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1.  Molecular chaperones and protein folding as therapeutic targets in Parkinson’s disease and other synucleinopathies 
Changes in protein metabolism are key to disease onset and progression in many neurodegenerative diseases. As a prime example, in Parkinson’s disease, folding, post-translational modification and recycling of the synaptic protein α-synuclein are clearly altered, leading to a progressive accumulation of pathogenic protein species and the formation of intracellular inclusion bodies. Altered protein folding is one of the first steps of an increasingly understood cascade in which α-synuclein forms complex oligomers and finally distinct protein aggregates, termed Lewy bodies and Lewy neurites. In neurons, an elaborated network of chaperone and co-chaperone proteins is instrumental in mediating protein folding and re-folding. In addition to their direct influence on client proteins, chaperones interact with protein degradation pathways such as the ubiquitin-proteasome-system or autophagy in order to ensure the effective removal of irreversibly misfolded and potentially pathogenic proteins. Because of the vital role of proper protein folding for protein homeostasis, a growing number of studies have evaluated the contribution of chaperone proteins to neurodegeneration. We herein review our current understanding of the involvement of chaperones, co-chaperones and chaperone-mediated autophagy in synucleinopathies with a focus on the Hsp90 and Hsp70 chaperone system. We discuss genetic and pathological studies in Parkinson’s disease as well as experimental studies in models of synucleinopathies that explore molecular chaperones and protein degradation pathways as a novel therapeutic target. To this end, we examine the capacity of chaperones to prevent or modulate neurodegeneration and summarize the current progress in models of Parkinson’s disease and related neurodegenerative disorders.
doi:10.1186/2051-5960-1-79
PMCID: PMC4046681  PMID: 24314025
Neurodegeneration; Parkinson’s disease; Alpha-synuclein; Molecular chaperone; Heat shock protein; Hsp70; Hsp90; Proteasome; Autophagy; Apoptosis
2.  Modulation of Heat Shock Transcription Factor 1 as a Therapeutic Target for Small Molecule Intervention in Neurodegenerative Disease 
PLoS Biology  2010;8(1):e1000291.
A yeast-based small molecule screen identifies a novel activator of human HSF1 and protein chaperone expression and which appears to alleviate the toxicity of protein misfolding diseases.
Neurodegenerative diseases such as Huntington disease are devastating disorders with no therapeutic approaches to ameliorate the underlying protein misfolding defect inherent to poly-glutamine (polyQ) proteins. Given the mounting evidence that elevated levels of protein chaperones suppress polyQ protein misfolding, the master regulator of protein chaperone gene transcription, HSF1, is an attractive target for small molecule intervention. We describe a humanized yeast-based high-throughput screen to identify small molecule activators of human HSF1. This screen is insensitive to previously characterized activators of the heat shock response that have undesirable proteotoxic activity or that inhibit Hsp90, the central chaperone for cellular signaling and proliferation. A molecule identified in this screen, HSF1A, is structurally distinct from other characterized small molecule human HSF1 activators, activates HSF1 in mammalian and fly cells, elevates protein chaperone expression, ameliorates protein misfolding and cell death in polyQ-expressing neuronal precursor cells and protects against cytotoxicity in a fly model of polyQ-mediated neurodegeneration. In addition, we show that HSF1A interacts with components of the TRiC/CCT complex, suggesting a potentially novel regulatory role for this complex in modulating HSF1 activity. These studies describe a novel approach for the identification of new classes of pharmacological interventions for protein misfolding that underlies devastating neurodegenerative disease.
Author Summary
The misfolding of proteins into a toxic state contributes to a variety of neurodegenerative diseases such as Huntington, Alzheimer, and Parkinson disease. Although no known cure exists for these afflictions, many studies have shown that increasing the levels of protein chaperones, proteins that assist in the correct folding of other proteins, can suppress the neurotoxicity of the misfolded proteins. As such, increasing the cellular concentration of protein chaperones might serve as a powerful therapeutic approach in treating protein misfolding diseases. Because the levels of protein chaperones in the cell are primarily controlled by the heat shock transcription factor 1 [HSF1], we have designed and implemented a pharmacological screen to identify small molecules that can promote human HSF1 activation and increase the expression of protein chaperones. Through these studies, we have identified HSF1A, a molecule capable of activating human HSF1, increasing the levels of protein chaperones and alleviating the toxicity of misfolded proteins in both cell culture as well as fruit fly models of neurodegenerative disease.
doi:10.1371/journal.pbio.1000291
PMCID: PMC2808216  PMID: 20098725
3.  Molecular Chaperones as Rational Drug Targets for Parkinson’s Disease Therapeutics 
Parkinson’s disease is a neurodegenerative movement disorder that is caused, in part, by the loss of dopaminergic neurons within the substantia nigra pars compacta of the basal ganglia. The presence of intracellular protein aggregates, known as Lewy bodies and Lewy neurites, within the surviving nigral neurons is the defining neuropathological feature of the disease. Accordingly, the identification of specific genes mutated in families with Parkinson’s disease and of genetic susceptibility variants for idiopathic Parkinson’s disease has implicated abnormalities in proteostasis, or the handling and elimination of misfolded proteins, in the pathogenesis of this neurodegenerative disorder. Protein folding and the refolding of misfolded proteins are regulated by a network of interactive molecules, known as the chaperone system, which is composed of molecular chaperones and co-chaperones. The chaperone system is intimately associated with the ubiquitin-proteasome system and the autophagy-lysosomal pathway which are responsible for elimination of misfolded proteins and protein quality control. In addition to their role in proteostasis, some chaperone molecules are involved in the regulation of cell death pathways. Here we review the role of the molecular chaperones Hsp70 and Hsp90, and the co-chaperones Hsp40, BAG family members such as BAG5, CHIP and Hip in modulating neuronal death with a focus on dopaminergic neurodegeneration in Parkinson’s disease. We also review current progress in preclinical studies aimed at targetting the chaperone system to prevent neurodegeneration. Finally, we discuss potential future chaperone-based therapeutics for the symptomatic treatment and possible disease modification of Parkinson’s disease.
PMCID: PMC3364514  PMID: 20942788
Bcl-2 associated athanogene (BAG) family; C-terminal Hsp70 interacting protein (CHIP); chaperones; co-chaperones; heat shock protein (Hsp); Hsp90 inhibitors; neurodegeneration; Parkinson’s disease
4.  Molecular Chaperones in Parkinson’s Disease – Present and Future 
Journal of Parkinson's disease  2011;1(4):299-320.
Parkinson’s disease, like many other neurodegenerative disorders, is characterized by the progressive accumulation of pathogenic protein species and the formation of intracellular inclusion bodies. The cascade by which the small synaptic protein α-synuclein misfolds to form distinctive protein aggregates, termed Lewy bodies and Lewy neurites, has been the subject of intensive research for more than a decade. Genetic and pathological studies in Parkinson’s disease patients as well as experimental studies in disease models have clearly established altered protein metabolism as a key element in the pathogenesis of Parkinson’s disease. Alterations in protein metabolism include misfolding and aggregation, post-translational modification and dysfunctional degradation of cytotoxic protein species.
Protein folding and re-folding are both mediated by a highly conserved network of molecules, called molecular chaperones and co-chaperones. In addition to the regulatory role in protein folding, molecular chaperone function is intimately associated with pathways of protein degradation, such as the ubiquitin-proteasome system and the autophagy-lysosomal pathway, to effectively remove irreversibly misfolded proteins. Because of the central role of molecular chaperones in maintaining protein homeostasis, we herein review our current knowledge on the involvement of molecular chaperones and co-chaperones in Parkinson’s disease. We further discuss the capacity of molecular chaperones to prevent or modulate neurodegeneration, an important concept for future neuroprotective strategies and summarize the current progress in preclinical studies in models of Parkinson’s disease and other neurodegenerative disorders. Finally we include a discussion on the future potential of using molecular chaperones as a disease modifying therapy.
PMCID: PMC3264060  PMID: 22279517
neurodegeneration; Parkinson’s disease; alpha-synuclein; Lewy body; molecular chaperone; proteasome; autophagy; lysosome; heat shock protein (Hsp); Hsp90 inhibitor
5.  Combining Comparative Proteomics and Molecular Genetics Uncovers Regulators of Synaptic and Axonal Stability and Degeneration In Vivo 
PLoS Genetics  2012;8(8):e1002936.
Degeneration of synaptic and axonal compartments of neurons is an early event contributing to the pathogenesis of many neurodegenerative diseases, but the underlying molecular mechanisms remain unclear. Here, we demonstrate the effectiveness of a novel “top-down” approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons. A series of comparative quantitative proteomic screens on synapse-enriched fractions isolated from the mouse brain following injury identified dynamic perturbations occurring within the proteome during both initiation and onset phases of degeneration. In silico analyses highlighted significant clustering of proteins contributing to functional pathways regulating synaptic transmission and neurite development. Molecular markers of degeneration were conserved in injury and disease, with comparable responses observed in synapse-enriched fractions isolated from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. An initial screen targeting thirteen degeneration-associated proteins using mutant Drosophila lines revealed six potential regulators of synaptic and axonal degeneration in vivo. Mutations in CALB2, ROCK2, DNAJC5/CSP, and HIBCH partially delayed injury-induced neurodegeneration. Conversely, mutations in DNAJC6 and ALDHA1 led to spontaneous degeneration of distal axons and synapses. A more detailed genetic analysis of DNAJC5/CSP mutants confirmed that loss of DNAJC5/CSP was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments. Our study has identified conserved molecular responses occurring within synapse-enriched fractions of the mouse brain during the early stages of neurodegeneration, focused on functional networks modulating synaptic transmission and incorporating molecular chaperones, cytoskeletal modifiers, and calcium-binding proteins. We propose that the proteins and functional pathways identified in the current study represent attractive targets for developing therapeutics aimed at modulating synaptic and axonal stability and neurodegeneration in vivo.
Author Summary
In diseases affecting the nervous system, such as Alzheimer's disease and motor neuron disease, the breakdown of synaptic connections between neurons is a critical early event, contributing to disease onset and progression. However, we still know very little about the molecular machinery present in synaptic and axonal compartments of neurons that regulate their stability and cause breakdown during neurodegeneration. In this study we examined the protein composition of healthy and degenerating synapse-enriched fractions isolated from the brains of mice in order to identify early molecular changes occurring during neurodegeneration. We identified a range of proteins and cellular pathways that were modulated in synapse-enriched fractions during the early phases of degeneration, many of which were already known to regulate synaptic function. Similar molecular alterations were found in synapse-enriched fractions prepared from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. Data from these proteomic studies were then used to design experiments in Drosophila, in which we found that at least six of the individual proteins modified in degenerating synapses from mice were capable of independently regulating neuronal stability and degeneration in vivo. Designing novel therapeutics to target these proteins and pathways may help to delay or prevent neurodegeneration across a range of diseases.
doi:10.1371/journal.pgen.1002936
PMCID: PMC3431337  PMID: 22952455
6.  Mechanisms of Altered Redox Regulation in Neurodegenerative Diseases—Focus on S-Glutathionylation 
Antioxidants & Redox Signaling  2012;16(6):543-566.
Abstract
Significance: Neurodegenerative diseases are characterized by progressive loss of neurons. A common feature is oxidative stress, which arises when reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) exceed amounts required for normal redox signaling. An imbalance in ROS/RNS alters functionality of cysteines and perturbs thiol–disulfide homeostasis. Many cysteine modifications may occur, but reversible protein mixed disulfides with glutathione (GSH) likely represents the common steady-state derivative due to cellular abundance of GSH and ready conversion of cysteine-sulfenic acid and S-nitrosocysteine precursors to S-glutathionylcysteine disulfides. Thus, S-glutathionylation acts in redox signal transduction and serves as a protective mechanism against irreversible cysteine oxidation. Reversal of protein-S-glutathionylation is catalyzed specifically by glutaredoxin which thereby plays a critical role in cellular regulation. This review highlights the role of oxidative modification of proteins, notably S-glutathionylation, and alterations in thiol homeostatic enzyme activities in neurodegenerative diseases, providing insights for therapeutic intervention. Recent Advances: Recent studies show that dysregulation of redox signaling and sulfhydryl homeostasis likely contributes to onset/progression of neurodegeneration. Oxidative stress alters the thiol–disulfide status of key proteins that regulate the balance between cell survival and cell death. Critical Issues: Much of the current information about redox modification of key enzymes and signaling intermediates has been gleaned from studies focused on oxidative stress situations other than the neurodegenerative diseases. Future Directions: The findings in other contexts are expected to apply to understanding neurodegenerative mechanisms. Identification of selectively glutathionylated proteins in a quantitative fashion will provide new insights about neuropathological consequences of this oxidative protein modification. Antioxid. Redox Signal. 16, 543–566.
I. Introduction
II. Neurodegenerative Diseases
A. Alzheimer 's disease
B. Parkinson's disease
C. Huntington's disease
D. Amyotrophic lateral sclerosis
E. Friedreich's ataxia
III. Production of Oxidants Within the Brain
A. Cytoplasmic sources of ROS
B. Mitochondrial sources of ROS
IV. Inflammation, Oxidative Stress, and Neurodegenerative Diseases
A. Inflammation and Parkinson's disease
B. Potential roles of glutaredoxin in inflammatory responses
V. Cellular Oxidant Defense and Sulfhydryl Homeostasis
A. Cellular functions of Grx
B. Glutaredoxin and neurodegeneration
C. Paradoxical pro-oxidant effects of therapy of Parkinson's disease
VI. Oxidative Stress and Apoptosis
A. Apoptosis signaling kinase 1 may be regulated directly or indirectly by Grx1, Trx1, and other effectors
1. Oxidation of negative and positive effectors of ASK1
B. Redox sensitivity of cytosolic proteins implicated in neuronal cell death
1. Glyceraldehyde-3-phosphate dehydrogenase
2. Tyrosine hydroxylase
3. p53
C. Apoptosis and modification of mitochondrial permeability pore proteins
1. Voltage-dependent anion channel
2. Adenosine nucleotide transporter
3. Redox sensitivity of calcium transporters
D. Oxidative modifications affecting the proteasome system, protein aggregation, and mitochondrial dynamics in neurodegeneration
VII. S-Glutathionylation and Plaque Formation
A. Actin
B. Tau
VIII. S-Glutathionylation of Proteins Involved with Mitochondrial Respiration
A. α-Ketoglutarate dehydrogenase
B. Mitochondrial NADP+-dependent isocitrate dehydrogenase
C. Complex 1
D. Complex 2
E. ATP synthase
F. Succinyl CoA transferase
IX. Potential Approaches to Therapy of the Neurodegenerative Diseases
X. Conclusions
doi:10.1089/ars.2011.4119
PMCID: PMC3270051  PMID: 22066468
7.  Molecular Chaperones and Co-Chaperones in Parkinson Disease 
Parkinson disease, a progressive neurodegenerative disorder, is caused by the pathological accumulation of proteins, including the ubiquitous presynaptic protein α-synuclein. Alterations in the metabolism of α-synuclein have clearly been linked to neurodegeneration, and early steps in the pathological sequence of this protein include the formation of oligomers, fibrils, and small aggregates. Targeting these early steps of oligomerization is one of the main therapeutic approaches in the quest to develop disease-modifying agents. Molecular chaperones, molecules that can mediate the proper folding and refolding of client proteins, are vital to cell function and survival and thus have been explored as potential therapeutic agents. Important to Parkinson disease, chaperones are capable of preventing α-synuclein misfolding, oligomerization, and aggregate formation as shown in vitro and in Parkinson disease animal models. Furthermore, chaperones and associated co-chaperones are closely linked to pathways of protein degradation, like the ubiquitin-proteasome system and autophagy, and are thus able to remove irreversibly misfolded proteins. In this review, we summarize the role of molecular chaperones in Parkinson disease models and discuss the importance of preserving protein homeostasis to prevent neurodegeneration. We also review the growing number of exciting studies that have targeted molecular chaperone function as a novel therapeutic approach.
doi:10.1177/1073858412441372
PMCID: PMC3904222  PMID: 22829394
neurodegeneration; Parkinson disease; αsynuclein; molecular chaperone; heat shock protein (Hsp); co-chaperone
8.  TRiC’s tricks inhibit huntingtin aggregation 
eLife  2013;2:e00710.
In Huntington’s disease, a mutated version of the huntingtin protein leads to cell death. Mutant huntingtin is known to aggregate, a process that can be inhibited by the eukaryotic chaperonin TRiC (TCP1-ring complex) in vitro and in vivo. A structural understanding of the genesis of aggregates and their modulation by cellular chaperones could facilitate the development of therapies but has been hindered by the heterogeneity of amyloid aggregates. Using cryo-electron microscopy (cryoEM) and single particle cryo-electron tomography (SPT) we characterize the growth of fibrillar aggregates of mutant huntingtin exon 1 containing an expanded polyglutamine tract with 51 residues (mhttQ51), and resolve 3-D structures of the chaperonin TRiC interacting with mhttQ51. We find that TRiC caps mhttQ51 fibril tips via the apical domains of its subunits, and also encapsulates smaller mhtt oligomers within its chamber. These two complementary mechanisms provide a structural description for TRiC’s inhibition of mhttQ51 aggregation in vitro.
DOI: http://dx.doi.org/10.7554/eLife.00710.001
eLife digest
Huntington’s disease is an inheritable neurodegenerative disorder that typically begins in mid-adulthood. It initially affects muscle coordination and progresses to include psychiatric symptoms and cognitive decline, leading to premature death. The disease is caused by a mutation in the huntingtin gene, which codes for the huntingtin protein, and all individuals who inherit a pathogenic form of the mutant gene will eventually develop the condition.
The huntingtin gene contains a series of repeats of the tri-nucleotide sequence CAG, which encodes for the amino acid glutamine. The number of repeats varies between individuals but if it exceeds 36, the huntingtin protein starts to form aggregates in the brain. Aggregation occurs when soluble protein precursors, known as oligomers, combine to form structures called fibrils, which in turn assemble into larger clusters. This phenomenon also occurs in several other tri-nucleotide diseases, each of which involves a mutated gene with an excess of tri-nucleotide repeats.
Inside cells, proteins called chaperones regulate the folding of other proteins and help to prevent aggregate formation. A chaperone protein known as TRiC, which interacts with approximately 10% of proteins in the cytosol, has been shown to inhibit the aggregation of mutant huntingtin proteins. However, it has not been possible to map the structural interactions between TRiC and huntingtin to date.
Now, Shahmoradian and Galaz-Montoya et al. have used cryo-electron tomography, combined with 3-D mapping and computer-aided reconstruction, to reveal the structure of a molecular complex consisting of TRiC and a pathogenic mutant huntingtin protein containing 51 CAG repeats. By imaging this system at different time points during the aggregation of mutant huntingtin, it was possible to characterize how the aggregates changed over time. They found that their shape differs in the presence and absence of TRiC, and that the chaperone interacts both with soluble huntingtin molecules—sequestering them so that they cannot join together—and with the tips of fibrils, preventing them from growing longer.
By providing the first direct demonstration of how TRiC inhibits the aggregation of mutant huntingtin, the results of Shahmoradian and Galaz-Montoya et al. could aid in the design of TRiC-based drugs to be used in the treatment of Huntington’s disease.
DOI: http://dx.doi.org/10.7554/eLife.00710.002
doi:10.7554/eLife.00710
PMCID: PMC3707056  PMID: 23853712
Cryo electron microscopy (cryoEM); Cryo electron tomography (cryoET); Single particle tomography (SPT); Huntingtin; TRiC chaperonin; Amyloid; None
9.  The Neuroendocrine Protein 7B2 Suppresses the Aggregation of Neurodegenerative Disease-related Proteins* 
The Journal of Biological Chemistry  2012;288(2):1114-1124.
Background: The neuroendocrine protein 7B2 blocks the aggregation of certain secreted proteins.
Results: 7B2 co-localizes with protein aggregates in Parkinson and Alzheimer disease brains; blocks the fibrillation of Aβ1–40, Aβ1–42, and α-synuclein; and blocks Aβ1–42-induced Neuro-2A cell death.
Conclusion: 7B2 inhibits the cytotoxicity of Aβ1–42 by modulation of oligomer formation.
Significance: 7B2 is a novel anti-aggregation secretory chaperone associated with neurodegenerative disease.
Neurodegenerative diseases such as Alzheimer (AD) and Parkinson (PD) are characterized by abnormal aggregation of misfolded β-sheet-rich proteins, including amyloid-β (Aβ)-derived peptides and tau in AD and α-synuclein in PD. Correct folding and assembly of these proteins are controlled by ubiquitously expressed molecular chaperones; however, our understanding of neuron-specific chaperones and their involvement in the pathogenesis of neurodegenerative diseases is limited. We here describe novel chaperone-like functions for the secretory protein 7B2, which is widely expressed in neuronal and endocrine tissues. In in vitro experiments, 7B2 efficiently prevented fibrillation and formation of Aβ1–42, Aβ1–40, and α-synuclein aggregates at a molar ratio of 1:10. In cell culture experiments, inclusion of recombinant 7B2, either in the medium of Neuro-2A cells or intracellularly via adenoviral 7B2 overexpression, blocked the neurocytotoxic effect of Aβ1–42 and significantly increased cell viability. Conversely, knockdown of 7B2 by RNAi increased Aβ1–42-induced cytotoxicity. In the brains of APP/PSEN1 mice, a model of AD amyloidosis, immunoreactive 7B2 co-localized with aggregation-prone proteins and their respective aggregates. Furthermore, in the hippocampus and substantia nigra of human AD- and PD-affected brains, 7B2 was highly co-localized with Aβ plaques and α-synuclein deposits, strongly suggesting physiological association. Our data provide insight into novel functions of 7B2 and establish this neural protein as an anti-aggregation chaperone associated with neurodegenerative disease.
doi:10.1074/jbc.M112.417071
PMCID: PMC3542996  PMID: 23172224
Aggregation; Amyloid; Chaperone Chaperonin; Neurodegeneration; Neurodegenerative Diseases; Protein Aggregation; 7B2; Alzheimer; Parkinson
10.  Accurate Prediction of DnaK-Peptide Binding via Homology Modelling and Experimental Data 
PLoS Computational Biology  2009;5(8):e1000475.
Molecular chaperones are essential elements of the protein quality control machinery that governs translocation and folding of nascent polypeptides, refolding and degradation of misfolded proteins, and activation of a wide range of client proteins. The prokaryotic heat-shock protein DnaK is the E. coli representative of the ubiquitous Hsp70 family, which specializes in the binding of exposed hydrophobic regions in unfolded polypeptides. Accurate prediction of DnaK binding sites in E. coli proteins is an essential prerequisite to understand the precise function of this chaperone and the properties of its substrate proteins. In order to map DnaK binding sites in protein sequences, we have developed an algorithm that combines sequence information from peptide binding experiments and structural parameters from homology modelling. We show that this combination significantly outperforms either single approach. The final predictor had a Matthews correlation coefficient (MCC) of 0.819 when assessed over the 144 tested peptide sequences to detect true positives and true negatives. To test the robustness of the learning set, we have conducted a simulated cross-validation, where we omit sequences from the learning sets and calculate the rate of repredicting them. This resulted in a surprisingly good MCC of 0.703. The algorithm was also able to perform equally well on a blind test set of binders and non-binders, of which there was no prior knowledge in the learning sets. The algorithm is freely available at http://limbo.vib.be.
Author Summary
Molecular chaperones are essential elements of the protein quality control machinery that governs translocation and folding of nascent polypeptides, refolding and degradation of misfolded proteins, and activation of a wide range of client proteins. This variety of functions results from the existence of multiple chaperones with different structures. Chaperones bind to exposed regions of proteins to fulfil their function. The chaperone must hereby recognise a certain signal sequence on the substrate protein. The nature of the sequence that is exposed will determine the types of chaperones that can interact with it, and in the end will also determine the fate of the substrate protein: refolding, translocation, degradation or activation. Knowledge of the binding sequence determinants of molecular chaperones will shed more light on the mechanism of how each chaperone contributes to the cellular protein quality control system.
In this study we have made an algorithm which accurately predicts binding sites for the well studied E. coli Hsp70 chaperone, DnaK, which is implicated in folding efficiency and prevention of aggregation. The ability to detect and design high-affinity DnaK binding sites enhances our understanding of chaperone-substrate recognition and opens great opportunities to enhance protein solubility using protein-DnaK binding motif fusions.
doi:10.1371/journal.pcbi.1000475
PMCID: PMC2717214  PMID: 19696878
11.  Hsp70 and Its Molecular Role in Nervous System Diseases 
Heat shock proteins (HSPs) are induced in response to many injuries including stroke, neurodegenerative disease, epilepsy, and trauma. The overexpression of one HSP in particular, Hsp70, serves a protective role in several different models of nervous system injury, but has also been linked to a deleterious role in some diseases. Hsp70 functions as a chaperone and protects neurons from protein aggregation and toxicity (Parkinson disease, Alzheimer disease, polyglutamine diseases, and amyotrophic lateral sclerosis), protects cells from apoptosis (Parkinson disease), is a stress marker (temporal lobe epilepsy), protects cells from inflammation (cerebral ischemic injury), has an adjuvant role in antigen presentation and is involved in the immune response in autoimmune disease (multiple sclerosis). The worldwide incidence of neurodegenerative diseases is high. As neurodegenerative diseases disproportionately affect older individuals, disease-related morbidity has increased along with the general increase in longevity. An understanding of the underlying mechanisms that lead to neurodegeneration is key to identifying methods of prevention and treatment. Investigators have observed protective effects of HSPs induced by preconditioning, overexpression, or drugs in a variety of models of brain disease. Experimental data suggest that manipulation of the cellular stress response may offer strategies to protect the brain during progression of neurodegenerative disease.
doi:10.1155/2011/618127
PMCID: PMC3049350  PMID: 21403864
12.  An interdomain sector mediating allostery in Hsp70 molecular chaperones 
The Hsp70 family of molecular chaperones provides a well defined and experimentally powerful model system for understanding allosteric coupling between different protein domains.New extensions to the statistical coupling analysis (SCA) method permit identification of a group of co-evolving amino-acid positions—a sector—in the Hsp70 that is associated with allosteric function.Literature-based and new experimental studies support the notion that the protein sector identified through SCA underlies the allosteric mechanism of Hsp70.This work extends the concept of protein sectors by showing that two non-homologous protein domains can share a single sector when the underlying biological function is defined by the coupled activity of the two domains.
Allostery is a biologically critical property by which distantly positioned functional surfaces on proteins functionally interact. This property remains difficult to elucidate at a mechanistic level (Smock and Gierasch, 2009) because long-range coupling within proteins arises from the cooperative action of groups of amino acids. As a case study, consider the Hsp70 molecular chaperones, a large and diverse family of two-domain allosteric proteins required for cellular viability in nearly every organism (Figure 1) (Mayer and Bukau, 2005). In the ADP-bound state, the two domains act independently, the C-terminal substrate-binding domain displays a stable configuration in which the so-called ‘lid' region is docked against the β-sandwich subdomain, and substrates bind with relatively high affinity (Figure 1A) (Moro et al, 2003; Swain et al, 2007; Bertelsen et al, 2009). Exchange of ADP for ATP in the N-terminal nucleotide-binding domain causes significant local and propagated conformational change, formation of an interface with the substrate-binding domain, opening of the lid subdomain, and a decrease in the binding affinity for substrates (Figure 1B) (Rist et al, 2006; Swain et al, 2007). Upon ATP hydrolysis by the nucleotide-binding domain, Hsp70 is returned to the ADP-bound configuration suitable for another round of substrate binding and release. This process of cyclical substrate binding and release underlies all biological functions of Hsp70 proteins.
What is the structural basis for the long-range functional coupling within Hsp70? When allostery is a conserved property of a protein family, one approach to this problem is to analyze the correlated evolution of amino acids in the family—the expected statistical signature of cooperative action of protein residues (Lockless and Ranganathan, 1999; Kass and Horovitz, 2002; Suel et al, 2003). Previous work using an implementation of this concept (the statistical coupling analysis or SCA) showed that proteins contain sparse networks of co-evolving amino acids termed ‘sectors' that link protein active sites with distinct functional surfaces through the protein core (Halabi et al, 2009). This architecture is consistent with known allosteric mechanisms in protein domains (Suel et al, 2003; Halabi et al, 2009).
However, the principle of co-evolution of protein residues need not be limited to the study of individual protein domains. Indeed, conserved allosteric coupling between two (or more) non-homologous domains implies the existence of shared sectors that span functional sites on different domains. Here, we test this concept by extending the SCA method to consider the allosteric mechanism acting between the two domains of the Hsp70 proteins. Hsp70-like proteins include not only the allosteric Hsp70s, but also the Hsp110s—homologs that contain both domains and are regarded as structural models for Hsp70s, but that do not exhibit allosteric coupling. In this study, we take advantage of the functional divergence between the Hsp70s and Hsp110s to reveal patterns of co-evolution between amino acids that are specifically associated with the allosteric mechanism.
To identify the allosteric sector in Hsp70, we used SCA to compute a weighted correlation matrix, C̃, that describes the co-evolution of every pair of amino-acids positions in a sequence alignment of 926 members of the Hsp70/110 family. We then applied a mathematical method known as singular value decomposition to simultaneously evaluate the pattern of divergence between sequences and the pattern of co-evolution between amino-acid positions. The basic idea is that if the pattern of sequence divergence is able to classify members of a protein family into distinct functional subgroups, then we can rigorously identify the group of co-evolving residues that correspond to the underlying mechanism. Figure 2A shows the principal axis of sequence variation in the Hsp70/110 family, showing a clear separation of the allosteric (Hsp70) and non-allosteric (Hsp110) members of this family. The corresponding axis of co-evolution between amino-acid positions reveals a subset of Hsp70/110 positions (∼20%, 115 residues out of 605 total) that underlie the divergence of Hsp70 and Hsp110 proteins (Figure 2B). These positions derive roughly equally from the nucleotide-binding domain (in blue, 56 positions) and the substrate-binding domain (in green, 59 positions) and are more conserved within the Hsp70 sub-family. These results define a protein sector that is predicted to underlie the allosteric mechanism of Hsp70.
What is the structural arrangement of the putative allosteric sector within the Hsp70 protein? Consistent with a function in allosteric coupling, the 115 sector residues form a physically contiguous network of atoms, linking the ATP-binding site on the nucleotide-binding domain to the substrate recognition site on the substrate-binding domain through the interdomain interface (Figure 2C). The physical connectivity is remarkable given that only ∼20% of overall Hsp70 residues is involved (Figure 2B). Thus, functionally coupled but non-homologous protein domains can share a single sector of co-evolving residues that connects their respective functional sites.
We compared the Hsp70 sector mapping with the large body of biochemical studies that have been carried out in this family. We find strong experimental support for the involvement of sector positions in the Hsp70 allosteric mechanism in several regions: (1) within the ATP-binding site, (2) at the interface linking the two domains, and (3) within the β-sandwich core of the substrate-binding domain. The sector analysis also makes predictions about the involvement of some previously untested residues; we show that mutations at two such sites in fact reduce the allosteric coupling within Hsp70 in vitro and fail to complement a DnaK knockout strain of E. coli in a stress-response assay. Taken together, we conclude that sector positions are associated with the allosteric mechanism of Hsp70.
This work also adds a new finding with regard to the concept of protein sectors. Previous work showed that multiple quasi-independent sectors, each of which contributes a different aspect of function, are possible within a single protein domain (Halabi et al, 2009). This work shows that a single sector can also span two different protein domains when biological function (here, nucleotide-dependent substrate binding) arises from their coupled action. This result emphasizes the point that sectors are units of functional selection and are not obviously related to traditional hierarchies of structural organization in proteins. An interesting possibility is that evolution of allostery between proteins might evolve through the joining of protein sectors, a conjecture that can be tested in future work.
Allosteric coupling between protein domains is fundamental to many cellular processes. For example, Hsp70 molecular chaperones use ATP binding by their actin-like N-terminal ATPase domain to control substrate interactions in their C-terminal substrate-binding domain, a reaction that is critical for protein folding in cells. Here, we generalize the statistical coupling analysis to simultaneously evaluate co-evolution between protein residues and functional divergence between sequences in protein sub-families. Applying this method in the Hsp70/110 protein family, we identify a sparse but structurally contiguous group of co-evolving residues called a ‘sector', which is an attribute of the allosteric Hsp70 sub-family that links the functional sites of the two domains across a specific interdomain interface. Mutagenesis of Escherichia coli DnaK supports the conclusion that this interdomain sector underlies the allosteric coupling in this protein family. The identification of the Hsp70 sector provides a basis for further experiments to understand the mechanism of allostery and introduces the idea that cooperativity between interacting proteins or protein domains can be mediated by shared sectors.
doi:10.1038/msb.2010.65
PMCID: PMC2964120  PMID: 20865007
allostery; chaperone; co-evolution; SCA; sector
13.  BiP Clustering Facilitates Protein Folding in the Endoplasmic Reticulum 
PLoS Computational Biology  2014;10(7):e1003675.
The chaperone BiP participates in several regulatory processes within the endoplasmic reticulum (ER): translocation, protein folding, and ER-associated degradation. To facilitate protein folding, a cooperative mechanism known as entropic pulling has been proposed to demonstrate the molecular-level understanding of how multiple BiP molecules bind to nascent and unfolded proteins. Recently, experimental evidence revealed the spatial heterogeneity of BiP within the nuclear and peripheral ER of S. cerevisiae (commonly referred to as ‘clusters’). Here, we developed a model to evaluate the potential advantages of accounting for multiple BiP molecules binding to peptides, while proposing that BiP's spatial heterogeneity may enhance protein folding and maturation. Scenarios were simulated to gauge the effectiveness of binding multiple chaperone molecules to peptides. Using two metrics: folding efficiency and chaperone cost, we determined that the single binding site model achieves a higher efficiency than models characterized by multiple binding sites, in the absence of cooperativity. Due to entropic pulling, however, multiple chaperones perform in concert to facilitate the resolubilization and ultimate yield of folded proteins. As a result of cooperativity, multiple binding site models used fewer BiP molecules and maintained a higher folding efficiency than the single binding site model. These insilico investigations reveal that clusters of BiP molecules bound to unfolded proteins may enhance folding efficiency through cooperative action via entropic pulling.
Author Summary
The misfolding of proteins carries important implications for diseases such as Alzheimer's, Parkinson's, cancer, and diabetes. Once misfolded, proteins tend to associate into aggregates that pose a toxic threat to the cell. Chaperones are proteins that rescue the cell from an accumulation of these maladjusted proteins through dissociation of toxic oligomers and proper (re)folding. The endoplasmic reticulum (ER) is an organelle that serves as the staging ground for the chaperone activities of protein transport, folding, and maturation in the early secretory pathway. We have developed a computational model to investigate potential mechanisms that enable multiple ER-resident molecules working in concert to effectively fold peptides and transport nascent proteins across the ER membrane. Although previous models focused on chaperone interactions with peptides, we have explored the influence of cooperativity among chaperone molecules to assist in protein folding and maturation. We found that chaperone cooperation led to a higher yield of folded molecules compared to when chaperones bound to peptides in a 1∶1 stoichiometry. We have concluded that the clustering or multiple binding of chaperones may facilitate protein folding in vivo.
doi:10.1371/journal.pcbi.1003675
PMCID: PMC4081015  PMID: 24991821
14.  Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens* 
The Journal of biological chemistry  2007;283(10):6330-6336.
Many diverse human diseases are associated with protein aggregation in ordered fibrillar structures called amyloid. Amyloid formation may mediate aberrant protein interactions that culminate in neurodegeneration in Alzheimer, Huntington, and Parkinson diseases and in prion encephalopathies. Studies of protein aggregation in the brain are hampered by limitations in imaging techniques and often require invasive methods that can only be performed postmortem. Here we describe transgenic mice in which aggregation-prone proteins that cause Huntington and Parkinson disease are expressed in the ocular lens. Expression of a mutant huntingtin fragment or α-synuclein in the lens leads to protein aggregation and cataract formation, which can be monitored in real time by noninvasive, highly sensitive optical techniques. Expression of a mutant huntingtin fragment in mice lacking the major lens chaperone, αB-crystallin, markedly accelerated the onset and severity of aggregation, demonstrating that the endogenous chaperone activity of αB-crystallin suppresses aggregation in vivo. These novel mouse models will facilitate the characterization of protein aggregation in vivo and are being used in efficient and economical screens for chemical and genetic modifiers of disease-relevant protein aggregation.
doi:10.1074/jbc.M709678200
PMCID: PMC2650484  PMID: 18167346
15.  Identification of common genetic modifiers of neurodegenerative diseases from an integrative analysis of diverse genetic screens in model organisms 
BMC Genomics  2012;13:71.
Background
An array of experimental models have been developed in the small model organisms C. elegans, S. cerevisiae and D. melanogaster for the study of various neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and expanded polyglutamine diseases as exemplified by Huntington's disease (HD) and related ataxias. Genetic approaches to determine the nature of regulators of the disease phenotypes have ranged from small scale to essentially whole genome screens. The published data covers distinct models in all three organisms and one important question is the extent to which shared genetic factors can be uncovered that affect several or all disease models. Surprisingly it has appeared that there may be relatively little overlap and that many of the regulators may be organism or disease-specific. There is, however, a need for a fully integrated analysis of the available genetic data based on careful comparison of orthologues across the species to determine the real extent of overlap.
Results
We carried out an integrated analysis using C. elegans as the baseline model organism since this is the most widely studied in this context. Combination of data from 28 published studies using small to large scale screens in all three small model organisms gave a total of 950 identifications of genetic regulators. Of these 624 were separate genes with orthologues in C. elegans. In addition, 34 of these genes, which all had human orthologues, were found to overlap across studies. Of the common genetic regulators some such as chaperones, ubiquitin-related enzymes (including the E3 ligase CHIP which directly links the two pathways) and histone deacetylases were involved in expected pathways whereas others such as the peroxisomal acyl CoA-oxidase suggest novel targets for neurodegenerative disease therapy
Conclusions
We identified a significant number of overlapping regulators of neurodegenerative disease models. Since the diseases have, as an underlying feature, protein aggregation phenotypes it was not surprising that some of the overlapping genes encode proteins involved in protein folding and protein degradation. Interestingly, however, some of the overlapping genes encode proteins that have not previously featured in targeted studies of neurodegeneration and this information will form a useful resource to be exploited in further studies of potential drug-targets.
doi:10.1186/1471-2164-13-71
PMCID: PMC3292922  PMID: 22333271
16.  Suppression of protein aggregation by chaperone modification of high molecular weight complexes 
Brain  2012;135(4):1180-1196.
Protein misfolding and aggregation are associated with many neurodegenerative diseases, including Huntington’s disease. The cellular machinery for maintaining proteostasis includes molecular chaperones that facilitate protein folding and reduce proteotoxicity. Increasing the protein folding capacity of cells through manipulation of DNAJ chaperones has been shown to suppress aggregation and ameliorate polyglutamine toxicity in cells and flies. However, to date these promising findings have not been translated to mammalian models of disease. To address this issue, we developed transgenic mice that over-express the neuronal chaperone HSJ1a (DNAJB2a) and crossed them with the R6/2 mouse model of Huntington’s disease. Over-expression of HSJ1a significantly reduced mutant huntingtin aggregation and enhanced solubility. Surprisingly, this was mediated through specific association with K63 ubiquitylated, detergent insoluble, higher order mutant huntingtin assemblies that decreased their ability to nucleate further aggregation. This was dependent on HSJ1a client binding ability, ubiquitin interaction and functional co-operation with HSP70. Importantly, these changes in mutant huntingtin solubility and aggregation led to improved neurological performance in R6/2 mice. These data reveal that prevention of further aggregation of detergent insoluble mutant huntingtin is an additional level of quality control for late stage chaperone-mediated neuroprotection. Furthermore, our findings represent an important proof of principle that DNAJ manipulation is a valid therapeutic approach for intervention in Huntington’s disease.
doi:10.1093/brain/aws022
PMCID: PMC3326252  PMID: 22396390
chaperones; Huntington’s disease; polyglutamine; aggregation; protein folding
17.  Therapeutic Approaches for Inhibition of Protein Aggregation in Huntington's Disease 
Experimental Neurobiology  2014;23(1):36-44.
Huntington's disease (HD) is a late-onset and progressive neurodegenerative disorder that is caused by aggregation of mutant huntingtin protein which contains expanded-polyglutamine. The molecular chaperones modulate the aggregation in early stage and known for the most potent protector of neurodegeneration in animal models of HD. Over the past decades, a number of studies have demonstrated molecular chaperones alleviate the pathogenic symptoms by polyQ-mediated toxicity. Moreover, chaperone-inducible drugs and anti-aggregation drugs have beneficial effects on symptoms of disease. Here, we focus on the function of molecular chaperone in animal models of HD, and review the recent therapeutic approaches to modulate expression and turn-over of molecular chaperone and to develop anti-aggregation drugs.
doi:10.5607/en.2014.23.1.36
PMCID: PMC3984955  PMID: 24737938
protein aggregation; Molecular chaperone; Huntington; anti-aggregation drug
18.  ATPase Subdomain IA Is a Mediator of Interdomain Allostery in Hsp70 Molecular Chaperones 
PLoS Computational Biology  2014;10(5):e1003624.
The versatile functions of the heat shock protein 70 (Hsp70) family of molecular chaperones rely on allosteric interactions between their nucleotide-binding and substrate-binding domains, NBD and SBD. Understanding the mechanism of interdomain allostery is essential to rational design of Hsp70 modulators. Yet, despite significant progress in recent years, how the two Hsp70 domains regulate each other's activity remains elusive. Covariance data from experiments and computations emerged in recent years as valuable sources of information towards gaining insights into the molecular events that mediate allostery. In the present study, conservation and covariance properties derived from both sequence and structural dynamics data are integrated with results from Perturbation Response Scanning and in vivo functional assays, so as to establish the dynamical basis of interdomain signal transduction in Hsp70s. Our study highlights the critical roles of SBD residues D481 and T417 in mediating the coupled motions of the two domains, as well as that of G506 in enabling the movements of the α-helical lid with respect to the β-sandwich. It also draws attention to the distinctive role of the NBD subdomains: Subdomain IA acts as a key mediator of signal transduction between the ATP- and substrate-binding sites, this function being achieved by a cascade of interactions predominantly involving conserved residues such as V139, D148, R167 and K155. Subdomain IIA, on the other hand, is distinguished by strong coevolutionary signals (with the SBD) exhibited by a series of residues (D211, E217, L219, T383) implicated in DnaJ recognition. The occurrence of coevolving residues at the DnaJ recognition region parallels the behavior recently observed at the nucleotide-exchange-factor recognition region of subdomain IIB. These findings suggest that Hsp70 tends to adapt to co-chaperone recognition and activity via coevolving residues, whereas interdomain allostery, critical to chaperoning, is robustly enabled by conserved interactions.
Author Summary
The Hsp70 family of molecular chaperones assists in protein folding, degradation, assembly/disassembly of some complexes, and intracellular trafficking. These activities in the cell are accomplished by coupled conformational switches/signals between their nucleotide-binding and substrate-binding domains (NBD and SBD), assisted by cognate co-chaperones. Despite significant progress in the field, the molecular basis of Hsp70 machinery and the key interactions that regulate interdomain communication are not fully understood. Using a combination of experimental and computational methods, including in vivo functional assays, sequence- and structure-based analyses and perturbation response scanning, we identified a network of conserved interactions in subdomain IA of the NBD, which plays a key (effector) role in propagating signals between the ATP-binding and substrate-binding sites. Subdomain IIA, on the other hand, appears to adapt to J-domain co-chaperone binding by virtue of a broadly distributed cluster of co-evolving residues on the recognition surface.
doi:10.1371/journal.pcbi.1003624
PMCID: PMC4022485  PMID: 24831085
19.  NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration 
Nature  2008;452(7189):887-891.
Neurodegeneration can be triggered by genetic or environmental factors. Although the precise cause is often unknown, many neurodegenerative diseases share common features such as protein aggregation and age dependence. Recent studies in Drosophila have uncovered protective effects of NAD synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) against activity-induced neurodegeneration and injury-induced axonal degeneration1,2. Here we show that NMNAT overexpression can also protect against spinocerebellar ataxia 1 (SCA1)-induced neurodegeneration, suggesting a general neuroprotective function of NMNAT. It protects against neurodegeneration partly through a proteasome-mediated pathway in a manner similar to heat-shock protein 70 (Hsp70). NMNAT displays chaperone function both in biochemical assays and cultured cells, and it shares significant structural similarity with known chaperones. Furthermore, it is upregulated in the brain upon overexpression of poly-glutamine expanded protein and recruited with the chaperone Hsp70 into protein aggregates. Our results implicate NMNAT as a stress-response protein that acts as a chaperone for neuronal maintenance and protection. Our studies provide an entry point for understanding how normal neurons maintain activity, and offer clues for the common mechanisms underlying different neurodegenerative conditions.
doi:10.1038/nature06721
PMCID: PMC3150538  PMID: 18344983
20.  Redox Proteomics in Selected Neurodegenerative Disorders: From Its Infancy to Future Applications 
Antioxidants & Redox Signaling  2012;17(11):1610-1655.
Abstract
Several studies demonstrated that oxidative damage is a characteristic feature of many neurodegenerative diseases. The accumulation of oxidatively modified proteins may disrupt cellular functions by affecting protein expression, protein turnover, cell signaling, and induction of apoptosis and necrosis, suggesting that protein oxidation could have both physiological and pathological significance. For nearly two decades, our laboratory focused particular attention on studying oxidative damage of proteins and how their chemical modifications induced by reactive oxygen species/reactive nitrogen species correlate with pathology, biochemical alterations, and clinical presentations of Alzheimer's disease. This comprehensive article outlines basic knowledge of oxidative modification of proteins and lipids, followed by the principles of redox proteomics analysis, which also involve recent advances of mass spectrometry technology, and its application to selected age-related neurodegenerative diseases. Redox proteomics results obtained in different diseases and animal models thereof may provide new insights into the main mechanisms involved in the pathogenesis and progression of oxidative-stress-related neurodegenerative disorders. Redox proteomics can be considered a multifaceted approach that has the potential to provide insights into the molecular mechanisms of a disease, to find disease markers, as well as to identify potential targets for drug therapy. Considering the importance of a better understanding of the cause/effect of protein dysfunction in the pathogenesis and progression of neurodegenerative disorders, this article provides an overview of the intrinsic power of the redox proteomics approach together with the most significant results obtained by our laboratory and others during almost 10 years of research on neurodegenerative disorders since we initiated the field of redox proteomics. Antioxid. Redox Signal. 17, 1610–1655.
I. Introduction
II. Protein (/Lipid) Oxidation and Protein Dysfunction
A. Protein carbonyls
B. Protein nitration
1. Peroxynitrite (ONOO−)
2. Nitrogen dioxide (NO2)
C. HNE adduction to proteins
D. Importance of clearance and detoxification systems
1. The proteasome, parkin, ubiquitin carboxy-terminal hydrolase-L1, and HSPs
2. Superoxide dismutase
3. Catalase
4. Peroxiredoxins
5. Trx and Trx reductase
6. Glutathione reductase
7. Vitamins in neurodegeneration
8. Involvement of iron in neurodegeneration
E. Role of iron in neurodegeneration
1. Fe homeostasis in AD
2. Fe homeostasis in PD
3. Fe homeostasis in ALS
4. Fe homeostasis in HD
F. Some known consequences of protein oxidation
III. Overview of Redox Proteomics
A. Global, gel-based approaches
B. Targeted, gel-free approach
1. Enrichment of PCO modified proteins
2. Enrichment of HNE modified proteins
3. Enrichment of 3-NT modified proteins
IV. Application of Redox Proteomics to Selected Neurodegenerative Disorders
A. Alzheimer's disease
1. PCO in AD
2. Identification of carbonylated proteins in brain of subjects with AD
a. Sample: the brain
b. Energy dysfunction
c. Excitotoxicity
d. Proteosomal dysfunction
e. Neuritic abnormalities
f. APP regulation, tau hyperphosphorylation, and cell cycle regulation
g. Synaptic abnormalities and LTP
h. pH maintenance
i. Mitochondrial abnormalities
3. Carbonylated proteins in brain of subjects with amnestic MCI
4. EAD carbonylated proteins
5. PCAD vs. amnestic MCI protein carbonylation in brain
6. Protein-bound HNE in brain and progression of Alzheimer's disease
7. Protein-bound 3-NT in brain and progression of Alzheimer's disease
8. Nitrated brain proteins in MCI
9. Nitrated proteins in EAD
B. Parkinson disease
1. Redox proteomics in PD
C. Amyotrophic lateral sclerosis
1. Redox proteomics studies in ALS transgenic mice
D. Huntington disease
1. Redox proteomics-transgenic mouse model of HD
2. Proteomics of HD brain
E. Down syndrome
1. Redox proteomics in DS transgenic mice
V. Conclusions and Future Directions
doi:10.1089/ars.2011.4109
PMCID: PMC3448942  PMID: 22115501
21.  Chemical Chaperone and Inhibitor Discovery: Potential Treatments for Protein Conformational Diseases 
Protein misfolding and aggregation cause a large number of neurodegenerative diseases in humans due to (i) gain of function as observed in Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and Prion’s disease or (ii) loss of function as observed in cystic fibrosis and α1-antitrypsin deficiency. These misfolded proteins could either lead to the formation of harmful amyloids that become toxic for the cells or to be recognized and prematurely degraded by the protein quality control system. An increasing number of studies has indicated that some low-molecular-weight compounds named as chemical chaperones can reverse the mislocalization and/or aggregation of proteins associated with human conformational diseases. These small molecules are thought to non-selectively stabilize proteins and facilitate their folding. In this review, we summarize the probable mechanisms of protein conformational diseases in humans and the use of chemical chaperones and inhibitors as potential therapeutic agents against these diseases. Furthermore, recent advanced experimental and theoretical approaches underlying the detailed mechanisms of protein conformational changes and current structure-based drug designs towards protein conformational diseases are also discussed. It is believed that a better understanding of the mechanisms of conformational changes as well as the biological functions of these proteins will lead to the development and design of potential interfering compounds against amyloid formation associated with protein conformational diseases.
PMCID: PMC2754919  PMID: 19812735
misfolding; Alzheimer’s disease; Prion’s disease; Parkinson’s disease; Huntington’s disease; amyloid; chemical chaperone; molecular dynamics simulation; structure-based drug design; protein conformational disease
22.  Role of ubiquitin-proteasome-mediated proteolysis in nervous system disease 
Biochimica et biophysica acta  2010;1809(2):128-140.
Proteolysis by the ubiquitin-proteasome pathway (UPP) is now widely recognized as a molecular mechanism controlling myriad normal functions in the nervous system. Also, this pathway is intimately linked to many diseases and disorders of the brain. Among the diseases connected to the UPP are neurodegenerative disorders such as Alzheimer’s, Parkinson’s and Huntington’s diseases. Perturbation in the UPP is also believed to play a causative role in mental disorders such as Angelman syndrome. The pathology of neurodegenerative diseases is characterized by abnormal deposition of insoluble protein aggregates or inclusion bodies within neurons. The ubiquitinated protein aggregates are believed to result from dysfunction of the UPP or from structural changes in the protein substrates which prevent their recognition and degradation by the UPP. An early effect of abnormal UPP in diseases of the nervous system is likely to be impairment of synaptic function. Here we discuss the UPP and its physiological roles in the nervous system and how alterations in the UPP relate to development of nervous system diseases.
doi:10.1016/j.bbagrm.2010.07.006
PMCID: PMC2995838  PMID: 20674814
23.  Heat shock transcription factor 1 as a therapeutic target in neurodegenerative diseases 
Nature reviews. Drug discovery  2011;10(12):930-944.
Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and prion-based neurodegeneration are associated with the accumulation of misfolded proteins, resulting in neuronal dysfunction and cell death. However, current treatments for these diseases predominantly address disease symptoms, rather than the underlying protein misfolding and cell death, and are not able to halt or reverse the degenerative process. Studies in cell culture, fruitfly, worm and mouse models of protein misfolding-based neurodegenerative diseases indicate that enhancing the protein-folding capacity of cells, via elevated expression of chaperone proteins, has therapeutic potential. Here, we review advances in strategies to harness the power of the natural cellular protein-folding machinery through pharmacological activation of heat shock transcription factor 1 — the master activator of chaperone protein gene expression — to treat neurodegenerative diseases.
doi:10.1038/nrd3453
PMCID: PMC3518299  PMID: 22129991
24.  Opportunities and Challenges for Molecular Chaperone Modulation to Treat Protein-Conformational Brain Diseases 
Neurotherapeutics  2013;10(3):416-428.
A common pathological hallmark of protein-conformational brain diseases is the formation of disease-specific protein aggregates. In Alzheimer’s disease, these are comprised of amyloid-β and Tau as opposed to α-synuclein in Parkinson’s disease and N-terminal fragments of mutant huntingtin in Huntington’s disease. Most aggregates also sequester molecular chaperones, a protein family that assists in the folding, refolding, stabilization, and processing of client proteins, including misfolded proteins in brain diseases. Molecular chaperone modulation has achieved remarkable therapeutic effects in some cellular and preclinical animal models of protein-conformational diseases. This has raised hope for chaperone-based strategies to combat these diseases. Here, we review briefly the functional diversity and medical significance of molecular chaperones, their therapeutic potential, and common and specific challenges towards clinical application.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-013-0186-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s13311-013-0186-5
PMCID: PMC3701765  PMID: 23536253
Molecular chaperones; Protein folding; Aggregation; Neurodegenerative diseases; Protein clearance
25.  Interaction of the Molecular Chaperone DNAJB6 with Growing Amyloid-beta 42 (Aβ42) Aggregates Leads to Sub-stoichiometric Inhibition of Amyloid Formation* 
The Journal of Biological Chemistry  2014;289(45):31066-31076.
Background: The origins of the inhibition of DNAJB6 against amyloid formation are unknown.
Results: DNAJB6 inhibits fibril formation of the Aβ42 peptide from Alzheimer disease at low sub-stoichiometric molar ratios through strong binding to aggregated species.
Conclusion: Such sequestration prevents the growth and the proliferation of the aggregates.
Significance: The efficacious action of the chaperone against amyloid formation involves interactions with multiple growing aggregates.
The human molecular chaperone protein DNAJB6 was recently found to inhibit the formation of amyloid fibrils from polyglutamine peptides associated with neurodegenerative disorders such as Huntington disease. We show in the present study that DNAJB6 also inhibits amyloid formation by an even more aggregation-prone peptide (the amyloid-beta peptide, Aβ42, implicated in Alzheimer disease) in a highly efficient manner. By monitoring fibril formation using Thioflavin T fluorescence and far-UV CD spectroscopy, we have found that the aggregation of Aβ42 is retarded by DNAJB6 in a concentration-dependent manner, extending to very low sub-stoichiometric molar ratios of chaperone to peptide. Quantitative kinetic analysis and immunochemistry studies suggest that the high inhibitory efficiency is due to the interactions of the chaperone with aggregated forms of Aβ42 rather than the monomeric form of the peptide. This interaction prevents the growth of such species to longer fibrils and inhibits the formation of new amyloid fibrils through both primary and secondary nucleation. A low dissociation rate of DNAJB6 from Aβ42 aggregates leads to its incorporation into growing fibrils and hence to its gradual depletion from solution with time. When DNAJB6 is eventually depleted, fibril proliferation takes place, but the inhibitory activity can be prolonged by introducing DNAJB6 at regular intervals during the aggregation reaction. These results reveal the highly efficacious mode of action of this molecular chaperone against protein aggregation, and demonstrate that the role of molecular chaperones can involve interactions with multiple aggregated species leading to the inhibition of both principal nucleation pathways through which aggregates are able to form.
doi:10.1074/jbc.M114.595124
PMCID: PMC4223311  PMID: 25217638
Alzheimer Disease; Amyloid-beta (Aβ); Chaperone DnaJ (DnaJ); Neurodegenerative Disease; Protein Aggregation; Hsp40; Aggregation Kinetics; Amyloid Fibril Formation; Inhibition Mechanism

Results 1-25 (1098469)