Folding Diseases Research Articles

Time-resolved interactome profiling deconvolutes secretory protein quality control dynamics

Many cellular processes are governed by protein–protein interactions that require tight spatial and temporal regulation. Accordingly, it is necessary to understand the dynamics of these interactions to fully comprehend and elucidate cellular processes and pathological disease states. To map de novo protein–protein interactions with time resolution at an organelle-wide scale, we developed a quantitative mass spectrometry method, time-resolved interactome profiling (TRIP). We apply TRIP to elucidate aberrant protein interaction dynamics that lead to the protein misfolding disease congenital hypothyroidism. We deconvolute altered temporal interactions of the thyroid hormone precursor thyroglobulin with pathways implicated in hypothyroidism pathophysiology, such as Hsp70-/90-assisted folding, disulfide/redox processing, and N-glycosylation. Functional siRNA screening identified VCP and TEX264 as key protein degradation components whose inhibition selectively rescues mutant prohormone secretion. Ultimately, our results provide novel insight into the temporal coordination of protein homeostasis, and our TRIP method should find broad applications in investigating protein-folding diseases and cellular processes.

Historic characteristics and mortality of patients in the Swiss Amyloidosis Registry.

Systemic amyloidoses are rare protein-folding diseases with heterogeneous, often nonspecific clinical presentations. To better understand systemic amyloidoses and to apply state-of-the-art diagnostic pathways and treatment, the interdisciplinary Amyloidosis Network was founded in 2013 at University Hospital Zurich. In this respect, a registry was implemented to study the characteristics and life expectancy of patients with amyloidosis within the area covered by the network. Patient data were collected retrospectively for the period 2005-2014 and prospectively from 2015 onwards. Patients aged 18 years or older diagnosed with any subtype of systemic amyloidosis were eligible for inclusion if they were treated in one of the four referring centres (Zurich, Chur, St Gallen, Bellinzona). Baseline data were captured at the time of diagnosis. Follow-up data were assessed half-yearly for the first two years, then annually. Between January 2005 and March 2020, 247 patients were screened, and 155 patients with confirmed systemic amyloidosis were included in the present analysis. The most common amyloidosis type was light-chain (49.7%, n = 77), followed by transthyretin amyloidosis (40%, n = 62) and amyloid A amyloidosis (5.2%, n = 8). Most patients (61.9%, n = 96) presented with multiorgan involvement. Nevertheless, single organ involvement was seen in all types of amyloidosis, most commonly in amyloid A amyloidosis (75%, n = 6). The median observation time of the surviving patients was calculated by the reverse Kaplan-Meier method and was 3.29 years (95% confidence interval [CI] 2.33-4.87); it was 4.87 years (95% CI 3.14-7.22) in light-chain amyloidosis patients and 1.85 years (95% CI 1.48-3.66) in transthyretin amyloidosis patients, respectively. The 1-, 3- and 5-year survival rates were 87.0% (95% CI 79.4-95.3%), 68.5% (95% CI 57.4-81.7%) and 66.0% (95% CI 54.6-79.9%) respectively for light-chain amyloidosis patients and 91.2% (95% CI 83.2-99.8%), 77.0% (95% CI 63.4-93.7%) and 50.6% (95% CI 31.8-80.3%) respectively for transthyretin amyloidosis patients. There was no significant difference between the two groups (p = 0.81). During registry set-up, a more comprehensive work-up of our patients suffering mainly from light-chain amyloidosis and transthyretin amyloidosis was implemented.Survival rates were remarkably high and similar between light-chain amyloidosis and transthyretin amyloidosis, a finding which was noted in similar historic registries of international centres. However, further studies are needed to depict morbidity and mortality as the amyloidosis landscape is changing rapidly.

Protein aggregation: A detrimental symptom or an adaptation mechanism?

Protein quality control mechanisms oversee numerous aspects of protein lifetime. From the point of protein synthesis, protein homeostasis machineries take part in folding, solubilization, and/or degradation of impaired proteins. Some proteins follow an alternative path upon loss of their solubility, thus are secluded from the cytosol and form protein aggregates. Protein aggregates differ in their function and composition, rendering protein aggregation a complex phenomenon that continues to receive plenty of attention in the scientific and medical communities. Traditionally, protein aggregates have been associated with aging and a large spectrum of protein folding diseases, such as neurodegenerative diseases, type 2 diabetes, or cataract. However, a body of evidence suggests that they may act as an adaptive mechanism to overcome transient stressful conditions, serving as a sink for the removal of misfolded proteins from the cytosol or storage compartments for machineries required upon stress release. In this review, we present examples and evidence elaborating different possible roles of protein aggregation and discuss their potential roles in stress survival, aging, and disease, as well as possible anti-aggregation interventions.

The deleterious variants of N-acetylgalactosamine-6-sulfatase (GalN6S) enzyme trigger Morquio a syndrome by disrupting protein foldings

Lysosomal enzymes degrade cellular macromolecules, while their inactivation causes human hereditary metabolic disorders. Mucopolysaccharidosis IVA (MPS IVA; Moquio A syndrome) is one of the lysosomal storage disorders caused by a defective Galactosamine-6-sulfatase (GalN6S) enzyme. In several populations, disease incidence is elevated due to missense mutations brought on by non-synonymous allelic variation in the GalN6S enzyme. Here, we studied the effect of non-synonymous single nucleotide polymorphism (nsSNPs) on the structural dynamics of the GalN6S enzyme and its binding with N-acetylgalactosamine (GalNAc) using all-atom molecular dynamics simulation and an essential dynamics approach. Consequently, in this study, we have identified three functionally disruptive mutations in domain-I and domain-II, that is, S80L, R90W, and S162F, which presumably contribute to post-translational modifications. The study delineated that both domains work cooperatively, and alteration in domain II (S80L, R90W) leads to conformational changes in the catalytic site in domain-I, while mutation S162F mainly provokes higher residual flexibility of domain II. These results show that these mutations impair the hydrophobic core, implying that Morquio A syndrome is caused by misfolding of the GalN6S enzyme. The results also show the instability of the GalN6S-GalNAc complex upon substitution. Overall, the structural dynamics resulting from point mutations give the molecular rationale for Moquio A syndrome and, more importantly, the Mucopolysaccharidoses (MPS) family of diseases, re-establishing MPS IVA as a protein-folding disease. Communicated by Ramaswamy H. Sarma

ABC-transporter CFTR folds with high fidelity through a modular, stepwise pathway

The question how proteins fold is especially pointed for large multi-domain, multi-spanning membrane proteins with complex topologies. We have uncovered the sequence of events that encompass proper folding of the ABC transporter CFTR in live cells by combining kinetic radiolabeling with protease-susceptibility assays. We found that CFTR folds in two clearly distinct stages. The first, co-translational, stage involves folding of the 2 transmembrane domains TMD1 and TMD2, plus one nucleotide-binding domain, NBD1. The second stage is a simultaneous, post-translational increase in protease resistance for both TMDs and NBD2, caused by assembly of these domains onto NBD1. Our assays probe every 2–3 residues (on average) in CFTR. This in-depth analysis at amino-acid level allows detailed analysis of domain folding and importantly also the next level: assembly of the domains into native, folded CFTR. Defects and changes brought about by medicines, chaperones, or mutations also are amenable to analysis. We here show that the well-known disease-causing mutation F508del, which established cystic fibrosis as protein-folding disease, caused co-translational misfolding of NBD1 but not TMD1 nor TMD2 in stage 1, leading to absence of stage-2 folding. Corrector drugs rescued stage 2 without rescuing NBD1. Likewise, the DxD motif in NBD1 that was identified to be required for export of CFTR from the ER we found to be required already upstream of export as CFTR mutated in this motif phenocopies F508del CFTR. The highly modular and stepwise folding process of such a large, complex protein explains the relatively high fidelity and correctability of its folding.

POS-212 RENAL AA AMYLOIDOSIS: A RETROSPECTIVE CLINICOPATHOLOGIC STUDY WITH EMPHASIS ON PROGNOSIS CONDUCTED IN 74 FRENCH PATIENTS DIAGNOSED BETWEEN 2010 AND 2021

AA amyloidosis is a protein folding disease characterized by extracellular deposition of amyloid A fibrils derived from the serum amyloid A inflammation protein, causing kidney dysfunction in patients with chronic inflammatory conditions. Several histological studies found a link between the topography or abundance of amyloid deposits and clinical presentation or renal prognosis. However, most of these studies are now outdated, present a non-exhaustive clinical collection and may no longer represent the current epidemiology, especially in France.

The Effect of Wnt Family Member 5a Gene Silencing on the Proliferation of Achondroplasia Using a DNAzymes-CoOOH Nanocomposite.

Achondroplasia is a kind of congenital dysplasia due to the defect of endochondral ossification. Achondroplasia is considered to be a protein folding disease leading to endoplasmic reticulum stress. Endoplasmic reticulum stress may lead to disease by affecting the function and survival state of chondrocytes, but the specific mechanism requires further study. In this study, bioinformatics methods, online database mining, screening of differentially expressed genes for pathway enrichment, and interaction analysis were conducted to detect the Wnt family member 5a (Wnt5a) gene. Additionally, we designed a novel DNAzymes-based nanocomposite that can simultaneously silence Wnt5a genes in chondrocytes. The nanocomposite was composed of amino-functionalized cobalt oxyhydroxide nanoflakes modified by DNAzymes that target the Wnt5a gene. Further, we conducted in vitro experiments to verify that Wnt5a can mediate the mitogen-activated protein kinase signaling pathway through the endoplasmic reticulum stress pathway to affect the proliferation of chondrocytes.

Reinforcement of the Unfolded Protein Response Mitigates Cytotoxicity Induced by Human Z‐Type α1‐Antitrypsin

Deficient human α1‐antitrypsin (AAT) variants are involved in pulmonary emphysema and liver cirrhosis. Especially, the Z‐type AAT (Z AAT) variant folds very slowly, and thus accumulates protein folding intermediates prone to aggregation in the endoplasmic reticulum (ER) of hepatocytes. Misfolded proteins accumulated in the ER lead to persistent ER stress and subsequent cell death. In this study, the contribution of unfolded protein response (UPR) in Z AAT‐induced cytotoxicity was investigated. Deletions of each UPR element severely hampered growth of Z AAT‐overexpressing yeast cells. Overexpression of UPR elements, except kar2, alleviated the slow growth phenotype of Z AAT‐overexpressing cells, possibly through augmentation of folding capacity. Furthermore, reinforcement of UPR elements promoted extracellular secretion of Z AAT, which may have mitigated the plasma deficiency of AAT. Our results therefore provide further information on therapeutic strategies to address protein folding diseases.

The heat shock response, determined by QuantiGene multiplex, is impaired in HD mouse models and not caused by HSF1 reduction

Huntington’s disease (HD) is a devastating neurodegenerative disorder, caused by a CAG/polyglutamine repeat expansion, that results in the aggregation of the huntingtin protein, culminating in the deposition of inclusion bodies in HD patient brains. We have previously shown that the heat shock response becomes impaired with disease progression in mouse models of HD. The disruption of this inducible arm of the proteostasis network is likely to exacerbate the pathogenesis of this protein-folding disease. To allow a rapid and more comprehensive analysis of the heat shock response, we have developed, and validated, a 16-plex QuantiGene assay that allows the expression of Hsf1 and nine heat shock genes, to be measured directly, and simultaneously, from mouse tissue. We used this QuantiGene assay to show that, following pharmacological activation in vivo, the heat shock response impairment in tibialis anterior, brain hemispheres and striatum was comparable between zQ175 and R6/2 mice. In contrast, although a heat shock impairment could be detected in R6/2 cortex, this was not apparent in the cortex from zQ175 mice. Whilst the mechanism underlying this impairment remains unknown, our data indicated that it is not caused by a reduction in HSF1 levels, as had been reported.

PDI-Regulated Disulfide Bond Formation in Protein Folding and Biomolecular Assembly.

Disulfide bonds play a pivotal role in maintaining the natural structures of proteins to ensure their performance of normal biological functions. Moreover, biological molecular assembly, such as the gluten network, is also largely dependent on the intermolecular crosslinking via disulfide bonds. In eukaryotes, the formation and rearrangement of most intra- and intermolecular disulfide bonds in the endoplasmic reticulum (ER) are mediated by protein disulfide isomerases (PDIs), which consist of multiple thioredoxin-like domains. These domains assist correct folding of proteins, as well as effectively prevent the aggregation of misfolded ones. Protein misfolding often leads to the formation of pathological protein aggregations that cause many diseases. On the other hand, glutenin aggregation and subsequent crosslinking are required for the formation of a rheologically dominating gluten network. Herein, the mechanism of PDI-regulated disulfide bond formation is important for understanding not only protein folding and associated diseases, but also the formation of functional biomolecular assembly. This review systematically illustrated the process of human protein disulfide isomerase (hPDI) mediated disulfide bond formation and complemented this with the current mechanism of wheat protein disulfide isomerase (wPDI) catalyzed formation of gluten networks.

MPN-274: Mpl is Activated by Dimers of MPN-Linked Calreticulin Mutants Stabilized by Disulfide Bonds and Ionic Interactions

Myeloproliferative neoplasms (MPNs) are frequently driven by insertions and deletions within the gene encoding calreticulin (CRT) that alter the sequence and change the net charge of the C-terminal domain. CRTDel52 (type-I) and CRTIns5 (type-II) are recurrent mutations. Although oncogenic transformation requires both mutated CRT and the myeloproliferative leukemia protein (Mpl), the molecular mechanism of CRT-mediated constitutive activation of Mpl is unknown. Our studies reveal that the novel C-terminal domain of CRTDel52 encodes specificity both for Mpl binding and for disulfide-mediated CRT dimerization. Disulfide-stabilized CRT dimers and multimers are observed in MPN patient-derived platelet lysates and in transfected mammalian cells. Cysteine mutations within both the C-terminal domain (C400A and C404A) and the N-terminal domain (C163A) of CRTDel52 diminish disulfide-mediated dimers and multimers of CRTDel52. Based on these data and published structures of crystalized CRT oligomers, we propose a structural model of the Mpl-activating CRTDel52 unit as a covalently linked dimer that is stabilized by disulfides and ionic interactions at both the C-terminal and N-terminal domains. In support of this model, we found that elimination of intermolecular disulfides and disruption of ionic interactions at the proposed dimerization interfaces are required to abrogate the ability of CRTDel52 to induce cytokine-independent proliferation of Ba/F3 cells via Mpl. Based on docking studies, we hypothesize that productive CRTDel52 dimers bind and activate Mpl dimers by inserting their C-terminal tails onto the ligand-binding pocket of Mpl and by engaging immature Mpl N-linked glycans via their conventional N-terminal glycan-binding site. Funding: This work was funded by NIH grant (R01 AI123957 to MR) and the University of Michigan Fast Forward Protein Folding Diseases Initiative. I.D.P. was supported by the NSF Division of Biological Infrastructure (award 1855425).

Tropane-Based Ibogaine Analog Rescues Folding-Deficient Serotonin and Dopamine Transporters.

Missense mutations that give rise to protein misfolding are rare, but collectively, defective protein folding diseases are consequential. Folding deficiencies are amenable to pharmacological correction (pharmacochaperoning), but the underlying mechanisms remain enigmatic. Ibogaine and its active metabolite noribogaine correct folding defects in the dopamine transporter (DAT), but they rescue only a very limited number of folding-deficient DAT mutant proteins, which give rise to infantile Parkinsonism and dystonia. Herein, a series of analogs was generated by reconfiguring the complex ibogaine ring system and exploring the structural requirements for binding to wild-type transporters, as well as for rescuing two equivalent synthetic folding-deficient mutants, SERT-PG601,602AA and DAT-PG584,585AA. The most active tropane-based analog (9b) was also an effective pharmacochaperone in vivo in Drosophila harboring the DAT-PG584,585AA mutation and rescued 6 out of 13 disease-associated human DAT mutant proteins in vitro. Hence, a novel lead pharmacochaperone has been identified that demonstrates medication development potential for patients harboring DAT mutations.

Cytosolic Ca2+ modulates Golgi structure through PKC‐mediated GRASP55 phosphorylation

It has been well documented that the endoplasmic reticulum (ER) responds to cellular stresses through the unfolded protein response (UPR), but it is unknown how the Golgi apparatus responds to similar stresses. In this study, we treated HeLa cells with ER stress inducers, thapsigargin (TG), tunicamycin (Tu) and Dithiothreitol (DTT), and found that only TG treatment resulted in Golgi fragmentation. TG induced Golgi fragmentation at a low dose and short time when UPR was undetectable, demonstrating that Golgi fragmentation occurs independently of ER stress. Further experiments demonstrated that Golgi fragmentation was through elevated intracellular Ca2+ and protein kinase Cα (PKCα) activity, which phosphorylates the Golgi stacking protein GRASP55. Significantly, activation of PKCα with other activating or inflammatory agents, including Phorbol 12‐myristate 13‐acetate (PMA) and histamine, modulates the Golgi structure in a similar fashion. Hence, our study revealed a novel mechanism through which increased cytosolic Ca2+ modulates Golgi structure and function.Support or Funding InformationThis work was supported by the National Institutes of Health (Grants GM112786, GM105920, and GM130331), MCubed and the Fastforward Protein Folding Disease Initiative of the University of Michigan to Y. Wang. S. Ireland is a graduate student who has been partially supported by the Mary Sue and Kenneth Coleman Endowed Fellowship Fund from the University of Michigan.

Ferrocene-modified peptides as inhibitors against insulin amyloid aggregation based on molecular simulation.

Peptide-based inhibitors have gradually been implicated as drugs for treating protein folding diseases because of their favorable biocompatibility and low toxicity. To develop potential therapeutic strategies for amyloid-related disorders, short peptides modified by Fc, ferrocene-l-Phe-l-Phe (Fc-FF) and ferrocene-l-Phe-l-Tyr (Fc-FY), were used as inhibitors for the investigation of the aggregation behavior of insulin. Firstly, molecular docking predicted the interaction between both Fc-peptides and insulin. Then, the experimental data from ThT, DLS, CD and TEM confirmed that Fc-FF and Fc-FY effectively inhibited insulin fibrillation and disaggregated mature insulin fibrils. Based on a dose-dependent manner, both Fc-peptides can strongly inhibit insulin fibrillation, extend lag phase time, reduce final fibril formation (beyond 99% by Fc-peptides of 400 µM), decrease the formation of high-content β-sheet structures and reduce the size of insulin fibrils. Additionally, we found that compared with Fc-FY, the better inhibitory effect of Fc-FF at concentration below 400 µM was mainly resulted from the difference in π-π interaction and hydrogen bonds between Fc-peptides and insulin, according to molecular dynamics analysis. Our results demonstrated Fc-peptides, Fc-FF and Fc-FY, may play effective roles in the development of new therapeutic drugs or strategies for amyloid-related disorders, and the molecular dynamics simulation may be helpful for designing appropriate inhibitors of anti-amyloidosis diseases.

Million-fold sensitivity enhancement in proteopathic seed amplification assays for biospecimens by Hofmeister ion comparisons

Recent work with prion diseases and synucleinopathies indicates that accurate diagnostic methods for protein-folding diseases can be based on the ultrasensitive, amplified measurement of pathological aggregates in biospecimens. A better understanding of the physicochemical factors that control the seeded polymerization of such aggregates, and their amplification in vitro, should allow improvements in existing assay platforms, as well as the development of new assays for other proteopathic aggregates. Here, we systematically investigated the effects of the ionic environment on the polymerization of tau, α-synuclein, and the prion protein (PrP) induced by aggregates in biospecimens. We screened salts of the Hofmeister series, a relative ordering of strongly and weakly hydrated salts that tend to precipitate or solubilize proteins. We found that sensitivities of tau-based assays for Alzheimer's seeds and PrP-based assays for prions were best in weakly hydrated anions. In contrast, we saw an inverse trend with different tau-based assays, improving detection sensitivity for progressive supranuclear palsy seeds by ≈106 Hofmeister analysis also improved detection of sporadic Creutzfeldt-Jakob disease prions in human nasal brushings and chronic wasting disease prions in deer-ear homogenates. Our results demonstrate strong and divergent influences of ionic environments on the amplification and detection of proteopathic seeds as biomarkers for protein-folding diseases.

Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers–Danlos syndrome–associated substitutions

Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin α2β1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly → Xaa (Xaa = Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin α2β1-binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin α2β1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin α2-inserted (α2I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly → Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly → Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly → Val change broke the essential Mg2+ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.

Activation of Heat Shock Protein 70 as a Potential Therapeutic Strategy for the Treatment of Protein Folding Disease

The heat shock protein 90 and 70 (Hsp90/70) chaperone system provides an essential protein quality control mechanism for the cell. Heme containing enzymes, such as cytochrome P450s and neuronal nitric oxide synthase (nNOS), are susceptible to damage by xenobiotics and inactivation by specific environmental toxins or drugs leading to the selective degradation of these proteins. In the course of studies on the inactivation of nNOS, we discovered that covalent alteration of the heme binding cleft leads to misfolding of the cleft, which is selectively recognized by Hsp90/70. Hsp70 recruits the c‐terminus of Hsp70 interacting protein (CHIP), an E3 ubiquitin ligase, leading to ubiquitination and proteasomal degradation of nNOS. In an analogous manner the Hsp90/70 chaperone system also regulates other client proteins that are prone to misfolding and aggregation, such as the polyglutamine‐androgen receptor (polyQ‐AR) a genetic mutant whose misfolding and aggregation leads to muscular‐ and neuro‐degeneration in Kennedy's disease. We have demonstrated that genetic and pharmacological activation of Hsp70 increases the ubiquitination and degradation of misfolded nNOS and polyQ‐AR in cells and alleviates neurotoxicity in a Drosophila model of Kennedy's disease. To advance the development of Hsp70 activation as a therapeutic strategy for the treatment of neurodegenerative diseases, we have established a workflow to identify novel Hsp70 modulators that enhance degradation of misfolded Hsp90/70 client proteins, utilizing nNOS as a model. We have developed a thermal shift high‐throughput screen to identify novel compounds that bind and thermostabilize Hsp70. Preliminary results suggest that this method can be utilized to identify compounds that increase nNOS ubiquitination in vitro and promote degradation of nNOS in HEK293 cells. We are currently working to develop high‐throughput ELISA‐based secondary assays for the quantification of nNOS ubiquitination in vitro and nNOS levels and ubiquitination in cells. This workflow has the potential to advance the development of therapeutics for the treatment of Kennedy's disease and other neurodegenerative diseases such as Alzheimer's, Huntington's, and Parkinson's disease which are caused by misfolding and aggregation of the Hsp90/70 client proteins tau, huntingtin, and α‐synuclein.Support or Funding InformationThis work was supported, in whole or in part, by National Institutes of Health Grant R01‐GMO77430 and R21‐NS101030, the University of Michigan Medical School's Protein Folding Diseases Initiative, and University of Michigan's Center for the Discovery of New Medicines. AD is an awardee of a PhRMA Foundation Pre‐Doctoral Fellowship in Pharmacology/Toxicology and a trainee of the University of Michigan, Pharmacological Sciences Training Program T32‐GM007767.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Human DnaJB6 Antiamyloid Chaperone Protects Yeast from Polyglutamine Toxicity Separately from Spatial Segregation of Aggregates.

Polyglutamine (polyQ) aggregates are associated with pathology in protein-folding diseases and with toxicity in the yeast Saccharomyces cerevisiae Protection from polyQ toxicity in yeast by human DnaJB6 coincides with sequestration of aggregates. Gathering of misfolded proteins into deposition sites by protein quality control (PQC) factors has led to the view that PQC processes protect cells by spatially segregating toxic aggregates. Whether DnaJB6 depends on this machinery to sequester polyQ aggregates, if this sequestration is needed for DnaJB6 to protect cells, and the identity of the deposition site are unknown. Here, we found DnaJB6-driven deposits share characteristics with perivacuolar insoluble protein deposition sites (IPODs). Binding of DnaJB6 to aggregates was necessary, but not enough, for detoxification. Focal formation required a DnaJB6-Hsp70 interaction and actin, polyQ could be detoxified without sequestration, and segregation of aggregates alone was not protective. Our findings suggest DnaJB6 binds to smaller polyQ aggregates to block their toxicity. Assembly and segregation of detoxified aggregates are driven by an Hsp70- and actin-dependent process. Our findings show sequestration of aggregates is not the primary mechanism by which DnaJB6 suppresses toxicity and raise questions regarding how and when misfolded proteins are detoxified during spatial segregation.

Natural Genetic Variation in Yeast Reveals That NEDD4 Is a Conserved Modifier of Mutant Polyglutamine Aggregation.

A feature common to late onset proteinopathic disorders is an accumulation of toxic protein conformers and aggregates in affected tissues. In the search for potential drug targets, many studies used high-throughput screens to find genes that modify the cytotoxicity of misfolded proteins. A complement to this approach is to focus on strategies that use protein aggregation as a phenotypic readout to identify pathways that control aggregate formation and maintenance. Here we use natural variation between strains of budding yeast to genetically map loci that influence the aggregation of a polyglutamine-containing protein derived from a mutant form of huntingtin, the causative agent in Huntington disease. Linkage analysis of progeny derived from a cross between wild and laboratory yeast strains revealed two polymorphic loci that modify polyglutamine aggregation. One locus contains the gene RFU1 which modifies ubiquitination states of misfolded proteins targeted by the E3-ubiquitin ligase complex Rsp5. Activity of the Rsp5 complex, and the mammalian homolog NEDD4, are critical in maintaining protein homeostasis in response to proteomic stress. Our analysis also showed linkage of the aggregation phenotype to a distinct locus containing a gene encoding the Rsp5-interacting Bul2 protein. Allele-swap experiments validated the impact of both RFU1 and BUL2 on huntingtin aggregation. Furthermore, we found that the nematode Caenorhabditis elegans’ ortholog of Rsp5, wwp-1, also negatively regulates polyglutamine aggregation. Knockdown of the NEDD4 in human cells likewise altered polyglutamine aggregation. Taken together, these results implicate conserved processes involving the ubiquitin regulation network that modify protein aggregation and provide novel therapeutic targets for polyglutamine and other protein folding diseases.

Biophysical and Spectroscopic Methods for Monitoring Protein Misfolding and Amyloid Aggregation.

Proteins exhibit a remarkable structural plasticity and may undergo conformational changes resulting in protein misfolding both in a biological context and upon perturbing physiopathological conditions. Such nonfunctional protein conformers, including misfolded states and aggregates, are often associated to protein folding diseases. Understanding the biology of protein folding diseases thus requires tools that allow the structural characterization of nonnative conformations of proteins and their interconversions. Here we present detailed procedures to monitor protein conformational changes and aggregation based on spectroscopic and biophysical methods that include circular dichroism, ATR-Fourier-transformed infrared spectroscopy, fluorescence spectroscopy and dynamic light scattering. To illustrate the application of these methods we report to our previous studies on misfolding, aggregation and amyloid fibril formation by superoxide dismutase 1 (SOD1), a protein whose toxic deposition is implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS).