Folding Diseases Research Articles

Modulation of the maladaptive stress response to manage diseases of protein folding.

Author SummaryThe function of all proteins is dependent on achieving the correct folded state, a process referred to as protein homeostasis or proteostasis. Cellular proteostasis is maintained by diverse signaling pathways, including the heat shock response (HSR), which protects proteins in the face of acute stress. However, genetic disorders are a challenge to cells, since the mutated protein will often fail to fold properly and function correctly. We have discovered that the chronic expression of such disease-causing proteins can trigger the sustained activation of the HSR in a failed attempt to correct the associated misfolding defect. Such chronic HSR activation presents an unanticipated challenge to the cell by initiating a sustained state of stress management, which leads to a general protein-folding deficiency. This in turn further exacerbates the disease phenotype—a condition we have termed maladaptive. We show that down-regulation of this maladaptive stress response (MSR) restores cellular protein folding and improves the disease condition in loss-of-function disorders such as cystic fibrosis, Niemann-Pick disease and alpha-1-antitrypsin deficiency, as well as gain-of-toxic-function diseases such as Alzheimer's disease. MSR management therefore potentially represents an important therapeutic first step in regulating the progression of human disease associated with chronic protein misfolding.

Living in constant crisis--when stress management becomes the problem.

Living in constant crisis--when stress management becomes the problem.

An Endoplasmic Reticulum Cyclophilin Cpr5p Rescues Z-type α1-Antitrypsin from Retarded Folding

Human α1-antitrypsin (α1-AT) is a natural inhibitor of neutrophil elastases and has several dozens of genetic variants. Most of the deficient genetic variants of human α1-AT are unstable and cause pulmonary emphysema. However, the most clinically significant variant, Z-type α1-AT, exhibits retarded protein folding that leads to accumulation of folding intermediates. These aggregate within the endoplasmic reticulum (ER) of hepatocytes, subsequently causing liver cirrhosis as well as emphysema. Here, we studied the role of an ER folding assistant protein Cpr5p on Z-type α1-AT folding. Cpr5p was induced > 2-fold in Z-type α1-AT-expressing yeast cells compared with the wild type. Knockout of CPR5 exacerbated cytotoxicity of Z-type α1-AT, and re-introduction of CPR5 rescued the knockout cells from aggravated cytotoxicity caused by the α1-AT variant. Furthermore, Cpr5p co-immunoprecipitated with Z-type α1-AT and facilitated its protein folding. Our results suggest that protein-folding diseases may be suppressed by folding assistant proteins at the site of causal protein biosynthesis.

Hereditary transthyretin amyloidosis

Hereditary amyloidosis is an autosomal dominant fatal multisystem disease caused by extracellular deposition of misfolded proteins and, therefore represents a hereditary protein folding or deposition disease that leads to progressive organ damage and eventually death. In most instances mutations within the transthyretin gene are the underlying cause. The main manifestation is a rapidly progressing axonal sensorimotor and autonomic polyneuropathy (familial amyloid polyneuropathy, FAP). Cardiac involvement is frequent in FAP and additional manifestations include the gastrointestinal tract and the eyes. A second manifestation type is cardiomyopathy with little or no polyneuropathy (familial amyloid cardiomyopathy, FAC). For therapy, orthotopic liver transplantation has been established for 25 years. Recently, the oral agent tafamidis, a transthyretin stabilizer, was licensed for treatment of stage 1 polyneuropathy. Additional treatment options are currently being studied.

A model in which heat shock protein 90 targets protein-folding clefts: rationale for a new approach to neuroprotective treatment of protein folding diseases.

In an EBM Minireview published in 2010, we proposed that the heat shock protein (Hsp)90/Hsp70-based chaperone machinery played a major role in determining the selection of proteins that have undergone oxidative or other toxic damage for ubiquitination and proteasomal degradation. The proposal was based on a model in which the Hsp90 chaperone machinery regulates signaling by modulating ligand-binding clefts. The model provides a framework for thinking about the development of neuroprotective therapies for protein-folding diseases like Alzheimer's disease (AD), Parkinson's disease (PD), and the polyglutamine expansion disorders, such as Huntington's disease (HD) and spinal and bulbar muscular atrophy (SBMA). Major aberrant proteins that misfold and accumulate in these diseases are "client" proteins of the abundant and ubiquitous stress chaperone Hsp90. These Hsp90 client proteins include tau (AD), α-synuclein (PD), huntingtin (HD), and the expanded glutamine androgen receptor (polyQ AR) (SBMA). In this Minireview, we update our model in which Hsp90 acts on protein-folding clefts and show how it forms a rational basis for developing drugs that promote the targeted elimination of these aberrant proteins.

Glycoprotein folding and quality-control mechanisms in protein-folding diseases.

Biosynthesis of proteins – from translation to folding to export – encompasses a complex set of events that are exquisitely regulated and scrutinized to ensure the functional quality of the end products. Cells have evolved to capitalize on multiple post-translational modifications in addition to primary structure to indicate the folding status of nascent polypeptides to the chaperones and other proteins that assist in their folding and export. These modifications can also, in the case of irreversibly misfolded candidates, signal the need for dislocation and degradation. The current Review focuses on the glycoprotein quality-control (GQC) system that utilizes protein N-glycosylation and N-glycan trimming to direct nascent glycopolypeptides through the folding, export and dislocation pathways in the endoplasmic reticulum (ER). A diverse set of pathological conditions rooted in defective as well as over-vigilant ER quality-control systems have been identified, underlining its importance in human health and disease. We describe the GQC pathways and highlight disease and animal models that have been instrumental in clarifying our current understanding of these processes.

The role of the cytosolic HSP70 chaperone system in diseases caused by misfolding and aberrant trafficking of ion channels

Protein-folding diseases are an ongoing medical challenge. Many diseases within this group are genetically determined, and have no known cure. Among the examples in which the underlying cellular and molecular mechanisms are well understood are diseases driven by misfolding of transmembrane proteins that normally function as cell-surface ion channels. Wild-type forms are synthesized and integrated into the endoplasmic reticulum (ER) membrane system and, upon correct folding, are trafficked by the secretory pathway to the cell surface. Misfolded mutant forms traffic poorly, if at all, and are instead degraded by the ER-associated proteasomal degradation (ERAD) system. Molecular chaperones can assist the folding of the cytosolic domains of these transmembrane proteins; however, these chaperones are also involved in selecting misfolded forms for ERAD. Given this dual role of chaperones, diseases caused by the misfolding and aberrant trafficking of ion channels (referred to here as ion-channel-misfolding diseases) can be regarded as a consequence of insufficiency of the pro-folding chaperone activity and/or overefficiency of the chaperone ERAD role. An attractive idea is that manipulation of the chaperones might allow increased folding and trafficking of the mutant proteins, and thereby partial restoration of function. This Review outlines the roles of the cytosolic HSP70 chaperone system in the best-studied paradigms of ion-channel-misfolding disease – the CFTR chloride channel in cystic fibrosis and the hERG potassium channel in cardiac long QT syndrome type 2. In addition, other ion channels implicated in ion-channel-misfolding diseases are discussed.

Protein Interactions and Transition Times that Influence the Pathogenesis of Protein Folding Diseases

Protein folding diseases occur when a specific protein fails to fold into its correct functional state as a consequence of mutation in the protein amino acid sequence. In this talk, I present a model of the folded and misfolded protein expression, processing and their interactions, which we have used to investigate how protein folding disease phenotypes develop from mutated genotypes. Modeling protein processing as a continuous flow reactor, we found that the pathogenesis of protein folding diseases can be modulated by a combination of the transition time of folded and misfolded proteins in the reactor, the ratio of folded and misfolded protein inflow rates in the reactor and a chemical interaction parameter between folded and misfolded proteins. Our analysis reveals therapeutic strategies targeting the modulation of protein folding diseases, which have been recently explored in cellular and animal models of Mutant INS-gene Induced Diabetes of Youth and Congenital Hypothyroidism with deficient thyroglobulin.

Creating a path from the heat shock response to therapeutics of protein-folding diseases: an interview with Rick Morimoto

Rick Morimoto Director of the Rice Institute of Biomedical Research and the Bill and Gayle Cook Professor of Biology at Northwestern University, is renowned for his insights into the heat shock response and its role in protein-folding diseases and aging. As part of Disease Models & Mechanisms’ (DMM’s) special series on protein-folding diseases, Rick recalls the karmic events that influenced his career and discusses the importance of invertebrate model systems to reveal the basic mechanisms underlying aging and protein misfolding, as well as efforts to discover therapies to reverse these biological processes.

The threads that tie protein-folding diseases

From unicellular organisms to humans, cells have evolved elegant systems to facilitate careful folding of proteins and the maintenance of protein homeostasis. Key modulators of protein homeostasis include a large, conserved family of proteins known as molecular chaperones, which augment the folding of nascent polypeptides and temper adverse consequences of cellular stress. However, errors in protein folding can still occur, resulting in the accumulation of misfolded proteins that strain cellular quality-control systems. In some cases, misfolded proteins can be targeted for degradation by the proteasome or via autophagy. Nevertheless, protein misfolding is a feature of many complex, genetically and clinically pleiotropic diseases, including neurodegenerative disorders and cancer. In recent years, substantial progress has been made in unraveling the complexity of protein folding using model systems, and we are now closer to being able to diagnose and treat the growing number of protein-folding diseases. To showcase some of these important recent advances, and also to inspire discussion on approaches to tackle unanswered questions, Disease Models & Mechanisms (DMM) presents a special collection of reviews from researchers at the cutting-edge of the field.

Mechanisms of protein-folding diseases at a glance

For a protein to function appropriately, it must first achieve its proper conformation and location within the crowded environment inside the cell. Multiple chaperone systems are required to fold proteins correctly. In addition, degradation pathways participate by destroying improperly folded proteins. The intricacy of this multisystem process provides many opportunities for error. Furthermore, mutations cause misfolded, nonfunctional forms of proteins to accumulate. As a result, many pathological conditions are fundamentally rooted in the protein-folding problem that all cells must solve to maintain their function and integrity. Here, to illustrate the breadth of this phenomenon, we describe five examples of protein-misfolding events that can lead to disease: improper degradation, mislocalization, dominant-negative mutations, structural alterations that establish novel toxic functions, and amyloid accumulation. In each case, we will highlight current therapeutic options for battling such diseases.

Molecular Assembly of Thyroglobulin Induced by In Vitro Nitric Oxide Treatments: Implication Its Role in Thyroid Cells

The formation of amyloid-like fibrils, which polymerize from various soluble proteins under physiological and acidic conditions, causes a wide range of protein-folding diseases, such as Alzheimer's disease and Parkinson's disease. Fibril assembly in in vitro solutions containing nitric oxide, a free radical that functions as an important signalling molecule involved in numerous physiological and pathological processes, has not been reported. Here, we investigated the protein assembly that occur in thyroglobulin under mildly acidic conditions in the presence of nitric oxide. Solution studies, size exclusion chromatography, dynamic light scattering and analytical ultracentrifugation, demonstrated the size changes of thyroglobulin oligomers after nitric oxide treatment. Following electron microscopic analysis visualized their structural changes and revealed that the molecules can morphologically form polymerized fibril assemblies with a length of 2-5μm and width 10-100nm. Taken together, these results provide suggestive evidence for the propensity of forming polymerized thyroglobulin fibrils implying their presence in thyroid cells, which may be related to the onset or progression of thyroid diseases.

Structural Determinants of Tau Aggregation Inhibitor Potency

Small-molecule Tau aggregation inhibitors are under investigation as potential therapeutic agents against Alzheimer disease. Many such inhibitors have been identified in vitro, but their potency-driving features, and their molecular targets in the Tau aggregation pathway, have resisted identification. Previously we proposed ligand polarizability, a measure of electron delocalization, as a candidate descriptor of inhibitor potency. Here we tested this hypothesis by correlating the ground state polarizabilities of cyanine, phenothiazine, and arylmethine derivatives calculated using ab initio quantum methods with inhibitory potency values determined in the presence of octadecyl sulfate inducer under reducing conditions. A series of rhodanine analogs was analyzed as well using potency values disclosed in the literature. Results showed that polarizability and inhibitory potency directly correlated within all four series. To identify putative binding targets, representative members of the four chemotypes were added to aggregation reactions, where they were found to stabilize soluble, but SDS-resistant Tau species at the expense of filamentous aggregates. Using SDS resistance as a secondary assay, and a library of Tau deletion and missense mutants as targets, interaction with cyanine was localized to the microtubule binding repeat region. Moreover, the SDS-resistant phenotype was completely dependent on the presence of octadecyl sulfate inducer, but not intact PHF6/PH6* hexapeptide motifs, indicating that cyanine interacted with a species in the aggregation pathway prior to nucleus formation. Together the data suggest that flat, highly polarizable ligands inhibit Tau aggregation by interacting with folded species in the aggregation pathway and driving their assembly into soluble but highly stable Tau oligomers.

Amyloidosis

Amyloidosis is the name for protein-folding diseases characterized by extracellular deposition of a specific soluble precursor protein that aggregates in the form of insoluble fibrils. The classification of amyloidosis is based on the chemical characterization of the precursor protein. Deposition of amyloid is localized or systemic. The 4 main types of systemic amyloidosis are AL, AA, ATTR, and Aβ2M type. A schematic approach is proposed for the clinical management of systemic amyloidosis. The importance of typing amyloid with confidence, the usefulness of imaging techniques, the principles of treatment, and the need for well-planned treatment monitoring during follow-up are discussed.

Protein Misfolding in Disease and Small Molecule Therapies

A large number of human disorders are caused by defects in protein folding resulting from genetic mutations or adverse physiological conditions, and these are collectively referred to protein misfolding diseases. Such disorders imply dysfunction of a cellular process either as a result of a toxic gain of function due to protein aggregation, or loss of function due to protein instability, inefficient folding or defective trafficking. For a number of cases, drugs acting directly on the affected protein have been found to prevent misfolding and rescue function. This brief review will illustrate molecular mechanisms through which small molecules acting as folding correctors can prevent excessive protein buildup or recover faulty protein conformers, thus acting as effective therapeutic pharmacological chaperones. As background, the principles underlying the thermodynamics and kinetics of the protein folding reaction will be overviewed, as well as pathways leading to the formation of misfolding. The mechanism of action of small molecule correctors will then be discussed in light of these basic principles using illustrative examples referring to drugs that are effective over proteins involved in trafficking and folding diseases, amyloid aggregation disorders and metabolic deficiencies. An outlook on synergistic effects between different folding correctors and their combination with proteostasis regulators will also be addressed, as a relevant strategy towards the design of more effective therapies against protein folding diseases.

On the design of broad based screening assays to identify potential pharmacological chaperones of protein misfolding diseases.

Correcting aberrant folds that develop during protein folding disease states is now an active research endeavor that is attracting increasing attention from both academic and industrial circles. One particular approach focuses on developing or identifying small molecule correctors or pharmacological chaperones that specifically stabilize the native fold. Unfortunately, the limited screening platforms available to rapidly identify or validate potential drug candidates are usually inadequate or slow because the folding disease proteins in question are often transiently folded and/or aggregation-prone, complicating and/or interfering with the assay outcomes. In this review, we outline and discuss the numerous platform options currently being employed to identify small molecule therapeutics for folding diseases. Finally, we describe a new stability screening approach that is broad based and is easily applicable toward a very large number of both common and rare protein folding diseases. The label free screening method described herein couples the promiscuity of the GroEL binding to transient aggregation-prone hydrophobic folds with surface plasmon resonance enabling one to rapidly identify potential small molecule pharmacological chaperones.

An Unusual Dimeric Small Heat Shock Protein Provides Insight into the Mechanism of This Class of Chaperones

Small heat shock proteins (sHSPs) are virtually ubiquitous stress proteins that are also found in many normal tissues and accumulate in diseases of protein folding. They generally act as ATP-independent chaperones to bind and stabilize denaturing proteins that can be later reactivated by ATP-dependent Hsp70/DnaK, but the mechanism of substrate capture by sHSPs remains poorly understood. A majority of sHSPs form large oligomers, a property that has been linked to their effective chaperone action. We describe AtHsp18.5 from Arabidopsis thaliana, demonstrating that it is dimeric and exhibits robust chaperone activity, which adds support to the model that suboligomeric sHSP forms are a substrate binding species. Notably, like oligomeric sHSPs, when bound to substrate, AtHsp18.5 assembles into large complexes, indicating that reformation of sHSP oligomeric contacts is not required for assembly of sHSP–substrate complexes. Monomers of AtHsp18.5 freely exchange between dimers but fail to coassemble in vitro with dodecameric plant cytosolic sHSPs, suggesting that AtHsp18.5 does not interact by coassembly with these other sHSPs in vivo. Data from controlled proteolysis and hydrogen–deuterium exchange coupled with mass spectrometry show that the N- and C-termini of AtHsp18.5 are highly accessible and lack stable secondary structure, most likely a requirement for substrate interaction. Chaperone activity of a series of AtHsp18.5 truncation mutants confirms that the N-terminal arm is required for substrate protection and that different substrates interact differently with the N-terminal arm. In total, these data imply that the core α-crystallin domain of the sHSPs is a platform for flexible arms that capture substrates to maintain their solubility.

Overcoming the Cellular Crowding Effect: VlsE-FRET is Destabilized Inside Cells

The understanding of protein folding dynamics inside cells is crucial for the development of new therapies against protein folding and aggregation diseases. In addition, many processes inside cells such as gene expression, association and dissociation processes, as well as protein degradation are controlled by protein stability and kinetics. Fluorescence Relaxation Imaging (FReI) instrument combines temperature jump, FRET and cell imaging to study dynamic processes inside cells from the microseconds to the seconds time scales. using FReI, we have carried out experiment to calculate the melting temperature (Tm) and relaxation time (τ) of a constructed FRET pair protein of the Variable Lyme Disease Surface Antigen E (VlsE). VlsE-FRET has an in vitro Tm= 38 ± 1 °C and τ = 0.5 ± 0.1 s. In contrast, VlsE-FRET has an in cell Tm = 35 ± 1 °C and τ = 2 ± 2 s. Also, the in vitro data support a single exponential unfolding process with β = 1.0 ± 0.1, but the in cell data does not with β = 0.8 ± 0.2. Interestingly, VlsE-FRET has two distinct populations in cell with melting temperatures of 33 ± 1 °C and 36 ± 1 °C. These data support that some proteins can overcome the crowding effect inside cells due to the contribution of chemical/electrostatic forces. VlsE has different regions that undergo antigenic variation in order to evade the immune system of the host. Probably, the VlsE-FRET destabilization in cell is part of the trigger mechanism for the extensive genetic and antigenic variation that allows VlsE to evolve.

Yeast Stress, Aging, and Death

Yeast Stress, Aging, and Death

A4 Development of disease modifying therapies in Huntington's disease

Dr Gillian Bates is Professor of Neurogenetics and Head of the Division of Genetics and Molecular Medicine at King9s College London School of Medicine. Her research interests are focused on understanding the molecular pathogenesis of Huntington9s disease (HD) and the development of therapeutic interventions. Her basic science projects include the proteolytic processing of huntingtin; huntingtin aggregation; chaperones, the heat shock response and impairment of protein homeostasis in HD. She uses a combination of pharmacology and genetic manipulation to validate therapeutic targets in mouse models of HD. She has been elected to the Royal Society (2007), EMBO (2002) and the Academy of Medical Sciences (1999). Huntington9s disease (HD) is a devastating neurodegenerative disorder for which there are no disease-modifying treatments. Since the Identification of the mutation as a CAG repeat expansion in 1993, a wide range of single cell (yeast and mammalian cell culture), invertebrate ( Drosophila melanogaster ) and Caenorhabditis elegans ) and mammalian (mouse and rat) models have been developed to better understand disease pathogenesis and identify targets for disease modification and allowed is a protein folding disease caused by a CAG/polyglutamine expansion in the huntingtin protein (HTT) and we are employing genetic and pharmacological approaches to evaluate mechanisms by which protein homeostasis might be restored.