SERF protein is a direct modifier of amyloid fiber assembly

The inherent cytotoxicity of aberrantly folded protein aggregates contributes substantially to the pathogenesis of amyloid diseases. It was recently shown that a class of evolutionary conserved proteins, called MOAG-4/SERF, profoundly alter amyloid toxicity via an autonomous but yet unexplained mode. We show that the biological function of human SERF1a originates from its atypical ability to specifically distinguish between amyloid and nonamyloid aggregation. This inherently unstructured protein directly affected the aggregation kinetics of a broad range of amyloidogenic proteins in vitro, while being inactive against nonamyloid aggregation. A representative biophysical analysis of the SERF1a:α-synuclein (aSyn) complex revealed that the amyloid-promoting activity resulted from an early and transient interaction, which was sufficient to provoke a massive increase of soluble aSyn amyloid nucleation templates. Therefore, the autonomous amyloid-modifying activity of SERF1a observed in living organisms relies on a direct and dedicated manipulation of the early stages in the amyloid aggregation pathway.     

The cellular modifier MOAG‐4/SERF drives amyloid formation through charge complementation

While aggregation‐prone proteins are known to accelerate aging and cause age‐related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG‐4, SERF1A, and SERF2 have recently been identified as cellular modifiers of such proteotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with protein segments enriched in negatively charged and hydrophobic, aromatic amino acids. The absence of such segments, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid‐promoting activity of SERF2. In protein aggregation models in the nematode worm Caenorhabditis elegans, protein aggregation and toxicity were suppressed by mutating the endogenous locus of MOAG‐4 to neutralize charge. Our data indicate that MOAG‐4 and SERF2 drive protein aggregation and toxicity by interactions with negatively charged segments in aggregation‐prone proteins. Such charge interactions might accelerate primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our study points at charge interactions between cellular modifiers and amyloidogenic proteins as potential targets for interventions to reduce age‐related protein toxicity.     

Molecular characterization of an aggregation-prone variant of alpha-synuclein used to model synucleinopathies

The misfolding and aggregation of alpha-synuclein (aSyn) are thought to be central events in synucleinopathies. The physiological function of aSyn has been related to vesicle binding and trafficking, but the precise molecular mechanisms leading to aSyn pathogenicity are still obscure. In cell models, aSyn does not readily aggregate, even upon overexpression. Therefore, cellular models that enable the study of aSyn aggregation are essential tools for our understanding of the molecular mechanisms that govern such processes. Here, we investigated the structural features of SynT, an artificial variant of aSyn that has been widely used as a model of aggregation in mammalian cell systems, since it is more prone to aggregation than aSyn. Using Nuclear Magnetic Resonance (NMR) spectroscopy we performed a detailed structural characterization of SynT through a systematic comparison with normal, unmodified aSyn. Interestingly, we found that the conformations adopted by SynT resemble those described for the unmodified protein, demonstrating the usefulness of SynT as a model for aSyn aggregation. However, subtle differences were observed at the N-terminal region involving transient intra and/or intermolecular interactions that are known to regulate aSyn aggregation. Importantly, our results indicate that disturbances in the N-terminal region of SynT, and the consequent decrease in membrane binding of the modified protein, might contribute to the observed aggregation behavior of aSyn, and validate the use of SynT, one of the few models of aSyn aggregation in cultured cells.     

Prolyl Oligopeptidase Enhances α-Synuclein Dimerization via Direct Protein-Protein Interaction

Prolyl oligopeptidase (PREP) accelerates the aggregation of α-synuclein (aSyn), a key protein involved in development of Parkinson disease and other synucleinopathies. PREP inhibitors reduce aSyn aggregation, but the mechanism has remained unknown. We have now used protein-fragment complementation assays (PCA) and microscale thermophoresis in parallel to show that PREP interacts directly with aSyn in both intact cells and in a cell-free system. Using split luciferase-based PCA, we first showed that PREP enhances the formation of soluble aSyn dimers in live Neuro-2A neuroblastoma cells. A PREP inhibitor, KYP-2047, reduced aSyn dimerization in PREP-expressing cells but not in cells lacking PREP expression. aSyn dimerization was also enhanced by PREP(S554A), an enzymatically inactive PREP mutant, but this was not affected by KYP-2047. PCA and microscale thermophoresis studies showed that aSyn interacts with both PREP and PREP(S554A) with low micromolar affinity. Neither the proline-rich, C-terminal domain of aSyn nor the hydrolytic activity of PREP was required for the interaction with PREP. Our results show that PREP binds directly to aSyn to enhance its dimerization and may thus serve as a nucleation point for aSyn aggregation. Native gel analysis showed that KYP-2047 shifts PREP to a compact monomeric form with reduced ability to promote aSyn nucleation. As PREP inhibition also enhances autophagic clearance of aSyn, PREP inhibitors may reduce accumulation of aSyn inclusions via a dual mechanism and are thus a novel therapeutic candidate for synucleinopathies. Our results also suggest that PREP has other cellular functions in addition to its peptidase activity.     

Assessing the Subcellular Dynamics of Alpha-synuclein Using Photoactivation Microscopy

Alpha-synuclein (aSyn) is implicated in Parkinson’s disease and several other neurodegenerative disorders. To date, the function and intracellular dynamics of aSyn are still unclear. Here, we tracked the dynamics of aSyn using photoactivatable green fluorescent protein as a reporter. We found that the availability of the aSyn N terminus modulates its shuttling into the nucleus. Interestingly, familial aSyn mutations altered the dynamics at which the protein distributes throughout the cell. Both the A30P and A53T aSyn mutations increase the speed at which the protein moves between the nucleus and cytoplasm, respectively. We also found that specific kinases potentiate the shuttling of aSyn between nucleus and cytoplasm. A mutant aSyn form that blocks S129 phosphorylation, S129A, results in the formation of cytoplasmic inclusions, suggesting phosphorylation modulates aggregation in addition to modulating aSyn intracellular dynamics. Finally, we found that the molecular chaperone HSP70 accelerates the entry of aSyn into the nuclear compartment.     

Intrinsically Semi-disordered State and Its Role in Induced Folding and Protein Aggregation

Intrinsically disordered proteins (IDPs) refer to those proteins without fixed three-dimensional structures under physiological conditions. Although experiments suggest that the conformations of IDPs can vary from random coils, semi-compact globules, to compact globules with different contents of secondary structures, computational efforts to separate IDPs into different states are not yet successful. Recently, we developed a neural-network-based disorder prediction technique SPINE-D that was ranked as one of the top performing techniques for disorder prediction in the biannual meeting of critical assessment of structure prediction techniques (CASP 9, 2010). Here, we further analyze the results from SPINE-D prediction by defining a semi-disordered state that has about 50 % predicted probability to be disordered or ordered. This semi-disordered state is partially collapsed with intermediate levels of predicted solvent accessibility and secondary structure content. The relative difference in compositions between semi-disordered and fully disordered regions is highly correlated with amyloid aggregation propensity (a correlation coefficient of 0.86 if excluding four charged residues and proline, 0.73 if not). In addition, we observed that some semi-disordered regions participate in induced folding, and others play key roles in protein aggregation. More specifically, a semi-disordered region is amyloidogenic in fully unstructured proteins (such as alpha-synuclein and Sup35) but prone to local unfolding that exposes the hydrophobic core to aggregation in structured globular proteins (such as SOD1 and lysozyme). A transition from full disorder to semi-disorder at about 30–40 Qs is observed in the poly-Q (poly-glutamine) tract of huntingtin. The accuracy of using semi-disorder to predict binding-induced folding and aggregation is compared with several methods trained for the purpose. These results indicate the usefulness of three-state classification (order, semi-disorder, and full-disorder) in distinguishing nonfolding from induced-folding and aggregation-resistant from aggregation-prone IDPs and in locating weakly stable, locally unfolding, and potentially aggregation regions in structured proteins. A comparison with five representative disorder-prediction methods showed that SPINE-D is the only method with a clear separation of semi-disorder from ordered and fully disordered states.     

The secondary structure of pressure- and temperature-induced aggregates of equine serum albumin studied by FT-IR spectroscopy

The protein aggregation is divided into amyloid fibrils and amorphous aggregates. Amyloid fibrils are composed of the 3-dimensional ordered structure and are bound to thioflavin T and Congo red dyes. The amorphous aggregates with the disordered structure do not bind to these dyes. We have investigated the pressure- and heat-induced aggregates of equine serum albumin (ESA) from the secondary structural viewpoint using FT-IR spectroscopy. We show the secondary structural differences between heat- and pressure-induced aggregates of ESA. The heat-induced irreversible aggregates of ESA are composed of the intermolecular beta-sheet structure without binding thioflavie T and Congo red to be amorphous form. On the other hand, the pressure-induced reversible aggregates are composed of the random structure to be also amorphous form. From the comparison of pressure effects on ESA in native and reducing conditions of disulfide bridges, we demonstrate that the restriction of structural flexibility by disulfide bridges is an important factor for the reversibility of the pressure-induced aggregation.     

shRNA-Based Screen Identifies Endocytic Recycling Pathway Components That Act as Genetic Modifiers of Alpha-Synuclein Aggregation,Secretion and Toxicity

Alpha-Synuclein (aSyn) misfolding and aggregation is common in several neurodegenerative diseases, including Parkinson’s disease and dementia with Lewy bodies, which are known as synucleinopathies. Accumulating evidence suggests that secretion and cell-to-cell trafficking of pathological forms of aSyn may explain the typical patterns of disease progression. However, the molecular mechanisms controlling aSyn aggregation and spreading of pathology are still elusive. In order to obtain unbiased information about the molecular regulators of aSyn oligomerization, we performed a microscopy-based large-scale RNAi screen in living cells. Interestingly, we identified nine Rab GTPase and kinase genes that modulated aSyn aggregation, toxicity and levels. From those, Rab8b, Rab11a, Rab13 and Slp5 were able to promote the clearance of aSyn inclusions and rescue aSyn induced toxicity. Furthermore, we found that endocytic recycling and secretion of aSyn was enhanced upon Rab11a and Rab13 expression in cells accumulating aSyn inclusions. Overall, our study resulted in the identification of new molecular players involved in the aggregation, toxicity, and secretion of aSyn, opening novel avenues for our understanding of the molecular basis of synucleinopathies.     

The effect of small molecules in modulating the chaperone activity of alphaB-crystallin against ordered and disordered protein aggregation

Protein aggregation can proceed via disordered or ordered mechanisms, with the latter being associated with amyloid fibril formation, which has been linked to a number of debilitating conditions including Alzheimer's, Parkinson's and Creutzfeldt-Jakob diseases. Small heat-shock proteins (sHsps), such as alphaB-crystallin, act as chaperones to prevent protein aggregation and are thought to play a key role in the prevention of protein-misfolding diseases. In this study, we have explored the potential for small molecules such as arginine and guanidine to affect the chaperone activity of alphaB-crystallin against disordered (amorphous) and ordered (amyloid fibril) forms of protein aggregation. The effect of these additives is highly dependent upon the target protein undergoing aggregation. Importantly, our results show that the chaperone action of alphaB-crystallin against aggregation of the disease-related amyloid fibril forming protein alpha-synucleinA53T is enhanced in the presence of arginine and similar positively charged compounds (such as lysine and guanidine). Thus, our results suggest that target protein identity plays a critical role in governing the effect of small molecules on the chaperone action of sHsps. Significantly, small molecules that regulate the activity of sHsps may provide a mechanism to protect cells from the toxic protein aggregation that is associated with some protein-misfolding diseases.     

The effects of the flavonoid baicalein and osmolytes on the Mg 2+ accelerated aggregation/fibrillation of carboxymethylated bovine 1SS-alpha-lactalbumin

Many protein conformational diseases arise when proteins form alternative stable conformations, resulting in aggregation and accumulation of the protein as fibrillar deposits, or amyloids. Interestingly, numerous proteins implicated in amyloid protein formation show similar structural and functional properties. Given this similarity, we tested the notion that carboxymethylated bovine alpha-lactalbumin (1SS-alpha-lac) could serve as a general amyloid fibrillation/aggregation model system. Like most amyloid forming systems, Mg2+ ions accelerate 1SS-alpha-lac amyloid fibril formation. While osmolytes such as trimethylamine N-oxide (TMAO), and sucrose enhanced thioflavin T detected aggregation, a mixture of trehalose and TMAO substantially inhibited aggregation. Most importantly however, the flavonoid, baicalein, known to inhibit alpha-synuclein amyloid fibril formation, also inhibits 1SS-alpha-lac amyloid with the same apparent efficacy. These data suggest that the easily obtainable 1SS-alpha-lac protein can serve as a general amyloid model and that some small molecule amyloid inhibitors may function successfully with many different amyloid systems.     

Glimpses of the molecular mechanisms of β2-microglobulin fibril formation in vitro: Aggregation on a complex energy landscape

β₂-microglobulin (β₂m) is a 99-residue protein that aggregates to form amyloid fibrils in dialysis-related amyloidosis. The protein provides a powerful model for exploration of the structural molecular mechanisms of fibril formation from a full-length protein in vitro. Fibrils have been assembled from β₂m under both low pH conditions, where the precursor is disordered, and at neutral pH where the protein is initially natively folded. Here we discuss the roles of sequence and structure in amyloid formation, the current understanding of the structural mechanisms of the early stages of aggregation of β₂m at both low and neutral pH, and the common and distinct features of these assembly pathways.     

Prevention of amyloid-like aggregation as a driving force of protein evolution

Uncontrolled protein aggregation is a constant challenge in all compartments of living organisms. The failure of a peptide or protein to remain soluble often results in pathology. So far, more than 40 human diseases have been associated with the formation of extracellular fibrillar aggregates - known as amyloid fibrils - or structurally related intracellular deposits. It is well known that molecular chaperones and elaborate quality control mechanisms exist in the cell to counteract aggregation. However, an increasing number of reports during the past few years indicate that proteins have also evolved structural and sequence-based strategies to prevent aggregation. This review describes these strategies and the selection pressures that exist on protein sequences to combat their uncontrolled aggregation. We will describe the different types of mechanism evolved by proteins that adopt different conformational states including normally folded proteins, intrinsically disordered polypeptide chains, elastomeric systems and multimodular proteins.     

FTY720/Fingolimod Reduces Synucleinopathy and Improves Gut Motility in A53T Mice: CONTRIBUTIONS OF PRO-BRAIN-DERIVED NEUROTROPHIC FACTOR (PRO-BDNF) AND MATURE BDNF*

Patients with Parkinson''s disease (PD) often have aggregated α-synuclein (aSyn) in enteric nervous system (ENS) neurons, which may be associated with the development of constipation. This occurs well before the onset of classic PD motor symptoms. We previously found that aging A53T transgenic (Tg) mice closely model PD-like ENS aSyn pathology, making them appropriate for testing potential PD therapies. Here we show that Tg mice overexpressing mutant human aSyn develop ENS pathology by 4 months. We then evaluated the responses of Tg mice and their WT littermates to the Food and Drug Administration-approved drug FTY720 (fingolimod, Gilenya) or vehicle control solution from 5 months of age. Long term oral FTY720 in Tg mice reduced ENS aSyn aggregation and constipation, enhanced gut motility, and increased levels of brain-derived neurotrophic factor (BDNF) but produced no significant change in WT littermates. A role for BDNF was directly assessed in a cohort of young A53T mice given vehicle, FTY720, the Trk-B receptor inhibitor ANA-12, or FTY720 + ANA-12 from 1 to 4 months of age. ANA-12-treated Tg mice developed more gut aSyn aggregation as well as constipation, whereas FTY720-treated Tg mice had reduced aSyn aggregation and less constipation, occurring in part by increasing both pro-BDNF and mature BDNF levels. The data from young and old Tg mice revealed FTY720-associated neuroprotection and reduced aSyn pathology, suggesting that FTY720 may also benefit PD patients and others with synucleinopathy. Another finding was a loss of tyrosine hydroxylase immunoreactivity in gut neurons with aggregated aSyn, comparable with our prior findings in the CNS.     

Design of model systems for amyloid formation: lessons for prediction and inhibition

The determination of the physico-chemical principles underlying amyloid deposition is fundamental to the identification of therapeutic strategies to prevent or cure amyloid-related disorders. Given the complexity of the molecular events involved in protein self-association, researchers have designed simplified systems that facilitate the discovery of factors that predispose polypeptides to amyloid formation and aggregation. These systems have provided valuable knowledge about the determinants underlying the structural transitions to the polymeric beta-sheet state present in amyloid fibers and in more disordered aggregates. The integration of this knowledge is crucial to the identification of the regions responsible for the amyloidogenic and aggregating behavior of a given protein. The reliable discovery of amyloid-promoting fragments in proteins should have a great impact on the development of anti-amyloid agents. Also, methods that identify aggregation-prone motifs have a broad range of biotechnological applications, such as the improvement of the solubility of recombinant proteins for pharmaceutical and industrial purposes, and peptide-based biomaterial engineering.     

Prolyl oligopeptidase inhibition reduces alpha-synuclein aggregation in a cellular model of multiple system atrophy

Multiple system atrophy (MSA) is a fatal neurodegenerative disease where the histopathological hallmark is glial cytoplasmic inclusions in oligodendrocytes, rich of aggregated alpha-synuclein (aSyn). Therefore, therapies targeting aSyn aggregation and toxicity have been studied as a possible disease-modifying therapy for MSA. Our earlier studies show that inhibition of prolyl oligopeptidase (PREP) with KYP-2047 reduces aSyn aggregates in several models. Here, we tested the effects of KYP-2047 on a MSA cellular models, using rat OLN-AS7 and human MO3.13 oligodendrocyte cells. As translocation of p25α to cell cytosol has been identified as an inducer of aSyn aggregation in MSA models, the cells were transiently transfected with p25α. Similar to earlier studies, p25α increased aSyn phosphorylation and aggregation, and caused tubulin retraction and impaired autophagy in OLN-AS7 cells. In both cellular models, p25α transfection increased significantly aSyn mRNA levels and also increased the levels of inactive protein phosphatase 2A (PP2A). However, aSyn or p25α did not cause any cellular death in MO3.13 cells, questioning their use as a MSA model. Simultaneous administration of 10 µM KYP-2047 improved cell viability, decreased insoluble phosphorylated aSyn and normalized autophagy in OLN-AS7 cells but similar impact was not seen in MO3.13 cells.     

SERF: in vitro election of random RNA fragments to identify protein binding sites within large RNAs

In vitro selection experiments have various goals depending on the composition of the initial pool and the selection method applied. We developed an in vitro selection variant (SERF, selection of random RNA fragments) that is useful for the identification of short RNA fragments originating from large RNAs that bind specifically to a protein. A pool of randomly fragmented RNA is constructed from a large RNA, which is the natural binding partner for a protein. Such a pool contains all the potential binding sites and is therefore used as starting material for affinity selection with the purified protein to find its natural target. Here we provide a detailed experimental protocol of the method. SERF has been developed for ribosomal systems and is a general approach providing a basis for functional and structural characterization of RNA-protein interactions in large ribonucleoprotein particles.     

Phosphorylation Modulates Clearance of Alpha-Synuclein Inclusions in a Yeast Model of Parkinson's Disease

Alpha-synuclein (aSyn) is the main component of proteinaceous inclusions known as Lewy bodies (LBs), the typical pathological hallmark of Parkinson''s disease (PD) and other synucleinopathies. Although aSyn is phosphorylated at low levels under physiological conditions, it is estimated that ∼90% of aSyn in LBs is phosphorylated at S129 (pS129). Nevertheless, the significance of pS129 in the biology of aSyn and in PD pathogenesis is still controversial. Here, we harnessed the power of budding yeast in order to assess the implications of phosphorylation on aSyn cytotoxicity, aggregation and sub-cellular distribution. We found that aSyn is phosphorylated on S129 by endogenous kinases. Interestingly, phosphorylation reduced aSyn toxicity and the percentage of cells with cytosolic inclusions, in comparison to cells expressing mutant forms of aSyn (S129A or S129G) that mimic the unphosphorylated form of aSyn. Using high-resolution 4D imaging and fluorescence recovery after photobleaching (FRAP) in live cells, we compared the dynamics of WT and S129A mutant aSyn. While WT aSyn inclusions were very homogeneous, inclusions formed by S129A aSyn were larger and showed FRAP heterogeneity. Upon blockade of aSyn expression, cells were able to clear the inclusions formed by WT aSyn. However, this process was much slower for the inclusions formed by S129A aSyn. Interestingly, whereas the accumulation of WT aSyn led to a marked induction of autophagy, cells expressing the S129A mutant failed to activate this protein quality control pathway. The finding that the phosphorylation state of aSyn on S129 can alter the ability of cells to clear aSyn inclusions provides important insight into the role that this posttranslational modification may have in the pathogenesis of PD and other synucleinopathies, opening novel avenues for investigating the molecular basis of these disorders and for the development of therapeutic strategies.     

Interpreting the aggregation kinetics of amyloid peptides

Amyloid fibrils are insoluble mainly beta-sheet aggregates of proteins or peptides. The multi-step process of amyloid aggregation is one of the major research topics in structural biology and biophysics because of its relevance in protein misfolding diseases like Alzheimer's, Parkinson's, Creutzfeld-Jacob's, and type II diabetes. Yet, the detailed mechanism of oligomer formation and the influence of protein stability on the aggregation kinetics are still matters of debate. Here a coarse-grained model of an amphipathic polypeptide, characterized by a free energy profile with distinct amyloid-competent (i.e. beta-prone) and amyloid-protected states, is used to investigate the kinetics of aggregation and the pathways of fibril formation. The simulation results suggest that by simply increasing the relative stability of the beta-prone state of the polypeptide, disordered aggregation changes into fibrillogenesis with the presence of oligomeric on-pathway intermediates, and finally without intermediates in the case of a very stable beta-prone state. The minimal-size aggregate able to form a fibril is generated by collisions of oligomers or monomers for polypeptides with unstable or stable beta-prone state, respectively. The simulation results provide a basis for understanding the wide range of amyloid-aggregation mechanisms observed in peptides and proteins. Moreover, they allow us to interpret at a molecular level the much faster kinetics of assembly of a recently discovered functional amyloid with respect to the very slow pathological aggregation.     

Electrostatic modulation of hnRNPA1 low‐complexity domain liquid–liquid phase separation and aggregation

Membrane‐less organelles and RNP granules are enriched in RNA and RNA‐binding proteins containing disordered regions. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a key regulating protein in RNA metabolism, localizes to cytoplasmic RNP granules including stress granules. Dysfunctional nuclear‐cytoplasmic transport and dynamic phase separation of hnRNPA1 leads to abnormal amyloid aggregation and neurodegeneration. The intrinsically disordered C‐terminal domain (CTD) of hnRNPA1 mediates both dynamic liquid–liquid phase separation (LLPS) and aggregation. While cellular phase separation drives the formation of membrane‐less organelles, aggregation within phase‐separated compartments has been linked to neurodegenerative diseases. To understand some of the underlying mechanisms behind protein phase separation and LLPS‐mediated aggregation, we studied LLPS of hnRNPA1 CTD in conditions that probe protein electrostatics, modulated specifically by varying pH conditions, and protein, salt and RNA concentrations. In the conditions investigated, we observed LLPS to be favored in acidic conditions, and by high protein, salt and RNA concentrations. We also observed that conditions that favor LLPS also enhance protein aggregation and fibrillation, which suggests an aggregation pathway that is LLPS‐mediated. The results reported here also suggest that LLPS can play a direct role in facilitating protein aggregation, and that changes in cellular environment that affect protein electrostatics can contribute to the pathological aggregation exhibited in neurodegeneration.     

Conformational transition occurring upon amyloid aggregation of the HET-s prion protein of Podospora anserina analyzed by hydrogen/deuterium exchange and mass spectrometry

The [Het-s] infectious element of the filamentous fungus Podospora anserina corresponds to the prion form of the HET-s protein. HET-s (289 amino acids in length) aggregates into amyloid fibers in vitro. Such fibers obtained in vitro are infectious, indicating that the [Het-s] prion can propagate as a self-perpetuating amyloid aggregate of the HET-s protein. Previous analyses have suggested that only a limited region of the HET-s protein is involved in amyloid formation and prion propagation. To document the conformational transition occurring upon amyloid aggregation of HET-s, we have developed a method involving hydrogen/deuterium exchange monitored by MALDI-MS. In a first step, a peptide mass fingerprint of the protein was obtained, leading to 87% coverage of the HET-s primary structure. Amyloid aggregates of HET-s were obtained, and H/D exchange was monitored on the soluble and on the amyloid form of HET-s. This study revealed that in the soluble form of HET-s, the C-terminal region (spanning from residues 240-289) displays a high solvent accessibility. In sharp contrast, solvent accessibility is drastically reduced in that region in the amyloid form. H/D exchange rates and levels in the N-terminal part of the protein (residues 1-220) are comparable in the soluble and the aggregated state. These results indicate that amyloid aggregation of HET-s involves a conformational transition of the C-terminal part of the protein from a mainly disordered to an aggregated state in which this region is highly protected from hydrogen exchange.