Recent structural and computational insights into conformational diseases

Protein aggregation correlates with the development of several deleterious human disorders such as Alzheimer's disease, Parkinson's disease, prion-associated transmissible spongiform encephalopathies and type II diabetes. The polypeptides involved in these disorders may be globular proteins with a defined 3D-structure or natively unfolded proteins in their soluble conformations. In either case, proteins associated with these pathogeneses all aggregate into amyloid fibrils sharing a common structure, in which beta-strands of polypeptide chains are perpendicular to the fibril axis. Because of the prominence of amyloid deposits in many of these diseases, much effort has gone into elucidating the structural basis of protein aggregation. A number of recent experimental and theoretical studies have significantly increased our understanding of the process. On the one hand, solid-state NMR, X-ray crystallography and single molecule methods have provided us with the first high-resolution 3D structures of amyloids, showing that they exhibit conformational plasticity and are able to adopt different stable tertiary folds. On the other hand, several computational approaches have identified regions prone to aggregation in disease-linked polypeptides, predicted the differential aggregation propensities of their genetic variants and simulated the early, crucial steps in protein self-assembly. This review summarizes these findings and their therapeutic relevance, as by uncovering specific structural or sequential targets they may provide us with a means to tackle the debilitating diseases linked to protein aggregation.     

Anti-aggregating antibodies,a new approach towards treatment of conformational diseases

More and more evidence shows that Alzheimer's and prion-related diseases belong to the family of conformational diseases characterized by protein self-association and tissue deposition as amyloid fibrils. Regardless of the nature of the protein constituent, all forms of amyloid are stable assemblies based on noncovalent interactions between subunits of crossed beta-sheet structure. Understanding the mechanism and molecular details of the pathological conformational conversion of amyloidogenic proteins may be of importance to the development of approaches towards prevention and treatment of such diseases. We previously found that monoclonal antibodies (mAbs) interact at strategic sites where protein unfolding is initiated, thereby stabilizing the protein and preventing further precipitation. Indeed, site-directed mAbs raised against the N-terminal region of Alzheimer's beta-peptide (A beta P) disaggregate A beta P fibrils, restore peptide solubility and prevent its neurotoxic effects. Similarly, selected mAbs raised against the human prion peptide 106-126 modulate conformational changes occurring in the prion peptide exposed to aggregating conditions, preventing its aggregation and related neurotoxicity on cultivated neural-like cells. All these data and related procedures bring more attention to the immunological concept in the treatment of conformational diseases, and the recent performance of such antibodies in transgenic mice, as a model for human diseases, suggests the development of vaccination approaches against such diseases.     

Neurodegenerative diseases caused by protein aggregation: a phenomenon at the borderline between molecular evolution and ageing.

A process of protein aggregation that causes intracellular or extracellular accumulation of insoluble protein deposits causes many important neurodegenerative diseases associated with the ageing. The recognition that protein aggregation plays a prominent role in pathogenesis of important pathologies such as Alzheimer's and Parkinson's diseases prompted the scientific community to focus on the molecular mechanism of protein aggregation. Many proteins with sophisticated functions can self-aggregate because their folding is complicate and abnormal intermolecular contacts can predominate over the normal intramolecular interactions. The review of biochemical functional and pathogenic implications attributed to alpha synuclein, A beta peptide, presenilin and apoE highlights for these proteins a common conformational plasticity and the capacity to adapt their secondary structure to surrounding solvent as well as to the contacted ligands. Their functions are not fully elucidated but there is an elevated number of metabolic pathways in which apparently they are involved as well as they generate functional contact with a remarkable number of other proteins. The mechanism by which alpha synuclein and A beta protein make fibrils is an example of conformational plasticity because both these polypeptides can visit a coil or helical structure, but otherwise they convert into a pathogenic beta sheet structure highly suitable for polymerisation and fibril formation. The emerging question in the puzzling pathogenic basis of these diseases is if protein aggregation associated with ageing has a role in molecular evolution of the species or if it just represents a calculated drawback.     

Neurodegenerative conformational disease and heat shock proteins

Many major neurodegenerative diseases are associated with proteins misfolding and aggregation, which are also called "neurodegenerative conformational disease". The interaction of gene mutation and environmental factors are probably primary events resulting in oligomer and aggregate formations of proteins. Moreover, the dysfunctions of protein control systems, i.e. the ubiquitin-proteasome system and autophagy-lysosomal system, also contribute to the neurodegenerative process. The present review mainly summarizes protein misfolding and aggregation in the development of neurodegenerative conformational disease and the underling mechanisms, as well as upregulation of heatshock proteins as a promising treatment method for this kind of disease.     

Protein conformational misfolding and amyloid formation: characteristics of a new class of disorders that include Alzheimer's and Prion diseases

The accumulation of proteinaceous deposits has been recognised to occur in several neurodegenerative conditions including Prion diseases, Alzheimer's disease, Parkinson's disease, and Huntington's disease. Over the last two decades interest in these conditions has increased markedly, fueled partially by an increasing prevalence of these diseases in the Western world. Evidence indicates that anomalous protein misfolding and aggregation, with an accompanying "toxic gain of function" is central to the neuropathogenesis of these diseases. An increased understanding of the similarities and differences in the production, aggregation and accumulation of the respective proteins involved in these diseases, and the associated mechanisms of neurodegeneration, should aid in the development of new therapeutic agents to treat this group of related disorders.     

Amyloid associated proteins in Alzheimer's and prion disease

Clustering of activated microglia in Abeta deposits is related to accumulation of amyloid associated factors and precedes the neurodegenerative changes in AD. Microglia-derived pro-inflammatory cytokines are suggested to be the driving force in AD pathology. Inflammation-related proteins, including complement factors, acute-phase proteins, pro-inflammatory cytokines, that normally are locally produced at low levels, are increasingly synthesized in Alzheimer's disease (AD) brain. Similar to AD, in prion diseases (Creutzfeldt-Jakob disease, Gerstmann-Str?ussler-Scheinker disease and experimentally scrapie infected mouse brain) amyloid associated factors and activated glial cells accumulate in amyloid deposits of conformational changed prion protein (PrPres). Biological properties of Abeta and prion (PrP) peptides, including their potential to activate microglia, relate to Abeta and PrP peptide fibrillogenic abilities that are influenced by certain amyloid associated factors. However, since small oligomers of amyloid forming peptides are more toxic to neurons than large fibrils, certain amyloid associated factors that enhance fibril formation, may sequester the potentially harmful Abeta and PrP peptides from the neuronal microenvironment. In this review the positive and negative actions of amyloid associated factors on amyloid peptide fibril formation and on the fibrillation state related activation of microglia will be discussed. Insight in these mechanisms will enable the design of specific therapies to prevent neurodegenerative diseases in which amyloid accumulation and glial activation are prominent early features.     

Aggregation of the neuroblastoma-associated mutant (S120G) of the human nucleoside diphosphate kinase-A/NM23-H1 into amyloid fibrils

The human nucleoside diphosphate (NDP) kinase A, product of the NME1 gene also named NM23-H1, is known as a metastasis suppressor protein. A naturally occurring variant, S120G, identified in neuroblastomas, possesses native three-dimensional structure and enzymatic activity but displays reduced conformational stability and a folding defect with the accumulation of a "molten globule" folding intermediate during refolding in vitro. As such intermediate has been postulated to be involved in amyloid formation, NDP kinase A may serve as a model protein for studying the relationship between folding intermediates and amyloid fibrils. The NDP kinase A S120G was heated in phosphate buffer (pH?7.0). The protein precipitated as amyloid fibrils, as demonstrated by electron microscopy, Congo red, and thioflavin T binding and FTIR spectroscopy. The NDP kinase A S120G, at neutral pH and at moderate temperature experiences a transition towards amyloid fibrils. The aggregation process was faster if seeded by preformed fibrils. The fibrils presented a large proteinase K-resistant core not including residue Gly 120, as shown by mass spectrometry. This suggests that the aggregation process is triggered by the reduced stability of the S120G variant and not by a specific increase in the kinase domain intrinsic aggregation propensity at the place of mutation. This constitutes one of the few reports on a protein involved in cancer biology able to aggregate into amyloid structures under mild conditions.     

Biophysical Aspects of Alzheimer’s Disease: Implications for Pharmaceutical Sciences

An increasing amount of findings suggests that the aggregation of soluble peptides and proteins into amyloid fibrils is a relevant upstream process in the complex cascade of events leading to the pathology of Alzheimer’s disease and several other neurodegenerative disorders. Nevertheless, several aspects of the correlation between the aggregation process and the onset and development of the pathology remain largely elusive. In this context, biophysical and biochemical studies in test tubes have proven extremely powerful in providing quantitative information about the structure and the reactivity of amyloids at the molecular level. In this review we use selected recent examples to illustrate the importance of such biophysical research to complement phenomenological studies based on cellular and molecular biology, and we discuss the implications for pharmaceutical applications associated with Alzheimer’s disease and other neurodegenerative disorders in both academic and industrial contexts.     

Controlling Protein Compartmentalization to Overcome Disease

Over the past decade, considerable progress has been made to improve our understanding of the intracellular transport of proteins. Mechanisms of nuclear import and export involving classical receptors have been studied. Signal sequences required for directing a protein molecule to a specific cellular compartment have been defined. Knowledge of subcellular trafficking of proteins has also increased our understanding of diseases caused due to mislocalization of proteins. A specific protein on deviating from its native cellular compartment may result in disease due to loss of its normal functioning and aberrant activity in the "wrong" compartment. Mislocalization of proteins results in diseases that range from metabolic disorders to cancer. In this review we discuss some of the diseases caused due to mislocalization. We further focus on application of nucleocytoplasmic transport to drug delivery. Various rationales to treat diseases by exploiting intracellular transport machinery have been proposed. Although the pathways for intracellular movement of proteins have been defined, these have not been adequately utilized for management of diseases involving mislocalized proteins. This review stresses the need for designing drug delivery systems utilizing these mechanisms as this area is least exploited but offers great potential.     

Production of reactive oxygen species from aggregating proteins implicated in Alzheimer's disease, Parkinson's disease and other neurodegenerative diseases

The deposition of abnormal protein fibrils is a prominent pathological feature of many different 'protein conformational' diseases, including some important neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), motor neurone disease and the 'prion' dementias. Some of the fibril-forming proteins or peptides associated with these diseases have been shown to be toxic to cells in culture. A clear understanding of the molecular mechanisms responsible for this toxicity should shed light on the probable link between protein deposition and cell loss in these diseases. In the case of the beta-amyloid (Abeta), which accumulates in the brain in AD, there is good evidence that the toxic mechanism involves the production of reactive oxygen species (ROS). By means of an electron spin resonance (ESR) spin-trapping method, we have shown recently that solutions of Abeta liberate readily detectable amounts of hydroxyl radicals upon incubation in vitro followed by the addition of small amounts of Fe(II). We have also obtained similar results with alpha-synuclein, which accumulates in Lewy bodies in PD. Our data suggest that hydrogen peroxide accumulates during Abeta or alpha-synuclein incubation and that this is subsequently converted to hydroxyl radicals, on addition of Fe (II), by Fenton's reaction. Consequently, we now support the idea that one of the fundamental molecular mechanisms underlying the pathogenesis of cell death in AD, PD, and possibly some other protein conformational diseases, could be the direct production of ROS during formation of the abnormal protein aggregates. This hypothesis suggests a novel approach to the therapy of this group of diseases.     

Proteus in the World of Proteins: Conformational Changes in Protein Kinases

The 512 protein kinases encoded by the human genome are a prime example of nature's ability to create diversity by introducing variations to a highly conserved theme. The activity of each kinase domain is controlled by layers of regulatory mechanisms involving different combinations of post‐translational modifications, intramolecular contacts, and intermolecular interactions. Ultimately, they all achieve their effect by favoring particular conformations that promote or prevent the kinase domain from catalyzing protein phosphorylation. The central role of kinases in various diseases has encouraged extensive investigations of their biological function and three‐dimensional structures, yielding a more detailed understanding of the mechanisms that regulate protein kinase activity by conformational changes. In the present review, we discuss these regulatory mechanisms and show how conformational changes can be exploited for the design of specific inhibitors that lock protein kinases in inactive conformations. In addition, we highlight recent developments to monitor ligand‐induced structural changes in protein kinases and for screening and identifying inhibitors that stabilize enzymatically incompetent kinase conformations.     

Structural and molecular basis of carbohydrate-protein interaction systems as potential therapeutic targets

The sugar chains covalently modifying proteins and lipids are recognized by a variety of proteins, thereby mediating a broad range of physiological and pathological events on cell surfaces as well as in cells. Hence, these carbohydrate-protein interaction systems could be potential therapeutic targets for various diseases, including viral infections, autoimmune diseases and neurodegenerative disorders. Cumulative crystallographic data of lectins complexed with their cognate carbohydrate ligands have elucidated the sugar recognition modes of these proteins, offering a structural basis for the design of drugs targeting carbohydrate-lectin interaction systems. In particular, structural and functional studies of animal L-type lectins, which possess a carbohydrate recognition domain with a structural resemblance to that of leguminous lectins such as concanavalin A, have demonstrated the molecular mechanisms underlying their distinct roles in sorting and trafficking of glycoproteins in cells, exemplifying the structure-based engineering that manipulates the sugar-binding properties of lectins. Furthermore, structural basis has been provided for the functional interplay between the L-type lectin ERGIC-53 and the EF-hand Ca2?-binding protein MCFD2 in the intracellular transport of the coagulation factors V and VIII. This article also deals with pathological carbohydrate-protein interactions involving ganglioside clusters on cell surfaces, particularly focusing on the interaction between amyloid β (Aβ) and GM1 ganglioside. This interaction triggers conformational transition and consequent aggregation of Aβ, and therefore, is considered to be a key step in Alzheimer's disease. The recently reported structural information of the Aβ-GM1 interaction is presented, underscoring the significance of assemblages of glycoconjugates as therapeutic targets.     

Nanomedicine and protein misfolding diseases

Misfolding and self assembly of proteins in nano-aggregates of different sizes and morphologies (nano-ensembles, primarily nanofilaments and nano-rings) is a complex phenomenon that can be facilitated, impeded, or prevented, by interactions with various intracellular metabolites, intracellular nanomachines controlling protein folding and interactions with other proteins. A fundamental understanding of molecular processes leading to misfolding and self-aggregation of proteins involved in various neurodegenerative diseases will provide critical information to help identify appropriate therapeutic routes to control these processes. An elevated propensity of misfolded protein conformation in solution to aggregate with the formation of various morphologies impedes the use of traditional physical chemical approaches for studies of misfolded conformations of proteins. In our recent alternative approach, the protein molecules were tethered to surfaces to prevent aggregation and AFM force spectroscopy was used to probe the interaction between protein molecules depending on their conformations. It was shown that formation of filamentous aggregates is facilitated at pH values corresponding to the maximum of rupture forces. In this paper, a novel surface chemistry was developed for anchoring of amyloid beta (Abeta) peptides at their N-terminal moieties. The use of the site specific immobilization procedure allowed to measure the rupture of Abeta-Abeta contacts at single molecule level. The rupture of these contacts is accompanied by the extension of the peptide chain detected by a characteristic elasto-mechanical component of the force-distance curves. Potential applications of the nanomechanical studies to understanding the mechanisms of development of protein misfolding diseases are discussed.     

Amyloid disease prevention by transthyretin native state complexation with carborane derivatives lacking cyclooxygenase inhibition

Misfolding and subsequent aggregation of any of a number of proteins leads to the accumulation of amyloid fibrils, which have been associated with a variety of diseases. One such amyloidogenic protein is transthyretin (TTR), a 55-kDa homotetrameric protein found in the blood plasma and cerebrospinal fluid where it binds and transports thyroxine. In humans, the T119M-TTR variant has been shown to be protective against familial amyloid polyneuropathy, a TTR amyloid disease, through kinetic stabilization of the unliganded tetrameric structure. Studies have indicated that a diverse range of small molecules may also bind TTR in the thyroxine-binding pocket and subsequently kinetically stabilize the protein's native conformation in vitro, preventing the misfolding that has been implicated in the progression of several diseases. However, cyclooxygenase inhibition is a common unwanted side effect among such small-molecule kinetic stabilizers. The recent development of transthyretin stabilizers not subject to cyclooxygenase inhibition may prove attractive for the long-term treatment of TTR misfolding diseases in humans. Such compounds are attained by incorporating aromatic carborane icosahedra at strategic points in their structures.     

Transgenic animal models of neurodegenerative diseases and their application to treatment development

Neurodegenerative disorders of the aging population affect over 5 million people in the US and Europe alone. The common feature is the progressive accumulation of misfolded proteins with the formation of toxic oligomers. Previous studies show that while in Alzheimer's disease (AD) misfolded amyloid-beta protein accumulates both in the intracellular and extracellular space, in Lewy body disease (LBD), Parkinson's disease (PD), Multiple System Atrophy (MSA), Fronto-Temporal dementia (FTD), prion diseases, amyotrophic lateral sclerosis (ALS) and trinucleotide repeat disorders (TNRD), the aggregated proteins accumulate in the plasma membrane and intracellularly. Protein misfolding and accumulation is the result of an altered balance between protein synthesis, aggregation rate and clearance. Based on these studies, considerable advances have been made in the past years in developing novel experimental models of neurodegenerative disorders. This has been in part driven by the identification of genetic mutations associated with familial forms of these conditions and gene polymorphisms associated with the more common sporadic variants of these diseases. Transgenic and knock out rodents and Drosophila as well as viral vector driven models of Alzheimer's disease (AD), PD, Huntington's disease (HD) and others have been developed, however the focus for this review will be on rodent models of AD, FTD, PD/LBD, and MSA. Promising therapeutic results have been obtained utilizing amyloid precursor protein (APP) transgenic (tg) models of AD to develop therapies including use of inhibitors of the APP-processing enzymes beta- and gamma-secretase as well as vaccine therapies.     

The role of eosinophils in non-parasitic infections

Eosinophils are a subset of leukocytes traditionally associated with Th2-related diseases and helminth infections. However, accumulating evidence suggests eosinophils play a more prominent role in the immune response to bacterial and viral pathogens than previously realized. Specifically, eosinophils possess antimicrobial properties against a broad range of pathogens, and release specific and secondary granules as a result of pathogen recognition. Pathogen recognition is accomplished through expression of Toll-like receptors, as well as other surface and intracellular receptors expressed by the eosinophil. Interestingly, specific killing mechanisms employed by each granule protein differ based on pathogen recognition, but ultimately release of eosinophil granules leads to direct killing of many different pathogens. The precise mechanisms of killing by granule proteins and the circumstances in which specific proteins are secreted are only now being determined. Future efforts to understand these mechanisms may lead toward clinical use of granule proteins as antimicrobial agents in humans, in addition to revealing implications regarding the use of eosinophil-depleting therapies for allergic disorders. This review will summarize the literature to date regarding the role of eosinophils in non-parasitic infections.     

Inhibition of protein misfolding and aggregation by small rationally-designed peptides

Several human diseases are associated with the presence of toxic fibrillar protein deposits. These diseases called protein misfolding disorders, are characterized by the accumulation of misfolded protein aggregates in diverse tissues. Strong evidence indicates that the conversion of a normal soluble protein into a beta-sheet-rich oligomeric structure and further fibrillar aggregation are the key events in the disease pathogenesis. Therefore, a promising therapeutic target consists of the prevention and dissolution of misfolded protein aggregates. Peptides designed to specifically bind to the pathogenic protein and block and/or reverse its abnormal conformational change constitute a new class of drugs. This article reviews this approach, describing diverse compounds reported to have this activity.     

Therapeutic approaches against common structural features of toxic oligomers shared by multiple amyloidogenic proteins

Impaired proteostasis is one of the main features of all amyloid diseases, which are associated with the formation of insoluble aggregates from amyloidogenic proteins. The aggregation process can be caused by overproduction or poor clearance of these proteins. However, numerous reports suggest that amyloid oligomers are the most toxic species, rather than insoluble fibrillar material, in Alzheimer's, Parkinson's, and Prion diseases, among others. Although the exact protein that aggregates varies between amyloid disorders, they all share common structural features that can be used as therapeutic targets. In this review, we focus on therapeutic approaches against shared features of toxic oligomeric structures and future directions.     

Spots, blots, peaks and chips: proteomic approaches in autoimmune diseases

During the past five years, investigations employing a variety of proteomic technologies have yielded a wealth of information on a number of autoimmune disorders. Animal models of autoimmune disease have been examined and have provided clues that can be useful in elucidating molecular pathways and mechanisms that play a role in autoimmune disorders. Human sera and body fluids have been analyzed and have resulted in the identification of autoantibodies that can be used as diagnostic markers in specific autoimmune diseases, and proteomic fingerprints of tissues and body fluids have resulted in the identification of individual proteins or patterns of protein expression that are deregulated in autoimmune diseases. The information provided by these proteomic studies are of diagnostic and therapeutic potential. This review provides an overview of the approaches used in the proteomic analyses of autoimmune disease.