ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification.

Prion-like self-perpetuating conformational conversion of proteins into amyloid aggregates is associated with both transmissible neurodegenerative diseases and non-Mendelian inheritance. The cellular energy currency ATP is known to indirectly regulate the formation, dissolution, or transmission of amyloid-like aggregates by providing energy to the molecular chaperones that maintain protein homeostasis. In this work, we demonstrate that ATP molecules, independent of any chaperones, modulate the formation and dissolution of amyloids from a yeast prion domain (NM domain of Saccharomyces cerevisiae Sup35) and restricts autocatalytic amplification by controlling the amount of fragmentable and seeding-competent aggregates. ATP, at (high) physiological concentrations in the presence of Mg2+, kinetically accelerates NM aggregation. Interestingly, ATP also promotes phase separation-mediated aggregation of a human protein harboring a yeast prion-like domain. We also show that ATP disaggregates preformed NM fibrils in a dose-independent manner. Our results indicate that ATP-mediated disaggregation, unlike the disaggregation by the disaggregase Hsp104, yields no oligomers that are considered one of the critical species for amyloid transmission. Furthermore, high concentrations of ATP delimited the number of seeds by giving rise to compact ATP-bound NM fibrils that exhibited nominal fragmentation by either free ATP or Hsp104 disaggregase to generate lower molecular weight amyloids. In addition, (low) pathologically relevant ATP concentrations restricted autocatalytic amplification by forming structurally distinct amyloids that are found seeding inefficient because of their reduced ß-content. Our results provide key mechanistic underpinnings of concentration-dependent chemical chaperoning by ATP against prion-like transmissions of amyloids.

The C-terminal 32-mer fragment of hemoglobin alpha is an amyloidogenic peptide with antimicrobial properties.

Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.

An intrinsically disordered pathological prion variant Y145Stop converts into self-seeding amyloids via liquid-liquid phase separation.

Biomolecular condensation via liquid-liquid phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) along with other biomolecules is proposed to control critical cellular functions, whereas aberrant phase transitions are associated with a range of neurodegenerative diseases. Here, we show that a disease-associated stop codon mutation of the prion protein (PrP) at tyrosine 145 (Y145Stop), resulting in a truncated, highly disordered, N-terminal IDR, spontaneously phase-separates into dynamic liquid-like droplets. Phase separation of this highly positively charged N-terminal segment is promoted by the electrostatic screening and a multitude of weak, transient, multivalent, intermolecular interactions. Single-droplet Raman measurements, in conjunction with an array of bioinformatic, spectroscopic, microscopic, and mutagenesis studies, revealed a highly mobile internal organization within the liquid-like condensates. The phase behavior of Y145Stop is modulated by RNA. Lower RNA:protein ratios promote condensation at a low micromolar protein concentration under physiological conditions. At higher concentrations of RNA, phase separation is abolished. Upon aging, these highly dynamic liquid-like droplets gradually transform into ordered, ß-rich, amyloid-like aggregates. These aggregates formed via phase transitions display an autocatalytic self-templating characteristic involving the recruitment and binding-induced conformational conversion of monomeric Y145Stop into amyloid fibrils. In contrast to this intrinsically disordered truncated variant, the wild-type full-length PrP exhibits a much lower propensity for both condensation and maturation into amyloids, hinting at a possible protective role of the C-terminal domain. Such an interplay of molecular factors in modulating the protein phase behavior might have much broader implications in cell physiology and disease.

NMR unveils an N-terminal interaction interface on acetylated-α-synuclein monomers for recruitment to fibrils.

Amyloid fibril formation of α-synuclein (αS) is associated with multiple neurodegenerative diseases, including Parkinson's disease (PD). Growing evidence suggests that progression of PD is linked to cell-to-cell propagation of αS fibrils, which leads to seeding of endogenous intrinsically disordered monomer via templated elongation and secondary nucleation. A molecular understanding of the seeding mechanism and driving interactions is crucial to inhibit progression of amyloid formation. Here, using relaxation-based solution NMR experiments designed to probe large complexes, we probe weak interactions of intrinsically disordered acetylated-αS (Ac-αS) monomers with seeding-competent Ac-αS fibrils and seeding-incompetent off-pathway oligomers to identify Ac-αS monomer residues at the binding interface. Under conditions that favor fibril elongation, we determine that the first 11 N-terminal residues on the monomer form a common binding site for both fibrils and off-pathway oligomers. Additionally, the presence of off-pathway oligomers within a fibril seeding environment suppresses seeded amyloid formation, as observed through thioflavin-T fluorescence experiments. This highlights that off-pathway αS oligomers can act as an auto-inhibitor against αS fibril elongation. Based on these data taken together with previous results, we propose a model in which Ac-αS monomer recruitment to the fibril is driven by interactions between the intrinsically disordered monomer N terminus and the intrinsically disordered flanking regions (IDR) on the fibril surface. We suggest that this monomer recruitment may play a role in the elongation of amyloid fibrils and highlight the potential of the IDRs of the fibril as important therapeutic targets against seeded amyloid formation.

Energy Landscapes and Heat Capacity Signatures for Monomers and Dimers of Amyloid-Forming Hexapeptides.

Amyloid formation is a hallmark of various neurodegenerative disorders. In this contribution, energy landscapes are explored for various hexapeptides that are known to form amyloids. Heat capacity (CV) analysis at low temperature for these hexapeptides reveals that the low energy structures contributing to the first heat capacity feature above a threshold temperature exhibit a variety of backbone conformations for amyloid-forming monomers. The corresponding control sequences do not exhibit such structural polymorphism, as diagnosed via end-to-end distance and a dihedral angle defined for the monomer. A similar heat capacity analysis for dimer conformations obtained using basin-hopping global optimisation shows clear features in end-to-end distance versus dihedral correlation plots, where amyloid-forming sequences exhibit a preference for larger end-to-end distances and larger positive dihedrals. These results hold true for sequences taken from tau, amylin, insulin A chain, a de novo designed peptide, and various control sequences. While there is a little overall correlation between the aggregation propensity and the temperature at which the low-temperature CV feature occurs, further analysis suggests that the amyloid-forming sequences exhibit the key CV feature at a lower temperature compared to control sequences derived from the same protein.

Cross-Linked α-Synuclein as Inhibitor of Amyloid Formation.

The aggregation and amyloid formation of α-synuclein is associated with Parkinson's disease and other synucleinopathies. In its native, monomeric form α-synuclein is an intrinsically disordered protein represented by highly dynamic conformational ensembles. Inhibition of α-synuclein aggregation using small molecules, peptides, or proteins has been at the center of interest in recent years. Our aim was to explore the effects of cross-linking on the structure and aggregation/amyloid formation properties of α-synuclein. Comparative analysis of available high-resolution amyloid structures and representative structural models and MD trajectory of monomeric α-synuclein revealed that potential cross-links in the monomeric protein are mostly incompatible with the amyloid forms and thus might inhibit fibrillation. Monomeric α-synuclein has been intramolecularly chemically cross-linked under various conditions using different cross-linkers. We determined the location of cross-links and their frequency using mass spectrometry and found that most of them cannot be realized in the amyloid structures. The inhibitory potential of cross-linked proteins has been experimentally investigated using various methods, including thioflavin-T fluorescence and transmission electron microscopy. We found that conformational constraints applied by cross-linking fully blocked α-synuclein amyloid formation. Moreover, DTSSP-cross-linked molecules exhibited an inhibitory effect on the aggregation of unmodified α-synuclein as well.

Amyloid fibril reduction through covalently modified lysine in HEWL and insulin.

Proteins possess a variety of nucleophiles, which can carry out different reactions in the functioning cells. Proteins endogenously and synthetically can be modified through their nucleophilic sites. The roles of these chemical modifications have not been completely revealed. These modifications can alter the protein folding process. Protein folding directly affects the function of proteins. If an error in protein folding occurs, it may cause protein malfunction leading to several neurodegenerative disorders such as Alzheimer's and Parkinson's. In this study, Hen Egg White Lysozyme (HEWL) and bovine insulin, as model proteins for studying the amyloid formation, were covalently attached with 5(6)-thiophenolfluorescein. The amyloid formation of the covalently labeled lysozyme and insulin were compared with the native proteins. Interestingly, the results indicated that the covalent attachment of fluorescein slowed down the amyloid formation of HEWL and insulin significantly. The amyloid formation was examined using Thioflavin T (ThT) fluorescence assay, circular dichroism, FTIR, and gel electrophoresis. Tandem mass spectrometry was employed to identify the sites of covalent modifications in HEWL. It turned out that two surface lysine residues (K97 and K 116) in HEWL were modified. Computational studies, including docking and molecular simulations, revealed that 5(6)-thiophenolfluorescein makes several non-covalent interactions with HEWL residues, including Lys 97, leading to the reduction of the ß-sheet in the protein. Additionally, AFM analysis confirmed the amyloid fibril reduction of lysine-modified bovine insulin and HEWL. Altogether, our results expand mechanistic insights into preventing amyloid formation by providing an approach for reducing amyloid formation by modifying specific lysine residues in the proteins.

A Palette of Fluorescent Aß42 Peptides Labelled at a Range of Surface-Exposed Sites.

Fluorescence-based single molecule techniques provide important tools towards understanding the molecular mechanism of complex neurodegenerative diseases. This requires efficient covalent attachment of fluorophores. Here we create a series of cysteine mutants (S8C, Y10C, S26C, V40C, and A42C) of Aß42, involved in Alzheimer's disease, based on exposed positions in the fibril structure and label them with the Alexa-fluorophores using maleimide chemistry. Direct stochastic optical reconstruction microscopy imaging shows that all the labelled mutants form fibrils that can be detected by virtue of Alexa fluorescence. Aggregation assays and cryo-electron micrographs establish that the careful choice of labelling position minimizes the perturbation of the aggregation process and fibril structure. Peptides labelled at the N-terminal region, S8C and Y10C, form fibrils independently and with wild-type. Peptides labelled at the fibril core surface, S26C, V40C and A42C, form fibrils only in mixture with wild-type peptide. This can be understood on the basis of a recent fibril model, in which S26, V40 and A42 are surface exposed in two out of four monomers per fibril plane. We provide a palette of fluorescently labelled Aß42 peptides that can be used to gain understanding of the complex mechanisms of Aß42 self-assembly and help to develop a more targeted approach to cure the disease.

Amyloid Formation in Nanoliter Droplets.

Processes that monitor the nucleation of amyloids and characterize the formation of amyloid fibrils are vital to medicine and pharmacology. In this study, we observe the nucleation and formation of lysozyme amyloid fibrils using a facile microfluidic system to generate nanoliter droplets that can control the flow rate and movement of monomer-in-oil emulsion droplets in a T-junction microchannel. Using a fluorescence assay, we monitor the nucleation and growth process of amyloids based on the volume of droplets. Using the microfluidic system, we demonstrate that the lag phase, which is vital to amyloid nucleation and growth, is reduced at a lower droplet volume. Furthermore, we report a peculiar phenomenon of high amyloid formation at the edge of a bullet-shaped droplet, which is likely due to the high local monomer concentration. Moreover, we discovered that amyloid fibrils synthesized in the nanoliter droplets are shorter and thicker than fibrils synthesized from a bulk solution via the conventional heating method. Herein, a facile procedure to observe and characterize the nucleation and growth of amyloid fibrils using nanoliter droplets is presented, which is beneficial for investigating new features of amyloid fibril formation as an unconventional synthetic method for amyloid fibrils.

Sulfated polysaccharide ascophyllan prevents amyloid fibril formation of human insulin and inhibits amyloid-induced hemolysis and cytotoxicity in PC12 cells.

We found that ascophyllan significantly inhibited the fibrillation of human insulin and was the most effective among the sulfated polysaccharides tested. Gel-filtration analysis suggested that ascophyllan was capable of forming a complex with insulin through a weak interaction. Secondary structure transition from native α-helix to ß-sheet predominant structure of insulin under the fibrillation conditions was suppressed in the presence of ascophyllan. Interestingly, ascophyllan attenuated insulin fibril-induced hemolysis of human erythrocytes. Moreover, ascophyllan attenuated insulin amyloid-induced cytotoxicity on rat pheochromocytoma PC12 cells and reduced the level of intracellular reactive oxygen species. This is the first report indicating that a sulfated polysaccharide, ascophyllan, can suppress the insulin amyloid fibril formation and inhibit the fibril-induced detrimental bioactivities.

Identification of Crocin as a New hIAPP Amyloid Inhibitor via a Simple Yet Highly Biospecific Screening System.

Amylin (hIAPP) amyloid formation plays an important role in the pathogenesis of type 2 diabetes (T2D), which makes it a promising therapeutic target for T2D. In this study, we established a screening tool for identifying chemicals affecting hIAPP amyloid formation based on a reported genetic tool, which constantly tracks protein aggregates in Saccharomyces cerevisiae. In order to obtain the hIAPP with better aggregation ability, the gene of hIAPP was tandemly ligated to create 1×, 2×, 4× or 6×-hIAPP expressing strains. By measuring the cell density and fluorescence intensity of green fluorescent protein (GFP) regulated by the aggregation status of hIAPP, it was found that four intramolecular ligated hIAPP (4×hIAPP) could form obvious amyloids with mild toxicity. The validity and reliability of the screening tool were verified by testing six reported hIAPP inhibitors, including curcumin, epigallocatechin gallate and so on. Combined with surface plasmon resonance (SPR) and the screening tool, which could be a screening system for hIAPP inhibitors, we found that crocin specifically binds to hIAPP and acts inhibit amyloid formation of hIAPP. The effect of crocin was further confirmed by Thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM) analysis. Thus, a screening system for hIAPP amyloid inhibitors and a new mechanism of crocin on anti-T2D were obtained as a result of this study.

Effects of the Toxic Metals Arsenite and Cadmium on α-Synuclein Aggregation In Vitro and in Cells.

Exposure to heavy metals, including arsenic and cadmium, is associated with neurodegenerative disorders such as Parkinson's disease. However, the mechanistic details of how these metals contribute to pathogenesis are not well understood. To search for underlying mechanisms involving α-synuclein, the protein that forms amyloids in Parkinson's disease, we here assessed the effects of arsenic and cadmium on α-synuclein amyloid formation in vitro and in Saccharomyces cerevisiae (budding yeast) cells. Atomic force microscopy experiments with acetylated human α-synuclein demonstrated that amyloid fibers formed in the presence of the metals have a different fiber pitch compared to those formed without metals. Both metal ions become incorporated into the amyloid fibers, and cadmium also accelerated the nucleation step in the amyloid formation process, likely via binding to intermediate species. Fluorescence microscopy analyses of yeast cells expressing fluorescently tagged α-synuclein demonstrated that arsenic and cadmium affected the distribution of α-synuclein aggregates within the cells, reduced aggregate clearance, and aggravated α-synuclein toxicity. Taken together, our in vitro data demonstrate that interactions between these two metals and α-synuclein modulate the resulting amyloid fiber structures, which, in turn, might relate to the observed effects in the yeast cells. Whilst our study advances our understanding of how these metals affect α-synuclein biophysics, further in vitro characterization as well as human cell studies are desired to fully appreciate their role in the progression of Parkinson's disease.

Chemical Chaperones Modulate the Formation of Metabolite Assemblies.

The formation of amyloid-like structures by metabolites is associated with several inborn errors of metabolism (IEMs). These structures display most of the biological, chemical and physical properties of protein amyloids. However, the molecular interactions underlying the assembly remain elusive, and so far, no modulating therapeutic agents are available for clinical use. Chemical chaperones are known to inhibit protein and peptide amyloid formation and stabilize misfolded enzymes. Here, we provide an in-depth characterization of the inhibitory effect of osmolytes and hydrophobic chemical chaperones on metabolite assemblies, thus extending their functional repertoire. We applied a combined in vivo-in vitro-in silico approach and show their ability to inhibit metabolite amyloid-induced toxicity and reduce cellular amyloid content in yeast. We further used various biophysical techniques demonstrating direct inhibition of adenine self-assembly and alteration of fibril morphology by chemical chaperones. Using a scaffold-based approach, we analyzed the physiochemical properties of various dimethyl sulfoxide derivatives and their role in inhibiting metabolite self-assembly. Lastly, we employed whole-atom molecular dynamics simulations to elucidate the role of hydrogen bonds in osmolyte inhibition. Our results imply a dual mode of action of chemical chaperones as IEMs therapeutics, that could be implemented in the rational design of novel lead-like molecules.

The protein-surfactant stoichiometry governs the conformational switching and amyloid nucleation kinetics of tau K18.

Amyloids are pathological hallmarks of a number of debilitating neurodegenerative diseases. Understanding the molecular mechanism of protein amyloid assembly with an emphasis on structural characterization of early, key prefibrillar species is important for targeted drug design and clinical interventions. Tau is an intrinsically disordered, microtubule-binding protein which is also implicated in various neurodegenerative disorders such as frontotemporal dementia, Down's syndrome, Alzheimer's disease, etc. Earlier reports have demonstrated that tau aggregation in vitro is triggered by anionic inducers, presumably due to charge compensation which facilitates intermolecular association between the tau polypeptide chains. However, the molecular mechanism of tau amyloid aggregation, involving the structural characterization of amyloidogenic intermediates formed especially during early key steps, remains elusive. In this work, we have employed a spectroscopic toolbox to elucidate the mechanism of anionic surfactant-induced disorder-to-order amyloid transition of a tau segment. This study revealed that the amyloid assembly is mediated via binding-induced conformational switching into an early partially helical amyloid-competent intermediate. Additionally, protein and inducer concentration-dependent studies indicated that at the higher protein and/or inducer concentrations, competing off-pathway intermediates dampen the amyloid assembly which implies that the stoichiometry of protein and inducer plays a key regulatory role in the amyloid nucleation and fibril elongation kinetics.

Is Porphyromonas gingivalis involved in Parkinson's disease?

Porphyromonas gingivalis, a major subgingival plaque bacterium in periodontitis, has recently attracted much attention as a possible microbial driver in Alzheimer's disease. In the present paper, another common neuroinflammatory disease, Parkinson's disease (PD), is discussed. A recent study found major virulence factors of P. gingivalis such as gingipain R1 (RgpA) and lipopolysaccharide in the blood circulation of a PD population. The current review reveals how features such as systemic inflammation, hypercoagulation, presence of amyloid fibrin(ogen) in plasma, and marked ultrastructural changes in platelets, probably induced by P. gingivalis, may affect the development of PD. Several other clinical studies have also demonstrated an association between periodontitis and PD. Even if the risk of periodontal diseases causing neurological disorders needs to be better substantiated, that should not keep us from trying to prevent them by performing careful daily dental hygiene.

Differential effects of Cu2+ and Fe3+ ions on in vitro amyloid formation of biologically-relevant α-synuclein variants.

Alterations in metal ion homeostasis appear coupled to neurodegenerative disorders but mechanisms are unknown. Amyloid formation of the protein α-synuclein in brain cells is a hallmark of Parkinson's disease. α-Synuclein can bind several metal ions in vitro and such interactions may affect the assembly process. Here we used biophysical methods to study the effects of micromolar concentrations of Cu2+ and Fe3+ ions on amyloid formation of selected α-synuclein variants (wild-type and A53T α-synuclein, in normal and N-terminally acetylated forms). As shown previously, Cu2+ speeds up aggregation of normal wild-type α-synuclein, but not the acetylated form. However, Cu2+ has a minimal effect on (the faster) aggregation of normal A53T α-synuclein, despite that Cu2+ binds to this variant. Like Cu2+, Fe3+ speeds up aggregation of non-acetylated wild-type α-synuclein, but with acetylation, Fe3+ instead slows down aggregation. In contrast, for A53T α-synuclein, regardless of acetylation, Fe3+ slows down aggregation with the effect being most dramatic for acetylated A53T α-synuclein. The results presented here suggest a correlation between metal-ion modulation effect and intrinsic aggregation speed of the various α-synuclein variants.

A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity.

The self-assembly of α-synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of α-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing α-synuclein, observing a dramatic reduction of α-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson's disease and related conditions.

Oligomerization and Conformational Change Turn Monomeric ß-Amyloid and Tau Proteins Toxic: Their Role in Alzheimer's Pathogenesis.

The structural polymorphism and the physiological and pathophysiological roles of two important proteins, ß-amyloid (Aß) and tau, that play a key role in Alzheimer's disease (AD) are reviewed. Recent results demonstrate that monomeric Aß has important physiological functions. Toxic oligomeric Aß assemblies (AßOs) may play a decisive role in AD pathogenesis. The polymorph fibrillar Aß (fAß) form has a very ordered cross-ß structure and is assumed to be non-toxic. Tau monomers also have several important physiological actions; however, their oligomerization leads to toxic oligomers (TauOs). Further polymerization results in probably non-toxic fibrillar structures, among others neurofibrillary tangles (NFTs). Their structure was determined by cryo-electron microscopy at atomic level. Both AßOs and TauOs may initiate neurodegenerative processes, and their interactions and crosstalk determine the pathophysiological changes in AD. TauOs (perhaps also AßO) have prionoid character, and they may be responsible for cell-to-cell spreading of the disease. Both extra- and intracellular AßOs and TauOs (and not the previously hypothesized amyloid plaques and NFTs) may represent the novel targets of AD drug research.

Structural Insight into IAPP-Derived Amyloid Inhibitors and Their Mechanism of Action.

Designed peptides derived from the islet amyloid polypeptide (IAPP) cross-amyloid interaction surface with Aß (termed interaction surface mimics or ISMs) have been shown to be highly potent inhibitors of Aß amyloid self-assembly. However, the molecular mechanism of their function is not well understood. Using solution-state and solid-state NMR spectroscopy in combination with ensemble-averaged dynamics simulations and other biophysical methods including TEM, fluorescence spectroscopy and microscopy, and DLS, we characterize ISM structural preferences and interactions. We find that the ISM peptide R3-GI is highly dynamic, can adopt a ß-like structure, and oligomerizes into colloid-like assemblies in a process that is reminiscent of liquid-liquid phase separation (LLPS). Our results suggest that such assemblies yield multivalent surfaces for interactions with Aß40. Sequestration of substrates into these colloid-like structures provides a mechanistic basis for ISM function and the design of novel potent anti-amyloid molecules.

A transcriptional signature of Alzheimer's disease is associated with a metastable subproteome at risk for aggregation.

It is well-established that widespread transcriptional changes accompany the onset and progression of Alzheimer's disease. Because of the multifactorial nature of this neurodegenerative disorder and its complex relationship with aging, however, it remains unclear whether such changes are the result of nonspecific dysregulation and multisystem failure or instead are part of a coordinated response to cellular dysfunction. To address this problem in a systematic manner, we performed a meta-analysis of about 1,600 microarrays from human central nervous system tissues to identify transcriptional changes upon aging and as a result of Alzheimer's disease. Our strategy to discover a transcriptional signature of Alzheimer's disease revealed a set of down-regulated genes that encode proteins metastable to aggregation. Using this approach, we identified a small number of biochemical pathways, notably oxidative phosphorylation, enriched in proteins vulnerable to aggregation in control brains and encoded by genes down-regulated in Alzheimer's disease. These results suggest that the down-regulation of a metastable subproteome may help mitigate aberrant protein aggregation when protein homeostasis becomes compromised in Alzheimer's disease.