Publications by authors named "Magdalena I Ivanova"

30 Publications

  • Page 1 of 1

Rational affinity maturation of anti-amyloid antibodies with high conformational and sequence specificity.

J Biol Chem 2021 Mar 3:100508. Epub 2021 Mar 3.

Department of Chemical Engineering,; Biointerfaces Institute,; Department of Pharmaceutical Sciences,; Department of Biomedical Engineering,; Protein Folding Disease Initiative,. Electronic address:

The aggregation of amyloidogenic polypeptides is strongly linked to several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Conformational antibodies that selectively recognize protein aggregates are leading therapeutic agents for selectively neutralizing toxic aggregates, diagnostic and imaging agents for detecting disease, and biomedical reagents for elucidating disease mechanisms. Despite their importance, it is challenging to generate high-quality conformational antibodies in a systematic and site-specific manner due to the properties of protein aggregates (hydrophobic, multivalent and heterogeneous) and limitations of immunization (uncontrolled antigen presentation and immunodominant linear epitopes). Toward addressing these challenges, we have developed a systematic directed evolution procedure for affinity maturing antibodies against Alzheimer's Aβ fibrils and selecting variants with strict conformational and sequence specificity. We first designed a library based on a lead conformational antibody by sampling combinations of amino acids in the antigen-binding site predicted to lead to high antibody specificity. Next, we displayed this library on the surface of yeast, sorted it against Aβ aggregates, and identified promising clones using deep sequencing. We identified several antibodies with similar or higher affinities than clinical-stage Aβ antibodies (aducanumab and crenezumab). Moreover, the affinity-matured antibodies retain high conformational specificity for Aβ aggregates, as observed for aducanumab and unlike crenezumab. Notably, the affinity-maturated antibodies display extremely low levels of non-specific interactions, as observed for crenezumab and unlike aducanumab. We expect that our systematic methods for generating antibodies with unique combinations of desirable properties will improve the generation of high-quality conformational antibodies specific for diverse types of aggregated conformers.
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http://dx.doi.org/10.1016/j.jbc.2021.100508DOI Listing
March 2021

Shared and divergent phase separation and aggregation properties of brain-expressed ubiquilins.

Sci Rep 2021 Jan 11;11(1):287. Epub 2021 Jan 11.

Department of Neurology, University of Michigan, Ann Arbor, MI, 48109-2200, USA.

The brain-expressed ubiquilins, UBQLNs 1, 2 and 4, are highly homologous proteins that participate in multiple aspects of protein homeostasis and are implicated in neurodegenerative diseases. Studies have established that UBQLN2 forms liquid-like condensates and accumulates in pathogenic aggregates, much like other proteins linked to neurodegenerative diseases. However, the relative condensate and aggregate formation of the three brain-expressed ubiquilins is unknown. Here we report that the three ubiquilins differ in aggregation propensity, revealed by in-vitro experiments, cellular models, and analysis of human brain tissue. UBQLN4 displays heightened aggregation propensity over the other ubiquilins and, like amyloids, UBQLN4 forms ThioflavinT-positive fibrils in vitro. Measuring fluorescence recovery after photobleaching (FRAP) of puncta in cells, we report that all three ubiquilins undergo liquid-liquid phase transition. UBQLN2 and 4 exhibit slower recovery than UBQLN1, suggesting the condensates formed by these brain-expressed ubiquilins have different compositions and undergo distinct internal rearrangements. We conclude that while all brain-expressed ubiquilins exhibit self-association behavior manifesting as condensates, they follow distinct courses of phase-separation and aggregation. We suggest that this variability among ubiquilins along the continuum from liquid-like to solid informs both the normal ubiquitin-linked functions of ubiquilins and their accumulation and potential contribution to toxicity in neurodegenerative diseases.
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http://dx.doi.org/10.1038/s41598-020-78775-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801659PMC
January 2021

Biophysical processes underlying cross-seeding in amyloid aggregation and implications in amyloid pathology.

Biophys Chem 2021 Feb 19;269:106507. Epub 2020 Nov 19.

Biophysics, University of Michigan, Ann Arbor, MI 48109, USA; Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA; Macromolecular Science and Engineering, University of Michigan, Ann Arbor, MI 48109, USA; Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

Abnormal aggregation of proteins into filamentous aggregates commonly associates with many diseases, such as Alzheimer's disease, Parkinson's disease and type-2 diabetes. These filamentous aggregates, also known as amyloids, can propagate their abnormal structures to either the same precursor molecules (seeding) or other protein monomers (cross-seeding). Cross-seeding has been implicated in the abnormal protein aggregation and has been found to facilitate the formation of physiological amyloids. It has risen to be an exciting area of research with a high volume of published reports. In this review article, we focus on the biophysical processes underlying the cross-seeding for some of the most commonly studied amyloid proteins. Here we will discuss the relevant literature related to cross-seeded polymerization of amyloid-beta, human islet amyloid polypeptide (hIAPP, or also known as amylin) and alpha-synuclein. SEVI (semen-derived enhancer of viral infection) amyloid formation by the cross-seeding between the bacterial curli protein and PAP is also briefly discussed.
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http://dx.doi.org/10.1016/j.bpc.2020.106507DOI Listing
February 2021

Small molecule induced toxic human-IAPP species characterized by NMR.

Chem Commun (Camb) 2020 Nov 2;56(86):13129-13132. Epub 2020 Oct 2.

Department of Chemistry, University of Michigan, Ann Arbor, MI, USA.

In this study, the effect of CurDAc, a water-soluble curcumin derivative, on the formation and stability of amyloid fibers is revealed. CurDAc interaction with amyloid is structurally selective, which is reflected in a strong interference with hIAPP aggregation while showing weaker interactions with human-calcitonin and amyloid-β in comparison. Remarkably, CurDAc also exhibited potent fiber disaggregation for hIAPP generating a toxic oligomeric species.
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http://dx.doi.org/10.1039/d0cc04803hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641245PMC
November 2020

High-Throughput Screening at the Membrane Interface Reveals Inhibitors of Amyloid-β.

Biochemistry 2020 06 5;59(24):2249-2258. Epub 2020 Jun 5.

Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.

Aggregation and the formation of oligomeric intermediates of amyloid-β (Aβ) at the membrane interface of neuronal cells are implicated in the cellular toxicity and pathology of Alzheimer's disease. Small molecule compounds have been shown to suppress amyloid aggregation and cellular toxicity, but often the presence of a lipid membrane negates their activity. A high-throughput screen of 1800 small molecules was performed to search for membrane active inhibitors, and 21 primary hits were discovered. Through the use of fluorescence-based assays, transmission electron microscopy, and dot blot assays, the initial 21 primary hits were narrowed down to five lead compounds. Nuclear magnetic resonance and circular dichroism experiments were used for further confirmation of amyloid inhibition at the membrane interface and to obtain insights into the secondary structure of amyloid-β, while size exclusion chromatography was used to characterize the size of Aβ species. Lastly, dye-leakage assays allowed us to understand how the addition of the five lead compounds affected amyloid-β's ability to permeate the lipid bilayer. These results provide insights into small molecules that stabilize small amyloid species in the presence of membranes for the development of tool compounds for deeper investigations of these transient species.
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http://dx.doi.org/10.1021/acs.biochem.0c00328DOI Listing
June 2020

Thiol-mediated and catecholamine-enhanced multimerization of a cerebrovascular disease enriched fragment of NOTCH3.

Exp Neurol 2020 06 28;328:113261. Epub 2020 Feb 28.

Departments of Neurology, University of Michigan, Ann Arbor, MI 48109-5622, USA; Neurology Service, VA Ann Arbor Healthcare System, Ann Arbor, MI 48105, USA. Electronic address:

Cerebral small vessel disease is a common condition linked to dementia and stroke. As an age-dependent brain pathology, cerebral SVD may share molecular processes with core neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Many neurodegenerative diseases feature abnormal protein accumulation and aberrant protein folding, resulting in multimerization of specific proteins. We investigated if a small NOTCH3 N-terminal fragment (NTF) that co-registers with pathologically affected cells in the inherited SVD, CADASIL, is capable of multimerization. We also characterized endogenous small molecule vascular enhancers and inhibitors of multimerization. NTF multimerizes spontaneously and also forms conjugates with vascular catecholamines, including dopamine and norepinephrine, which avidly promote multimerization of the protein. Inhibition of catecholamine-dependent multimerization by vitamin C and reversal by reducing agents implicate an essential role of oxidation in NTF multimerization. Antibodies that react with degenerating arteries in CADASIL tissue preferentially bind to multimerized forms of NTF. These studies suggest that multimerization of proteins in the aging brain is not restricted to neuronal molecules and may participate in age-dependent vascular pathology.
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http://dx.doi.org/10.1016/j.expneurol.2020.113261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146869PMC
June 2020

High-throughput screening yields several small-molecule inhibitors of repeat-associated non-AUG translation.

J Biol Chem 2019 12 23;294(49):18624-18638. Epub 2019 Oct 23.

Department of Neurology, University of Michigan, Ann Arbor, Michigan 48109; Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, Michigan 48109; Veterans Affairs Ann Arbor Healthcare System, Ann Arbor, Michigan 48105. Electronic address:

Repeat-associated non-AUG (RAN) translation is a noncanonical translation initiation event that occurs at nucleotide-repeat expansion mutations that are associated with several neurodegenerative diseases, including fragile X-associated tremor ataxia syndrome (FXTAS), ALS, and frontotemporal dementia (FTD). Translation of expanded repeats produces toxic proteins that accumulate in human brains and contribute to disease pathogenesis. Consequently, RAN translation constitutes a potentially important therapeutic target for managing multiple neurodegenerative disorders. Here, we adapted a previously developed RAN translation assay to a high-throughput format to screen 3,253 bioactive compounds for inhibition of RAN translation of expanded CGG repeats associated with FXTAS. We identified five diverse small molecules that dose-dependently inhibited CGG RAN translation, while relatively sparing canonical translation. All five compounds also inhibited RAN translation of expanded GGGGCC repeats associated with ALS and FTD. Using CD and native gel analyses, we found evidence that three of these compounds, BIX01294, CP-31398, and propidium iodide, bind directly to the repeat RNAs. These findings provide proof-of-principle supporting the development of selective small-molecule RAN translation inhibitors that act across multiple disease-causing repeats.
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http://dx.doi.org/10.1074/jbc.RA119.009951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6901296PMC
December 2019

Mechanistic insights into the protective roles of polyphosphate against amyloid cytotoxicity.

Life Sci Alliance 2019 10 18;2(5). Epub 2019 Sep 18.

Department of Molecular, Cellular and Developmental Biology University of Michigan, Ann Arbor, MI, USA

The universally abundant polyphosphate (polyP) accelerates fibril formation of disease-related amyloids and protects against amyloid cytotoxicity. To gain insights into the mechanism(s) by which polyP exerts these effects, we focused on α-synuclein, a well-studied amyloid protein, which constitutes the major component of Lewy bodies found in Parkinson's disease. Here, we demonstrate that polyP is unable to accelerate the rate-limiting step of α-synuclein fibril formation but effectively nucleates fibril assembly once α-synuclein oligomers are formed. Binding of polyP to α-synuclein either during fibril formation or upon fibril maturation substantially alters fibril morphology and effectively reduces the ability of α-synuclein fibrils to interact with cell membranes. The effect of polyP appears to be α-synuclein fibril specific and successfully prevents the uptake of fibrils into neuronal cells. These results suggest that altering the polyP levels in the extracellular space might be a potential therapeutic strategy to prevent the spreading of the disease.
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http://dx.doi.org/10.26508/lsa.201900486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6751573PMC
October 2019

Tau's Three-Repeat Domain and EFhd2 Co-incubation Leads to Increased Thioflavin Signal.

Front Neurosci 2018 3;12:879. Epub 2018 Dec 3.

Department of Neurology, University of Michigan, Ann Arbor, MI, United States.

Aggregation of the protein tau is a pathological hallmark of Alzheimer's disease (AD) and related disorders. However, the molecular mechanisms that lead to tau protein aggregation are still unclear. Previously, we showed that EFhd2 protein is associated with pathological aggregated forms of tau in AD brain. Further, immuno-gold analyses of purified tau aggregates showed that EFhd2 co-localized with filamentous tau structures. We demonstrated that EFhd2's coiled-coil domain is required for its association with tau proteins. However, it is unknown the role that EFhd2 plays in tau aggregation. Here, we show that incubation of K19-tau with substoichiometric amount of EFhd2 promote the formation of amyloid structures . The result suggests that EFhd2 may play a role in the biogenesis of aggregated tau.
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http://dx.doi.org/10.3389/fnins.2018.00879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286997PMC
December 2018

Mutant UBQLN2 promotes toxicity by modulating intrinsic self-assembly.

Proc Natl Acad Sci U S A 2018 10 17;115(44):E10495-E10504. Epub 2018 Oct 17.

Department of Neurology, University of Michigan, Ann Arbor, MI 48109-2200;

UBQLN2 is one of a family of proteins implicated in ubiquitin-dependent protein quality control and integrally tied to human neurodegenerative disease. Whereas wild-type UBQLN2 accumulates in intraneuronal deposits in several common age-related neurodegenerative diseases, mutations in the gene encoding this protein result in X-linked amyotrophic lateral sclerosis/frontotemporal dementia associated with TDP43 accumulation. Using in vitro protein analysis, longitudinal fluorescence imaging and cellular, neuronal, and transgenic mouse models, we establish that UBQLN2 is intrinsically prone to self-assemble into higher-order complexes, including liquid-like droplets and amyloid aggregates. UBQLN2 self-assembly and solubility are reciprocally modulated by the protein's ubiquitin-like and ubiquitin-associated domains. Moreover, a pathogenic UBQLN2 missense mutation impairs droplet dynamics and favors amyloid-like aggregation associated with neurotoxicity. These data emphasize the critical link between UBQLN2's role in ubiquitin-dependent pathways and its propensity to self-assemble and aggregate in neurodegenerative diseases.
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http://dx.doi.org/10.1073/pnas.1810522115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217421PMC
October 2018

Inhibition of curli assembly and biofilm formation by the human systemic amyloid precursor transthyretin.

Proc Natl Acad Sci U S A 2017 11 30;114(46):12184-12189. Epub 2017 Oct 30.

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-1048;

During biofilm formation, and other Enterobacteriaceae produce an extracellular matrix consisting of curli amyloid fibers and cellulose. The precursor of curli fibers is the amyloidogenic protein CsgA. The human systemic amyloid precursor protein transthyretin (TTR) is known to inhibit amyloid-β (Aβ) aggregation in vitro and suppress the Alzheimer's-like phenotypes in a transgenic mouse model of Aβ deposition. We hypothesized that TTR might have broad antiamyloid activity because the biophysical properties of amyloids are largely conserved across species and kingdoms. Here, we report that both human WT tetrameric TTR (WT-TTR) and its engineered nontetramer-forming monomer (M-TTR, F87M/L110M) inhibit CsgA amyloid formation in vitro, with M-TTR being the more efficient inhibitor. Preincubation of WT-TTR with small molecules that occupy the T4 binding site eliminated the inhibitory capacity of the tetramer; however, they did not significantly compromise the ability of M-TTR to inhibit CsgA amyloidogenesis. TTR also inhibited amyloid-dependent biofilm formation in two different bacterial species with no apparent bactericidal or bacteriostatic effects. These discoveries suggest that TTR is an effective antibiofilm agent that could potentiate antibiotic efficacy in infections associated with significant biofilm formation.
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http://dx.doi.org/10.1073/pnas.1708805114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5699053PMC
November 2017

Flow-aligned, single-shot fiber diffraction using a femtosecond X-ray free-electron laser.

Cytoskeleton (Hoboken) 2017 Dec 19;74(12):472-481. Epub 2017 Jun 19.

Center for Free Electron Laser Science, DESY, Notkestrasse 85, Hamburg, 22607, Germany.

A major goal for X-ray free-electron laser (XFEL) based science is to elucidate structures of biological molecules without the need for crystals. Filament systems may provide some of the first single macromolecular structures elucidated by XFEL radiation, since they contain one-dimensional translational symmetry and thereby occupy the diffraction intensity region between the extremes of crystals and single molecules. Here, we demonstrate flow alignment of as few as 100 filaments (Escherichia coli pili, F-actin, and amyloid fibrils), which when intersected by femtosecond X-ray pulses result in diffraction patterns similar to those obtained from classical fiber diffraction studies. We also determine that F-actin can be flow-aligned to a disorientation of approximately 5 degrees. Using this XFEL-based technique, we determine that gelsolin amyloids are comprised of stacked β-strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual α-synuclein amyloids.
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http://dx.doi.org/10.1002/cm.21378DOI Listing
December 2017

Reduced Lipid Bilayer Thickness Regulates the Aggregation and Cytotoxicity of Amyloid-β.

J Biol Chem 2017 03 1;292(11):4638-4650. Epub 2017 Feb 1.

From the Department of Chemistry,

The aggregation of amyloid-β (Aβ) on lipid bilayers has been implicated as a mechanism by which Aβ exerts its toxicity in Alzheimer's disease (AD). Lipid bilayer thinning has been observed during both oxidative stress and protein aggregation in AD, but whether these pathological modifications of the bilayer correlate with Aβ misfolding is unclear. Here, we studied peptide-lipid interactions in synthetic bilayers of the short-chain lipid dilauroyl phosphatidylcholine (DLPC) as a simplified model for diseased bilayers to determine their impact on Aβ aggregate, protofibril, and fibril formation. Aβ aggregation and fibril formation in membranes composed of dioleoyl phosphatidylcholine (DOPC) or 1- palmitoyl-2-oleoyl phosphatidylcholine mimicking normal bilayers served as controls. Differences in aggregate formation and stability were monitored by a combination of thioflavin-T fluorescence, circular dichroism, atomic force microscopy, transmission electron microscopy, and NMR. Despite the ability of all three lipid bilayers to catalyze aggregation, DLPC accelerates aggregation at much lower concentrations and prevents the fibrillation of Aβ at low micromolar concentrations. DLPC stabilized globular, membrane-associated oligomers, which could disrupt the bilayer integrity. DLPC bilayers also remodeled preformed amyloid fibrils into a pseudo-unfolded, molten globule state, which resembled on-pathway, protofibrillar aggregates. Whereas the stabilized, membrane-associated oligomers were found to be nontoxic, the remodeled species displayed toxicity similar to that of conventionally prepared aggregates. These results provide mechanistic insights into the roles that pathologically thin bilayers may play in Aβ aggregation on neuronal bilayers, and pathological lipid oxidation may contribute to Aβ misfolding.
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http://dx.doi.org/10.1074/jbc.M116.764092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5377779PMC
March 2017

Neurotoxicity of the Parkinson Disease-Associated Pesticide Ziram Is Synuclein-Dependent in Zebrafish Embryos.

Environ Health Perspect 2016 Nov 15;124(11):1766-1775. Epub 2016 Jun 15.

Department of Neurology, University of Los Angeles (UCLA), Los Angeles, California, USA.

Background: Exposure to the commonly used dithiocarbamate (DTC) pesticides is associated with an increased risk of developing Parkinson disease (PD), although the mechanisms by which they exert their toxicity are not completely understood.

Objective: We studied the mechanisms of ziram's (a DTC fungicide) neurotoxicity in vivo.

Methods: Zebrafish (ZF) embryos were utilized to determine ziram's effects on behavior, neuronal toxicity, and the role of synuclein in its toxicity.

Results: Nanomolar-range concentrations of ziram caused selective loss of dopaminergic (DA) neurons and impaired swimming behavior. Because ziram increases α-synuclein (α-syn) concentrations in rat primary neuronal cultures, we investigated the effect of ziram on ZF γ-synuclein 1 (γ1). ZF express 3 synuclein isoforms, and ZF γ1 appears to be the closest functional homologue to α-syn. We found that recombinant ZF γ1 formed fibrils in vitro, and overexpression of ZF γ1 in ZF embryos led to the formation of neuronal aggregates and neurotoxicity in a manner similar to that of α-syn. Importantly, knockdown of ZF γ1 with morpholinos and disruption of oligomers with the molecular tweezer CLR01 prevented ziram's DA toxicity.

Conclusions: These data show that ziram is selectively toxic to DA neurons in vivo, and this toxicity is synuclein-dependent. These findings have important implications for understanding the mechanisms by which pesticides may cause PD. Citation: Lulla A, Barnhill L, Bitan G, Ivanova MI, Nguyen B, O'Donnell K, Stahl MC, Yamashiro C, Klärner FG, Schrader T, Sagasti A, Bronstein JM. 2016. Neurotoxicity of the Parkinson disease-associated pesticide ziram is synuclein-dependent in zebrafish embryos. Environ Health Perspect 124:1766-1775; http://dx.doi.org/10.1289/EHP141.
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http://dx.doi.org/10.1289/EHP141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5089875PMC
November 2016

Distinct C9orf72-Associated Dipeptide Repeat Structures Correlate with Neuronal Toxicity.

PLoS One 2016 24;11(10):e0165084. Epub 2016 Oct 24.

Department of Neurology, University of Michigan, Ann Arbor, Michigan, United States of America.

Hexanucleotide repeat expansions in C9orf72 are the most common inherited cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansions elicit toxicity in part through repeat-associated non-AUG (RAN) translation of the intronic (GGGGCC)n sequence into dipeptide repeat-containing proteins (DPRs). Little is known, however, about the structural characteristics and aggregation propensities of the dipeptide units comprising DPRs. To address this question, we synthesized dipeptide units corresponding to the three sense-strand RAN translation products, analyzed their structures by circular dichroism, electron microscopy and dye binding assays, and assessed their relative toxicity when applied to primary cortical neurons. Short, glycine-arginine (GR)3 dipeptides formed spherical aggregates and selectively reduced neuronal survival compared to glycine-alanine (GA)3 and glycine-proline (GP)3 dipeptides. Doubling peptide length had little effect on the structure of GR or GP peptides, but (GA)6 peptides formed β-sheet rich aggregates that bound thioflavin T and Congo red yet lacked the typical fibrillar morphology of amyloids. Aging of (GA)6 dipeptides increased their β-sheet content and enhanced their toxicity when applied to neurons. We also observed that the relative toxicity of each tested dipeptide was proportional to peptide internalization. Our results demonstrate that different C9orf72-related dipeptides exhibit distinct structural properties that correlate with their relative toxicity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0165084PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5077081PMC
June 2017

Unbiased screen identifies aripiprazole as a modulator of abundance of the polyglutamine disease protein, ataxin-3.

Brain 2016 11;139(11):2891-2908

Department of Neurology, University of Michigan, Ann Arbor, MI, USA.

No disease-modifying treatment exists for the fatal neurodegenerative polyglutamine disease known both as Machado-Joseph disease and spinocerebellar ataxia type 3. As a potential route to therapy, we identified small molecules that reduce levels of the mutant disease protein, ATXN3. Screens of a small molecule collection, including 1250 Food and Drug Administration-approved drugs, in a novel cell-based assay, followed by secondary screens in brain slice cultures from transgenic mice expressing the human disease gene, identified the atypical antipsychotic aripiprazole as one of the hits. Aripiprazole increased longevity in a Drosophila model of Machado-Joseph disease and effectively reduced aggregated ATXN3 species in flies and in brains of transgenic mice treated for 10 days. The aripiprazole-mediated decrease in ATXN3 abundance may reflect a complex response culminating in the modulation of specific components of cellular protein homeostasis. Aripiprazole represents a potentially promising therapeutic drug for Machado-Joseph disease and possibly other neurological proteinopathies.
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http://dx.doi.org/10.1093/brain/aww228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5840879PMC
November 2016

Structure of the toxic core of α-synuclein from invisible crystals.

Nature 2015 Sep 9;525(7570):486-90. Epub 2015 Sep 9.

Howard Hughes Medical Institute, UCLA-DOE Institute, Departments of Biological Chemistry and Chemistry and Biochemistry, Box 951570, UCLA, Los Angeles, California 90095-1570, USA.

The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy. As the crystals are thousands of times too small for structure determination by synchrotron X-ray diffraction, we use micro-electron diffraction to determine the structure at atomic resolution. The 1.4 Å resolution structure demonstrates that this method can determine previously unknown protein structures and here yields, to our knowledge, the highest resolution achieved by any cryo-electron microscopy method to date. The structure exhibits protofibrils built of pairs of face-to-face β-sheets. X-ray fibre diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. The NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, presenting opportunities for the design of inhibitors of α-synuclein fibrils.
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http://dx.doi.org/10.1038/nature15368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791177PMC
September 2015

Competitive inhibition reaction mechanisms for the two-step model of protein aggregation.

Biophys Chem 2014 Sep-Oct;193-194:9-19. Epub 2014 Jul 5.

Department of Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI 48109, USA; Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, MI 48109, USA; Brehm Center for Diabetes Research, University of Michigan Medical School, Ann Arbor, MI 48105, USA. Electronic address:

We propose three new reaction mechanisms for competitive inhibition of protein aggregation for the two-step model of protein aggregation. The first mechanism is characterized by the inhibition of native protein, the second is characterized by the inhibition of aggregation-prone protein and the third mechanism is characterized by the mixed inhibition of native and aggregation-prone proteins. Rate equations are derived for these mechanisms, and a method is described for plotting kinetic results to distinguish these three types of inhibitors. The derived rate equations provide a simple way of estimating the inhibition constant of native or aggregation-prone protein inhibitors in protein aggregation. The new approach is used to estimate the inhibition constants of different peptide inhibitors of insulin aggregation.
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http://dx.doi.org/10.1016/j.bpc.2014.06.006DOI Listing
May 2015

Molecular basis for preventing α-synuclein aggregation by a molecular tweezer.

J Biol Chem 2014 Apr 24;289(15):10727-10737. Epub 2014 Feb 24.

Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan 48823. Electronic address:

Recent work on α-synuclein has shown that aggregation is controlled kinetically by the rate of reconfiguration of the unstructured chain, such that the faster the reconfiguration, the slower the aggregation. In this work we investigate this relationship by examining α-synuclein in the presence of a small molecular tweezer, CLR01, which binds selectively to Lys side chains. We find strong binding to multiple Lys within the chain as measured by fluorescence and mass-spectrometry and a linear increase in the reconfiguration rate with concentration of the inhibitor. Top-down mass-spectrometric analysis shows that the main binding of CLR01 to α-synuclein occurs at the N-terminal Lys-10/Lys-12. Photo-induced cross-linking of unmodified proteins (PICUP) analysis shows that under the conditions used for the fluorescence analysis, α-synuclein is predominantly monomeric. The results can be successfully modeled using a kinetic scheme in which two aggregation-prone monomers can form an encounter complex that leads to further oligomerization but can also dissociate back to monomers if the reconfiguration rate is sufficiently high. Taken together, the data provide important insights into the preferred binding site of CLR01 on α-synuclein and the mechanism by which the molecular tweezer prevents self-assembly into neurotoxic aggregates by α-synuclein and presumably other amyloidogenic proteins.
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http://dx.doi.org/10.1074/jbc.M113.524520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036189PMC
April 2014

Reduction of the C191-C220 disulfide of α-chymotrypsinogen A reduces nucleation barriers for aggregation.

Biophys Chem 2014 Jan 28;185:79-87. Epub 2013 Nov 28.

Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, United States. Electronic address:

Proper disulfide formation can be essential for the conformational stability of natively folded proteins. For proteins that must unfold in order to aggregate, disruption of native disulfides may therefore promote aggregation. This study characterizes differences in the aggregation process for wild-type (WT) α-chymostrypsinogen A (aCgn) and the same molecule with one of its native disulfides (C191-C220) reduced to free thiols (aCgnSH) at acidic pH, where WT aCgn forms semi-flexible amyloid polymers. Loss of the disulfide leads to no discernable differences in folded monomer secondary or tertiary structure based on circular dichroism (CD) or intrinsic fluorescence (FL), and causes a small decrease in the free energy change upon unfolding. After unfolding-mediated aggregation, the resulting amyloid morphology and structure are similar or indistinguishable for aCgn and aCgnSH by CD, FL, ThT binding, multi-angle laser light scattering, and transmission electron microscopy. Aggregates of aCgn and aCgnSH are also able to cross-seed with monomers of the other species. However, aggregates of aCgnSH are more resistive than aCgn aggregates to urea-mediated dissociation, suggesting some degree of structural differences in the aggregated species that was not resolvable in detail without higher resolution methods. Mechanistic analyses of aggregation kinetics indicate that the initiation or nucleation of new aggregates from aCgnSH involves a mono-molecular rate limiting step, possibly the unfolding step. In contrast, that for aCgn involves an oligomeric intermediate, suggesting native disulfide linkages help to hinder non-native protein aggregation by providing conformational barriers to key nucleation event(s).
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http://dx.doi.org/10.1016/j.bpc.2013.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4108794PMC
January 2014

Aggregation-triggering segments of SOD1 fibril formation support a common pathway for familial and sporadic ALS.

Proc Natl Acad Sci U S A 2014 Jan 16;111(1):197-201. Epub 2013 Dec 16.

Howard Hughes Medical Institute, UCLA-DOE Institute for Genomics and Proteomics, and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1570.

ALS is a terminal disease of motor neurons that is characterized by accumulation of proteinaceous deposits in affected cells. Pathological deposition of mutated Cu/Zn superoxide dismutase (SOD1) accounts for ∼20% of the familial ALS (fALS) cases. However, understanding the molecular link between mutation and disease has been difficult, given that more than 140 different SOD1 mutants have been observed in fALS patients. In addition, the molecular origin of sporadic ALS (sALS) is unclear. By dissecting the amino acid sequence of SOD1, we identified four short segments with a high propensity for amyloid fibril formation. We find that fALS mutations in these segments do not reduce their propensity to form fibrils. The atomic structures of two fibril-forming segments from the C terminus, (101)DSVISLS(107) and (147)GVIGIAQ(153), reveal tightly packed β-sheets with steric zipper interfaces characteristic of the amyloid state. Based on these structures, we conclude that both C-terminal segments are likely to form aggregates if available for interaction. Proline substitutions in (101)DSVISLS(107) and (147)GVIGIAQ(153) impaired nucleation and fibril growth of full-length protein, confirming that these segments participate in aggregate formation. Our hypothesis is that improper protein maturation and incompletely folded states that render these aggregation-prone segments available for interaction offer a common molecular pathway for sALS and fALS.
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http://dx.doi.org/10.1073/pnas.1320786110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890817PMC
January 2014

A novel "molecular tweezer" inhibitor of α-synuclein neurotoxicity in vitro and in vivo.

Neurotherapeutics 2012 Apr;9(2):464-76

Department of Neurology, UCLA David Geffen School of Medicine, Los Angeles, CA 90095, USA.

Aggregation of α-synuclein (α-syn) is implicated as being causative in the pathogenesis of Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies. Despite several therapies that improve symptoms in these disorders, none slow disease progression. Recently, a novel "molecular tweezer" (MT) termed CLR01 has been described as a potent inhibitor of assembly and toxicity of multiple amyloidogenic proteins. Here we investigated the ability of CLR01 to inhibit assembly and toxicity of α-syn. In vitro, CLR01 inhibited the assembly of α-syn into β-sheet-rich fibrils and caused disaggregation of pre-formed fibrils, as determined by thioflavin T fluorescence and electron microscopy. α-Syn toxicity was studied in cell cultures and was completely mitigated by CLR01 when α-syn was expressed endogenously or added exogenously. To determine if CLR01 was also protective in vivo, we used a novel zebrafish model of α-syn toxicity (α-syn-ZF), which expresses human, wild-type α-syn in neurons. α-Syn-ZF embryos developed severe deformities due to neuronal apoptosis and most of them died within 48 to 72 h. CLR01 added to the water significantly improved zebrafish phenotype and survival, suppressed α-syn aggregation in neurons, and reduced α-syn-induced apoptosis. α-Syn expression was found to inhibit the ubiquitin proteasome system in α-syn-ZF neurons, resulting in further accumulation of α-syn. Treatment with CLR01 almost completely mitigated the proteasome inhibition. The data suggest that CLR01 is a promising therapeutic agent for the treatment of Parkinson's disease and other synucleinopathies.
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http://dx.doi.org/10.1007/s13311-012-0105-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3337029PMC
April 2012

Molecular level insights into thermally induced α-chymotrypsinogen A amyloid aggregation mechanism and semiflexible protofibril morphology.

Biochemistry 2010 Dec 18;49(49):10553-64. Epub 2010 Nov 18.

Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22904, United States.

Understanding nonnative protein aggregation is critical not only to a number of amyloidosis disorders but also for the development of effective and safe biopharmaceuticals. In a series of previous studies [Weiss et al. (2007) Biophys. J. 93, 4392-4403; Andrews et al. (2007) Biochemistry 46, 7558-7571; Andrews et al. (2008) Biochemistry 47, 2397-2403], α-chymotrypsinogen A (aCgn) and bovine granulocyte colony stimulating factor (bG-CSF) have been shown to exhibit the kinetic and morphological features of other nonnative aggregating proteins at low pH and ionic strength. In this study, we investigated the structural mechanism of aCgn aggregation. The resultant aCgn aggregates were found to be soluble and exhibited semiflexible filamentous aggregate morphology under transmission electron microscopy. In addition, the filamentous aggregates were demonstrated to possess amyloid characteristics by both Congo red binding and X-ray diffraction. Peptide level hydrogen exchange (HX) analysis suggested that a buried native β-sheet comprised of three peptide segments (39-46, 51-64, and 106-114) reorganizes into the cross-β amyloid core of aCgn aggregates and that at least ∼50% of the sequence adopts a disordered structure in the aggregates. Furthermore, the equimolar, bimodal HX labeling distribution observed for three reported peptides (65-102, 160-180, and 229-245) suggested a heterogeneous assembly of two molecular conformations in aCgn aggregates. This demonstrates that extended β-sheet interactions typical of the amyloid are sufficiently strong that a relatively small fraction of polypeptide sequence can drive formation of filamentous aggregates even under conditions favoring colloidal stability.
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http://dx.doi.org/10.1021/bi1014216DOI Listing
December 2010

Molecular basis for insulin fibril assembly.

Proc Natl Acad Sci U S A 2009 Nov 28;106(45):18990-5. Epub 2009 Oct 28.

Howard Hughes Medical Institute, UCLA-DOE Institute for Genomics and Proteomics, Los Angeles CA 90095-1570, USA.

In the rare medical condition termed injection amyloidosis, extracellular fibrils of insulin are observed. We found that the segment of the insulin B-chain with sequence LVEALYL is the smallest segment that both nucleates and inhibits the fibrillation of full-length insulin in a molar ratio-dependent manner, suggesting that this segment is central to the cross-beta spine of the insulin fibril. In isolation from the rest of the protein, LVEALYL forms microcrystalline aggregates with fibrillar morphology, the structure of which we determined to 1 A resolution. The LVEALYL segments are stacked into pairs of tightly interdigitated beta-sheets, each pair displaying the dry steric zipper interface typical of amyloid-like fibrils. This structure leads to a model for fibrils of human insulin consistent with electron microscopic, x-ray fiber diffraction, and biochemical studies.
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http://dx.doi.org/10.1073/pnas.0910080106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2776439PMC
November 2009

Atomic structures of amyloid cross-beta spines reveal varied steric zippers.

Nature 2007 May 29;447(7143):453-7. Epub 2007 Apr 29.

Howard Hughes Medical Institute, UCLA-DOE Institute of Genomics and Proteomics, Los Angeles, California 90095-1570, USA.

Amyloid fibrils formed from different proteins, each associated with a particular disease, contain a common cross-beta spine. The atomic architecture of a spine, from the fibril-forming segment GNNQQNY of the yeast prion protein Sup35, was recently revealed by X-ray microcrystallography. It is a pair of beta-sheets, with the facing side chains of the two sheets interdigitated in a dry 'steric zipper'. Here we report some 30 other segments from fibril-forming proteins that form amyloid-like fibrils, microcrystals, or usually both. These include segments from the Alzheimer's amyloid-beta and tau proteins, the PrP prion protein, insulin, islet amyloid polypeptide (IAPP), lysozyme, myoglobin, alpha-synuclein and beta(2)-microglobulin, suggesting that common structural features are shared by amyloid diseases at the molecular level. Structures of 13 of these microcrystals all reveal steric zippers, but with variations that expand the range of atomic architectures for amyloid-like fibrils and offer an atomic-level hypothesis for the basis of prion strains.
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http://dx.doi.org/10.1038/nature05695DOI Listing
May 2007

The structural biology of protein aggregation diseases: Fundamental questions and some answers.

Acc Chem Res 2006 Sep;39(9):568-75

Howard Hughes Medical Institute, UCLA-DOE Institute of Genomics and Proteomics, Los Angeles, California 90095-1570, USA.

Amyloid fibrils are found in association with at least two dozen fatal diseases. The tendency of numerous proteins to convert into amyloid-like fibrils poses fundamental questions for structural biology and for protein science in general. Among these are the following: What is the structure of the cross-beta spine, common to amyloid-like fibrils? Is there a sequence signature for proteins that form amyloid-like fibrils? What is the nature of the structural conversion from native to amyloid states, and do fibril-forming proteins have two distinct stable states, the native state and the amyloid state? What is the basis of protein complementarity, in which a protein chain can bind to itself? We offer tentative answers here, based on our own recent structural studies.
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http://dx.doi.org/10.1021/ar0500618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017558PMC
September 2006

A systematic screen of beta(2)-microglobulin and insulin for amyloid-like segments.

Proc Natl Acad Sci U S A 2006 Mar 7;103(11):4079-82. Epub 2006 Mar 7.

Howard Hughes Medical Institute and University of California--Department of Energy Institute of Genomics and Proteomics, University of California, Los Angeles, CA 90095, USA.

Identifying sequence determinants of fibril-forming proteins is crucial for understanding the processes causing >20 proteins to form pathological amyloid depositions. Our approach to identifying which sequences form amyloid-like fibrils is to screen the amyloid-forming proteins human insulin and beta(2)-microglobulin for segments that form fibrils. Our screen is of 60 sequentially overlapping peptides, 59 being six residues in length and 1 being five residues, covering every noncysteine-containing segment in these two proteins. Each peptide was characterized as amyloid-like or nonfibril-forming. Amyloid-like peptides formed fibrils visible in electron micrographs or needle-like microcrystals showing a cross-beta diffraction pattern. Eight of the 60 peptides (three from insulin and five from beta(2)-microglobulin) were identified as amyloid-like. The results of the screen were used to assess the computational method, and good agreement between prediction and experiments was found. This agreement suggests that the pair-of-sheets, zipper spine model on which the computational method is based is at least approximately correct for the structure of the fibrils and suggests the nature of the sequence signal for formation of amyloid-like fibrils.
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http://dx.doi.org/10.1073/pnas.0511298103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449649PMC
March 2006

The 3D profile method for identifying fibril-forming segments of proteins.

Proc Natl Acad Sci U S A 2006 Mar 7;103(11):4074-8. Epub 2006 Mar 7.

Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095, USA.

Based on the crystal structure of the cross-beta spine formed by the peptide NNQQNY, we have developed a computational approach for identifying those segments of amyloidogenic proteins that themselves can form amyloid-like fibrils. The approach builds on experiments showing that hexapeptides are sufficient for forming amyloid-like fibrils. Each six-residue peptide of a protein of interest is mapped onto an ensemble of templates, or 3D profile, generated from the crystal structure of the peptide NNQQNY by small displacements of one of the two intermeshed beta-sheets relative to the other. The energy of each mapping of a sequence to the profile is evaluated by using ROSETTADESIGN, and the lowest energy match for a given peptide to the template library is taken as the putative prediction. If the energy of the putative prediction is lower than a threshold value, a prediction of fibril formation is made. This method can reach an accuracy of approximately 80% with a P value of approximately 10(-12) when a conservative energy threshold is used to separate peptides that form fibrils from those that do not. We see enrichment for positive predictions in a set of fibril-forming segments of amyloid proteins, and we illustrate the method with applications to proteins of interest in amyloid research.
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http://dx.doi.org/10.1073/pnas.0511295103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449648PMC
March 2006

An amyloid-forming segment of beta2-microglobulin suggests a molecular model for the fibril.

Proc Natl Acad Sci U S A 2004 Jul 12;101(29):10584-9. Epub 2004 Jul 12.

Howard Hughes Medical Institute and University of California-Department of Energy Institute of Genomics and Proteomics, Box 951570, University of California, Los Angeles, CA 90095, USA.

In humans suffering from dialysis-related amyloidosis, the protein beta2-microglobulin (beta2M) is deposited as an amyloid; however, an amyloid of beta2M is unknown in mice. beta2M sequences from human and mouse are 70% identical, but there is a seven-residue peptide in which six residues differ. This peptide from human beta2M forms amyloid in vitro, whereas the mouse peptide does not. Substitution of the human peptide for its counterpart in the mouse sequence results in the formation of amyloid in vitro. These results show that a seven-residue segment of human beta2M is sufficient to convert beta2M to the amyloid state, and that specific residue interactions are crucial to the conversion. These observations are consistent with a proposed Zipper-spine model for beta2M amyloid, in which the spine of the fibril consists of an anhydrous beta-sheet.
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http://dx.doi.org/10.1073/pnas.0403756101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC489978PMC
July 2004

Role of the C-terminal 28 residues of beta2-microglobulin in amyloid fibril formation.

Biochemistry 2003 Nov;42(46):13536-40

Howard Hughes Medical Institute and UCLA-DOE Institute of Genomics and Proteomics, 90095-1571, USA.

Beta2microglobulin (beta2m) is the major protein component of the fibrillar amyloid deposits isolated from patients diagnosed with dialysis-related amyloidosis (DRA). While investigating the molecular mechanism of amyloid fibril formation by beta2m, we found that the beta2m C-terminal peptide of 28 residues (cbeta2m) itself forms amyloid fibrils. When viewed by electron microscopy, cbeta2m aggregates appear as elongated unbranched fibers, the morphology typical for amyloids. Cbeta2m fibers stain with Congo red and show apple-green birefringence in polarized light, characteristic of amyloids. The observation that the beta2m C-terminal fragment readily forms amyloid fibrils implies that beta2m amyloid fibril formation proceeds via interactions of amyloid forming segments, which become exposed when the beta2m subunit is partially unfolded.
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http://dx.doi.org/10.1021/bi0301486DOI Listing
November 2003