all but three chaperone proteins did exhibit a significant change in expression. HSP 70 RY, heat shock cognate (HSC) 71 and glucose-regulated protein (GRP) 75 showed a significant decrease (P < 0.05) in DS temporal cortex whereas HSP 70.1 and GRP 78 were significantly increased (P<0.05) in cerebellum. Whilst T-complex 1 (TCP-1) epsilon subunit showed a significant decrease (P< 0.05) in parietal cortex, a similar extent of increase (P<0.05) as that observed in cerebellum was obtained in parietal levels of GRP 78. Alpha-crystallin B, HSP 60 and GRP 94 did not show any detectable changes in expression patterns. This report presents the first approach to quantify nine different chaperones simultaneously at the protein level in different brain regions and provides evidence for aberrant chaperone expression patterns in DS. The relevance of this aberrant expression patterns are discussed in relation to the biochemical and neuropathological abnormalities in DS brain.     

Synthetic "chaperones": nanoparticle-mediated refolding of thermally denatured proteins

Thermally denatured chymotrypsin, lysozyme and papain are substantially refolded towards their native conformation by gold nanoparticle bearing dicarboxylate sidechains.     

Digital microfluidics and delivery of molecular payloads with magnetic porous silicon chaperones

Digital microfluidics involves the manipulation of molecules and materials in discrete packages. This paper reviews our work using amphiphilic magnetic microparticles constructed from porous silicon. An individual porous particle can be used to carry a nanomole or smaller quantities of a reagent, and assemblies of the particles can encapsulate and transport microliter droplets of liquid containing inorganic, organic, or biological molecules. The tracking and identification of each particle can be accomplished with spectral labels that are encoded into the particles during their synthesis. When used to chaperone liquid droplets, the labels can identify the separate droplets prior to mixing and also the combined droplets after mixing. Magnetic iron oxide nanoparticles encapsulated in the porous matrix allow the manipulation of the particles or whole droplet assemblies with a magnetic field, and they also allow heating of the particle's payload by means of an externally applied RF field. Examples of organic, inorganic, and biomolecular addition reactions, catalytic reactions, and thermolysis reactions are described.     

On the molecular mechanism of stabilization of proteins by cosolvents: role of Lifshitz electrodynamic forces

Several ionic and nonionic additives are known to affect structural stability of proteins in aqueous solutions. At a fundamental level, the mechanism of stabilization or destabilization of proteins by cosolvents must be related to three-body interactions between the protein, additive, and the water medium. In this study, the role of the Lifshitz-van der Waals electrodynamic interaction between various additives (sucrose, glycerol, urea, poly(ethylene glycol)-200, betaine, taurine, proline, and valine) and bovine serum albumin (BSA) in water medium was examined. The electrodynamic interaction energy was attractive for all of the additives studied here when both far ultraviolet and infrared relaxations of the additives were included in their dielectric susceptibility representations. However, when only the infrared contribution was included for structure stabilizers and both far ultraviolet and infrared contributions for the structure destabilizers, the resulting electrodynamic interaction energy (E/kT) followed the structure stabilizing and/or destabilizing behavior of the additives; that is, the interaction was attractive for urea and PEG200 (structure destabilizers), whereas it was repulsive for sucrose, glycerol, betaine, taurine, alanine, valine, and proline (structure stabilizers). The electrodynamic interaction energy E/kT at any given surface-to-surface separation distance between the additives and BSA was positively correlated (r(2) = 0.92) with the experimental thermal denaturation temperature (T(d)) of BSA in 1 M solutions of the additives. These analyses provided a mechanistic basis for the experimental observations of exclusion of the structure-stabilizing additives from the protein-water interface and binding of the structure-destabilizing additives to the protein surface. The role of water structure in the three-body electrodynamic interaction is discussed. It is hypothesized that in the case of additives that enhance water structure the hydration shells formed around the additives effectively dampen the contribution of ultraviolet frequencies to the dielectric susceptibility of the additives and thus impart repulsive electrodyanamic interaction between the additive and the protein, whereas the opposite occurs in the case of additives that breakdown the hydrogen-bonded structure of water.     

Controlled hierarchical self-assembly of networked coordination nanocapsules via the use of molecular chaperones

Supramolecular chaperones play an important role in directing the assembly of multiple protein subunits and redox-active metal ions into precise, complex and functional quaternary structures. Here we report that hydroxyl tailed C-alkylpyrogallol[4]arene ligands and redox-active Mn^II ions, with the assistance of proline chaperone molecules, can assemble into two-dimensional (2D) and/or three-dimensional (3D) networked nanocapsules. Dimensionality is controlled by coordination between the exterior of nanocapsule subunits, and endohedral functionalization within the 2D system is achieved via chaperone guest encapsulation. The tailoring of surface properties of nanocapsules via coordination chemistry is also shown as an effective method for the fine-tuning magnetic properties, and electrochemical and spectroscopic studies support that the nanocapsule is an effective homogeneous water-oxidation electrocatalyst, operating at pH 6.07 with an exceptionally low overpotential of 368 mV.

Molecular chaperones play a critical role in directing the assembly of nanocapsules that assemble into 2D or 3D coordination networks.     

Surface-supported bilayers with transmembrane proteins: role of the polymer cushion revisited

Protein lateral mobility in surface-supported bilayers is often much lower than the mobility of the lipids. In the present study we explore whether the incorporation of a PEG cushion between the bilayer and the substrate increases the lateral mobility of transmembrane proteins in bilayers produced via directed assembly, a method based on Langmuir-Blodgett deposition techniques. In our experiments, the PEG cushions were incorporated by adding PEG lipids to the protein/lipid monolayer at the air/water interface, at the first step of bilayer assembly. The protein and lipid mobilities in 160 different bilayers, with various PEG molecular weights and PEG lipid concentrations, were measured and compared. We found that the measured diffusion coefficients do not depend on the PEG molecular weight or the PEG lipid concentration and are very similar to the values measured in the absence of PEG. Therefore, contrary to our expectations, we found that a PEG cushion does not necessarily increase protein mobility, suggesting that the low protein mobility is not a consequence of protein-substrate interactions. Furthermore, we showed that the low protein mobility is not due to protein aggregation. The major determinant of protein mobility in surface-supported bilayer systems appears to be the method of bilayer assembly. While proteins were always mobile if the bilayers were prepared using the directed assembly method, in the presence and absence of a PEG cushion, other bilayer assembly protocols resulted in complete lack of protein mobility.     

Simulation of the opening and closing of Hsp70 chaperones by coarse-grained molecular dynamics

Heat-shock proteins 70 (Hsp70s) are key molecular chaperones which assist in the folding and refolding/disaggregation of proteins. Hsp70s, which consist of a nucleotide-binding domain (NBD, consisting of NBD-I and NBD-II subdomains) and a substrate-binding domain [SBD, further split into the β-sheet (SBD-β) and α-helical (SBD-α) subdomains], occur in two major conformations having (a) a closed SBD, in which the SBD and NBD domains do not interact, (b) an open SBD, in which SBD-α interacts with NBD-I and SBD-β interacts with the top parts of NBD-I and NBD-II. In the SBD-closed conformation, SBD is bound to a substrate protein, with release occurring after transition to the open conformation. While the transition from the closed to the open conformation is triggered efficiently by binding of adenosine triphosphate (ATP) to the NBD, it also occurs, although less frequently, in the absence of ATP. The reverse transition occurs after ATP hydrolysis. Here, we report canonical and multiplexed replica exchange simulations of the conformational dynamics of Hsp70s using a coarse-grained molecular dynamics approach with the UNRES force field. The simulations were run in the following three modes: (i) with the two halves of the NBD unrestrained relative to each other, (ii) with the two halves of the NBD restrained in an "open" geometry as in the SBD-closed form of DnaK (2KHO), and (iii) the two halves of NBD restrained in a "closed" geometry as in known experimental structures of ATP-bound NBD forms of Hsp70. Open conformations, in which the SBD interacted strongly with the NBD, formed spontaneously during all simulations; the number of transitions was largest in simulations carried out with the "closed" NBD domain, and smallest in those carried out with the "open" NBD domain; this observation is in agreement with the experimentally-observed influence of ATP-binding on the transition of Hsp70's from the SBD-closed to the SBD-open form. Two kinds of open conformations were observed: one in which SBD-α interacts with NBD-I and SBD-β interacts with the top parts of NBD-I and NBD-II (as observed in the structures of nucleotide exchange factors), and another one in which this interaction pattern is swapped. A third type of motion, in which SBD-α binds to NBD without dissociating from SBD-β was also observed. It was found that the first stage of interdomain communication (approach of SBD-β, to NBD) is coupled with the rotation of the long axes of NBD-I and NBD-II towards each other. To the best of our knowledge, this is the first successful simulation of the full transition of an Hsp70 from the SBD-closed to the SBD-open conformation.     

Dealing with the unknown: Metabolomics and Metabolite Atlases

Metabolomics is the comprehensive profiling of the small molecule composition of a biological sample. Since metabolites are often the indirect products of gene expression, this approach is being used to provide new insights into a variety of biological systems (clinical, bioenergy, etc.). A grand challenge for metabolomics is the complexity of the data, which often include many experimental artifacts. This is compounded by the tremendous chemical diversity of metabolites. Identification of each uncharacterized metabolite is in many ways its own puzzle (compared with proteomics, which is based on predictable fragmentation patterns of polypeptides). Therefore, effective data reduction/prioritization strategies are critical for this rapidly developing field. Here we review liquid chromatography electrospray ionization mass spectrometry (LC/MS)-based metabolomics, methods for feature finding/prioritization, approaches for identifying unknown metabolites, and construction of method specific ‘Metabolite Atlases’.     

Cobalt complexes with tripodal ligands: implications for the design of drug chaperones

Extensive research is currently being conducted into metal complexes that can selectively deliver cytotoxins to hypoxic regions in tumours. The development of pharmacologically suitable agents requires an understanding of appropriate ligand-metal systems for chaperoning cytotoxins. In this study, cobalt complexes with tripodal tren (tris-(2-aminoethyl)amine) and tpa (tris-(2-pyridylmethyl)amine) ligands were prepared with ancillary hydroxamic acid, β-diketone and catechol ligands and several parameters, including: pK(a), reduction potential and cytotoxicity were investigated. Fluorescence studies demonstrated that only tpa complexes with β-diketones showed any reduction by ascorbate in situ and similarly, cellular cytotoxicity results demonstrated that ligation to cobalt masked the cytotoxicity of the ancillary groups in all complexes except the tpa diketone derivative [Co(naac)tpa](ClO(4))(2) (naac = 1-methyl-3-(2-naphthyl)propane-1,3-dione). Additionally, it was shown that the hydroxamic acid complexes could be isolated in both the hydroxamate and hydroximate form and the pK(a) values (5.3-8.5) reveal that the reversible protonation/deprotonation of the complexes occurs at physiologically relevant pHs. These results have clear implications for the future design of prodrugs using cobalt moieties as chaperones, providing a basis for the design of cobalt complexes that are both more readily reduced and more readily taken up by cells in hypoxic and acidic environments.     

Local control of molecular fragmentation: the role of orientation

Local control theory, where the instantaneous response of a system to an external field determines the control field, is employed for the purpose of inducing molecular fragmentation processes via infrared excitation. In particular, the effects of the orientational motion are investigated and compared with the idealized case of a frozen rotation. It is shown that the rotational degree of freedom is crucial for the applicability of the employed local control algorithm. The addition of an additional static electric field which induces a molecular preorientation offers an efficient way for the local control. In particular, with increasing static field strength, the fragmentation yield approaches unity so that the idealized rotationless case is recovered. Numerical results are presented for the NaI molecule.     

Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins fromE. coli

The high-level expression of recombinant gene products in the gramnegative bacteriumEscherichia coli often results in the misfolding of the protein of interest and its subsequent degradation by cellular proteases or its deposition into biologically inactive aggregates known as inclusion bodies. It has recently become clear that in vivo protein folding is an energy-dependent process mediated by two classes of folding modulators. Molecular chaperones, such as the DnaK-DnaJ-GrpE and GroEL-GroES systems, suppress off-pathway aggregation reactions and facilitate proper folding through ATP-coordinated cycles of binding and release of folding intermediates. On the other hand, folding catalysts (foldases) accelerate rate-limiting steps along the protein folding pathway such as thecis/trans isomerization of peptidyl-prolyl bonds and the formation and reshuffling of disulfide bridges. Manipulating the cytoplasmic folding environment by increasing the intracellular concentration of all or specific folding modulators, or by inactivating genes encoding these proteins, holds great promise in facilitating the production and purification of heterologous proteins. Purified folding modulators and artificial systems that mimic their mode of action have also proven useful in improving the in vitro refolding yields of chemically denatured polypeptides. This review examines the usefulness and limitations of molecular chaperones and folding catalysts in both in vivo and in vitro folding processes.     

Towards molecular imaging and treatment of disease with radionuclides: the role of inorganic chemistry

Molecular imaging and radiotherapy using radionuclides is a rapidly expanding field of medicine and medical research. This article highlights the development of the role of inorganic chemistry in designing and producing the radiopharmaceuticals on which this interdisciplinary science depends.     

Classical molecular electrostatics: recognition of ligands in proteins and the vibrational Stark effect

It is shown that classical electrostatics quantitatively describes both the binding of the diatomic ligands XO (X = C, N, O) to the heme group in myoglobin and the dependence of their vibrational frequencies upon an external field, the vibrational Stark effect. The key is a proper treatment of induced dipoles. The results suggest that ligand binding occurs via an "electrostatic bond", a generalization of the standard ionic bond to include induction, and, more generally, that classical electrostatics can replace quantum mechanics for a considerable simplification of some complex problems.     

Dealing with the Ambiguity of Glycan Substructure Search

The level of ambiguity in describing glycan structure has significantly increased with the upsurge of large-scale glycomics and glycoproteomics experiments. Consequently, an ontology-based model appears as an appropriate solution for navigating these data. However, navigation is not sufficient and the model should also enable advanced search and comparison. A new ontology with a tree logical structure is introduced to represent glycan structures irrespective of the precision of molecular details. The model heavily relies on the GlycoCT encoding of glycan structures. Its implementation in the GlySTreeM knowledge base was validated with GlyConnect data and benchmarked with the Glycowork library. GlySTreeM is shown to be fast, consistent, reliable and more flexible than existing solutions for matching parts of or whole glycan structures. The model is also well suited for painless future expansion.     

The role of molecular diffusion in the adhesion of elastomers

Butyl rubber (polyisobutylene-co-isoprene) mixed with polyisobutylene was crosslinked to yield elastomeric macromolecular networks containing dissolved linear macromolecules. Adhesion of these materials to themselves (self-adhesion) and to an inert substrate was investigated over a wide range of peel rates and test temperatures. Greatly enhanced self-adhesion was found when linear polyisobutylene molecules of high molecular weight were present, but the strength of adhesion to a rigid inert substrate was hardly affected. The enhancement of self-adhesion is attributed to interdiffusion of polyisobutylene molecules. It was greatest at intermediate peel rates and temperatures, becoming insignificant at extremely low rates, probably because the diffusing species can then migrate readily, and at high effective rates of peel when the polymer approaches the glassy state and the strength of adhesion is high in all cases. A transition to somewhat lower levels of adhesion at relatively high rates of peel is tentatively ascribed to the onset of molecular fracture in place of pullout. The presence of large amounts of low-molecular-weight polyisobutylene (M?_v = 50,000 g/mol) increased the level of self-adhesion and of adhesion to an inert substrate to a similar degree, over a broad range of peel rates. This effect is attributed primarily to enhanced viscous losses in the elastomeric layer during separation. Application of these results to crack and weld-line healing in glassy plastics is discussed.     

Glycosides, depression and suicidal behaviour: the role of glycoside-linked proteins

Nowadays depression and suicide are two of the most important worldwide public health problems. Although their specific molecular mechanisms are still largely unknown, glycosides can play a fundamental role in their pathogenesis. These molecules act presumably through the up-regulation of plasticity-related proteins: probably they can have a presynaptic facilitatory effect, through the activation of several intracellular signaling pathways that include molecules like protein kinase A, Rap-1, cAMP, cADPR and G proteins. These proteins take part in a myriad of brain functions such as cell survival and synaptic plasticity. In depressed suicide victims, it has been found that their activity is strongly decreased, primarily in hippocampus and prefrontal cortex. These studies suggest that glycosides can regulate neuroprotection through Rap-1 and other molecules, and may play a crucial role in the pathophysiology of depression and suicide.