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A non-invasive and sensitive blood test has long been a goal for early stage disease diagnosis and treatment for Alzheimers disease (AD) and other proteinopathy diseases. We previously reported that preeclampsia (PE), a severe pregnancy complication, is another proteinopathy disorder with impaired autophagy. We hypothesized that induced autophagy deficiency would promote accumulation of pathologic protein aggregates. Here, we describe a novel, sensitive assay that detects serum protein aggregates from patients with PE (n=33 early onset and 33 late onset) and gestational age-matched controls (n=77) as well as AD in both dementia and prodromal mild cognitive impairment (MCI, n=24) stages with age-matched controls (n=19). The assay employs exposure of genetically engineered, autophagy-deficient human trophoblasts (ADTs) to serum from patients. The aggregated protein complexes and their individual components, including transthyretin, amyloid beta -42, alpha -synuclein, and phosphorylated tau231, can be detected and quantified by co-staining with ProteoStat, a rotor dye with affinity to aggregated proteins, and respective antibodies. Detection of protein aggregates in ADTs was not dependent on transcriptional upregulation of these biomarkers. The ROC curve analysis validated the robustness of the assay for its specificity and sensitivity (PE; AUC: 1, CI: 0.949-1.00; AD; AUC: 0.986, CI: 0.832-1.00). In conclusion, we have developed a novel, noninvasive diagnostic and predictive assay for AD, MCI and PE. Funding Agencies|NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [P20 GM121298, 3P20GM121298-04W1, P30 GM114750]; Brown University DEANS Award; Brown University Seed Award; William and Mary Oh-William and Elsa Zopfi Professorship Award

Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is a multitasking RNA/DNA binding protein. FUS aggregation is implicated in various neurodegenerative diseases. RNA was suggested to modulate phase transition of FUS. Here, we found that FUS transforms into the amorphous aggregation state as an instant response to the shear stress caused by usual pipetting even at a low FUS concentration, 100 nM. It was revealed that non-coding RNA can suppress the transformation of FUS into aggregates. The suppressive effect of RNA on FUS aggregation is sequence-dependent. These results suggested that the non-coding RNA could be a prospective suppressor of FUS aggregation caused by mechanistic stress in cells. Our finding might pave the way for more research on the role of RNAs as aggregation inhibitors, which could facilitate the development of therapies for neurodegenerative diseases.

Microcapsules and microgels consisting of macromolecular networks have received increasing attention due to their biomedical and pharmaceutical applications. Protein microgels and in particular silk-based microcapsules have desirable properties due to their biocompatibility and lack of toxicity. Typically such structures formed through emulsion templating are spherical in geometry due to interfacial tension. However, approaches to synthesis particles with more complex and non-spherical geometries are sought due to their packing properties and cargo release characteristics. Here, we describe a droplet-microfluidic strategy for generating asymmetric tubular-like microgels from reconstituted silk fibroin; a major component of native silk. It was determined using fluorescence microscopy, that the shear stress within the microchannel promotes surface protein aggregation, resulting in the asymmetric morphology of the microgels. Moreover, the structural transition that the protein undergoes was confirmed using FTIR. Crucially, the core of the microgels remains liquid, while the surface has fully aggregated into a fibrillar network. Additionally, we show that microgel morphology could be controlled by varying the dispersed to continuous phase flow rates, while it was determined that the radius of curvature of the asymmetric microgels is correlated to the wall shear stress. By comparing the surface fluorescence intensity of the microgels as a function of radius of curvature, the effect of the shear stress on the amount of aggregation could be quantified. Finally, the potential use of these asymmetric microgels as carriers of cargo molecules is showcased. As the core of the microgel remains liquid but the shell has gelled, this approach is highly suitable for the storage of bio-active cargo molecules such as antibodies, making such a delivery system attractive in the context of biomedical and pharmaceutical applications.

Distinct mutations in the secreted extracellular matrix protein, fibulin-3 (F3), have been associated with a number of ocular diseases ranging from primary open angle glaucoma to cuticular age-related macular degeneration to a rare macular dystrophy, Malattia Leventinese (ML). The R345W F3 mutation that causes ML leads to F3 misfolding, inefficient secretion and accumulation at higher intracellular steady state levels in cultured cells. Herein, we determined whether fifteen other clinically-identified F3 mutations also led to similar levels of misfolding and secretion defects, which might provide insight into their potential pathogenicity. Surprisingly, we found that only a single F3 variant, L451F, presented with a significant secretion defect (69.5 ± 2.4% of wild-type (WT) F3 levels) and a corresponding increase in intracellular levels (226.8 ± 25.4% of WT F3 levels). Upon follow-up studies, when this conserved residue (L451) was mutated to a charged (Asp or Arg) or bulky (Pro, Trp, Tyr) residue, F3 secretion was also compromised, indicating the importance of small side chains (Leu, Ala, or Gly) at this residue. To uncover potential inherent F3 instability not easily observed under typical culture conditions, we genetically eliminated the sole stabilizing N-linked glycosylation site (N249) from select clinically-identified F3 mutants. This removal exacerbated R345W and L451F secretion defects (19.8 ± 3.0% and 12.4 ± 1.2% of WT F3 levels, respectively), but also revealed a previously undiscovered secretion defect in another C-terminal variant, Y397H (42.0 ± 10.1% of WT F3 levels). Yet, glycan removal did not change the relative secretion of the N-terminal mutants tested (D49A, R140W, I220F). These results highlight the uniqueness and molecular similarities between the R345W and L451F variants and also suggest that previously identified disease-associated mutations (e.g., R140W) are indistinguishable from WT with respect to secretion, hinting that they may lead to disease by an alternative mechanism.

Type 2 diabetes (T2D), alike Parkinson’s disease (PD), belongs to the group of protein misfolding diseases (PMDs), which share aggregation of misfolded proteins as a hallmark. Although the major aggregating peptide in β-cells of T2D patients is Islet Amyloid Polypeptide (IAPP), alpha-synuclein (αSyn), the aggregating peptide in substantia nigra neurons of PD patients, is expressed also in β-cells. Here we show that αSyn, encoded by Snca, is a component of amyloid extracted from pancreas of transgenic mice overexpressing human IAPP (denoted hIAPPtg mice) and from islets of T2D individuals. Notably, αSyn dose-dependently promoted IAPP fibril formation in vitro and tail-vein injection of αSyn in hIAPPtg mice enhanced β-cell amyloid formation in vivo whereas β-cell amyloid formation was reduced in hIAPPtg mice on a Snca −/− background. Taken together, our findings provide evidence that αSyn and IAPP co-aggregate both in vitro and in vivo, suggesting a role for αSyn in β-cell amyloid formation.

Amyloidogenic protein assembly into insoluble fibrillar aggregates is linked with several neurodegenerative disorders, such as Alzheimer’s or Parkinson’s disease, affecting millions of people worldwide. The search for a potential anti-amyloid drug has led to the discovery of hundreds of compounds, none of which have passed all clinical trials. Gallic acid has been shown to both modulate factors leading to the onset of neurodegenerative disorders, as well as directly inhibit amyloid formation. However, the conditions under which this effect is seen could lead to oxidation of this polyphenol, likely changing its properties. Here we examine the effect of gallic acid and its oxidised form on the aggregation of a model amyloidogenic protein–insulin at low pH conditions. We show a vastly higher inhibitory potential of the oxidised form, as well as an alteration in the aggregation pathway, leading to the formation of a specific fibril conformation.

Human pancreatic islet amyloid polypeptide (hIAPP) and beta amyloid (Aβ) can accumulate in Type 2 diabetes (T2D) and Alzheimer’s disease (AD) brains and evidence suggests that interaction between the two amyloidogenic proteins can lead to the formation of heterocomplex aggregates. However, the structure and consequences of the formation of these complexes remains to be determined. The main objective of this study was to characterise the different types and morphology of Aβ-hIAPP heterocomplexes and determine if formation of such complexes exacerbate neurotoxicity. We demonstrate that hIAPP promotes Aβ oligomerization and formation of small oligomer and large aggregate heterocomplexes. Co-oligomerized Aβ42-hIAPP mixtures displayed distinct amorphous structures and a 3-fold increase in neuronal cell death as compared to Aβ and hIAPP alone. However, in contrast to hIAPP, non-amyloidogenic rat amylin (rIAPP) reduced oligomer Aβ-mediated neuronal cell death. rIAPP exhibited reductions in Aβ induced neuronal cell death that was independent of its ability to interact with Aβ and form heterocomplexes; suggesting mediation by other pathways. Our findings reveal distinct effects of IAPP peptides in modulating Aβ aggregation and toxicity and provide new insight into the potential pathogenic effects of Aβ-IAPP hetero-oligomerization and development of IAPP based therapies for AD and T2D.

The human tau is a microtubule-associated intrinsically unstructured protein that forms intraneuronal cytotoxic deposits in neurodegenerative diseases, like tauopathies. Recent studies indicate that in Alzheimer’s disease, ribosomal dysfunction might be a crucial event in the disease pathology. Our earlier studies had demonstrated that amorphous protein aggregation in the presence of ribosome can lead to sequestration of the ribosomal components. The present study aims at determining the effect of incubation of the full-length tau protein (Ht40) and its microtubule binding 4-repeat domain (K18) on the eukaryotic ribosome. Our in vitro studies show that incubation of Ht40 and the K18 tau variants with isolated non-translating yeast ribosome can induce a loss of ribosome physical integrity resulting in formation of tau-rRNA-ribosomal protein aggregates. Incubation with the tau protein variants also led to a disappearance of the peak indicating the ribosome profile of the HeLa cell lysate and suppression of translation in the human in vitro translation system. The incubation of tau protein with the ribosomal RNA leads to the formation of tau-rRNA aggregates. The effect of K18 on the yeast ribosome can be mitigated in the presence of cellular polyanions like heparin and tRNA, thereby indicating the electrostatic nature of the aggregation process.

Intrinsically disordered proteins (IDPs) are involved in various important biological processes, such as cell signalling, transcription, translation, cell division regulation etc. Many IDPs need to maintain their disordered conformation for proper function. Osmolytes, natural organic compounds responsible for maintaining osmoregulation, have been believed to regulate the functional activity of macromolecules including globular proteins and IDPs due to their ability of modulating the macromolecular structure, conformational stability, and functional integrity. In the present study, we have investigated the effect of all classes of osmolytes on two model IDPs, α- and β-casein. It was observed that osmolytes can serve either as folding inducers or folding evaders. Folding evaders, in general, do not induce IDP folding and therefore had no significant effect on structural and functional integrity of IDPs. On the other hand, osmolytes taurine and TMAO serve as folding inducers by promoting structural collapse of IDPs that eventually leads to altered structural and functional integrity of IDPs. This study sheds light on the osmolyte-induced regulation of IDPs and their possible role in various disease pathologies.

Autophagy is a lysosome-dependent degradation program to maintain cellular homeostasis in response to a variety of stressful conditions, such as long-lived or non-functional subcellular organelles, protein aggregates, nutrient limitation, and virus/bacteria infection. Accordingly, dysregulation of autophagy is closely associated with many human pathophysiological conditions, such as neurodegenerative diseases, aging, and cancer, and autophagy is highlighted as an important therapeutic target for these human diseases. In autophagy process, PIK3C3/VPS34 complex plays important roles in autophagosome biogenesis. Accumulating evidences that inhibition of PIK3C3/VPS34 complex successfully blocks autophagy make the complex as an attractive target for the development of autophagy-specific inhibitors. However, considering that various forms of PIK3C3/VPS34 complex exist and they are involved in many different cellular functions, the targeting of the pro-autophagy PIK3C3/VPS34 complex is required to specifically inhibit autophagy. To identify autophagy inhibitors targeting the pro-autophagy complex, we have performed the screening of a customized natural product library consisting of 35 herbal extracts which are widely used in the oriental medicine as anti-inflammation and/or anti-tumor reagents. We discovered that an alcoholic extract of Thuja orientalis L. leaves inhibits pro-autophagy complex formation by disrupting the interaction between autophagy-specific factor, ATG14L, and the complex core unit Vps34-Beclin 1 in vitro. Also, it inhibits the nutrient starvation induced autophagy and diminished pro-autophagy PIK3C3/VPS34 complex containing either ATG14L or UVRAG in several cell lines. Our results strongly suggest that Thuja orientalis L. leave extract functions as an autophagy-specific inhibitor not decreasing the complex activity nor the protein level, but preventing protein–protein interaction between autophagy-specific factor (ATG14L and UVRAG) and PIK3C3/VPS34 complex core unit, Vps34-Beclin 1, thereby specifically depleting the pro-autophagy complex to inhibit autophagy.

In human cells under stress conditions, misfolded polypeptides can form potentially cytotoxic insoluble aggregates. To eliminate aggregates, the HSP70 chaperone machinery extracts and resolubilizes polypeptides for triage to refolding or degradation. Yeast and bacterial chaperones of the small heat-shock protein (sHSP) family can bind substrates at early stages of misfolding, during the aggregation process. The co-aggregated sHSPs then facilitate downstream disaggregation by HSP70. Because it is unknown whether a human sHSP has this activity, we investigated the disaggregation role of human HSPB1. HSPB1 co-aggregated with unfolded protein substrates, firefly luciferase and mammalian lactate dehydrogenase. The co-aggregates formed with HSPB1 were smaller and more regularly shaped than those formed in its absence. Importantly, co-aggregation promoted the efficient disaggregation and refolding of the substrates, led by HSP70. HSPB1 itself was also extracted during disaggregation, and its homo-oligomerization ability was not required. Therefore, we propose that a human sHSP is an integral part of the chaperone network for protein disaggregation.

Triosephosphate isomerase (TIM) is an enzyme of the glycolysis pathway which exists in almost all types of cells. Its structure is the prototype of a motif called TIM-barrel or (α/β)8 barrel, which is the most common fold of all known enzyme structures. The simplest form in which TIM is catalytically active is a homodimer, in many species of bacteria and eukaryotes, or a homotetramer in some archaea. Here we show that the purified homodimeric TIMs from nine different species of eukaryotes and one of an extremophile bacterium spontaneously form higher order aggregates that can range from 3 to 21 dimers per macromolecular complex. We analysed these aggregates with clear native electrophoresis with normal and inverse polarity, blue native polyacrylamide gel electrophoresis, liquid chromatography, dynamic light scattering, thermal shift assay and transmission electron and fluorescence microscopies, we also performed bioinformatic analysis of the sequences of all enzymes to identify and predict regions that are prone to aggregation. Additionally, the capacity of TIM from Trypanosoma brucei to form fibrillar aggregates was characterized. Our results indicate that all the TIMs we studied are capable of forming oligomers of different sizes. This is significant because aggregation of TIM may be important in some of its non-catalytic moonlighting functions, like being a potent food allergen, or in its role associated with Alzheimer’s disease.

Type 2 diabetes (T2DM) is associated with aggregation of the human islet amyloid polypeptide (hIAPP) into cytotoxic amyloid species. Here we tested the effect of a diphenylpyrazole (DPP)-derived small molecule inhibitor, anle145c, on cytotoxicity and on aggregation properties of hIAPP. We demonstrate that incubation of hIAPP with the inhibitor yields ~10 nm-sized non-toxic oligomers, independent of the initial aggregation state of hIAPP. This suggests that anle145c has a special mode of action in which anle145c-stabilized oligomers act as a thermodynamic sink for the preferred aggregation state of hIAPP and anle145c. We also demonstrate that the inhibitor acts in a very efficient manner, with sub-stoichiometric concentrations of anle145c being sufficient to (i) inhibit hIAPP-induced death of INS-1E cells, (ii) prevent hIAPP fibril formation in solution, and (iii) convert preformed hIAPP fibrils into non-toxic oligomers. Together, these results indicate that anle145c is a promising candidate for inhibition of amyloid formation in T2DM.

Amyloids are β-sheets-rich protein fibrils that cause neurodegenerative and other incurable human diseases affecting millions of people worldwide. However, a number of proteins is functional in the amyloid state in various organisms from bacteria to humans. Using an original proteomic approach, we identified a set of proteins forming amyloid-like aggregates in the brain of young healthy rats. One of them is the FXR1 protein, which is known to regulate memory and emotions. We showed that FXR1 clearly colocalizes in cortical neurons with amyloid-specific dyes Congo-Red, Thioflavines S and T. FXR1 extracted from brain by immunoprecipitation shows yellow-green birefringence after staining with Congo red. This protein forms in brain detergent-resistant amyloid oligomers and insoluble aggregates. RNA molecules that are colocalized with FXR1 in cortical neurons are insensitive to treatment with RNase A. All these data suggest that FXR1 functions in rat brain in amyloid form. The N-terminal amyloid-forming fragment of FXR1 is highly conserved across mammals. We assume that the FXR1 protein may be presented in amyloid form in brain of different species of mammals, including humans.

12 pages, 7 figures.-- This article is licensed under a Creative Commons Attribution 4.0 International License, Most fish-allergic patients have anti-β-parvalbumin (β-PV) immunoglobulin E (IgE), which cross-reacts among fish species with variable clinical effects. Although the β-PV load is considered a determinant for allergenicity, fish species express distinct β-PV isoforms with unknown pathogenic contributions. To identify the role various parameters play in allergenicity, we have taken Gadus morhua and Scomber japonicus models, determined their β-PV isoform composition and analyzed the interaction of the IgE from fish-allergic patient sera with these different conformations. We found that each fish species contains a major and a minor isoform, with the total PV content four times higher in Gadus morhua than in Scomber japonicus. The isoforms showing the best IgE recognition displayed protease-sensitive globular folds, and if forming amyloids, they were not immunoreactive. Of the isoforms displaying stable globular folds, one was not recognized by IgE under any of the conditions, and the other formed highly immunoreactive amyloids. The results showed that Gadus morhua muscles are equipped with an isoform combination and content that ensures the IgE recognition of all PV folds, whereas the allergenic load of Scomber japonicus is under the control of proteolysis. We conclude that the consideration of isoform properties and content may improve the explanation of fish species allergenicity differences, This work was supported by grants from the Spanish AEI/EU-FEDER SAF2014-52661-C3 (MG) and BFU2015- 72271-EXP (MG), Angulas Aguinaga (MG, RRP) contract and the Ramón Areces Foundation (MC). MC is a Ramón y Cajal fellow

The inherited form of open angle glaucoma arises due to a toxic gain-of-function intracellular misfolding event involving a mutated myocilin olfactomedin domain (OLF). Mutant myocilin is recognized by the endoplasmic reticulum (ER)-resident heat shock protein 90 paralog, glucose regulated protein 94 (Grp94), but their co-aggregation precludes mutant myocilin clearance by ER-associated degradation. When the Grp94-mutant myocilin interaction is abrogated by inhibitors or siRNA, mutant myocilin is efficiently degraded. Here we dissected Grp94 into component domains (N, NM, MC) to better understand the molecular factors governing its interaction with OLF. We show that the Grp94 N-terminal nucleotide-binding N domain is responsible for accelerating OLF aggregation in vitro. Upon inhibiting the isolated N domain pharmacologically or removing the Pre-N terminal 57 residues from full-length Grp94, OLF aggregation rates revert to those seen for OLF alone, but only pharmacological inhibition rescues co-aggregation. The Grp94-OLF interaction is below the detection limit of fluorescence polarization measurements, but chemical crosslinking paired with mass spectrometry analyses traps a reproducible interaction between OLF and the Grp94 N domain, as well as between OLF and the Grp94 M domain. The emerging molecular-level picture of quinary interactions between Grp94 and myocilin points to a role for the far N-terminal sequence of the Grp94 N domain and a cleft in the M domain. Our work further supports drug discovery efforts to inhibit these interactions as a strategy to treat myocilin-associated glaucoma.

The aberrant accumulation of ubiquitinated protein aggregates in cells plays a critical role in the pathogenesis of several degenerative diseases, including Parkinson disease (PD) and cystic fibrosis (CF). In this study, we found that Ras GTPase-activating protein-binding protein 1 (G3BP1) inhibits ubiquitinated protein aggregations induced by p62 and USP10 in cultured cells. p62 is a ubiquitin receptor, and p62 and its binding partner USP10 have been shown to augment ubiquitinated protein aggregation. G3BP1 interacted with p62 and USP10 and inhibited p62/USP10-induced protein aggregation. The G3BP1 inhibition of protein aggregations targeted two aggregation-prone proteins, α-synuclein and CFTR-ΔF508, which are causative factors of PD and CF, respectively. G3BP1 depletion increased the amounts of ubiquitinated α-synuclein and CFTR-ΔF508 protein. A proteasome reporter indicated that G3BP1 depletion inhibits the proteasome activity. We herein present evidence that G3BP1, p62 and USP10 together control ubiquitinated protein toxicity by controlling both ubiquitination and aggregation. Taken together, these results suggest that G3BP1, p62 and USP10 could be therapeutic targets for ubiquitinated protein aggregation disorders, including PD and CF.

Recent studies have proposed that nucleic acids act as potential cofactors for protein aggregation and prionogenesis. By means of sedimentation, transmission electron microscopy, circular dichroism, static and dynamic light scattering, we have studied how RNA can influence the aggregation of the murine recombinant prion protein (rPrP). We find that RNA, independent of its sequence, source and size, modulates rPrP aggregation in a bimodal fashion, affecting both the extent and the rate of rPrP aggregation in a concentration dependent manner. Analogous to RNA-induced liquid-liquid phase transitions observed for other proteins implicated in neurodegenerative diseases, high protein to RNA ratios stimulate rPrP aggregation, while low ratios suppress it. However, the latter scenario also promotes formation of soluble oligomeric aggregates capable of seeding de novo rPrP aggregation. Furthermore, RNA co-aggregates with rPrP and thereby gains partial protection from RNase digestion. Our results also indicate that rPrP interacts with the RNAs with its N-terminus. In summary, this study elucidates the proposed adjuvant role of RNA in prion protein aggregation and propagation, and thus advocates an auxiliary role of the nucleic acids in protein aggregation in general. Manuscript title: Direct involvement of RNA in mammalian prion protein aggregation: Involvement of RNA in rPrP aggregation

Cadmium(Cd) induces cytotoxicity via autophagy-induced apoptosis in non-activated mouse monocytes; however, the molecular mechanism remains unclear. Here, we show that autophagy induces Fas (CD95/APO-1)-mediated apoptosis by promoting accumulation of p62/SQSTM1 in response to Cd. Cd produced tumor necrosis factor (TNF)-α, peaking at 6 h, and exhibiting a concentration-dependent increase. Immunoblot analysis revealed polyubiquitinated (polyUb) full-length Fas (antibody clone G-9) and reduced cytosolic Fas (antibody clone M-20) in Cd-exposed RAW264.7 cells. The accumulation of polyUb-Fas was transient and positively correlated with polyUb-p62 and polyUb-proteins. Autophagy inhibition via chemical and genetic modulation suppressed Cd-induced polyUb-p62, polyUb-Fas, and polyUb-protein levels, whereas the level of cytosolic Fas recovered to that of the control. Immunofluorescence (IF) staining for full-length Fas, p62, and ubiquitin revealed an aggregated pattern in Cd-induced apoptotic cells, which was inhibited by blocking autophagy. Fas colocalized with microtubule-associated protein 1 light chain (LC)-3B. IF staining and immunoprecipitation assays revealed colocalization and interaction among p62, Ub, and Fas. Knockdown of p62 reduced the binding of Ub and Fas. Together, these data suggest that polyUb-p62 targets Fas and recruits it to autophagosomes, where Fas transiently aggregates to promote apoptosis and is degraded with polyUb-p62. In conclusion, autophagy regulates C-terminal cytosolic Fas aggregation via p62 polyubiquitination, which is required for apoptosis and may play a critical role in the production of select cytokines.

Hybrid cells of Nicotiana suaveolens x N. tabacum grow normally at 36 °C, but immediately express lethality due to probable autoimmune response when transferred from 36 to 28 °C. Our recent study showed that the temperature-sensitive lethality of these hybrid cells occurs through autolytic programmed cell death (PCD). However, what happens in hybrid cells following the induction of autoimmune response to autolytic PCD is unclear. We hypothesized that accumulation of protein aggregates in hybrid cells induces autolytic PCD and examined detergent-insoluble protein (protein aggregates) isolated from hybrid cells expressing lethality. The amount of insoluble proteins increased in hybrid cells. Sodium 4-phenylbutyrate, a chemical chaperone, inhibited both the accumulation of insoluble proteins and irreversible progression of cell death. In contrast, E-64, a cysteine protease inhibitor, accelerated both the accumulation of insoluble proteins and cell death. Moreover, proteome analysis revealed that proteasome-component proteins were accumulated specifically in cells treated with E-64, and proteasome activity of hybrid cells decreased after induction of lethality. These findings demonstrate that accumulation of protein aggregates, including proteasome subunits, eventually cause autolytic PCD in hybrid cells. This suggests a novel process inducing plant PCD by loss of protein homeostasis and provides clues to future approaches for elucidating the whole process.

Oligomers of Aβ peptide are implicated as the most probable causative agent in Alzheimer’s disease. However, their structural properties remain elusive due to the dynamic and heterogeneous character of oligomeric species coexisting in solution. Nevertheless, new approaches, mainly based on mass spectrometry, provide unique access to these different structural forms. Using these methods, we previously showed that the N-terminal, non-amyloidogenic region of Aβ is involved in the network of interactions specifically stabilizing oligomers. In the present study, we identified three histidine residues as active participants in this network. Detailed knowledge of the structural features that are potentially important for oligomer-mediated neurotoxicity is a prerequisite for the rational design of oligomerization modifiers.

A potential mechanism of cytotoxicity attributed to Alzheimer’s Aβ peptides postulates that their aggregation disrupts membrane structure causing uncontrollable permeation of Ca2+ ions. To gain molecular insights into these processes, we have performed all-atom explicit solvent replica exchange with solute tempering molecular dynamics simulations probing aggregation of the naturally occurring Aβ fragment Aβ25-35 within the DMPC lipid bilayer. To compare the impact produced on the bilayer by Aβ25-35 oligomers and monomers, we used as a control our previous simulations, which explored binding of Aβ25-35 monomers to the same bilayer. We found that compared to monomeric species aggregation results in much deeper insertion of Aβ25-35 peptides into the bilayer hydrophobic core causing more pronounced disruption in its structure. Aβ25-35 peptides aggregate by incorporating monomer-like structures with stable C-terminal helix. As a result the Aβ25-35 dimer features unusual helix head-to-tail topology supported by a parallel off-registry interface. Such topology affords further growth of an aggregate by recruiting additional peptides. Free energy landscape reveals that inserted dimers represent the dominant equilibrium state augmented by two metastable states associated with surface bound dimers and inserted monomers. Using the free energy landscape we propose the pathway of Aβ25-35 binding, aggregation, and insertion into the lipid bilayer.

The yeast Hsp104 protein disaggregase is often used as a reporter for misfolded or damaged protein aggregates and protein quality control and ageing research. Observing Hsp104 fusions with fluorescent proteins is a popular approach to follow post stress protein aggregation, inclusion formation and disaggregation. While concerns that bigger protein tags, such as genetically encoded fluorescent tags, may affect protein behaviour and function have been around for quite some time, experimental evidence of how exactly the physiology of the protein of interest is altered within fluorescent protein fusions remains limited. To address this issue, we performed a comparative assessment of endogenously expressed Hsp104 fluorescent fusions function and behaviour. We provide experimental evidence that molecular behaviour may not only be altered by introducing a fluorescent protein tag but also varies depending on such a tag within the fusion. Although our findings are especially applicable to protein quality control and ageing research in yeast, similar effects may play a role in other eukaryotic systems.

Physical instabilities of proteins in the form of protein aggregation continue to be a major challenge in the development of protein drug candidates. Aggregation can occur during different stages of product lifecycle such as freeze–thaw, manufacturing, shipping, and storage, and can potentially delay commercialization of candidates. A lack of clear understanding of the underlying mechanism(s) behind protein aggregation and the potential immunogenic reactions renders the presence of aggregates in biotherapeutic products undesirable. Understanding and minimizing aggregation can potentially reduce immunogenic responses and make protein therapeutics safer. Therefore, it is imperative to identify, understand, and control aggregation during early formulation development and develop reliable and orthogonal analytical methodologies to detect and monitor levels of aggregation. Freezing and thawing are typical steps involved in the manufacturing of drug product and could result in complex physical and chemical changes, which in turn could potentially cause protein aggregation. This study provides a systematic approach in understanding and selecting the ideal freeze–thaw conditions for manufacturing of protein-based therapeutics. It identifies the importance of balancing different excipients with an overall goal of sufficiently reducing or eliminating aggregation and developing a stable and scalable formulation. The results demonstrated that the freeze–thaw damage of mAb-1 in aqueous solutions was significantly reduced by identification of optimal freeze–thaw conditions using first a small-scale model with subsequent at-scale verifications. The work provides a framework for successful transfer of drug product manufacturing process from small-scale to the manufacturing scale production environment especially for molecules that are susceptible to freeze–thaw induced degradations.

Polyglutamine (polyQ) expansion of proteins can trigger protein misfolding and amyloid-like aggregation, which thus lead to severe cytotoxicities and even the respective neurodegenerative diseases. However, why polyQ aggregation is toxic to cells is not fully elucidated. Here, we took the fragments of polyQ-expanded (PQE) ataxin-7 (Atx7) and huntingtin (Htt) as models to investigate the effect of polyQ aggregates on the cellular proteostasis of endogenous ataxin-3 (Atx3), a protein that frequently appears in diverse inclusion bodies. We found that PQE Atx7 and Htt impair the cellular proteostasis of Atx3 by reducing its soluble as well as total Atx3 level but enhancing formation of the aggregates. Expression of these polyQ proteins promotes proteasomal degradation of endogenous Atx3 and accumulation of its aggregated form. Then we verified that the co-chaperone HSJ1 is an essential factor that orchestrates the balance of cellular proteostasis of Atx3; and further discovered that the polyQ proteins can sequester HSJ1 into aggregates or inclusions in a UIM domain-dependent manner. Thereby, the impairment of Atx3 proteostasis may be attributed to the sequestration and functional loss of cellular HSJ1. This study deciphers a potential mechanism underlying how PQE protein triggers proteinopathies, and also provides additional evidence in supporting the hijacking hypothesis that sequestration of cellular interacting partners by protein aggregates leads to cytotoxicity or neurodegeneration.

The pigment cell-specific protein PMEL forms a functional amyloid matrix in melanosomes onto which the pigment melanin is deposited. The amyloid core consists of a short proteolytic fragment, which we have termed the core-amyloid fragment (CAF) and perhaps additional parts of the protein, such as the PKD domain. A highly O-glycosylated repeat (RPT) domain also derived from PMEL proteolysis associates with the amyloid and is necessary to establish the sheet-like morphology of the assemblies. Excluded from the aggregate is the regulatory N-terminus, which nevertheless must be linked in cis to the CAF in order to drive amyloid formation. The domain is then likely cleaved away immediately before, during, or immediately after the incorporation of a new CAF subunit into the nascent amyloid. We had previously identified a 21 amino acid long region, which mediates the regulatory activity of the N-terminus towards the CAF. However, many mutations in the respective segment caused misfolding and/or blocked PMEL export from the endoplasmic reticulum, leaving their phenotype hard to interpret. Here, we employ a saturating mutagenesis approach targeting the motif at single amino acid resolution. Our results confirm the critical nature of the PMEL N-terminal region and identify several residues essential for PMEL amyloidogenesis.

The pollination services provided by the honey bee are critical in both natural and agricultural ecosystems. Honey bee colonies in the United States have suffered from an increased rate of die-off in recent years, stemming from a complex set of interacting stresses that remain poorly described. Defining specific common cellular processes and cellular stress responses impacted by multiple stressors represent a key step in understanding these synergies. Proteotoxic stresses negatively impact protein synthesis, folding, and degradation. Diverse proteotoxic stresses induce expression of genes encoding small heat shock proteins (sHSP) of the expanded lethal (2) essential for life (l(2)efl) gene family. In addition to upregulation by the Integrated Stress Response (ISR), the Heat Shock Response (HSR), and the Oxidative Stress Response (OSR), our data provide first evidence that sHSP genes are upregulated by the Unfolded Protein Response (UPR). As these genes appear to be part of a core stress response that could serve as a useful biomarker for cellular stress in honey bees, we designed and tested an RT-LAMP assay to detect increased l(2)efl gene expression in response to heat-stress. While this assay provides a powerful proof of principle, further work will be necessary to link changes in sHSP gene expression to colony-level outcomes, to adapt our preliminary assay into a Point of Care Testing (POCT) assay appropriate for use as a diagnostic tool for use in the field, and to couple assay results to management recommendations.

One of the hallmarks of the most common neurodegenerative disease, Alzheimer’s disease (AD), is the extracellular deposition and aggregation of Amyloid Beta (Aβ)-peptides in the brain. Previous studies have shown that select metal ions, most specifically copper (Cu) and zinc (Zn) ions, have a synergistic effect on the aggregation of Aβ-peptides. In the present study, inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the metal content of a commercial recombinant human Aβ40 peptide. Cu and Zn were among the metals detected; unexpectedly, nickel (Ni) was one of the most abundant elements. Using a fluorescence-based assay, we found that Aβ40 peptide in vitro aggregation was enhanced by addition of Zn2+ and Ni2+, and Ni2+-induced aggregation was facilitated by acidic conditions. Nickel binding to Aβ40 peptide was confirmed by isothermal titration calorimetry. Addition of the Ni-specific chelator dimethylglyoxime (DMG) inhibited Aβ40 aggregation in absence of added metal, as well as in presence of Cu2+ and Ni2+, but not in presence of Zn2+. Finally, mass spectrometry analysis revealed that DMG can coordinate Cu or Ni, but not Fe, Se or Zn. Taken together, our results indicate that Ni2+ ions enhance, whereas nickel chelation inhibits, Aβ peptide in vitro aggregation. Hence, DMG-mediated Ni-chelation constitutes a promising approach towards inhibiting or slowing down Aβ40 aggregation.

Misfolded protein oligomers are increasingly recognized as highly cytotoxic agents in a wide range of human disorders associated with protein aggregation. In this study, we assessed the possible uptake and resulting toxic effects of model protein oligomers administered to C. elegans through the culture medium. We used an automated machine-vision, high-throughput screening procedure to monitor the phenotypic changes in the worms, in combination with confocal microscopy to monitor the diffusion of the oligomers, and oxidative stress assays to detect their toxic effects. Our results suggest that the oligomers can diffuse from the intestinal lumen to other tissues, resulting in a disease phenotype. We also observed that pre-incubation of the oligomers with a molecular chaperone (αB-crystallin) or a small molecule inhibitor of protein aggregation (squalamine), reduced the oligomer absorption. These results indicate that exogenous misfolded protein oligomers can be taken up by the worms from their environment and spread across tissues, giving rise to pathological effects in regions distant from their place of absorbance.

Equine pituitary pars intermedia dysfunction (PPID) is a common endocrine disease of aged horses that shows a similar pathophysiology as Parkinson’s Disease (PD) with increased levels of α-synuclein (α-syn). While α-syn is thought to play a pathogenic role in horses with PPID, it is unclear if α-syn is also misfolded in the pars intermedia and could similarly promote self-aggregation and propagation. Consequently, α-syn was isolated from the pars intermedia from groups of healthy young and aged horses, and aged PPID-afflicted horses. Seeding experiments confirmed the prion-like properties of α-syn isolated from PPID-afflicted horses. Next, detection of α-syn fibrils in pars intermedia via transmission electron microscopy (TEM) was exclusive to PPID-afflicted horses. A bank of fragment peptides was designed to further characterize equine α-syn misfolding. Region 62–87 of equine and human α-syn peptides was found to be most prone to aggregation according to Tango bioinformatic program and kinetics of aggregation via a thioflavin T fluorescence assay. In both species, fragment peptide 62–87 is capable of generating mature fibrils as demonstrated by TEM. The combined animal, bioinformatic, and biophysical studies provide evidence that equine α-syn is misfolded in PPID horses.

Misfolding of the cellular prion protein, PrPC, into the amyloidogenic isoform, PrPSc, which forms infectious protein aggregates, the so-called prions, is a key pathogenic event in prion diseases. No pathogens other than prions have been identified to induce misfolding of PrPC into PrPSc and propagate infectious prions in infected cells. Here, we found that infection with a neurotropic influenza A virus strain (IAV/WSN) caused misfolding of PrPC into PrPSc and generated infectious prions in mouse neuroblastoma cells through a hit-and-run mechanism. The structural and biochemical characteristics of IAV/WSN-induced PrPSc were different from those of RML and 22L laboratory prions-evoked PrPSc, and the pathogenicity of IAV/WSN-induced prions were also different from that of RML and 22L prions, suggesting IAV/WSN-specific formation of PrPSc and infectious prions. Our current results may open a new avenue for the role of viral infection in misfolding of PrPC into PrPSc and formation of infectious prions.

Plants have evolved genetic and physiological mechanisms to mitigate the adverse effects of high temperature. CARBOXYL TERMINUS OF THE HSC70-INTERACTING PROTEINS (CHIP) is a conserved chaperone-dependent ubiquitin E3 ligase that targets misfolded proteins. Here, we report functional analysis of the SlCHIP gene from tomato (Solanum lycopersicum) in heat tolerance. SlCHIP encodes a CHIP protein with three tandem tetracopeptide repeat (TPR) motifs and a C-terminal U box domain. Phylogenetic analysis of CHIP homologs from animals, spore-bearing and seed plants revealed a tree topology similar to the evolutionary tree of the organisms. Expression of SlCHIP was induced under high temperature and was also responsive to plant stress hormones. Silencing of SlCHIP in tomato reduced heat tolerance based on increased heat stress symptoms, reduced photosynthetic activity, elevated electrolyte leakage and accumulation of insoluble protein aggregates. The accumulated protein aggregates in SlCHIP-silenced plants were still highly ubiquitinated, suggesting involvement of other E3 ligases in ubiquitination. SlCHIP restored the heat tolerance of Arabidopsis chip mutant to the wild type levels. These results indicate that tomato SlCHIP plays a critical role in heat stress responses most likely by targeting degradation of misfolded proteins that are generated during heat stress.

TDP-43 and hnRNPA1 contain tandemly-tethered RNA-recognition-motif (RRM) domains, which not only functionally bind an array of nucleic acids, but also participate in aggregation/fibrillation, a pathological hallmark of various human diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), alzheimer's disease (AD) and Multisystem proteinopathy (MSP). Here, by DSF, NMR and MD simulations we systematically characterized stability, ATP-binding and conformational dynamics of TDP-43 and hnRNPA1 RRM domains in both tethered and isolated forms. The results reveal three key findings: (1) upon tethering TDP-43 RRM domains become dramatically coupled and destabilized with Tm reduced to only 49 °C. (2) ATP specifically binds TDP-43 and hnRNPA1 RRM domains, in which ATP occupies the similar pockets within the conserved nucleic-acid-binding surfaces, with the affinity slightly higher to the tethered than isolated forms. (3) MD simulations indicate that the tethered RRM domains of TDP-43 and hnRNPA1 have higher conformational dynamics than the isolated forms. Two RRM domains become coupled as shown by NMR characterization and analysis of inter-domain correlation motions. The study explains the long-standing puzzle that the tethered TDP-43 RRM1–RRM2 is particularly prone to aggregation/fibrillation, and underscores the general role of ATP in inhibiting aggregation/fibrillation of RRM-containing proteins. The results also rationalize the observation that the risk of aggregation-causing diseases increases with aging.

Amyloid-β (Aβ), reported as a significant constituent of drusen, was implicated in the pathophysiology of age-related macular degeneration (AMD), yet the identity of the major pathogenic Aβ species in the retina has remained hitherto unclear. Here, we examined the in-vivo retinal impact of distinct supramolecular assemblies of Aβ. Fibrillar (Aβ40, Aβ42) and oligomeric (Aβ42) preparations showed clear biophysical hallmarks of amyloid assemblies. Measures of retinal structure and function were studied longitudinally following intravitreal administration of the various Aβ assemblies in rats. Electroretinography (ERG) delineated differential retinal neurotoxicity of Aβ species. Oligomeric Aβ42 inflicted the major toxic effect, exerting diminished ERG responses through 30 days post injection. A lesser degree of retinal dysfunction was noted following treatment with fibrillar Aβ42, whereas no retinal compromise was recorded in response to Aβ40 fibrils. The toxic effect of Aβ42 architectures was further reflected by retinal glial response. Fluorescence labelling of Aβ42 species was used to detect their accumulation into the retinal tissue. These results provide conceptual evidence of the differential toxicity of particular Aβ species in-vivo, and promote the mechanistic understanding of their retinal pathogenicity. Stratifying the impact of pathological Aβ aggregation in the retina may merit further investigation to decipher the pathophysiological relevance of processes of molecular self-assembly in retinal disorders.

Protein aggregation is a major concern in biotherapeutic applications of monoclonal antibodies. Introducing charged mutations is among the promising strategies to improve aggregation resistance. However, the impact of such mutations on solubilizing activity depends largely on the inserting location, whose mechanism is still not well understood. Here, we address this issue from a solvation viewpoint, and this is done by analyzing how the change in solvation free energy upon charged mutation is composed of individual contributions from constituent residues. To this end, we perform molecular dynamics simulations for a number of antibody mutants and carry out the residue-wise decomposition of the solvation free energy. We find that, in addition to the previously identified “global” principle emphasizing the key role played by the protein total net charge, a local net charge within $$\sim$$ ∼ 15 Å from the mutation site exerts significant effects. For example, when the net charge of an antibody is positive, the global principle states that introducing a positively charged mutation will lead to more favorable solvation. Our finding further adds that an even more optimal mutation can be done at the site around which more positively charged residues and fewer negatively charged residues are present. Such a “local” design principle accounts for the location dependence of charged mutations, and will be useful in producing aggregation-resistant antibodies.

Parkinson's disease (PD) is a neurodegenerative disorder for which only symptomatic treatments are available. Repurposing drugs that target α-synuclein aggregation, considered one of the main drivers of PD progression, could accelerate the development of disease-modifying therapies. In this work, we focused on chemically modified tetracycline 3 (CMT-3), a derivative with reduced antibiotic activity that crosses the blood–brain barrier and is pharmacologically safe. We found that CMT-3 inhibited α-synuclein amyloid aggregation and led to the formation of non-toxic molecular species, unlike minocycline. Furthermore, CMT-3 disassembled preformed α-synuclein amyloid fibrils into smaller fragments that were unable to seed in subsequent aggregation reactions. Most interestingly, disaggregated species were non-toxic and less inflammogenic on brain microglial cells. Finally, we modelled the interactions between CMT-3 and α-synuclein aggregates by molecular simulations. In this way, we propose a mechanism for fibril disassembly. Our results place CMT-3 as a potential disease modifier for PD and possibly other synucleinopathies.

Lewy bodies (LBs) and glial cytoplasmic inclusions (GCIs) are specific aggregates found in Parkinson’s disease (PD) and multiple system atrophy (MSA), respectively. These aggregates mainly consist of α-synuclein (α-syn) and have been reported to propagate in the brain. In animal experiments, the fibrils of α-syn propagate similarly to prions but there is still insufficient evidence to establish this finding in humans. Here, we analysed the protein structure of these aggregates in the autopsy brains of patients by synchrotron Fourier-transform infrared micro-spectroscopy (FTIRM) analysis without extracting or artificially amplifying the aggregates. As a result, we found that the content of the β-sheet structure in LBs in patients with PD was significantly higher than that in GCIs in patients with MSA (52.6 ± 1.9% in PD vs. 38.1 ± 0.9% in MSA, P

Natural products have played a dominant role in the discovery of lead compounds for the development of drugs aimed at the treatment of human diseases. This electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS)—based study demonstrates that dietary antioxidants, isolated components from the stigmas of saffron (Crocus sativus L.) may be effective in inhibiting Aβ fibrillogenesis, a neuropathological hallmark of Alzheimer’s Disease (AD). This study reveals a substantial alteration in the monomer/oligomer distribution of Aβ1-40, concomitant with re-direction of fibril formation, induced by the natural product interaction. These alterations on the Aβ1-40 aggregation pathway are most prominent for trans-crocin-4 (TC4). Use of ESI-IMS-MS, electron microscopy alongside Thioflavin-T kinetics, and the interpretation of 3-dimensional Driftscope plots indicate a correlation of these monomer/oligomer distribution changes with alterations to Aβ1-40 amyloid formation. The latter could prove instrumental in the development of novel aggregation inhibitors for the prevention, or treatment of AD.

Aggregation of α-synuclein (α-syn) is associated with the manifestation of various pathogenic synucleinopathies, including Parkinson’s disease attributed to both genetic and environmental stress factors. The initial events triggering α-syn aggregation and disease initiation due to environmental stress factors are still largely unknown. Here, to understand the mechanism of misfolding and aggregation initiation, we induced α-syn aggregation with rotenone, an established chemical inducer of PD like symptoms. We found that rotenone accelerates the formation of structurally distinct oligomers and fibrils that act as templates and increase the formation of conformers capable of spreading to the neighboring neuronal cells. Molecular dynamics simulations and NMR studies revealed the involvement of NAC region and formation of helical conformations resulting in structural variations in oligomers and fibrils. These structural variations affect the cytotoxic potential of oligomers and fibrils, where, the beta sheet rich oligomers and fibrils alter the membrane potential of neuronal cells and lead to early apoptosis. Our results describe the initial mechanistic events in pathogenic protein aggregation, where initial structural alterations in response to external stress factors dictate the toxicity of resulting conformers. This information will further provide insights in the understanding of protein aggregation, disease progression and pathogenesis.

Cataract is the leading cause of blindness worldwide but the mechanisms involved in the process of cataractogenesis are not yet fully understood. Two most prevalent types of age-related cataracts are nuclear (N) and cortical (C) cataracts. A common environmental factor in most age-related cataracts is believed to be oxidative stress. The lens epithelium, the first physical and biological barrier in the lens, is build from lens epithelial cells (LECs). LECs are important for the maintenance of lens transparency as they control energy production, antioxidative mechanisms and biochemical transport for the whole lens. The purpose of this study is to characterize compounds in LECs originated from N and C cataracts, by using the synchrotron radiation-based Fourier Transform Infrared (SR-FTIR) microspectroscopy, in order to understand the functional importance of their different bio-macromolecules in cataractogenesis. We used the SR-FTIR microspectroscopy setup installed on the beamline MIRAS at the Spanish synchrotron light source ALBA, where measurements were set to achieve single cell resolution, with high spectral stability and high photon flux. The results showed that protein aggregation in form of fibrils was notably pronounced in LECs of N cataracts, while oxidative stress and the lipids peroxidation were more pronounced in LECs of C cataracts.

Individuals with Down syndrome (DS) commonly show unique pathological phenotypes throughout their life span. Besides the specific effects of dosage-sensitive genes on chromosome 21, recent studies have demonstrated that the gain of a chromosome exerts an adverse impact on cell physiology, regardless of the karyotype. Although dysregulated transcription and perturbed protein homeostasis are observed in common in human fibroblasts with trisomy 21, 18, and 13, whether and how this aneuploidy-associated stress acts on other cell lineages and affects the pathophysiology are unknown. Here, we investigated cellular stress responses in human trisomy 21 and 13 neurons differentiated from patient-derived induced pluripotent stem cells. Neurons of both trisomies showed increased vulnerability to apoptotic cell death, accompanied by dysregulated protein homeostasis and upregulation of the endoplasmic reticulum stress pathway. In addition, misfolded protein aggregates, comprising various types of neurodegenerative disease-related proteins, were abnormally accumulated in trisomic neurons. Intriguingly, treatment with sodium 4-phenylbutyrate, a chemical chaperone, successfully decreased the formation of protein aggregates and prevented the progression of cell apoptosis in trisomic neurons. These results suggest that aneuploidy-associated stress might be a therapeutic target for the neurodegenerative phenotypes in DS.