Reconstitutive refolding of diacylglycerol kinase, an integral membrane protein

While the formation of kinetically trapped misfolded structural states by membrane proteins is related to a number of diseases, relatively few studies of misfolded membrane proteins in their purified state have been carried out and few methods for refolding such proteins have been reported. In this paper, misfolding of the trimeric integral membrane protein diacylglycerol kinase (DAGK) is documented and a method for refolding the protein is presented; 65 single-cysteine mutants of DAGK were examined. A majority were found to have lower-than-expected activities when purified into micellar solutions, with additional losses in activity often being observed following membrane reconstitution. A variety of evidence indicates that the low activities observed for most of these mutants results from kinetically based misfolding of the protein, with misfolding often being manifested by the formation of aberrant oligomeric states. A method referred to as "reconstitutive refolding" for correcting misfolded DAGK is presented. This method is based upon reconstituting DAGK into multilamellar POPC vesicles by dialyzing the detergent dodecylphosphocholine out of mixed micellar mixtures. For 55 of the 65 mutants tested, there was a gain of DAGK activity during reconstitutive refolding. In 33 of these cases, the gain in activity was greater than 2-fold. The refolding results for cysteine replacement mutants at DAGK sites known to be highly conserved provide teleological insight into whether sites are conserved, because they are critical for catalysis, for maintenance of the proper folding pathway, or for some other reason.     

1H, 13C and 15N resonance assignments of human muscle acylphosphatase

Human muscle acylphosphatase (mAcP) is an enzyme with a ferrodoxin-like topology whose primary role is to hydrolyze the carboxyl-phosphate bonds of acylphosphates. The protein has been widely used as a model system for elucidating the molecular determinants of protein folding and misfolding. We present here the full NMR assignments of the backbone and side chains resonances of mAcP complexed with phosphate, thus providing an important resource for future solution-state NMR spectroscopic studies of the structure and dynamics of this protein in the contexts of protein folding and misfolding.     

A model of threshold behavior reveals rescue mechanisms of bystander proteins in conformational diseases

Conformational diseases result from the failure of a specific protein to fold into its correct functional state. The misfolded proteins can lead to the toxic aggregation of proteins. Protein misfolding in conformational diseases often displays a threshold behavior characterized by a sudden shift between nontoxic to toxic levels of misfolded proteins. In some conformational diseases, evidence suggests that misfolded proteins interact with bystander proteins (unfolded and native folded proteins), eliciting a misfolded phenotype. These bystander isomers would follow their normal physiological pathways in absence of misfolded proteins. In this article, we present a general mechanism of bystander and misfolded protein interaction which we have used to investigate how the threshold behavior in protein misfolding is triggered in conformational diseases. Using a continuous flow reactor model of the endoplasmic reticulum, we found that slight changes in the bystander protein residence time in the endoplasmic reticulum or the ratio of basal misfolded to bystander protein inflow rates can trigger the threshold behavior in protein misfolding. Our analysis reveals three mechanisms to rescue bystander proteins in conformational diseases. The results of our model can now help direct experiments to understand the threshold behavior and develop therapeutic strategies targeting the modulation of conformational diseases.     

Gene expression response to misfolded protein as a screen for soluble recombinant protein

Proper protein folding is key to producing recombinant proteins for structure determination. We have examined the effect of misfolded recombinant protein on gene expression in Escherichia coli. Comparison of expression patterns indicates a unique set of genes responding to translational misfolding. The response is in part analogous to heat shock and suggests a translational component to the regulation. We have further utilized the expression information to generate reporters responsive to protein misfolding. These reporters were used to identify properly folded recombinant proteins and to create soluble domains of insoluble proteins for structural studies.     

A Single Mutation at Residue 25 Populates the Folding Intermediate of E. coli RNase H and Reveals a Highly Dynamic Partially Folded Ensemble

Understanding the nature of partially folded intermediates transiently populated during protein folding is important for understanding both protein folding and misfolding. These ephemeral species, however, often elude direct experimental characterization. The well-characterized protein ribonuclease H (RNase H) from Escherichia coli populates an on-pathway intermediate identified in both bulk studies and single-molecule mechanical unfolding experiments. Here, we set out to trap the transient intermediate of RNase H at equilibrium by selectively destabilizing the region of the protein known to be unfolded in this species. Surprisingly, a single change at Ile25 (I25A) resulted in the equilibrium population of the intermediate under near-native conditions. The intermediate was undetectable in a series of heteronuclear single quantum coherences, revealing the dynamic nature of this partially unfolded form on the timescale of NMR detection. This result is in contrast to studies in which the structures of trapped intermediates are solved by NMR, indicating that they are well packed and native-like. The dynamic nature of the RNase H intermediate may be important for its role as an on-pathway, productive species that promotes efficient folding.     

Observing folding pathways and kinetics of a single sodium-proton antiporter from Escherichia coli

Mechanisms of folding and misfolding of membrane proteins are of interest in cell biology. Recently, we have established single-molecule force spectroscopy to observe directly the stepwise folding of the Na+/H+ antiporter NhaA from Escherichia coli in vitro. Here, we improved this approach significantly to track the folding intermediates of a single NhaA polypeptide forming structural segments such as the Na+-binding site, transmembrane alpha-helices, and helical pairs. The folding rates of structural segments ranged from 0.31 s(-1) to 47 s(-1), providing detailed insight into a distinct folding hierarchy of an unfolded polypeptide into the native membrane protein structure. In some cases, however, the folding chain formed stable and kinetically trapped non-native structures, which could be assigned to misfolding events of the antiporter.     

Iterative annealing mechanism explains the functions of the GroEL and RNA chaperones

Molecular chaperones are ATP‐consuming machines, which facilitate the folding of proteins and RNA molecules that are kinetically trapped in misfolded states. Unassisted folding occurs by the kinetic partitioning mechanism according to which folding to the native state, with low probability as well as misfolding to one of the many metastable states, with high probability, occur rapidly. GroEL is an all‐purpose stochastic machine that assists misfolded substrate proteins to fold. The RNA chaperones such as CYT‐19, which are ATP‐consuming enzymes, help the folding of ribozymes that get trapped in metastable states for long times. GroEL does not interact with the folded proteins but CYT‐19 disrupts both the folded and misfolded ribozymes. The structures of GroEL and RNA chaperones are strikingly different. Despite these differences, the iterative annealing mechanism (IAM) quantitatively explains all the available experimental data for assisted folding of proteins and ribozymes. Driven by ATP binding and hydrolysis and GroES binding, GroEL undergoes a catalytic cycle during which it samples three allosteric states, T (apo), R (ATP bound), and R^″ (ADP bound). Analyses of the experimental data show that the efficiency of the GroEL–GroES machinery and mutants is determined by the resetting rate k _{R ″ → T} , which is largest for the wild‐type (WT) GroEL. Generalized IAM accurately predicts the folding kinetics of Tetrahymena ribozyme and its variants. Chaperones maximize the product of the folding rate and the steady‐state native state fold by driving the substrates out of equilibrium. Neither the absolute yield nor the folding rate is optimized.     

Early steps in oxidation-induced SOD1 misfolding: implications for non-amyloid protein aggregation in familial ALS

Among the diseases of protein misfolding, amyotrophic lateral sclerosis (ALS) is unusual in that the proteinaceous neuronal inclusions that are the hallmark of the disease have neither the classic fibrillar appearance of amyloid by transmission electron microscopy nor the affinity for the dye Congo red that is a defining feature of amyloid. Mutations in the Cu, Zn superoxide dismutase (SOD1) cause the largest subset of inherited ALS cases. The mechanism by which this highly stable enzyme misfolds to form non-amyloid aggregates is currently poorly understood, as are the stresses that initiate misfolding. The oxidative damage hypothesis proposes that SOD1's normal free radical scavenger role puts it at risk of oxidative damage and that it is this damage that triggers the misfolding primed by mutation. Here, we present evidence that hydrogen peroxide treatment, which generates free radical species at the SOD1 active site, causes oxidative damage to active-site histidine residues, leading to major structural changes and non-amyloid aggregation similar to that seen in ALS. Time-resolved measurements of release of bound metal ligands, exposure of hydrophobic surface area, and alterations in the SOD1 proton NMR spectrum have allowed us to model the early structural changes occurring as SOD1 misfolds, prior to aggregation. ALS-causing SOD1 mutations apparently alter this pathway by increasing exposure of buried epitopes in misfolded species populated at endpoint. We have identified a well-populated early misfolding intermediate that could serve as a target for therapies designed to block downstream misfolding and aggregation events and thereby treat SOD1-associated ALS.     

19F NMR studies of plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1) is a 43 kDa protein involved in the regulation of fibrinolysis. PAI-1 is the principal inhibitor of tissue-type plasminogen activator (t-PA), trapping the proteinase as an acyl-enzyme covalent complex (approximately 105 kDa). Four single tryptophan mutants of PAI-1 have been constructed in which three of the four tryptophan residues (Trp86, Trp139, Trp175, and Trp262) were replaced with phenylalanine. Biosynthetic incorporation of 5-fluorotryptophan (5F-Trp) into wild-type PAI-1 (5FW wtPAI-1) and the single tryptophan mutants (5FW86, 5FW139, 5FW175, and 5FW262) was achieved, allowing a (19)F NMR spectroscopic study of PAI-1 in its active and cleaved forms and in complex with t-PA. The (19)F NMR spectrum of active 5FW wtPAI-1 shows four clearly resolved peaks at -39.20, -49.26, -50.74, and -52.57 ppm relative to trifluoroacetic acid at 0 ppm. Unequivocal assignments of these four resonances in the spectrum of 5FW wtPAI-1 to specific tryptophan residues were accomplished by measuring the chemical shifts of the (19)F resonances of the single tryptophan mutants. There was close agreement between the resonances observed in 5FW wtPAI-1 and of those in the mutants for all three protein forms. This would imply little structural perturbation in the local structures of the tryptophan residues resulting from substitution by phenylalanine. The 5FW wtPAI-1 was observed to have lower second-order rate constant (k(app)) for the inhibition of t-PA than the natural tryptophan wtPAI-1, suggesting that the decreased activity may result from a small structural effect of the fluorine substituent of the indole ring. Further alterations in the k(app) and the stoichiometry of inhibition (SI) were observed in each of the mutants indicating an effect of the three tryptophan to phenylalanine mutations. Detailed interpretation of the (19)F NMR spectra of the PAI-1 mutants provides insights into the local segmental structure of the active form of the proteins and the structural changes that occur in the cleaved and t-PA complexed forms.     

Calnexin Improves the Folding Efficiency of Mutant Rhodopsin in the Presence of Pharmacological Chaperone 11-cis-Retinal

The lectin chaperone calnexin (Cnx) is important for quality control of glycoproteins, and the chances of correct folding of a protein increase the longer the protein interacts with Cnx. Mutations in glycoproteins increase their association with Cnx, and these mutant proteins are retained in the endoplasmic reticulum. However, until now, the increased interaction with Cnx was not known to increase the folding of mutant glycoproteins. Because many human diseases result from glycoprotein misfolding, a Cnx-assisted folding of mutant glycoproteins could be beneficial. Mutations of rhodopsin, the glycoprotein pigment of rod photoreceptors, cause misfolding resulting in retinitis pigmentosa. Despite the critical role of Cnx in glycoprotein folding, surprisingly little is known about its interaction with rhodopsin or whether this interaction could be modulated to increase the folding of mutant rhodopsin. Here, we demonstrate that Cnx preferentially associates with misfolded mutant opsins associated with retinitis pigmentosa. Furthermore, the overexpression of Cnx leads to an increased accumulation of misfolded P23H opsin but not the correctly folded protein. Finally, we demonstrate that increased levels of Cnx in the presence of the pharmacological chaperone 11-cis-retinal increase the folding efficiency and result in an increase in correct folding of mutant rhodopsin. These results demonstrate that misfolded rather than correctly folded rhodopsin is a substrate for Cnx and that the interaction between Cnx and mutant, misfolded rhodopsin, can be targeted to increase the yield of folded mutant protein.     

NMR evidence for progressive stabilization of native-like structure upon aggregation of acid-denatured LysN

The acid-denatured form of the protein LysN aggregates reversibly at pH 2.0. The strength of self-association increases with increasing Cl(-) anion concentration. At low concentrations of protein or Cl(-) anion, resonances of denatured LysN are in slow exchange with a minor form of the protein, which shows native-like NMR chemical shifts. The minor native-like resonances increase in intensity with increasing protein concentration, demonstrating that a native-like monomer fold is stabilized on aggregation of the acid-denatured protein. At high concentrations of protein or Cl(-) anion, interconversion between the major and minor resonances appears to shift from slow to intermediate exchange on the NMR timescale. NMR line-broadening is more pronounced for the major resonances of the denatured protein, which show sigmoidal decay curves with increasing Cl(-) concentration. The mid-points of the decay curves for residues in different parts of the molecule are non-coincident. We propose that differences in the NMR line-broadening transitions of individual residues reflect a stepwise stabilization of native-like structure on aggregation, starting with the segments of the protein that form the initial association interface. The resonances of the denatured protein with the greatest sensitivity to self-association correspond roughly to those that are most perturbed in the native protein on binding of the natural substrate tRNA(Lys). This suggests that the hydrophobic surfaces that promote intermolecular misfolding of acid-denatured LysN, may resemble those used for substrate binding by the native protein.     

Identification and Characterization of a Misfolded Monomeric Serpin Formed at Physiological Temperature

The native serpin state is kinetically trapped. However, under mildly destabilizing conditions, the conformational landscape changes, and a number of nonnative conformations with increased stability can be readily formed. The ability to undergo structural change is due to intrinsic strain within the serpin's tertiary fold, which is utilized for proteinase inhibition but renders the protein susceptible to aberrant folding and self-association. The relationship between these various conformations is poorly understood. Antichymotrypsin (ACT) is an inhibitory serpin that readily forms a number of inactive conformations, induced via either environmental stress or interaction with proteinases. Here we have used a variety of biophysical and structural techniques to characterize the relationship between some of these conformations. Incubation of ACT at physiological temperature results in the formation of a range of conformations, including both polymer and misfolded monomer. The ability to populate these nonnative states and the native conformation reflects an energy landscape that is very sensitive to the solution conditions. X-ray crystallography reveals that the misfolded monomeric conformation is in the delta conformation. Further polymerization and seeding experiments show that the delta conformation is an end point in the misfolding pathway of ACT and not an on-pathway intermediate formed during polymerization. The observation that ACT readily forms this inactive conformation at physiological temperature and pH suggests that it may have a role in both health and disease.     

Folding or holding?—Hsp70 and Hsp90 chaperoning of misfolded proteins in neurodegenerative disease

The toxic accumulation of misfolded proteins as inclusions, fibrils, or aggregates is a hallmark of many neurodegenerative diseases. However, how molecular chaperones, such as heat shock protein 70 kDa (Hsp70) and heat shock protein 90 kDa (Hsp90), defend cells against the accumulation of misfolded proteins remains unclear. The ATP-dependent foldase function of both Hsp70 and Hsp90 actively transitions misfolded proteins back to their native conformation. By contrast, the ATP-independent holdase function of Hsp70 and Hsp90 prevents the accumulation of misfolded proteins. Foldase and holdase functions can protect against the toxicity associated with protein misfolding, yet we are only beginning to understand the mechanisms through which they modulate neurodegeneration. This review compares recent structural findings regarding the binding of Hsp90 to misfolded and intrinsically disordered proteins, such as tau, α-synuclein, and Tar DNA-binding protein 43. We propose that Hsp90 and Hsp70 interact with these proteins through an extended and dynamic interface that spans the surface of multiple domains of the chaperone proteins. This contrasts with many other Hsp90–client protein interactions for which only a single bound conformation of Hsp90 is proposed. The dynamic nature of these multidomain interactions allows for polymorphic binding of multiple conformations to vast regions of Hsp90. The holdase functions of Hsp70 and Hsp90 may thus allow neuronal cells to modulate misfolded proteins more efficiently by reducing the long-term ATP running costs of the chaperone budget. However, it remains unclear whether holdase functions protect cells by preventing aggregate formation or can increase neurotoxicity by inadvertently stabilizing deleterious oligomers.     

Engineering enhanced protein disaggregases for neurodegenerative disease

Abstract

Protein misfolding and aggregation underpin several fatal neurodegenerative diseases, including Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). There are no treatments that directly antagonize the protein-misfolding events that cause these disorders. Agents that reverse protein misfolding and restore proteins to native form and function could simultaneously eliminate any deleterious loss-of-function or toxic gain-of-function caused by misfolded conformers. Moreover, a disruptive technology of this nature would eliminate self-templating conformers that spread pathology and catalyze formation of toxic, soluble oligomers. Here, we highlight our efforts to engineer Hsp104, a protein disaggregase from yeast, to more effectively disaggregate misfolded proteins connected with PD, ALS, and FTD. Remarkably subtle modifications of Hsp104 primary sequence yielded large gains in protective activity against deleterious α-synuclein, TDP-43, FUS, and TAF15 misfolding. Unusually, in many cases loss of amino acid identity at select positions in Hsp104 rather than specific mutation conferred a robust therapeutic gain-of-function. Nevertheless, the misfolding and toxicity of EWSR1, an RNA-binding protein with a prion-like domain linked to ALS and FTD, could not be buffered by potentiated Hsp104 variants, indicating that further amelioration of disaggregase activity or sharpening of substrate specificity is warranted. We suggest that neuroprotection is achievable for diverse neurodegenerative conditions via surprisingly subtle structural modifications of existing chaperones.     

Cell death: protein misfolding and neurodegenerative diseases

Several chronic neurodegenerative disorders manifest deposits of misfolded or aggregated proteins. Genetic mutations are the root cause for protein misfolding in rare families, but the majority of patients have sporadic forms possibly related to environmental factors. In some cases, the ubiquitin-proteasome system or molecular chaperones can prevent accumulation of aberrantly folded proteins. Recent studies suggest that generation of excessive nitric oxide (NO) and reactive oxygen species (ROS), in part due to overactivity of the NMDA-subtype of glutamate receptor, can mediate protein misfolding in the absence of genetic predisposition. S-Nitrosylation, or covalent reaction of NO with specific protein thiol groups, represents one mechanism contributing to NO-induced protein misfolding and neurotoxicity. Here, we present evidence suggesting that NO contributes to protein misfolding via S-nitrosylating protein-disulfide isomerase or the E3 ubiquitin ligase parkin. We discuss how memantine/NitroMemantine can inhibit excessive NMDA receptor activity to ameliorate NO production, protein misfolding, and neurodegeneration.     

Contribution of glutamatergic signaling to nitrosative stress-induced protein misfolding in normal brain aging and neurodegenerative diseases

Glutamatergic hyperactivity, associated with Ca2+ influx and consequent production of nitric oxide (NO), is potentially involved in both normal brain aging and age-related neurodegenerative disorders. Many neurodegenerative diseases are characterized by conformational changes in proteins that result in their misfolding and aggregation. Normal protein degradation by the ubiquitin-proteasome system can prevent accumulation of aberrantly folded proteins. Our recent studies have linked nitrosative stress to protein misfolding and neuronal cell death. In particular, molecular chaperones - such as protein disulfide isomerase, glucose regulated protein 78, and heat shock proteins - can provide neuroprotection from misfolded proteins by facilitating proper folding and thus preventing aggregation. Here, we present evidence for the hypothesis that NO contributes to normal brain aging and degenerative conditions by S-nitrosylating specific chaperones that would otherwise prevent accumulation of misfolded proteins.     

Pharmacologic rescue of conformationally-defective proteins: implications for the treatment of human disease

The process of quality control in the endoplasmic reticulum involves a variety of mechanisms which ensure that only correctly folded proteins enter the secretory pathway. Among these are conformation-screening mechanisms performed by molecular chaperones that assist in protein folding and prevent non-native (or misfolded) proteins from interacting with other misfolded proteins. Chaperones play a central role in the triage of newly formed proteins prior to their entry into the secretion, retention, and degradation pathways. Despite this stringent quality control mechanism, gain- or loss-of-function mutations that affect protein folding in the endoplasmic reticulum can manifest themselves as profound effects on the health of an organism. Understanding the molecular, cellular, and energetic mechanisms of protein routing could prevent or correct the structural abnormalities associated with disease-causing misfolded proteins. Rescue of misfolded, "trafficking-defective", but otherwise functional, proteins is achieved by a variety of physical, chemical, genetic, and pharmacological approaches. Pharmacologic chaperones (or "pharmacoperones") are template molecules that may potentially arrest or reverse diseases by inducing mutant proteins to adopt native-type-like conformations instead of improperly folded ones. Such restructuring leads to a normal pattern of cellular localization and function. This review focuses on protein misfolding and misrouting related to various disease states and describes promising approaches to overcoming such defects. Special attention is paid to the gonadotropin-releasing hormone receptor, since there is a great deal of information about this receptor, which has recently emerged as a particularly instructive model.     

Alzheimer disease

Alzheimer disease (AD) has traditionally been thought to involve the misfolding and aggregation of two different factors that contribute in parallel to pathogenesis: amyloid-β (Aβ) peptides, which represent proteolytic fragments of the transmembrane amyloid precursor protein, and tau, which normally functions as a neuronally enriched, microtubule-associated protein that predominantly accumulates in axons. Recent evidence has challenged this model, however, by revealing numerous functional interactions between Aβ and tau in the context of pathogenic mechanisms for AD. Moreover, the propagation of toxic, misfolded Aβ and tau bears a striking resemblance to the propagation of toxic, misfolded forms of the canonical prion protein, PrP, and misfolded Aβ has been shown to induce tau misfolding in vitro through direct, intermolecular interaction. In this review we discuss evidence for the prion-like properties of both Aβ and tau individually, as well as the intriguing possibility that misfolded Aβ acts as a template for tau misfolding in vivo.     

Urea-dependent unfolding of murine adenosine deaminase: sequential destabilization as measured by 19F NMR

Murine adenosine deaminase (mADA) is a 40 kDa (beta/alpha)(8)-barrel protein consisting of eight central beta-strands and eight peripheral alpha-helices containing four tryptophan residues. In this study, we investigated the urea-dependent behavior of the protein labeled with 6-fluorotryptophan (6-(19)F-Trp). The (19)F NMR spectrum of 6-(19)F-Trp-labeled mADA reveals four distinct resonances in the native state and three partly overlapped resonances in the unfolded state. The resonances were assigned unambiguously by site-directed mutagenesis. Equilibrium unfolding of 6-(19)F-Trp-labeled mADA was monitored using (19)F NMR based on these assignments. The changes in intensity of folded and unfolded resonances as a function of urea concentration show transition midpoints consistent with data observed by far-UV CD and fluorescence spectroscopy, indicating that conformational changes in mADA during urea unfolding can be followed by (19)F NMR. Chemical shifts of the (19)F resonances exhibited different changes between 1.0 and 6.0 M urea, indicating that local structures around 6-(19)F-Trp residues change differently. The urea-induced changes in local structure around four 6-(19)F-Trp residues of mADA were analyzed on the basis of the tertiary structure and chemical shifts of folded resonances. The results reveal that different local regions in mADA have different urea-dependent behavior, and that local regions of mADA change sequentially from native to intermediate topologies on the unfolding pathway.     

A rugged free energy landscape separates multiple functional RNA folds throughout denaturation

The dynamic mechanisms by which RNAs acquire biologically functional structures are of increasing importance to the rapidly expanding fields of RNA therapeutics and biotechnology. Large energy barriers separating misfolded and functional states arising from alternate base pairing are a well-appreciated characteristic of RNA. In contrast, it is typically assumed that functionally folded RNA occupies a single native basin of attraction that is free of deeply dividing energy barriers (ergodic hypothesis). This assumption is widely used as an implicit basis to interpret experimental ensemble-averaged data. Here, we develop an experimental approach to isolate persistent sub-populations of a small RNA enzyme and show by single molecule fluorescence resonance energy transfer (smFRET), biochemical probing and high-resolution mass spectrometry that commitment to one of several catalytically active folds occurs unexpectedly high on the RNA folding energy landscape, resulting in partially irreversible folding. Our experiments reveal the retention of molecular heterogeneity following the complete loss of all native secondary and tertiary structure. Our results demonstrate a surprising longevity of molecular heterogeneity and advance our current understanding beyond that of non-functional misfolds of RNA kinetically trapped on a rugged folding-free energy landscape.