Protein Aggregation Diseases Research Articles

Aggregation and disaggregation features of the human proteome

Protein aggregates have negative implications in disease. While reductionist experiments have increased our understanding of aggregation processes, the systemic view in biological context is still limited. To extend this understanding, we used mass spectrometry‐based proteomics to characterize aggregation and disaggregation in human cells after non‐lethal heat shock. Aggregation‐prone proteins were enriched in nuclear proteins, high proportion of intrinsically disordered regions, high molecular mass, high isoelectric point, and hydrophilic amino acids. During recovery, most aggregating proteins disaggregated with a rate proportional to the aggregation propensity: larger loss in solubility was counteracted by faster disaggregation. High amount of intrinsically disordered regions were associated with faster disaggregation. However, other characteristics enriched in aggregating proteins did not correlate with the disaggregation rates. In addition, we analyzed changes in protein thermal stability after heat shock. Soluble remnants of aggregated proteins were more thermally stable compared with control condition. Therefore, our results provide a rich resource of heat stress‐related protein solubility data and can foster further studies related to protein aggregation diseases.

Rational Design of Monodispersed Mutants of Proteins by Identifying Aggregation Contact Sites Using Solubilizing Agents.

Suppression of protein aggregation is a subject of growing importance in the treatment of protein aggregation diseases, an urgent worldwide human health problem, and the production of therapeutic proteins, such as antibody drugs. We previously reported a method to identify compounds that suppress aggregation, based on screening using multiple terminal deletion mutants. We now present a method to determine the aggregation contact sites of proteins, using such solubilizing compounds, to design monodispersed mutants. We applied this strategy to the chemokine receptor-binding domain (CRBD) of FROUNT, which binds to the membrane-proximal C-terminal intracellular region of CCR2. Initially, the backbone NMR signals were assigned to a certain extent by available methods, and the putative locations of five α-helices were identified. Based on NMR chemical shift perturbations upon varying the protein concentrations, the first and third helices were found to contain the aggregation contact sites. The two helices are amphiphilic, and based on an NMR titration with 1,6-hexanediol, a CRBD solubilizing compound, the contact sites were identified as the hydrophobic patches located on the hydrophilic sides of the two helices. Subsequently, we designed multiple mutants targeting amino acid residues on the contact sites. Based on their NMR spectra, a doubly mutated CRBD (L538E/P612S) was selected from the designed mutants, and its monodispersed nature was confirmed by other biophysical methods. We then assessed the CCR2-binding activities of the mutants. Our method is useful for the protein structural analyses, the treatment of protein aggregation diseases, and the improvement of therapeutic proteins.

Impact of Four Common Hydrogels on Amyloid-β (Aβ) Aggregation and Cytotoxicity: Implications for 3D Models of Alzheimer's Disease.

The physiochemical properties of hydrogels utilized in 3D culture can be used to modulate cell phenotype and morphology with a striking resemblance to cellular processes that occur in vivo. Indeed, research areas including regenerative medicine, tissue engineering, in vitro cancer models, and stem cell differentiation have readily utilized 3D biomaterials to investigate cell biological questions. However, cells are only one component of this biomimetic milieu. In many models of disease such as Alzheimer’s disease (AD) that could benefit from the in vivo-like cell morphology associated with 3D culture, other aspects of the disease such as protein aggregation have yet to be methodically considered in this 3D context. A hallmark of AD is the accumulation of the peptide amyloid-β (Aβ), whose aggregation is associated with neurotoxicity. We have previously demonstrated the attenuation of Aβ cytotoxicity when cells were cultured within type I collagen hydrogels versus on 2D substrates. In this work, we investigated the extent to which this phenomenon is conserved when Aβ is confined within hydrogels of varying physiochemical properties, notably mesh size and bioactivity. We investigated the Aβ structure and aggregation kinetics in solution and hydrogels composed of type I collagen, agarose, hyaluronic acid, and polyethylene glycol using fluorescence correlation spectroscopy and thioflavin T assays. Our results reveal that all hydrogels tested were associated with enhanced Aβ aggregation and Aβ cytotoxicity attenuation. We suggest that confinement itself imparts a profound effect, possibly by stabilizing Aβ structures and shifting the aggregate equilibrium toward larger species. If this phenomenon of altered protein aggregation in 3D hydrogels can be generalized to other contexts including the in vivo environment, it may be necessary to reevaluate aspects of protein aggregation disease models used for drug discovery.

Organismal Protein Homeostasis Mechanisms.

Sustaining a healthy proteome is a lifelong challenge for each individual cell of an organism. However, protein homeostasis or proteostasis is constantly jeopardized since damaged proteins accumulate under proteotoxic stress that originates from ever-changing metabolic, environmental, and pathological conditions. Proteostasis is achieved via a conserved network of quality control pathways that orchestrate the biogenesis of correctly folded proteins, prevent proteins from misfolding, and remove potentially harmful proteins by selective degradation. Nevertheless, the proteostasis network has a limited capacity and its collapse deteriorates cellular functionality and organismal viability, causing metabolic, oncological, or neurodegenerative disorders. While cell-autonomous quality control mechanisms have been described intensely, recent work on Caenorhabditis elegans has demonstrated the systemic coordination of proteostasis between distinct tissues of an organism. These findings indicate the existence of intricately balanced proteostasis networks important for integration and maintenance of the organismal proteome, opening a new door to define novel therapeutic targets for protein aggregation diseases. Here, we provide an overview of individual protein quality control pathways and the systemic coordination between central proteostatic nodes. We further provide insights into the dynamic regulation of cellular and organismal proteostasis mechanisms that integrate environmental and metabolic changes. The use of C. elegans as a model has pioneered our understanding of conserved quality control mechanisms important to safeguard the organismal proteome in health and disease.

Differential activation of eMI by distinct forms of cellular stress

ABSTRACT As one of the major, highly conserved catabolic pathways, autophagy delivers cytosolic components to lysosomes for degradation. It is essential for development, cellular homeostasis, and coping with stress. Reduced autophagy increases susceptibility to protein aggregation diseases and leads to phenotypes associated with aging. Of the three major forms of autophagy, macroautophagy (MA) can degrade organelles or aggregated proteins, and chaperone-mediated autophagy is specific for soluble proteins containing KFERQ-related targeting motifs. During endosomal microautophagy (eMI), cytoplasmic proteins are engulfed into late endosomes in an ESCRT machinery-dependent manner. eMI can be KFERQ-specific or occur in bulk and be induced by prolonged starvation. Its physiological regulation and function, however, are not understood. Here, we show that eMI in the Drosophila fat body, akin to the mammalian liver, is induced upon oxidative or genotoxic stress in an ESCRT and partially Hsc70-4-dependent manner. Interestingly, eMI activation is selective, as ER stress fails to elicit a response. Intriguingly, we find that reducing MA leads to a compensatory enhancement of eMI, suggesting a tight interplay between these degradative processes. Furthermore, we show that mutations in DNA damage response genes are sufficient to trigger eMI and that the response to oxidative stress is under the control of MAPK/JNK signaling. Our data suggest that, controlled by various signaling pathways, eMI allows an organ to react and adapt to specific types of stress and is thus likely critical to prevent disease. Abbreviations: Atg: autophagy-related; CMA: chaperone-mediated autophagy; DDR: DNA damage repair; Df: deficiency (deletion); (E)GFP: (enhanced) green fluorescent protein; eMI: endosomal microautophagy; ER: endoplasmatic reticulum; ESCRT: endosomal sorting complexes required for transport; Eto: etoposide; FLP: flipase; Hsc: heat shock cognate protein; LAMP2A: lysosomal-associated membrane protein 2A; LE: late endosome; MA: macroautophagy; MI: microautophagy; MVB: multivesicular body; PA: photoactivatable; Para: paraquat; ROS: reactive oxygen species; SEM: standard error of means; Tor: target of rapamycin [serine/threonine kinase]; UPR: unfolded protein response; Vps: vacuolar protein sorting.

Liquid-liquid phase separation in autophagy.

Liquid-liquid phase separation (LLPS) compartmentalizes and concentrates biomacromolecules into distinct condensates. Liquid-like condensates can transition into gel and solid states, which are essential for fulfilling their different functions. LLPS plays important roles in multiple steps of autophagy, mediating the assembly of autophagosome formation sites, acting as an unconventional modulator of TORC1-mediated autophagy regulation, and triaging protein cargos for degradation. Gel-like, but not solid, protein condensates can trigger formation of surrounding autophagosomal membranes. Stress and pathological conditions cause aberrant phase separation and transition of condensates, which can evade surveillance by the autophagy machinery. Understanding the mechanisms underlying phase separation and transition will provide potential therapeutic targets for protein aggregation diseases.

Comparative analysis of the intracellular responses to disease-related aggregation-prone proteins

Aggregation-prone proteins (APPs) have been implicated in numerous human diseases but the underlying mechanisms are incompletely understood. Here we comparatively analysed cellular responses to different APPs. Our study is based on a systematic proteomic and phosphoproteomic analysis of a set of yeast proteotoxicity models expressing different human disease-related APPs, which accumulate intracellular APP inclusions and exhibit impaired growth. Clustering and functional enrichment analyses of quantitative proteome-level data reveal that the cellular response to APP expression, including the chaperone response, is specific to the APP, and largely differs from the response to a more generalized proteotoxic insult such as heat shock. We further observe an intriguing association between the subcellular location of inclusions and the location of the cellular response, and provide a rich dataset for future mechanistic studies. Our data suggest that care should be taken when designing research models to study intracellular aggregation, since the cellular response depends markedly on the specific APP and the location of inclusions. Further, therapeutic approaches aimed at boosting protein quality control in protein aggregation diseases should be tailored to the subcellular location affected by inclusion formation. SignificanceWe have examined the global cellular response, in terms of protein abundance and phosphorylation changes, to the expression of five human neurodegeneration-associated, aggregation-prone proteins (APPs) in a set of isogenic yeast models. Our results show that the cellular response to each APP is unique to that protein, is different from the response to thermal stress, and is associated with processes at the subcellular location of APP inclusion formation. These results further our understanding of how cells, in a model organism, respond to expression of APPs implicated in neurodegenerative diseases like Parkinson's, Alzheimer's, and ALS. They have implications for mechanisms of toxicity as well as of protective responses in the cell. The specificity of the response to each APP means that research models of these diseases should be tailored to the APP in question. The subcellular localization of the response suggest that therapeutic interventions should also be targeted within the cell.

Human γS-Crystallin-Copper Binding Helps Buffer against Aggregation Caused by Oxidative Damage.

Divalent metal cations can play a role in protein aggregation diseases, including cataract. Here we compare the aggregation of human γS-crystallin, a key structural protein of the eye lens, via mutagenesis, ultraviolet light damage, and the addition of metal ions. All three aggregation pathways result in globular, amorphous-looking structures that do not elongate into fibers. We also investigate the molecular mechanism underlying copper(II)-induced aggregation. This work was motivated by the observation that zinc(II)-induced aggregation of γS-crystallin is driven by intermolecular bridging of solvent-accessible cysteine residues, while in contrast, copper(II)-induced aggregation of this protein is exacerbated by the removal of solvent-accessible cysteines via mutation. Here we find that copper(II)-induced aggregation results from a complex mechanism involving multiple interactions with the protein. The initial protein-metal interactions result in the reduction of Cu(II) to Cu(I) with concomitant oxidation of γS-crystallin. In addition to the intermolecular disulfides that represent a starting point for aggregation, intramolecular disulfides also occur in the cysteine loop, a region of the N-terminal domain that was previously found to mediate the early stages of cataract formation. This previously unobserved ability of γS-crystallin to transfer disulfides intramolecularly suggests that it may serve as an oxidation sink for the lens after glutathione levels have become depleted during aging. γS-Crystallin thus serves as the last line of defense against oxidation in the eye lens, a result that underscores the chemical functionality of this protein, which is generally considered to play a purely structural role.

The actin cytoskeletal network plays a role in yeast prion transmission and contributes to prion stability.

Chaperone networks are required for the shearing and generation of transmissible propagons from pre-existing prion aggregates. However, other cellular networks needed for maintaining yeast prions are largely uncharacterized. Here, we establish a novel role for actin networks in prion maintenance. The [PIN+ ] prion, also known as [RNQ+ ], exists as stable variants dependent upon the chaperone machinery for the transmission of propagons to daughter cells during cell division and cytoplasmic transfer. Loss of the Hsp104 molecular chaperone leads to the growth of prion particles until they are too large to be transmitted. Here, we isolated a unique [PIN+ ] variant, which is unstable in actin mutants. This prion loss is observed over many generations, and coincides with the detection of both high molecular weight species of Rnq1 and large visible aggregates that are asymmetrically retained during cell division. Our data suggest that the irregular actin networks found in these mutants may influence propagon number by slowly permitting aggregate growth over time, resulting in the generation of nontransmissible large aggregates. Thus, we show the potential contribution of cytoskeletal networks in the transmission of prion propagons, which parallels models that have been proposed for cell-to-cell transmission of small amyloids in neurodegenerative protein aggregation diseases.

Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover.

The inability to remove protein aggregates in post-mitotic cells such as muscles or neurons is a cellular hallmark of aging cells and is a key factor in the initiation and progression of protein misfolding diseases. While protein aggregate disorders share common features, the molecular level events that culminate in abnormal protein accumulation cannot be explained by a single mechanism. Here we show that loss of the serine/threonine kinase NUAK causes cellular degeneration resulting from the incomplete clearance of protein aggregates in Drosophila larval muscles. In NUAK mutant muscles, regions that lack the myofibrillar proteins F-actin and Myosin heavy chain (MHC) instead contain damaged organelles and the accumulation of select proteins, including Filamin (Fil) and CryAB. NUAK biochemically and genetically interacts with Drosophila Starvin (Stv), the ortholog of mammalian Bcl-2-associated athanogene 3 (BAG3). Consistent with a known role for the co-chaperone BAG3 and the Heat shock cognate 71 kDa (HSC70)/HSPA8 ATPase in the autophagic clearance of proteins, RNA interference (RNAi) of Drosophila Stv, Hsc70-4, or autophagy-related 8a (Atg8a) all exhibit muscle degeneration and muscle contraction defects that phenocopy NUAK mutants. We further demonstrate that Fil is a target of NUAK kinase activity and abnormally accumulates upon loss of the BAG3-Hsc70-4 complex. In addition, Ubiquitin (Ub), ref(2)p/p62, and Atg8a are increased in regions of protein aggregation, consistent with a block in autophagy upon loss of NUAK. Collectively, our results establish a novel role for NUAK with the Stv-Hsc70-4 complex in the autophagic clearance of proteins that may eventually lead to treatment options for protein aggregate diseases.

Targeting Inflammation, PHA-767491 Shows a Broad Spectrum in Protein Aggregation Diseases

Many protein aggregation diseases (PAD) affect the nervous system. Deposits of aggregated disease-specific proteins are found within or around the neuronal cells of neurodegenerative diseases. Although the main protein component is disease-specific, oligomeric aggregates are presumed to be the key agents causing the neurotoxicity. Evidence has shown that protein aggregates cause a chronic inflammatory reaction in the brain, resulting in neurodegeneration. Therefore, strategies targeting anti-inflammation could be beneficial to the therapeutics of PAD. PHA-767491 was originally identified as an inhibitor of CDC7/CDK9 and was found to reduce TDP-43 phosphorylation and prevent neurodegeneration in TDP-43 transgenic animals. We recently identified PHA-767491 as a GSK-3β inhibitor. In this study, we established mouse hippocampal primary culture with tau-hyperphosphorylation through the activation of GSK-3β using Wortmannin and GF109203X. We found that PHA-767491 significantly improved the neurite outgrowth of hippocampal primary neurons against the neurotoxicity induced by GSK-3β. We further showed that PHA-767491 had neuroprotective ability in hippocampal primary culture under oligomeric Aβ treatment. In addition, PHA-767491 attenuated the neuroinflammation in mouse cerebellar slice culture with human TBP-109Q agitation. Further study of SCA17 transgenic mice carrying human TBP-109Q showed that PHA-767491 ameliorated the gait ataxia and the inflammatory response both centrally and peripherally. Our findings suggest that PHA-767491 has a broad spectrum of activity in the treatment of different PAD and that this activity could be based on the anti-inflammation mechanism.

Mitophagy in the Retinal Pigment Epithelium of Dry Age-Related Macular Degeneration Investigated in the NFE2L2/PGC-1α-/- Mouse Model.

Increased oxidative stress and mitochondrial damage are observed in protein aggregation diseases, such as age-related macular degeneration (AMD). We have recently reported elevated levels of oxidative stress markers, damaged mitochondria, accumulating lysosomal lipofuscin and extracellular drusen-like structures in the retinal pigment epithelial cells (RPE) of the dry AMD-resembling NFE2L2/PGC1α double knockout (dKO) mouse model. Here, we provide evidence of a disturbance in the autolysosomal machinery handling mitochondrial clearance in the RPE cells of one-year-old NFE2L2/PGC1α-deficient mice. Confocal immunohistochemical analysis revealed an upregulation of autophagosome marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) as well as numerous mitophagy markers, such as PTE-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase (PARKIN) together with damaged mitochondria. However, we detected no evidence of increased autolysosome formation in transmission electron micrographs or of colocalization of lysosomal marker LAMP2 (lysosome-associated membrane protein 2) and the mitochondrial marker ATP synthase β in confocal micrographs. Interestingly, we observed an upregulation of late autolysosomal fusion Ras-related protein (Rab7) in the perinuclear space of RPE cells together with autofluorescence aggregates. Our results reveal that there is at least a relative decrease of mitophagy in the RPE cells of NFE2L2/PGC1α dKO mice. This further supports the hypothesis that mitophagy is a putative therapy target in AMD-like pathology.

Unexpected cell type-dependent effects of autophagy on polyglutamine aggregation revealed by natural genetic variation in C. elegans

BackgroundMonogenic protein aggregation diseases, in addition to cell selectivity, exhibit clinical variation in the age of onset and progression, driven in part by inter-individual genetic variation. While natural genetic variants may pinpoint plastic networks amenable to intervention, the mechanisms by which they impact individual susceptibility to proteotoxicity are still largely unknown.ResultsWe have previously shown that natural variation modifies polyglutamine (polyQ) aggregation phenotypes in C. elegans muscle cells. Here, we find that a genomic locus from C. elegans wild isolate DR1350 causes two genetically separable aggregation phenotypes, without changing the basal activity of muscle proteostasis pathways known to affect polyQ aggregation. We find that the increased aggregation phenotype was due to regulatory variants in the gene encoding a conserved autophagy protein ATG-5. The atg-5 gene itself conferred dosage-dependent enhancement of aggregation, with the DR1350-derived allele behaving as hypermorph. Surprisingly, increased aggregation in animals carrying the modifier locus was accompanied by enhanced autophagy activation in response to activating treatment. Because autophagy is expected to clear, not increase, protein aggregates, we activated autophagy in three different polyQ models and found a striking tissue-dependent effect: activation of autophagy decreased polyQ aggregation in neurons and intestine, but increased it in the muscle cells.ConclusionsOur data show that cryptic natural variants in genes encoding proteostasis components, although not causing detectable phenotypes in wild-type individuals, can have profound effects on aggregation-prone proteins. Clinical applications of autophagy activators for aggregation diseases may need to consider the unexpected divergent effects of autophagy in different cell types.

Desmin Forms Toxic, Seeding Competent Amyloid Aggregates that Persist in Muscle Fibers

Desmin-associated myofibrillar myopathy (MFM) has pathologic similarities to neurodegeneration-associated protein aggregate diseases. Desmin is an abundant muscle-specific intermediate filament, and disease mutations lead to its aggregation in cells, animals, and patients. We reasoned that similar to neurodegeneration-associated proteins, desmin itself may form amyloid. Desmin peptides corresponding to putative amyloidogenic regions formed seeding-competent amyloid fibrils. Amyloid formation was increased when disease-associated mutations were made within the peptide, and this conversion was inhibited by the anti-amyloid compound epigallocatechin-gallate. Moreover, a purified desmin fragment (aa 117 to 348) containing both amyloidogenic regions formed amyloid fibrils under physiologic conditions. Desmin fragment-derived amyloid coaggregated with full-length desmin and was able to template its conversion into fibrils in vitro. Desmin amyloids were cytotoxic to myotubes and disrupted their myofibril organization compared with desmin monomer or other nondesmin amyloids. Finally, desmin fragment amyloid persisted when introduced into mouse skeletal muscle. These data suggest that desmin forms seeding-competent amyloid that is toxic to myofibers. Moreover, small molecules known to interfere with amyloid formation and propagation may have therapeutic potential in MFM.

Single-residue physicochemical characteristics kinetically partition membrane protein self-assembly and aggregation.

Ninety-five percent of all transmembrane proteins exist in kinetically trapped aggregation-prone states that have been directly linked to neurodegenerative diseases. Interestingly, the primary sequence almost invariably avoids off-pathway aggregate formation, by folding reliably into its native, thermodynamically stabilized structure. However, with the rising incidence of protein aggregation diseases, it is now important to understand the underlying mechanism(s) of membrane protein aggregation. Micromolecular physicochemical and biochemical alterations in the primary sequence that trigger the formation of macromolecular cross-β aggregates can be measured only through combinatorial spectroscopic experiments. Here, we developed spectroscopic thermal perturbation with 117 experimental variables to assess how subtle protein sequence variations drive the molecular transition of the folded protein to oligomeric aggregates. Using the Yersinia pestis outer transmembrane β-barrel Ail as a model, we delineated how a single-residue substitution that alters the membrane-anchoring ability of Ail significantly contributes to the kinetic component of Ail stability. We additionally observed a stabilizing role for interface aliphatics, and that interface aromatics physicochemically contribute to Ail self-assembly and aggregation. Moreover, our method identified the formation of structured oligomeric intermediates during Ail aggregation. We show that the self-aggregation tendency of Ail is offset by the evolution of a thermodynamically compromised primary sequence that balances folding, stability, and oligomerization. Our approach provides critical information on how subtle changes in protein primary sequence trigger cross-β fibril formation, with insights that have direct implications for deducing the molecular progression of neurodegeneration and amyloidogenesis in humans.

Aggregate Interactome Based on Protein Cross-linking Interfaces Predicts Drug Targets to Limit Aggregation in Neurodegenerative Diseases.

SummaryDiagnosis of neurodegenerative diseases hinges on “seed” proteins detected in disease-specific aggregates. These inclusions contain diverse constituents, adhering through aberrant interactions that our prior data indicate are nonrandom. To define preferential protein-protein contacts mediating aggregate coalescence, we created click-chemistry reagents that cross-link neighboring proteins within human, APPSw-driven, neuroblastoma-cell aggregates. These reagents incorporate a biotinyl group to efficiently recover linked tryptic-peptide pairs. Mass-spectroscopy outputs were screened for all possible peptide pairs in the aggregate proteome. These empirical linkages, ranked by abundance, implicate a protein-adherence network termed the “aggregate contactome.” Critical hubs and hub-hub interactions were assessed by RNAi-mediated rescue of chemotaxis in aging nematodes, and aggregation-driving properties were inferred by multivariate regression and neural-network approaches. Aspirin, while disrupting aggregation, greatly simplified the aggregate contactome. This approach, and the dynamic model of aggregate accrual it implies, reveals the architecture of insoluble-aggregate networks and may reveal targets susceptible to interventions to ameliorate protein-aggregation diseases.

Fine Tuning: Effects of Post-Translational Modification on Hsp70 Chaperones.

The discovery of heat shock proteins shaped our view of protein folding in the cell. Since their initial discovery, chaperone proteins were identified in all domains of life, demonstrating their vital and conserved functional roles in protein homeostasis. Chaperone proteins maintain proper protein folding in the cell by utilizing a variety of distinct, characteristic mechanisms to prevent aberrant intermolecular interactions, prevent protein aggregation, and lower entropic costs to allow for protein refolding. Continued study has found that chaperones may exhibit alternative functions, including maintaining protein folding during endoplasmic reticulum (ER) import and chaperone-mediated degradation, among others. Alternative chaperone functions are frequently controlled by post-translational modification, in which a given chaperone can switch between functions through covalent modification. This review will focus on the Hsp70 class chaperones and their Hsp40 co-chaperones, specifically highlighting the importance of post-translational control of chaperones. These modifications may serve as a target for therapeutic intervention in the treatment of diseases of protein misfolding and aggregation.

Desmin forms toxic, seeding-competent amyloid aggregates that persist in muscle fibers

Desmin-associated myofibrillar myopathy (MFM) has pathologic similarities to neurodegeneration-associated protein aggregate diseases. Desmin is an abundant muscle-specific intermediate filament, and disease mutations lead to its aggregation in cells, animals, and patients. We reasoned that similar to neurodegeneration-associated proteins, desmin itself may form amyloid. Desmin peptides corresponding to putative amyloidogenic regions formed seeding-competent amyloid fibrils. Amyloid formation was increased when disease-associated mutations were made within the peptide, and this conversion was inhibited by the anti-amyloid compound epigallocatechin-gallate. Moreover, a purified desmin fragment (aa 117 to 348) containing both amyloidogenic regions formed amyloid fibrils under physiologic conditions. Desmin fragment-derived amyloid coaggregated with full-length desmin and was able to template its conversion into fibrils in vitro. Desmin amyloids were cytotoxic to myotubes and disrupted their myofibril organization compared with desmin monomer or other nondesmin amyloids. Finally, desmin fragment amyloid persisted when introduced into mouse skeletal muscle. These data suggest that desmin forms seeding-competent amyloid that is toxic to myofibers. Moreover, small molecules known to interfere with amyloid formation and propagation may have therapeutic potential in MFM.

The Role of Protein Thermodynamics and Primary Structure in Fibrillogenesis of Variable Domains from Immunoglobulin Light Chains

Immunoglobulin light-chain amyloidosis is a protein aggregation disease that leads to proteinaceous deposits in a variety of organs in the body and, if untreated, ultimately results in death. The mechanisms by which light-chain aggregation occurs are not well understood. Here we have used solution NMR spectroscopy and biophysical studies to probe immunoglobulin variable domain λV6-57 VL aggregation, a process that appears to drive the degenerative phenotypes in amyloidosis patients. Our results establish that aggregation proceeds via the unfolded state. We identify, through NMR relaxation experiments recorded on the unfolded domain ensemble, a series of hotspots that could be involved in the initial phases of aggregate formation. Mutational analysis of these hotspots reveals that the region that includes K16-R24 is particularly aggregation prone. Notably, this region includes the site of the R24G substitution, a mutation that is found in variable domains of λ light-chain deposits in 25% of patients. The R24G λV6-57 VL domain aggregates more rapidly than would be expected on the basis of thermodynamic stability alone, while substitutions in many of the aggregation-prone regions significantly slow down fibril formation.

XBP1 signalling is essential for alleviating mutant protein aggregation in ER-stress related skeletal disease.

The unfolded protein response (UPR) is a conserved cellular response to the accumulation of proteinaceous material in endoplasmic reticulum (ER), active both in health and disease to alleviate cellular stress and improve protein folding. Multiple epiphyseal dysplasia (EDM5) is a genetic skeletal condition and a classic example of an intracellular protein aggregation disease, whereby mutant matrilin-3 forms large insoluble aggregates in the ER lumen, resulting in a specific ‘disease signature’ of increased expression of chaperones and foldases, and alternative splicing of the UPR effector XBP1. Matrilin-3 is expressed exclusively by chondrocytes thereby making EDM5 a perfect model system to study the role of protein aggregation in disease. In order to dissect the role of XBP1 signalling in aggregation-related conditions we crossed a p.V194D Matn3 knock-in mouse model of EDM5 with a mouse line carrying a cartilage specific deletion of XBP1 and analysed the resulting phenotype. Interestingly, the growth of mice carrying the Matn3 p.V194D mutation compounded with the cartilage specific deletion of XBP1 was severely retarded. Further phenotyping revealed increased intracellular retention of amyloid-like aggregates of mutant matrilin-3 coupled with dramatically decreased cell proliferation and increased apoptosis, suggesting a role of XBP1 signalling in protein accumulation and/or degradation. Transcriptomic analysis of chondrocytes extracted from wild type, EDM5, Xbp1-null and compound mutant lines revealed that the alternative splicing of Xbp1 is crucial in modulating levels of protein aggregation. Moreover, through detailed transcriptomic comparison with a model of metaphyseal chondrodysplasia type Schmid (MCDS), an UPR-related skeletal condition in which XBP1 was removed without overt consequences, we show for the first time that the differentiation-state of cells within the cartilage growth plate influences the UPR resulting from retention of a misfolded mutant protein and postulate that modulation of XBP1 signalling pathway presents a therapeutic target for aggregation related conditions in cells undergoing proliferation.