doi:10.1016/j.jmb.2012.04.017 J. Mol. Biol. (2012) 421, 378–389

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Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

Nucleobindin 1 Caps Human Islet Amyloid PolypeptideProtofibrils to Prevent Amyloid Fibril Formation

Ruchi Gupta 1, Neeraj Kapoor 1, Daniel P. Raleigh 2⁎and Thomas P. Sakmar 1⁎1Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10065, USA2Department of Chemistry, Graduate Program in Biochemistry and Structural Biology, and Graduate Program inBiophysics, State University of New York at Stony Brook, Stony Brook, NY 11794, USA

Received 29 September 2011;received in revised form9 April 2012;accepted 14 April 2012Available online24 April 2012

Edited by S. Radford

Keywords:amylin;islet amyloid polypeptide;type 2 diabetes mellitus;amyloid;nucleobindin 1

*Corresponding authors. E-mail addraleigh@notes.cc.sunysb.edu; sakmAbbreviations used: AFM, atomic

DLS, dynamic light scattering; ER, enhIAPP, human islet amyloid polypeangle light scattering; M/L, mass penucleobindin 1; sNUCB1, soluble Nexclusion chromatography; STEM, selectron microscopy; TEM, transmismicroscopy; thio-T, thioflavin-T; TMvirus; UPR, unfolded protein respon

0022-2836/$ - see front matter © 2012 E

Many human diseases are associated with amyloid fibril deposition,including type 2 diabetes mellitus where human islet amyloid polypeptide(hIAPP) forms fibrils in the pancreas. We report here that engineered,soluble forms of the human Ca2+-binding protein nucleobindin 1 (NUCB1)prevent hIAPP fibril formation and disaggregate preexisting hIAPP fibrils.Scanning transmission electron microscopy (STEM) and atomic forcemicroscopy indicate that NUCB1 binds to and stabilizes heterogeneousprefibrillar hIAPP species. The NUCB1-stabilized prefibrillar species wereisolated by size-exclusion chromatography and analyzed by STEM,dynamic light scattering, and multi-angle light scattering. The stabilizedprefibrillar species show a size range of 2–6 million Da and have othersimilarities to hIAPP protofibrils, but they do not progress to becomemature fibrils. The effects of NUCB1 are absent in the presence of Ca2+. Wepostulate that the engineered forms of NUCB1 prevent hIAPP fibrilformation by a mechanism where protofibril-like species are “capped” toprevent further fibril assembly andmaturation. This mode of action appearsto be different from other protein-based inhibitors, suggesting that NUCB1may offer a new approach to inhibiting amyloid formation and disag-gregating amyloid fibrils.

© 2012 Elsevier Ltd. All rights reserved.

Introduction

Amyloid fibril deposition is a pathophysiologicalhallmark of a number of severe degenerative

dresses:ar@rockefeller.edu.force microscopy;doplasmic reticulum;ptide; MALS, multi-r unit length; NUCB1,UCB1; SEC, size-canning transmissionsion electronV, tobacco mosaicse.

lsevier Ltd. All rights reserve

disorders, including Alzheimer's disease, Creutz-feldt–Jakob disease, Parkinson's disease, and Hun-tington's disease. Amyloid is also associated withtype 2 diabetes mellitus and the systemicamyloidoses.1–5 Amyloidogenic peptides and pro-teins, including unfolded, misfolded, or partiallydenatured proteins, share no primary structuralhomology, but the general process of amyloidformation follows a characteristic pathway.6,7

Peptides adopt β-sheet secondary structure andself-assemble into protofibrils with cross-β-sheetquaternary structure. The protofibrils pack to formfilaments and mature fibrils. Amyloid fibrils, andespecially their precursor protofibrillar species,appear to be toxic to cells and tissues.8,9 Proteinsand peptides with amyloidogenic potential arepresent normally, and their unaggregated forms

d.

379Nucleobindin 1 Inhibits Amyloid Formation by hIAPP

often play important roles in cell physiology andregulation. For example, human islet amyloidpolypeptide (hIAPP) is a natively unstructuredpancreatic endocrine hormone that regulates foodintake and fat storage.10 It is co-secreted withinsulin from pancreatic β-cells in response toglucose uptake.11 hIAPP is a very hydrophobicpeptide with a significantly high propensity toaggregate rapidly in aqueous solution in vitro evenat low micromolar concentrations. Physiologically,the concentration of hIAPP in secretory granulescan vary from 100 μM to 4 mM, but even at suchhigh concentrations, hIAPP does not aggregateunder the normal environment of secretorygranules.12 However, in type 2 diabetes mellitus,aggregation of hIAPP results in islet amyloiddeposits, which are toxic to the insulin-producingβ-cells.11,13

Several peptide fragments and derivatives ofhIAPP that inhibit fibril formation in vitro havebeen identified and a number of small-moleculeinhibitors have been reported, but new inhibitors aredesired, especially since many existing ones workonly in molar excess.14–20 We investigated the abilityof nucleobindin 1 (NUCB1), a 55-kDa Ca2+-bindingprotein that is widely expressed and is conservedfrom flies to humans, to inhibit hIAPP fibrilformation.21 We show that a form of NUCB1engineered to be soluble, sNUCB1, inhibits hIAPPaggregation at substoichiometric levels and effec-tively disaggregates preformed fibrils in the absenceof Ca2+. The anti-amyloidogenic activity of NUCB1is lost in the presence of Ca2+. Using scanningtransmission electron microscopy (STEM) andatomic force microscopy (AFM) in conjunctionwith other biochemical methods, we demonstratethat Ca2+-free sNUCB1 binds to and stabilizes aprefibrillar species of hIAPP. NUCB1 appears to“cap” a protofibril-like species, which inhibits itsfurther assembly and growth. The sNUCB1-boundprefibrillar hIAPP species appears to be a “dead-end” complex that is incapable of seeding furtheraggregation of hIAPP.

Results

A soluble form of NUCB1 inhibits amyloid fibrilformation by hIAPP

NUCB1 is nominally considered to be a Golgi-resident Ca2+-binding protein, but it is also found inthe cytoplasm and nucleus and can be secreted aswell.22 We first engineered a soluble form ofNUCB1, called sNUCB1, for heterologous expres-sion in Escherichia coli as described earlier.23 We thenmeasured the aggregation kinetics of hIAPP aloneand in the presence of different amounts of sNUCB1

using a conventional thioflavin-T (thio-T) fluores-cence-binding assay (Fig. 1a). For hIAPP alone,aggregation starts with a nucleation phase ofapproximately 5 min under the conditions of ourassay and extends into a growth/elongation phasebetween 5 and 20 min. The growth phase of thecurve plateaus into a stationary phase where fibrilsare in equilibrium with the soluble peptide. WhensNUCB1 was incubated with hIAPP in the absenceof Ca2+, we observed a significant decrease in theoverall enhancement of thio-T fluorescence and achange in the kinetics of fluorescence enhancement.The effect was maximal in the presence of anequimolar amount (32 μM) of sNUCB1 and hIAPP.The inhibition of the thio-T fluorescence increasewas observed even at substoichiometric ratios ofsNUCB1 to hIAPP (Fig. 1a).We used transmission electron microscopy (TEM)

as a direct probe of amyloid fibril formation. hIAPPalone (32 μM) aggregates within 7 min to formoligomers, which continue to grow and form higher-order intermediates, protofibrils, and protofila-ments, which can be observed as early as about13 min. The protofibrillar intermediates assembleand extend into mature amyloid fibrils within60 min (Fig. 1b). Reaction mixtures with stoichio-metric or lower concentrations of sNUCB1 werepreincubated with hIAPP (32 μM) and analyzedusing TEM (Fig. 1c). The samples withdrawn at7 min and 13 min from reaction mixtures thatcontain 32 μM, 10.7 μM, 6.4 μM, or 3.2 μM sNUCB1,respectively, show oligomeric species, and the 60-min sample shows the presence of ordered prefi-brillar species, which clearly differ from amyloidfibrils. Thus, sNUCB1 alters the normal amyloidassembly process and leads to the formation ofprefibrillar aggregates that do not go on to formamyloid. The formation of prefibrillar species ineach of these reactions explains the observed initialincrease in the thio-T fluorescence shown in Fig. 1.The absence of an apparent lag phase in the presenceof sNUCB1 indicates that the protein might drivehIAPP to form prefibrillar species. However, con-ventional hIAPP fibrils were not observed in thepresence of sNUCB1 in the TEM assay except atlimiting concentrations of sNUCB1. In a controlexperiment, 32 μM Ca2+-free sNUCB1 alone doesnot aggregate when incubated at room temperature(Fig. 1b).NUCB1 is a dimeric multi-domain protein with

two Ca2+-binding EF hand domains. Ca2+ readilybinds to sNUCB1 and induces conformationalchanges within the protein subunit.23 We testedthe effect of Ca2+-bound sNUCB1 on hIAPPamyloid formation under conditions where Ca2+-free sNUCB1 effectively inhibited fibril formation.Ca2+-bound sNUCB1 had no significant effect onformation of hIAPP fibrils. TEM images of hIAPPincubated in the presence of sNUCB1 and Ca2+

Fig. 1. Ca2+-free sNUCB1 inhibits hIAPP fibril formation in a dose-dependent manner. (a) The kinetics of hIAPPaggregation was monitored using fluorescence-based thio-T binding assays. Aggregation of hIAPP alone shows an initialnucleation phase, which then extends into a rapid growth phase. The aggregation continues and reaches a plateau,indicating the formation of amyloid fibrils. Incubation of hIAPP with stoichiometric or substoichiometric concentrationsof Ca2+-free sNUCB1 shows significant inhibition of fibril formation. Each reaction was also monitored using TEM. As acontrol, (b) hIAPP and Ca2+-free sNUCB1 were incubated alone and samples were withdrawn at 7 min, 13 min, and60 min. hIAPP alone aggregates rapidly to form fibrils within 60 min, whereas Ca2+-free sNUCB1 alone does not formhigher-order aggregates, (c) TEM analysis of the reaction mixtures presented in (a) confirms that Ca2+-free sNUCB1inhibits hIAPP amyloid formation. Scale bars represent 200 nm.

380 Nucleobindin 1 Inhibits Amyloid Formation by hIAPP

(3 mM) show the presence of protofibrils andprotofilaments at 15 min, which continue to growinto fibrillar structures. Mature hIAPP amyloid

fibrils are seen in the 60-min sample (Fig. 2). Fibrilswere not observed under the identical conditions inthe absence of Ca2+.

Fig. 2. The anti-amyloidogenicactivity of sNUCB1 is Ca2+ depen-dent. The aggregation of hIAPPwas monitored in the presence ofequimolar sNUCB1. In the absenceof Ca2+, sNUCB1 effectively in-hibits hIAPP fibril formation whenadded at T=0. When excess Ca2+

(3 mM) is present in the reactionmixture, sNUCB1 does not inhibithIAPP fibril formation.

Fig. 3. Ca2+-free sNUCB1 disaggregates of hIAPP fibrils. In the absence of Ca2+, sNUCB1 effectively disaggregatesamyloid fibrils within 15 min of the addition. Disaggregation persists even in samples withdrawn at 30 min and 60min. Inthe presence of excess Ca2+ (3 mM), sNUCB1 does not disaggregate hIAPP fibrils. TEM analysis shows the presence offibrillar aggregates at all monitored time points. Scale bars represent 200 nm.

381Nucleobindin 1 Inhibits Amyloid Formation by hIAPP

Evidence that sNUCB1 disaggregates hIAPPfibrils

We investigated the ability of sNUCB1 to disag-gregate preformed hIAPP fibrils. In the absence ofCa2+, sNUCB1 effectively disaggregates hIAPPfibrils into smaller species within 15 min. Thesespecies do not reassociate to form fibrils even after45 min of additional incubation (Fig. 3). As observedin the inhibition experiments, Ca2+ eliminated theability of sNUCB1 to disaggregate hIAPP fibrils.

graphs corresponding to samples withdrawn at 60 min afteinhibits the progression of seeds into mature fibrils and disa

TEM images corresponding to samples withdrawnafter addition of equimolar amounts of sNUCB1 tofibrils in the presence of 3 mM Ca2+ show fibrillarspecies in the micrographs at all time points (Fig. 3).The observation that sNUCB1 inhibits amyloid

formation at substoichiometric concentrations sug-gests that it may bind to oligomeric hIAPP species,which implies that sNUCB1 should inhibit amyloidformation when added at the lag phase of theamyloid formation pathway. sNUCB1 was added atdifferent points during the hIAPP aggregation

Fig. 4. Ca2+-free sNUCB1 in-hibits hIAPP amyloidogenesis in astate-independent manner. hIAPP(32 μM) aggregation was moni-tored using thio-T (32 μM). The lagphase is shown in blue, the growthphase is in green, and the station-ary phase is in yellow. Aggrega-tion was allowed to proceed to (a)the nucleation phase at 7 min, (b)the midpoint of growth phase at15 min, and (c) the initiation ofstationary phase at 40 min. At theindicated time, samples were re-moved and an equimolar amount(32 μM) of Ca2+-free sNUCB1 wasadded. The pink circles indicatethe time points at which sNUCB1was added. TEM images werecollected immediately before addi-tion of sNUCB1 and 60 min afteraddition of sNUCB1. The micro-

r addition show that Ca2+-free sNUCB1 both effectivelyggregates mature fibrils. Scale bars represent 200 nm.

Fig. 5. Biophysical characterization of Ca2+-free sNUCB1-stabilized hIAPP prefibrillar species. (a) SEC was used toanalyze the products of the 1:1 mixture of hIAPP and Ca2+-free sNUCB1. Ca2+-free sNUCB1 alone elutes at 12 ml (reddotted curve) while the sample from a 1:1 reaction mixture removed after 60 min gives an additional peak, Peak 1, elutingat 7.8 ml. (b) Dot blot assay shows that sNUCB1 is present in both peaks, hIAPP is only present in the Peak 1 fraction. (c)TEM analysis of the Peak 1 fraction shows the presence of oligomeric and prefibrillar species whereas no higher-orderaggregates were observed in the Peak 2 fraction. Inset: TEM image of fibrils alone. (d) MALS analysis (left) across Peak 1estimates the molecular mass in the range of 2–6 million Da and DLS (right) predicts hydrodynamic radii in the range of22–42 nm. MALS and DLS analysis of Peak 2 predicts a molecular mass and hydrodynamic radii characteristic for a dimerof sNUCB1.

382 Nucleobindin 1 Inhibits Amyloid Formation by hIAPP

process, in the lag phase (7 min), in the middle of thegrowth phase (13 min), and in the steady-stateplateau (40 min), and then incubated for anadditional 60 min. TEM showed no amyloid fibrilsin any of the samples. The 13-min time pointshowed prefibrillar species, which were differentfrom the filaments observed in the absence ofsNUCB1. As a control, TEM micrographs wererecorded for each sample prior to the addition ofsNUCB1 (Fig. 4). These results suggest that Ca2+-free sNUCB1 possesses the ability to inhibit hIAPPamyloid fibril formation at any stage of thepathway.

sNUCB1 stabilizes hIAPP prefibrillar species

We characterized a mixture of sNUCB1 andhIAPP by size-exclusion chromatography (SEC)

(Fig. 5a). Since hIAPP contains only one Tyr andno Trp residues, sNUCB1 dominates the absorbanceat 280 nm. The SEC chromatogram shows peaks at7.6 ml (Peak 1) and 11.8 ml (Peak 2). Injection of a 32-μM solution of Ca2+-free sNUCB1 alone onto thecolumn generates a peak centered at 11.8 ml (Fig.5a). The peaks were analyzed by dot blot analysisusing an anti-NUCB1 antibody and an anti-IAPPantibody. The results of the dot blot analysis showthat sNUCB1 was present in both the Peak 1 andPeak 2 fractions, whereas hIAPP was present only inthe Peak 1 fraction (Fig. 5b). Control experimentsshowed no cross reactivity for either antibody. Bothfractions were further analyzed by TEM. Themicrographs show the presence of only prefibrillarspecies in the Peak 1 fraction, whereas the Peak 2fraction reveals an image similar to that observed forsNUCB1 in the absence of hIAPP (Fig. 5c).

Fig. 6. sNUCB1 stabilizes hIAPP protofibrillar species. STEM was used to determine the M/L distribution ofprefibrillar species in the Peak 1 fraction. (a) The image shows a typical dark-field cryo-STEM image of the Peak 1 fraction.The adjacent panel shows images representing prefibrillar species observed in STEM. The scale bar or width of each boxrepresents 100 nm. (b) Quantitative analysis shows the presence of prefibrillar species with M/L ranging from 1 to 7 kDa/Å with the predominant species ranging from 2 to 5 kDa/Å.

383Nucleobindin 1 Inhibits Amyloid Formation by hIAPP

Multi-angle light scattering (MALS) and dynamiclight scattering (DLS) analysis were performed toestimate the average molecular mass and hydrody-namic radii of the eluting species. MALS analysis ofPeak 1 gives an estimated molecular mass in therange of 2–6 million Da, while the DLS analysisyields hydrodynamic radii of 22–42 nm (Fig. 5d).The data suggest that sNUCB1 binds to andstabilizes a high-molecular-mass prefibrillar hIAPPspecies. Analysis of Peak 2 shows a 97- to 99-kDamolecular mass species with a hydrodynamic radiusof 6.2 nm (Fig. 5d), consistent with the valueobserved for a dimer of sNUCB1.STEM was used to estimate the mass per unit

length (M/L) value of the observed prefibrillarspecies. Dark-field images were recorded for thePeak 1 fraction using tobacco mosaic virus (TMV) asan internal standard (Fig. 6). The M/L data werepooled and are presented as histograms. STEManalysis of Peak 1 fraction gave a M/L value rangingfrom 1–7 kDa/Å with the peak of the distribution at2 kDa/Å (Fig. 6). Goldsbury et al. have analyzedhIAPP fibrils using STEM and proposed that 100-nm-long protofibrils have a M/L value of approxi-mately 1 kDa/Å and thereby a mass of 1000 kDa.24

The sNUCB1-stabilized prefibrillar species are 40–80 nm long, and a mean M/L value of 2 kDa/Åassigns an average mass of 1200 kDa to these species.The difference of around 200 kDa is most likely amass contribution of bound sNUCB1. Peak 1 fractionthus consists of protofibril-like species bound tosNUCB1 and will be referred to in the followingdiscussion as prefibrillar species. The data stronglyargue that Ca2+-free sNUCB1 inhibits hIAPP fibrilformation by stabilizing prefibrillar species.As a control,we tested the ability of sNUCB1 tobind

a monomeric analogue of hIAPP. Pramlintide is a

variant of hIAPP that contains three Pro substitutionsand is known not to aggregate. Isothermal titrationcalorimetry and SEC experiments showed that pram-lintide does not bind Ca2+-free sNUCB1 (Fig. S1),suggesting that sNUCB1 recognizes a higher-orderstructure of hIAPP, and not hIAPP monomers.We investigated the stability of the sNUCB1-

bound prefibrillar species by incubating the Peak 1fraction at room temperature for 7 days withconstant stirring followed by SEC. The same elutionprofile was observed as had been previouslydetected for Peak 1. TEM images were recorded onday 1 and on day 7 after elution from the column.The same prefibrillar species were detected for bothsamples, demonstrating that the sNUCB1-stabilizedprefibrillar species are stable for at least 1 week (Fig.S2). These experiments confirm that the sNUCB1-bound prefibrillar species are highly stable and donot associate further to form fibrils.

sNUCB1 “caps” the ends of prefibrillarintermediates

NUCB1 has no Cys residues, which provides anopportunity to introduce a site-specific reactivesulfhydryl group for labeling with nanogold. AnS44C mutant of sNUCB1, sNUCB1(S44C), wasprepared. The mutant was stable in solution and didnot aggregate. Control experiments showed thatsNUCB1(S44C) effectively inhibits hIAPP fibril for-mation (Fig. S3). We purified nano-Au maleimide-labeled sNUCB1(S44C) using SEC (Fig. 7a) andobserved a labeling efficiency of 1.02 mol/mol,suggesting that each sNUCB1(S44C) monomer has asingle nano-Au label (Fig. S3). TEManalysis of samplesof labeled sNUCB1(S44C) shows density for twojuxtaposed nano-Au molecules, consistent with the

(a)

(b)

(e)(d)(c)

Fig. 7. Nano-Au-labeled sNUCB1(S44C) caps hIAPP prefibrillar species. (a) Nano-Au-labeled sNUCB1(S44C) waspurified via SEC. The chromatogram shows the co-elution of the 280-nm protein peak (black) and the 420-nm nano-Aupeak (red). The inset shows a schematic of the conjugation reaction that covalently links the nano-Au to a Cys residue. (b)TEM analysis of the labeled sample shows the presence of two typical nano-Au densities corresponding to a dimer of alabeled sNUCB1(S44C). Nano-Au-labeled sNUCB1(S44C) was incubated with equimolar hIAPP for 60 min, after whichsamples werewithdrawn. TEM analysis shows the presence of high-molecular-mass prefibrillar species with labeled ends.The scale bar represents 100 nm. AFM in non-contact mode was used to corroborate the TEM studies. (c) The micrographsreveal sNUCB1-stabilized hIAPP prefibrillar species with the height of prefibrillar species varying from 1.0 to 2.0 nm. Theinhibition reaction was repeated using purified sNUCB1(S44C)-[Nano-Au]. (d) AFM images confirm that sNUCB1(S44C)-[Nano-Au] caps the ends of prefibrillar species of hIAPP. The presence of Nano-Au clusters was confirmed by an increase inheight of the ends of these prefibrillar species. (e) AFMmicrographs show hIAPP protofibrils studded with sNUCB1(S44C)-[Nano-Au]. The image illustrates the interaction of sNUCB1(S44C) with preformed protofibrils of hIAPP. The scale bar ineach AFM image represents 100 nm. The color contour scale on the right of each AFM panel represents the height of eachsample in nanometers. AFM images of mature fibrils are included in the Supporting Information.

384 Nucleobindin 1 Inhibits Amyloid Formation by hIAPP

385Nucleobindin 1 Inhibits Amyloid Formation by hIAPP

labeling of eachmonomer subunit in a sNUCB1(S44C)dimer (Fig. 7b). We incubated an equimolar amount ofnano-Au-labeled sNUCB1(S44C) with hIAPP andanalyzed the resulting mixture after 60 min usingTEM. The micrograph shows that the nanogold-labeled mutant inhibits fibril formation by trappingspecies that appear to be identical with thosesequestered by sNUCB1. Strikingly, nano-Au densi-ty was present only at the ends of the structures andno significant density was observed at any otherlocations (Fig. 7b). This suggests that sNUCB1(S44C)inhibits hIAPP fibril formation by interacting withand capping the ends of the high-molecular-massprefibrillar species.AFM studies were conducted to test the proposed

“capping” mechanism. sNUCB1 was incubated withequimolar hIAPP, and the mixture was analyzedafter 60 min using non-contact mode AFM (Fig. 7c).The micrographs show the presence of prefibrillarspecies with heights varying from 1 to 2 nm andlengths ranging from 40 to 100 nm. Their height isconsistent with the protofibrillar species of severalaggregating peptides and proteins.25,26 Control AFMexperiments on hIAPP mature fibrils show that theirheights varied from 3 to 5 nm (Fig. S4), which issignificantly different from that of the prefibrillar orprotofibrillar species observed in Fig. 7c. The AFMmeasurements were repeated using nano-Au-labeledsNUCB1(S44C). The protein was incubated withhIAPP for 60 min. AFM images show the presenceof prefibrillar species with nano-Au density presentat the ends as indicated by an increase in height at theends of the prefibrillar species (Fig. 7d). The AFMdata thus strongly suggest that sNUCB1-stabilizedprefibrillar species are similar to protofibrils ofhIAPP, but with sNUCB1 capping the ends.To test whether or not sNUCB1 can interact with

preformed hIAPP protofibrils, we incubated hIAPPalone for 10 min with constant stirring and thenadded an equimolar amount of nano-Au-labeledsNUCB1(S44C). The 10-min time point correspondsto the growth phase of hIAPP aggregation whereprotofibrils and filaments are formed as monomersare added to the growing nuclei. The reactionmixture was analyzed using AFM. The micrographsreveal hIAPP protofibrils studded with nano-Au-labeled sNUCB1(S44C) (Fig. 7e). The protofibril inthe micrograph is 2 nm in height, consistent with theheight reported for typical protofibrils formed fromother amyloidogenic proteins.25,26 The presence ofsNUCB1(S44C) along the length of the preformedprotofibril shows the interaction of protein withpreexisting protofibrils or protofilaments of hIAPP.

Prefibrillar species in complex with sNUCB1 donot seed amyloid fibril formation

We tested the ability of sNUCB1-bound prefi-brillar species to seed hIAPP fibril formation.

Seeding refers to the addition of preformed nucleiinto a solution of unaggregated protein. Addition ofthe seed shortens or bypasses the lag phase.Incubation of hIAPP with seeds generated fromsonication of normal hIAPP fibrils shortens the lagphase of the aggregation process and increases therate of fibril formation (Fig. 8). We then tested theability of the sNUCB1-stabilized species to seedamyloid formation. In this experiment, sNUCB1wasincubated with equimolar hIAPP, and the reactionmixture was fractionated using SEC. The sNUCB1-stabilized prefibrillar species isolated as Peak 1 werethen used to “seed” an hIAPP fibrillization reaction.Incubation of hIAPP with increasing amounts of thePeak 1 fraction had no significant effect on the lagphase of the process (Fig. 8). This shows thatsNUCB1-stabilized prefibrillar species cannot“seed” hIAPP amyloid formation and suggest thatthey are a “dead-end” entity unable to supportfurther growth.A second complementary set of seeding studies

was preformed using nano-Au-labeled hIAPP seeds.Unlabeled hIAPP fibrils were sonicated to generatesmaller fragments and labeled with nano-Au underreducing conditions. These labeled fibril fragmentswere then used to “seed” amyloid formation byunlabeled hIAPP. Figure 8 shows the incorporationof labeled protofibrils into a growing hIAPPamyloid fibril. Bidirectional growth was observed.The bidirectional growth of nano-Au-labeled seedsinto amyloid fibrils has been demonstrated for NMfibers.27 The experiment was repeated using thesNUCB1-stabilized hIAPP prefibrillar complex as aseed. The Peak 1 fraction was collected and labeledwith nano-Au under reducing conditions. SincesNUCB1 contains no Cys residues, only hIAPP islabeled with nano-Au in this experiment (Fig. 8).The nano-Au-labeled material was added to unla-beled hIAPP. TEM images of samples withdrawnfrom the aggregation reaction show that no nano-AU is incorporated into the fibrils and thus confirmsthat the sNUCB1-stabilized complex does not seedfibril formation (Fig. 8). This result is consistent withthe thio-T monitored seeding experiment andconfirms that the sNUCB1-stabilized prefibrillarspecies are dead-end products that cannot seedhIAPP aggregation.We ruled out proteolytic activity of sNUCB1 as a

mechanism of action using a fluorescence substrate-based protease assay with BODIPY FL-casein as thesubstrate. Addition of even a 10-fold molar excess ofsNUCB1 did not result in any notable enhancementin fluorescence intensity (Fig. S5). SDS-PAGEanalyses of the samples withdrawn from thereaction mixtures confirm that sNUCB1 does notcleave the BODIPY FL-casein substrate (Fig. S5).Liquid chromatography–mass spectrometry of mix-tures of Ca2+-free sNUCB1 and hIAPP provides athird independent test. The mass spectrum shows

(a)

(b)

(c)(d)

Fig. 8. Ca2+-free sNUCB1-stabilized hIAPPprefibrillar species are not incorporated into growingfibrils. (a) Aggregationkinetics of hIAPP alone (black) or in the presence of 50 μl (orange), 100 μl (purple), or 200 μl (green) of the Peak 1 fractionmonitored using thio-T binding assays. No noticeable shortening of the lag phasewas observed. The inset shows a seedingreaction with pure preformed hIAPP amyloid fibers. A significant shortening of the lag phase was observed when 20 μl(blue) or 50 μl (red) pure hIAPP seedwas added. Nano-gold labeling was used to visualize seeding. (b) Nano-gold-labeledhIAPP seeds are incorporated into hIAPP fibrils. Preformed hIAPP fibrils were sonicated to generate seeds labeled withnano-Au, and used to seed hIAPP fibrillization. TEM analysis shows incorporation of the nano-Au-labeled aggregate. (c)Nano-gold-labeled, sNUCB1-stabilized prefibrillar species are not incorporated into hIAPP fibrils. The material from Peak1 was labeled with nano-Au and analyzed using TEM. (d) The nano-Au-labeled prefibrillar species were then incubatedwith hIAPP, and sample was withdrawn at 60 min and analyzed by TEM. Themature hIAPP fibrils do not incorporate thenano-Au-labeled sNUCB1-stabilized prefibrillar species. Scale bars represent 200 nm.

386 Nucleobindin 1 Inhibits Amyloid Formation by hIAPP

peaks due to sNUCB1 and hIAPP but no apparentlower molecular weight peaks (Fig. S5).

Discussion

The data presented here show that sNUCB1inhibits hIAPP fibril formation in a dose-dependentmanner. Importantly, our results show that Ca2+-free sNUCB1 can inhibit fibril formation whenadded during either the nucleation or the growth

phase of amyloidogenesis. Thus, if aggregation ofhIAPP has been initiated, sNUCB1 can still preventthe growth of existing amyloidogenic intermediatesinto fibrils. Intriguingly, sNUCB1 also efficientlydisaggregates the preformed fibrils. Functionally,molecules with these properties of stage-indepen-dent inhibition and disaggregation offer the excit-ing possibility of being able to clear existingamyloid aggregates, while preventing further se-questration of functional monomers into amyloiddeposits.

387Nucleobindin 1 Inhibits Amyloid Formation by hIAPP

Our mechanistic studies show that sNUCB1interacts with prefibrillar aggregates of hIAPP toform stable 2–6 million Da complexes that can beisolated by SEC. The heterogeneous complexes havehydrodynamic radii ranging from 22 to 42 nm, witha predominant mass density similar to the distribu-tion observed for normal hIAPP protofibrils.24 Thenano-Au labeling studies demonstrate that sNUCB1stabilizes these prefibrillar aggregates by cappingtheir ends. The capped prefibrillar species do notaggregate even when incubated for more than1 week and are unable to seed the aggregation ofhIAPP monomers to form amyloid fibrils, indicatingthat the capped prefibrillar complex is inert anddoes not get incorporated into a growing fibril. Asimilar capping mechanism has been proposed forsmall-molecule inhibitors of amyloid formation, butto the best of our knowledge, sNUCB1 is the firstprotein-based inhibitor which has been shown to actby capping protofibrils.28,29

The AFM studies show that preformed protofibrilsinteract with nano-Au-labeled sNUCB1(S44C). Thispossibly provides a prototypical snapshot of howsNUCB1may interactwith protofibrils and eventuallyfibrils to facilitate disaggregation. For a self-assemblyprocess such as amyloidogenesis, the kinetics ofgrowth can often be dominated by secondary nucle-ation events resulting from fibril fragmentation.30–33

sNUCB1 can effectively disaggregate amyloid fibrils,and this might be accomplished through interactionwith products of hIAPP fibril fragmentation orsecondary nuclei. The binding of sNUCB1 to thesesecondary nuclei would shift the equilibrium of theprocess in the favor of fibril fragmentation.The inhibition of hIAPP fibril formation and

disaggregation of preformed fibrils by sNUCB1occur only in the absence of Ca2+. We recentlyreported that sNUCB1 is asymmetric in shape andthe dimeric sNUCB1 undergoes secondary-structuretransition from predominantly α-helical to β-sheetas a function of concentration.23 This structuraltransition might facilitate the interaction of sNUCB1with prefibrillar aggregates of hIAPP, which alsopossess a β-sheet structure. Intriguingly, the C-terminal fragment of NUCB1 has a polyglutamine(poly-Q) stretch. Poly-Q stretches are unstructuredmotifs that facilitate protein–protein interaction. Infuture work, we will characterize an engineeredmutant of sNUCB1 that does not bind to Ca2+.The precise relationship of our findings to the

physiological role of NUCB1 is, as of yet, unknown.However, NUCB1 has been reported to be upregu-lated in the endoplasmic reticulum (ER)-mediatedunfolded protein response (UPR). NUCB1 apparentlyinhibits site-1-protease-mediated activating transcrip-tion factor 6 cleavage without affecting activatingtranscription factor 6 transport and thus preventsUPR activation. ER stress induces NUCB1 secretionfrom Golgi and increased intracellular NUCB1

expression.29 It is conceivable that NUCB1 controlsthe UPR by sensing the presence of unfolded proteinsin the ER. The observed anti-amyloidogenic activityof sNUCB1 is nucleotide independent. However, it ispossible that NUCB1 caps these prefibrillar speciesin vivo and assists the chaperone machinery inclearing these complexes while they are still soluble.

Methods

Thio-T binding assay

Enhancement in thio-T fluorescence was used tomonitor the kinetics of hIAPP aggregation in the absenceand presence of Ca2+-free sNUCB1. All fluorescencemeasurements were performed on a Jobin-Yvon Horibafluorescence spectrophotometer using an excitation wave-length of 450 nm and an emission wavelength of 485 nm.The emission and excitation slits were both set to 5 nmand a 1.0-cm cuvette was used. Experiments wereperformed by diluting an hexafluoroisopropanol stocksolution of hIAPP into 20 mM Tris–HCl, pH 7.5. Eachstock solution was filtered through a 0.45-μm-pore-sizeGHP Acrodisc syringe filter prior to the experiment. Thefinal reaction mixture contained hIAPP with or withoutCa2+-free sNUCB1 along with 32 μM thio-T and 2%hexafluoroisopropanol in the reaction buffer of 20 mMTris–HCl, pH 7.5. Experiments were conducted at 25 °Cwith constant stirring. The fluorescence intensity observedfor binding of thio-T to Ca2+-free sNUCB1 alone wassubtracted from each kinetic curve.

Transmission electron microscopy

TEM was performed at the Bioimaging facility atRockefeller University. Samples were prepared by placing5 μl of solution onto formvar-coated 300-mesh coppergrids and counterstained with 2% aqueous uranyl acetatesolution. Samples were viewed with an FEI Tecnai12BioTwinG2 transmission electron microscope at 80 kV.Digital images were acquired with an AMT XR-60 CCDDigital Camera System and compiled using AdobePhotoshop CS2 and ImageJ.

Atomic force microscopy

Samples were adsorbed onto freshly cleaved mica froma dilute buffer solution of 20 mM Tris–HCl, pH 7.5, andleft to dry. AFM imaging was performed in air once thesamples were completely dry. AFM measurements weretaken on a Park Systems XE-100 in True Non-ContactMode. Rectangular silicon cantilevers were used (Nano-sensors, Neuchatel, Switzerland) (NCHR, f=330 kHz,k=42 N/m). Non-contact mode images were taken atscan rates between 0.3 and 1 Hz.

Size-exclusion chromatography

SEC was performed using a Superose6 10/30 GLcolumn attached to an AKTA explorer FPLC. Twohundred microliters of a reaction mixture with Ca2+-freesNUCB1 (32 μM) in 20 mM Tris–HCl, pH 7.5, or Ca2+-free

388 Nucleobindin 1 Inhibits Amyloid Formation by hIAPP

sNUCB1 preincubated with equimolar amount of hIAPPin reaction buffer was injected onto a buffer-equilibratedcolumn. The chromatogram corresponding to absorbanceat 280 nm was plotted as a function of elution volume foreach reaction.

Dot blot assay

Dot blots were performed by blotting samples onto anactivated polyvinylidene fluoride membrane, allowingthem to dry and then blocking themwith 5%milk solutionin TBS (20 mM Tris–HCl, pH 7.5, 150 mM NaCl) at roomtemperature. The membrane was incubated with asolution of rabbit polyclonal primary antibody againsthIAPP (1:2 dilution of prediluted stock) or NUCB1 (1:500dilution of 1 μg/μl stock) in TBS for 1 h at roomtemperature followed by three washes with TBS-T(20 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 0.05%Tween-20) for 5 min each. The membranes were thenincubated with horseradish-peroxidase-conjugated anti-rabbit secondary antibody (1:8000 dilution of the stock) inTBS for 30 min at room temperature followed by twowashes with TBS-T for 5 min each and a subsequent washwith TBS for 5 min. The blots were developed using theSuperSignal West Femto Maximum Sensitivity Substrate(Thermo Scientific).

STEM and M/L measurements

Dark-field micrographs of unstained freeze-dried spec-imens together with TMV particles as a mass standardwere prepared according to the standard “wet film”method of the Brookhaven STEM facility. This involvedfreeze-drying the sample with TMV on titanium gridssupporting a 2- to 3-nm carbon film. Grids were scannedat −150 °C on a custom-built STEM at 40 kV with a probefocused to 0.25 nm with a dwell time of 30 μs/pixel.Digital dark-field micrographs of freeze-dried specimenswere recorded with 512×512 pixels at raster steps of 1.0 or2.0 nm per pixel and an average radiation dose of 1×102 to5×102 electrons/nm2. The images were analyzed usingthe PCMass30 software (Brookhaven STEM resource). Theresulting data were normalized to the known M/L ofTMV (131.4 kDa/nm).

Light scattering

Light scattering data were collected using a Superose610/30 GL SEC column (GE Healthcare, Piscataway, NJ),connected to a high-performance liquid chromatographysystem, Agilent 1200 (Agilent Technologies, Wilmington,DE), equipped with an autosampler. The elution from SECwas monitored by a photodiode array UV/VIS detector(Agilent Technologies), differential refractometer (OPT-Lab rEx, Wyatt Corp., Santa Barbara, CA), and static anddynamic multi-angle laser light scattering (LS) detector(HELEOS II with QELS capability, Wyatt Corp.). The SEC-UV/LS/RI system was pre-equilibrated in buffer (20 mMTris–HCl, pH 7.5), and 200 μl of the reaction mixturecontaining Ca2+-free sNUCB1 alone or incubated with anequimolar amount of hIAPPwas injected onto the column.Chemstation software (Agilent Technologies) controlledthe high-performance liquid chromatography operation

and data collection from the multi-wavelength UV/VISdetector, while the ASTRA software (Wyatt Corp.)collected data from the refractive index detector and thelight-scattering detectors and recorded the UV trace at280 nm sent from the photodiode array detector.Molecular masses were determined across the entireelution profile for each observed peak in intervals of 1 sfrom static LS measurement using ASTRA software aspreviously described.34 DLS measurements were made“on line” at an angle of 100° with a 2-s collection timeacross the elution peaks. Time-resolved scatter intensityfluctuations were analyzed using ASTRA Software (WyattCorp.), which implements the cumulants method35 todetermine the intensity correlation function.

Acknowledgements

This work was supported by National Institutes ofHealth grant GMO78114 (to D.P.R) and by theEleanor Schwartz Charitable Trust and the Bridgesto Better Medicine Fund (to T.P.S). We thank Dr. W.Vallen Graham for helpful discussions. We alsothank Ms. Trisha Barua, Dr. Fanling Meng, and Ms.Ping Cao of Stony Brook University for preparinghIAPP. We gratefully acknowledge the assistance ofDr. Ewa Folta-Stogniew of the Keck FoundationBiotechnology Resource Laboratory (Yale Universi-ty School of Medicine), Dr. JosephWall (BrookhavenNational Laboratory), Dr. Hiro Uryu (RockefellerUniversity Electron Microscopy Resource Center),and Dr. Cynthia Buenviaje-Coggins and Ying Xiongof Park AFM Systems.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2012.04.017

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