Full Paper DOI:10.1002/ijch.201600076 Formation of Apoptosis-Inducing Amyloid Fibrils by Tryptophan Shira Shaham-Niv,[a] Pavel Rehak,[b] Lela Vukovic´,[c] Lihi Adler-Abramovich,[d] Petr Krµl,[b,e,f] and Ehud Gazit*[a, g] Abstract:Manymajor degenerative disorders are associated Furthermore, the assembliesshow significant cytotoxicity with the formationofamyloidfibrils by proteins and pep- triggered by an apoptosis mechanism, similar to that known tides. Recent studies have extended the repertoire of amyloi- for amyloids.Asacontrol, the non-amyloidogenic amino dogenic building blocks to non-proteinaceous entities, in- acid alanine was used under the same conditionsand did cluding aminoacids and nucleobases. Here, based on the not show any toxicity.Molecular dynamics simulationswere high propensity of tryptophan-containing proteins and pep- used to explore the possible growth mechanism, molecular tides to form amyloid fibrils, we explored the self-assembly organization,and stability of tryptophan amyloidalfibrils. profile of this amino acid. We discovered that tryptophan Taken together,weprovide further extensiontothe amyloid forms fibrillary assemblieswith adiameterof15–75 nm. hypothesis and additional indication for aknwonmecha- These fibrils bind the thioflavin Tamyloid-specific dye and nism of toxicity for both amyloid-associated and metabolic show atypicalspectrum of amyloid proteins uponbinding. disorders. Keywords: aggregation · amino acids · amyloids · cytotoxicity · metabolic disorders 1. Introduction brillar supramolecular assemblies have adiameter of 5– Over the pastdecade,the phenomenon of soluble protein 20 nm, are predominantly rich in b-sheet secondary struc- and peptide misfolding, leading to the formation of or- ture,and specifically bind dyes,such as thioflavin T dered amyloid fibrils,has been increasingly associated (ThT) and Congo red.[2] Interestingly,analysis of short with agreat variety of notable human disorders with un- functional fragmentsfrom unrelated amyloid-forming related etiology,includingAlzheimersdisease,Parkin- proteins and polypeptidesidentified aremarkable occur- sonsdisease,and type II diabetes.[1] All amyloid fibrils rence of aromatic residues.[3] Thearomatic residues most share auniqueset of similar biophysical and structural likely have an important role in the amyloidogenic pro- properties,despite being formed by adiverse and struc- cess and in the stabilizationofamyloidal structures by turally unrelated group of proteins and peptides.These fi- [a] S. Shaham-Niv,E.Gazit [e] P. Krµl Department of Molecular Microbiology and Biotechnology Department of Physics George S. Wise Faculty of Life Sciences University of Illinois at Chicago TelAviv University Chicago (USA) TelAviv 69978 (Israel) [f]P.Krµl [b] P. Rehak, P. Krµl Department of Biopharmaceutical Sciences Department of Chemistry University of Illinois at Chicago University of Illinois at Chicago Chicago (USA) Chicago (USA) [g] E. Gazit [c] L. Vukovic´ Department of Materials Science and Engineering Department of Chemistry Iby and Aladar Fleischman Faculty of Engineering University of Texas at El Paso TelAviv University El Paso (USA) TelAviv 69978 (Israel) [d] L. Adler-Abramovich Tel.:(+972) 3-640-9030 Department of Oral Biology e-mail:[email protected] The Goldschleger School of Dental Medicine Supporting information for this article is available on the WWW TelAviv University under http://dx.doi.org/10.1002/ijch.201600076. TelAviv 69978 (Israel) Isr.J.Chem. 2017,57, 729 –737 2017 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim 729 Full Paper geometricallyrestricted interactions betweenplanar aro- teine,phenylalanine,and tyrosinehave high aggregation matic chemical entities.Theseresidues can affect the potential,[11] in agreement with their potential aggregative morphology of the assemblies,accelerate their formation, property as isolated amino acids.[10] However, the highest improve their stability and reduce the minimal association propensity was calculated for tryptophan,[11] whichwas concentration.[4] It was previously shown that penta- and not found in our initial screen. In addition,the high ag- tetrapeptidescan form typical amyloid fibrils.Moreover, gregation ability of tryptophan was also shown in the con- the diphenylalaninedipeptide,the core recognition motif text of tripeptides,where the aminoacid exhibited high within the b-amyloid polypeptide,was found to form aggregation propensity when positioned in the N-termi- well-ordered nanotubular assemblies in aqueous solu- nal, middle,orC-terminal positions.[12] Thearomatic tion.[5] Lateron, these dipeptideassemblies were shown amino acid tryptophan (see Figure 1A) is essential for to share opticaland functional properties with amyloids humans, playing acrucial role in protein stability and rec- that were assembled from the full-length polypeptide.[6] ognition, despite its rarity in protein sequences.[13] Fur- Thepentapeptide- and tetrapeptide-based amyloid nano- thermore,tryptophan is acritical component of numerous assemblies were foundtobecytotoxic via an apoptotic metabolic pathways,being abiochemical precursor for se- cell death pathway,which indicates abiological generic rotonin, melatonin, and niacin.[14] Tryptophan is accumu- property and acommon mechanism of toxicity.[7] Thus lated in pathological conditions, such as several metabolic the minimal peptideassemblies appear to reflect both the disorders.The accumulation of tryptophan has been re- physicalaswell as functional properties of natural amy- ported in two inborn errors of metabolism, hypertrypto- loid fibrils. phanemia and Hartnup disease (see Table 1), both shown While the formationofcytotoxic supramolecular enti- to be rare and inherited autosomal recessive disorders. ties has previously been linked to proteins and peptides, Hypertryptophanemia occurs due to the inabilityofthe it was first demonstrated by our groupand later by others body to process tryptophan.Asaresult, there is amassive that phenylalanine,asasingle amino acid, can also self- buildupoftryptophan in the bloodand urine, which fur- assemble to form amyloid-like fibrils showing typical ul- ther leads to musculoskeletal effects and to behavioral trastructural, biophysical, and biochemical properties.[8] and developmental abnormalities.[15] Hartnup disease is Moreover, we revealed the connectionbetween these as- caused by damage to aneutral amino acid transporter, semblies and the accumulation of phenylalanine in phe- limited to the kidneys and small intestine, which affects nylketonuria (PKU), aknownmetabolic disorder.We the absorption of nonpolar amino acids,mainlytrypto- demonstrated that these phenylalanine assemblies are cy- phan. Thus,increasedlevels of tryptophanand indolic totoxic and that antibodies raised towardsthese species compounds are detectedinthe patients urine.Common deplete fibril toxicity.The generation of antibodies in symptoms include the development of arash on parts of aPKU mice model and the identification of aggregate de- the body exposed to the sun, mental retardation, head- posits in patients brains post mortem suggested apatho- aches,collapsing and fainting.[16] logical role of these assemblies.[8a] In light of the above findings,wescreened for condi- Metabolicdisorders,such as PKU,and more specifical- tions in whichtryptophan could form visible structures ly inbornerrors of metabolism, are the result of aflaw in and foundthat by increasingtryptophan concentration, asingle gene encodingfor metabolic enzymes.As using the same conditionsfor self-assembly as with other aresult, accumulating metabolites may interfere with the metabolites,wecould detect the formation of assemblies normal function of cells and tissues and thus cause severe in the test tube.The properties of these assemblies were abnormalities.Unlessthese inheriteddisorders are treat- further examined usinglower concentrations,similar to ed with avery strict diet,they may result in mentalretar- the ones used in our previousscreening. Herewepresent, dation and other developmental problems.Although for the first time,the ability of the single amino acid tryp- these disorders are individually considered very rare,col- tophan to form amyloid supramolecular assemblies.We lectively they constituteavery substantial part of pedia- characterized the tryptophan aggregates usingacombina- tric genetic diseases.[9] Recently,wehave extended the ge- tion of diverse biophysicaland biological assays,inaddi- neric amyloid hypothesis to includeadditional non-protei- tion to the use of molecular dynamics (MD)simulations naceous entities,includingamino acids and nucleobases. to modelthe fibril formationand stability.This work fur- We demonstrated the ability of these small metabolites to ther extends the generic amyloid hypothesis and gives fur- form amyloidal fibrils,sharingthe same biophysical prop- ther supporting evidence for the associationbetween me- erties,aspresented by electronmicroscopy and the ThT tabolite amyloidformationand metabolic pathologies. binding assay, and their clear apoptoticeffect on aneuro- nal cell model. These new findings introduceanew possi- ble amyloid-like mechanism of metabolic disorders,sug- 2. Experimental Results and Discussion gesting anew paradigm for these rare maladies.[10] Previous work that examined the amyloid aggregation In previous work that calculated the amyloidaggregation propensity of all 20 coded amino acids revealedthat cys- potential of all amino acids in the context of proteins and Isr.J.Chem. 2017,57, 729 –737 2017 Wiley-VCH