Searching for the Best Transthyretin Aggregation Protocol to Study Amyloid Fibril Disruption

  Ci a ion: Fe ei a, E.; Almeida, Z.L.; C uz, P.F.; Sil a e Sousa, M.; Ve íssimo, P.; B i o, R.M.M. Sea ching o he Bes T ans hy e in Agg ega ion P o ocol o S udy Amyloid Fib il Dis up ion. In . J. Mol. Sci. 2022,23, 391. h ps://doi.o g/ 10.3390/ijms23010391 Academic Edi o : Vy au as Smi no as Recei ed: 31 Augus 2021 Accep ed: 20 Decembe 2021 Published: 30 Decembe 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). In e na ional Jou nal o Molecula Sciences A icle Sea ching o he Bes T ans hy e in Agg ega ion P o ocol o S udy Amyloid Fib il Dis up ion Elisabe e Fe ei a 1,†, Zaida L. Almeida 1,† , Ped o F. C uz 1, Ma a Sil a e Sousa 1, Paula Ve íssimo 2 and Rui M. M. B i o 1,* 1Chemis y Depa men and Coimb a Chemis y Cen e-Ins i u e o Molecula Sciences (CQC-IMS), Uni e si y o Coimb a, 3004-535 Coimb a, Po ugal; [email p o ec ed] (E.F.); [email p o ec ed] (Z.L.A.); [email p o ec ed] (P.F.C.); [email p o ec ed] (M.S.e.S.) 2Cen e o Neu oscience and Cell Biology, Uni e si y o Coimb a, 3004-517 Coimb a, Po ugal; [email p o ec ed] *Co espondence: [email p o ec ed] † These au ho s con ibu ed equally o his wo k. Abs ac : Se e al degene a i e amyloid diseases, wi h no ully e ec i e ea men , a ec millions o people wo ldwide. These pa hologies—amyloidoses—a e known o be associa ed wi h he o ma ion o o de ed p o ein agg ega es and highly s able and insoluble amyloid ib ils, which a e deposi ed in mul iple issues and o gans. The dis up ion o p e o med amyloid agg ega es and ib ils is one possible he apeu ic s a egy agains amyloidosis; howe e , only a ew compounds ha e been iden i ied as possible ib il dis up o s in i o o da e. To p ope ly iden i y chemical compounds as po en ial ib il dis up o s, a eliable, as , and economic sc eening p o ocol mus be de eloped. Fo his pu pose, h ee amyloid ib il o ma ion p o ocols using ans hy e in (TTR), a plasma p o ein in ol ed in se e al amyloidoses, we e s udied using hio la in-T luo escence assays, ci cula dich oism (CD), u bidi y, dynamic ligh sca e ing (DLS), and ansmission elec on mic oscopy (TEM), in o de o cha ac e ize and selec he mos app op ia e ib il o ma ion p o ocol. Sa u a ion ans e di e ence nuclea magne ic esonance spec oscopy (STD NMR) was success ully used o s udy he in e ac ion o doxycycline, a known amyloid ib il dis up o , wi h p e o med wild- ype TTR (TTRw ) agg ega es and ib ils. DLS and TEM we e also used o cha ac e ize he e ec o doxycycline on TTRw amyloid species disagg ega ion. A compa ison o he TTR amyloid mo phology o med in di e en expe imen al condi ions is also p esen ed. Keywo ds: ans hy e in; p o ein agg ega ion; amyloid ib ils; amyloidosis; amyloid dis up o s; ib il disagg ega ion; sc eening p o ocol 1. In oduc ion Cu en ly, conside able a en ion is gi en o a g oup o p o ein mis olding diseases known as amyloidosis [ 1 – 4 ]. The e a e app oxima ely 50 o hese pa hologies, in ol ing di e en p ecu so p o eins and associa ed wi h he o ma ion o ex acellula amyloid ib ils o in acellula inclusions wi h amyloid-like cha ac e is ics [ 3 , 5 ]. Consequen ly, amyloidoses can a ec di e en o gans, namely he hea , li e , kidneys, ne ous sys em, spleen, skin, and gas oin es inal ac , among o he s. Se e e amyloidosis can lead o li e- h ea ening o gan ailu e and dea h. No e ec i e disease modi ying he apies a e a ailable o mos amyloid diseases, including Alzheime ’s disease (AD) and Pa kinson’s disease (PD). Amyloid- β pep ide, p ion p o ein, α -synuclein, and ans hy e in (TTR) a e examples o he mo e han 40 di e en human pep ides and p o eins iden i ied in amyloid deposi s ha a e a he o igin o he known amyloidoses [3]. In e es ingly, amyloid ib ils p esen common mo phologies and s uc u al p ope ies, despi e being o igina ed by qui e di e en p ecu so p o eins. Amyloid ib ils a e long unb anched s uc u es wi h a high β -shee con en s uc u al co e and he modynamically In . J. Mol. Sci. 2022,23, 391. h ps://doi.o g/10.3390/ijms23010391 h ps://www.mdpi.com/jou nal/ijms In . J. Mol. Sci. 2022,23, 391 2 o 17 e y s able, which may be de ec ed h ough binding o dyes, such as Congo ed, o he luo escen p obe hio la in-T (ThT) [1,6]. Amyloids o med by he p o ein ans hy e in (TTR) a e a he o igin o se e al amy- loidoses (ATTR) ha can be di ided in wo main ypes: spo adic, age- ela ed amyloidosis, and he edi a y o amilial amyloidosis. Wild- ype TTR amyloidosis (ATTRw ), o me ly known as senile sys emic amyloidosis (SSA), is an idiopa hic disease cha ac e ized by he deposi ion o wild- ype (w ) TTR amyloid mainly in he hea . ATTRw is s ongly co e- la ed wi h ageing, a ec ing an es ima ed 25% o he wo ld popula ion o e he age o 80 [ 7 ], and may gi e ise o hea y deposi ion and in il a ion o amyloid in he ca diac issue, esul ing in conges i e hea ailu e [ 8 ]. Mu an TTR amyloidosis (ATTRm) comp ises a g oup o amilial/he edi a y and a e mul isys em diseases wi h a wide spec um o clinical mani es a ions, such as amilial amyloid polyneu opa hy (hATTR-PN o FAP) [ 9 ], amilial amyloid ca diomyopa hy (hATTR-CM o FAC) [ 10 ], and amilial lep omeningeal amyloidosis [ 11 ], which may occu in isola ion o in combina ion wi h amilial ocula amyloidosis [ 12 ] and addi ional clinical mani es a ions. These ATTRm amyloidoses a e caused by mo e han 130 di e en known TTR a ian s [13,14]. Human TTR is a 55 kDa homo e ame ic plasma p o ein wi h a high p opo ion o β -shee s uc u e ha is mainly biosyn hesized in he li e [ 15 ], bu also syn hesized in he cho oid plexus o he b ain [ 16 ] and in he e inal pigmen ous epi helium o he eye [ 17 ]. The main known unc ions o TTR a e he anspo o hy oid ho mones and e inol in associa ion wi h e inol-binding p o ein. Addi ionally, TTR is also known o ha e a neu op o ec i e ole [18]. O e he yea s, he molecula mechanisms o amyloid o ma ion ha e been he subjec o conside able scien i ic in e es [ 19 , 20 ]. In i o ib il o ma ion has been a powe ul ool in he s udy o he mechanism ha leads o amyloid o ma ion, helping o explain he e en s occu ing in i o . Rega ding he TTR agg ega ion p ocess, TTR ins abili y caused by mu a ions o by o he ac o s, leads o he dissocia ion o he na i e e ame in o monome s ha unde go pa ial un olding [ 21 – 23 ]. These non-na i e monome s can associa e in o soluble oligome s ha sel -assemble, leading o he inal amyloid ib il [ 20 , 24 ]. Howe e , he clinical and gene ic he e ogenei y o ATTR, oge he wi h he di e en s uc u al and unc ional cha ac e is ics o he TTR a ian s, sugges s ha TTR ib il o ma ion may in ol e di e se molecula pa hways [ 25 ]. Amyloid deposi s may be cons i u ed no only by ull-leng h TTR ib ils, bu also by C- e minal TTR agmen s and amo phous agg ega es [26–28]. Al hough li e ansplan a ion and he apies [ 29 , 30 ] such as TTR s abiliza ion [ 27 ] o supp ession o TTR exp ession [ 31 – 36 ] a e cu en ly in use, he e is no ully e ec i e ea - men o all he clinical mani es a ions o ATTR amyloidosis ye . Conside ing he cu en knowledge o TTR and TTR amyloid o ma ion, a ious he apeu ic s a egies a ge ing di e en s eps o he agg ega ion pa hway may be de ised [ 28 – 30 ]. The dis up ion o amyloid agg ega es and ib ils is one o hese s a egies, and has gained impo ance in bo h expe imen al assays and clinical s udies [ 37 – 46 ] in he las wo decades. Howe e , o da e, only a ew compounds ha e been iden i ied as possible amyloid ib il dis up o s o ATTR. Among hese a e some polyphenols, such as EGCG (epigalloca echin-3-galla e) [37,38,45] , IDOX (40-iodo-40-deoxydoxo ubicin) [39,40], and doxycycline [42,43,46]. In o de o disco e new amyloid ib il dis up o s agains ATTR, we aimed o de elop an app op ia e in i o sc eening p o ocol o TTRw disagg ega ion. Fo his pu pose, he i s s ep was he cha ac e iza ion and selec ion o an app op ia e agg ega ion p o ocol and iden i ica ion o he espec i e amyloid agg ega e/ ib il model. Ideally, he in i o o med amyloid species would ha e simila s uc u e and mo phology o in i o o med species, and he agg ega ion p o ocol expe imen al condi ions would be as close as possible o he in i o p ocess o TTR amyloid o ma ion, main aining physiological condi ions, such as a e age p o ein concen a ion, pH, ionic s eng h, and empe a u e. Howe e , he na i e e ame ic o m o TTRw has high con o ma ional s abili y [ 47 – 49 ], and he amyloid o - ma ion p ocess in hese condi ions is leng hy and hus is unsui able o be used in sc eening In . J. Mol. Sci. 2022,23, 391 3 o 17 p o ocols. A e a ca e ul analysis o he a ailable li e a u e [ 21 , 50 , 51 ], h ee agg ega e and ib il o ma ion p o ocols we e selec ed o compa ison: acidi ica ion a pH 2.0 and pH 4.4, and hea ing a pH 7.4. Fu he mo e, he e ec o changing he pH om 2.0 o 4.4 o physiological condi ions (pH 7.4), a e agg ega ion, was also s udied. Thio la in-T (ThT) luo escence assays, ci cula dich oism (CD), u bidi y, dynamic ligh sca e ing (DLS), and ansmission elec on mic oscopy (TEM) we e used o cha ac e ise he iming and p oduc s o med wi h hese p o ocols and he e ec o pH modi ica ion. The choice o app op ia e expe imen al condi ions and me hods o p o ide ele an in o ma ion on he e ec o pa icula compounds on ib il in e ac ion and disagg ega ion is o pa amoun impo ance in a sc eening p o ocol. Sa u a ion ans e di e ence nuclea magne ic esonance spec oscopy (STD NMR) was used o s udy he in e ac ion o TTR ib illa s uc u es and a known TTR ib il dis up o —doxycycline—in mo e de ail. DLS, TEM, and u bidi y expe imen s we e used o cha ac e ize he e ec o his compound on TTR amyloid ib il dis up ion. Addi ionally, doxycycline was chosen o alida e he expe imen al p o ocols due o i s well demons a ed an i-amyloidogenic ac i i y in i o [ 46 ] and in i o . I has been shown ha doxycycline ac s as L55P TTR amyloid ib il dis up o in i o [ 40 ] and disagg ega es amyloid deposi s in V30M TTR ansgenic mice wi h a concomi an dec ease o a ious amyloidosis issue ma ke s [ 41 , 42 ]. This compound was e en used in clinical ials, some imes in combina ion wi h TUDCA ( au ou sodeoxycholic acid) [ 43 ] and o he imes in combina ion wi h UDCA (u sodeoxycholic acid) [52]. 2. Resul s 2.1. Cha ac e iza ion o TTRw Fib il Fo ma ion P o ocols In o de o summa ize he h ee agg ega ion p o ocols o TTRw used in his s udy, Table 1shows he main equi emen s o hese p o ocols in e ms o expe imen al condi ions, and Table 2summa izes he main cha ac e is ics o he agg ega es and ib ils o med du ing agg ega ion and he esul o pH changes on hese amyloid s uc u es. Table 1. Expe imen al condi ions o h ee TTRw agg ega ion p o ocols. pH Tempe a u e Incuba ion Pe iod P o ein Concen a ion S i ing 2 25 ◦C A leas 1 week 80 µM and hen dilu ed o 3.6 µMNo 4.4 37 ◦C 3 days 3.6 µM No 7.4 60 ◦C 6 days 3.6 µM No Table 2. Cha ac e iza ion o TTRw amyloid species o med by di e en agg ega ion p o ocols. Agg ega ion P o ocol Mo phology o he Agg ega es and Fib ils Leng h/Diame e o Agg ega ed Species Thio la in-T Assay E ec o pH Adjus men o pH 7.4 pH 2.0 Long, unb anched, ma u e ib ils 50 o 170 nm/5 o 9 nm Posi i e Al e a ions in he size and seconda y s uc u e o ib ils Fib ils emain ThT posi i e pH 4.4 Mix u e o sphe oid s uc u es, amo phous agg ega es, and sho unb anched ib ils Sphe oid agg ega es (4 o 6 nm); ib ils (25 nm/4–5 nm) Posi i e Sligh e ec on size, seconda y s uc u e, and mo phology o agg ega es o mo e amo phous and less ib illa species Fib ils emain ThT posi i e pH 7.4 Unb anched ib ils 10 o 50 nm/4 o 6 nm Posi i e No necessa y Acco ding o Tables 1and 2, he ib il o ma ion p o ocol pe o med a pH 2.0 wi h 0.1 M NaCl was mo e ime-consuming and equi ed highe concen a ions o p o ein. Amyloid ib ils obse ed by TEM appea ed as long unb anched ilamen ous s uc u es 5 o 9 nm in diame e wi h a iable leng h, indica ing a ma u e g ow h o hese ib illa In . J. Mol. Sci. 2022,23, 391 4 o 17 s uc u es [ 1 ] which we e ThT posi i e. The a -UV CD spec um also showed a minimum a ound 213 nm, and a CD spec um ypical o TTRw amyloid ib ils [ 20 ] wi h high β -shee con en (Table 3). In an a emp o mimic he in i o condi ions o TTRw amyloid ib ils, pH adjus men o pH 7.4 o p e o med ib ils a pH 2.0 was ca ied ou , and he e ec on ib il size, s uc u e, and mo phology was analysed by DLS, CD, and TEM, espec i ely. DLS esul s showed ha a e 11 days o ib il incuba ion a pH 7.4, he sample had la ge popula ions o smalle pa icles (be ween 100 and 1000 nm) compa ed o ib il incuba ion a pH 2.0 (Figu e S1). The changes in seconda y s uc u e o p e o med TTRw ib ils we e analysed by a -UV CD and he minimum a 213 nm was no as p onounced, indica ing a clea loss in β -shee and a gain o uno de ed seconda y s uc u es (Figu e 1A and Table 3). TEM obse a ions indica ed ha , a pH 7.4, ib ils los hei ex ended ib illa s uc u e and became mo e sphe ical and amo phous wi h diame e s be ween 4 and 13 nm when compa ed o ib ils a pH 2.0 (Figu e 2A, pH 2.0 o 7.4), co obo a ing DLS and CD esul s; howe e , hey s ill emained ThT posi i e. Table 3. Seconda y s uc u e es ima ion by Ci cula Dich oism using he web se e BeS Sel. Agg ega ion P o ocol pH 2.0 pH 2.0 → pH 7.4 pH 4.4 pH 4.4 → pH 7.4 pH 7.4 (60 ◦C) pH 7.4 (37 ◦C) α-Helix 0.14 0.03 0.07 0.08 0.11 0.09 β-Shee 0.43 0.32 0.32 0.24 0.4 0.39 Tu n 0.09 0.13 0.16 0.17 0.11 0.13 Uno de ed 0.34 0.52 0.45 0.51 0.38 0.39 In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 o 17 ib ils amo phous and less ib illa species pH 7.4 Unb anched ib ils 10 o 50 nm/4 o 6 nm Posi i e No necessa y Acco ding o Tables 1 and 2, he ib il o ma ion p o ocol pe o med a pH 2.0 wi h 0.1 M NaCl was mo e ime-consuming and equi ed highe concen a ions o p o ein. Am- yloid ib ils obse ed by TEM appea ed as long unb anched ilamen ous s uc u es 5 o 9 nm in diame e wi h a iable leng h, indica ing a ma u e g ow h o hese ib illa s uc- u es [1] which we e ThT posi i e. The a -UV CD spec um also showed a minimum a ound 213 nm, and a CD spec um ypical o TTRw amyloid ib ils [20] wi h high β- shee con en (Table 3). In an a emp o mimic he in i o condi ions o TTRw amyloid ib ils, pH adjus men o pH 7.4 o p e o med ib ils a pH 2.0 was ca ied ou , and he e ec on ib il size, s uc u e, and mo phology was analysed by DLS, CD, and TEM, e- spec i ely. DLS esul s showed ha a e 11 days o ib il incuba ion a pH 7.4, he sample had la ge popula ions o smalle pa icles (be ween 100 and 1000 nm) compa ed o ib il incuba ion a pH 2.0 (Figu e S1). The changes in seconda y s uc u e o p e o med TTRw ib ils we e analysed by a -UV CD and he minimum a 213 nm was no as p onounced, indica ing a clea loss in β-shee and a gain o uno de ed seconda y s uc u es (Figu e 1A and Table 3). TEM obse a ions indica ed ha , a pH 7.4, ib ils los hei ex ended ib illa s uc u e and became mo e sphe ical and amo phous wi h diame e s be ween 4 and 13 nm when compa ed o ib ils a pH 2.0 (Figu e 2A, pH 2.0 o 7.4), co obo a ing DLS and CD esul s; howe e , hey s ill emained ThT posi i e. Table 3. Seconda y s uc u e es ima ion by Ci cula Dich oism using he web se e BeS Sel. Agg ega ion P o ocol pH 2.0 pH 2.0 → pH 7.4 pH 4.4 pH 4.4 → pH 7.4 pH 7.4 (60 °C) pH 7.4 (37 °C) α-Helix 0.14 0.03 0.07 0.08 0.11 0.09 β-Shee 0.43 0.32 0.32 0.24 0.4 0.39 Tu n 0.09 0.13 0.16 0.17 0.11 0.13 Uno de ed 0.34 0.52 0.45 0.51 0.38 0.39 Figu e 1. Fa -UV CD spec a o TTRw ib ils om di e en ib il o ma ion p o ocols. (A) TTRw ib ils assembled a pH 2.0 and a e pH adjus men o 7.4, (B) a pH 4.4 and a e pH adjus men o 7.4, and (C) a pH 7.4 a 60 °C and a e empe a u e change o 37 °C. All TTRw samples we e analysed a 3.6 μM and all CD spec a we e acqui ed a he end o each ib il o ma ion p o ocol. Rega ding he agg ega ion a pH 4.4, his p o ocol was he leas ime-consuming, had low p o ein equi emen s, and was sui able o pH adjus men o 7.4 (Tables 1 and 2). The TEM images o he amyloid s uc u es o med showed sho e ib ils ha we e 5 o 6 nm in diame e and a leas 25 nm in leng h, as well as some amo phous agg ega es and se - e al oligome ic species ha we e 4 o 6 nm in diame e (Figu e 2A, pH 4.4). The CD spec- um also indica ed a less p onounced β-shee p o ile, co obo a ing he TEM esul s (Fig- u e 1B and Table 3). In addi ion, he DLS and CD expe imen s showed ha pH adjus men Figu e 1. Fa -UV CD spec a o TTRw ib ils om di e en ib il o ma ion p o ocols. ( A ) TTRw ib ils assembled a pH 2.0 and a e pH adjus men o 7.4, ( B ) a pH 4.4 and a e pH adjus men o 7.4, and ( C ) a pH 7.4 a 60 ◦ C and a e empe a u e change o 37 ◦ C. All TTRw samples we e analysed a 3.6 µM and all CD spec a we e acqui ed a he end o each ib il o ma ion p o ocol. Rega ding he agg ega ion a pH 4.4, his p o ocol was he leas ime-consuming, had low p o ein equi emen s, and was sui able o pH adjus men o 7.4 (Tables 1and 2). The TEM images o he amyloid s uc u es o med showed sho e ib ils ha we e 5 o 6 nm in diame e and a leas 25 nm in leng h, as well as some amo phous agg ega es and se e al oligome ic species ha we e 4 o 6 nm in diame e (Figu e 2A, pH 4.4). The CD spec um also indica ed a less p onounced β -shee p o ile, co obo a ing he TEM esul s (Figu e 1B and Table 3). In addi ion, he DLS and CD expe imen s showed ha pH adjus men o 7.4 sligh ly modi ied he pa icle size (Figu es S2 and 2B, pH 4.4 o 7.4) and seconda y s uc u e (Figu e 1B). The TEM images e ealed mo e sphe oid and amo phous mo phologies and less ib illa s uc u es (4 o 5 nm in diame e , and up o 25 nm in leng h), which is in o al ag eemen wi h a small loss o β -shee s uc u e and an inc ease in uno de ed s uc u e (Table 3) when compa ed wi h he ib ils o med a 4.4, and e en a pH 2.0 (Figu e 2A, pH 4.4 o 7.4); howe e , hey emained ThT posi i e (Table 2). In . J. Mol. Sci. 2022,23, 391 5 o 17 In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 o 17 o 7.4 sligh ly modi ied he pa icle size (Figu es S2 and 2B, pH 4.4 o 7.4) and seconda y s uc u e (Figu e 1B). The TEM images e ealed mo e sphe oid and amo phous mo phol- ogies and less ib illa s uc u es (4 o 5 nm in diame e , and up o 25 nm in leng h), which is in o al ag eemen wi h a small loss o β-shee s uc u e and an inc ease in uno de ed s uc u e (Table 3) when compa ed wi h he ib ils o med a 4.4, and e en a pH 2.0 (Fig- u e 2A, pH 4.4 o 7.4); howe e , hey emained ThT posi i e (Table 2). Figu e 2. Analysis o he e ec o doxycycline on p e o med TTRw agg ega es and ib ils. TTRw amyloid species (3.6 μM) assembled a pH 2.0 (pH 2.0), pH 2.0 ollowed by pH adjus men o 7.4 (pH 2.0 o 7.4), pH 4.4 (pH 4.4), pH 4.4 ollowed by pH adjus men o 7.4 (pH 4.4 o 7.4), and induced by hea ing a pH 7.4 (pH 7.4) we e incuba ed bo h in he absence (con ol) and p esence o 50× mola excess o doxycycline (180 μM) o he indica ed pe iod o ime and analysed by (A) TEM and (B) DLS. The scale ba s ep esen 200 nm, and he a ows indica e some o he amyloid s uc u es ob- se ed. Figu e 2. Analysis o he e ec o doxycycline on p e o med TTRw agg ega es and ib ils. TTRw amyloid species (3.6 µ M) assembled a pH 2.0 (pH 2.0), pH 2.0 ollowed by pH adjus men o 7.4 (pH 2.0 o 7.4), pH 4.4 (pH 4.4), pH 4.4 ollowed by pH adjus men o 7.4 (pH 4.4 o 7.4), and induced by hea ing a pH 7.4 (pH 7.4) we e incuba ed bo h in he absence (con ol) and p esence o 50 × mola excess o doxycycline (180 µ M) o he indica ed pe iod o ime and analysed by ( A ) TEM and ( B ) DLS. The scale ba s ep esen 200 nm, and he a ows indica e some o he amyloid s uc u es obse ed. Rega ding he hea -induced ib il o ma ion a pH 7.4, his p o ocol had a du a ion o 6 days and equi ed low concen a ions o p o ein (Table 1). The esul ing species we e ThT posi i e (Table 2), and TEM images showed unb anched ib illa s uc u es ha we e sho e and na owe han he ib ils o med a pH 2.0, wi h a diame e o 4 o 6 nm and a leng h o 10 o 50 nm (Figu e 2A, pH 7.4). The CD spec um also displayed he ypical β -shee p o ile (Figu e 1C and Table 3). To be e cha ac e ize his p o ocol, u bidi y was also used o ollow he agg ega ion p ocess. An inc ease in u bidi y was obse ed in he 6 days o hea ing (Figu e S3), which implies agg ega ion. The ansi ion om 60 ◦ C o 37 ◦ C did no induce a dec ease in u bidi y, indica ing ha he dec ease o empe a u e In . J. Mol. Sci. 2022,23, 391 6 o 17 nei he causes ib il b eakdown (Figu e S3, 8 h day) no an al e a ion in seconda y s uc u e (Figu e 1C and Table 3). 2.2. In e ac ion o Doxycycline wi h P e o med TTRw Fib ils The in e ac ion be ween doxycycline (Figu e 3A) and p e o med TTRw agg ega es and/o ib ils, p epa ed acco ding o he p e iously desc ibed agg ega ion p o ocols, was s udied using 1 H STD NMR. The 1 H NMR spec um o doxycycline (Figu e 3B, ligh g ey), 1 H STD NMR spec um o doxycycline in he p esence o TTRw ib ils (Figu e 3B, black), and he o - esonance spec um (Figu e 3B, g ey) o each expe imen al condi ion is ep esen ed in Figu e 3B. The signals p esen in he STD spec um, iden i ied by hei chemical shi s, ep esen he doxycycline p o ons ha a e in close con ac wi h TTR amyloid s uc u es. In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 o 17 Rega ding he hea -induced ib il o ma ion a pH 7.4, his p o ocol had a du a ion o 6 days and equi ed low concen a ions o p o ein (Table 1). The esul ing species we e ThT posi i e (Table 2), and TEM images showed unb anched ib illa s uc u es ha we e sho e and na owe han he ib ils o med a pH 2.0, wi h a diame e o 4 o 6 nm and a leng h o 10 o 50 nm (Figu e 2A, pH 7.4). The CD spec um also displayed he ypical β-shee p o ile (Figu e 1C and Table 3). To be e cha ac e ize his p o ocol, u bidi y was also used o ollow he agg ega ion p ocess. An inc ease in u bidi y was obse ed in he 6 days o hea ing (Figu e S3), which implies agg ega ion. The ansi ion om 60 °C o 37 °C did no induce a dec ease in u bidi y, indica ing ha he dec ease o empe a u e nei- he causes ib il b eakdown (Figu e S3, 8 h day) no an al e a ion in seconda y s uc u e (Figu e 1C and Table 3). 2.2. In e ac ion o Doxycycline wi h P e o med TTRw Fib ils The in e ac ion be ween doxycycline (Figu e 3A) and p e o med TTRw agg ega es and/o ib ils, p epa ed acco ding o he p e iously desc ibed agg ega ion p o ocols, was s udied using 1 H STD NMR. The 1 H NMR spec um o doxycycline (Figu e 3B, ligh g ey), 1 H STD NMR spec um o doxycycline in he p esence o TTRw ib ils (Figu e 3B, black), and he o - esonance spec um (Figu e 3B, g ey) o each expe imen al condi ion is ep- esen ed in Figu e 3B. The signals p esen in he STD spec um, iden i ied by hei chem- ical shi s, ep esen he doxycycline p o ons ha a e in close con ac wi h TTR amyloid s uc u es. Figu e 3. ( A ) Molecula s uc u e o doxycycline and ( B ) 1 H STD NMR o doxycycline (2 mM) in he p esence o p e o med TTRw ib ils (20 µ M) assembled a pH 2.0 (pH 2.0), pH 2.0 ha unde wen pH adjus men o 7.4 (pH 2.0 o 7.4), pH 4.4 (pH 4.4), and assembled by hea ing a pH 7.4 (pH 7.4). The e e ence 1 H NMR spec um o doxycycline a he co esponding pH is ep esen ed in ligh g ey, he 1 H STD NMR spec um is ep esen ed in black, and he o - esonance spec um is ep esen ed in g ey. A pH 2.0, he doxycycline signals om he a oma ic p o ons (i.e., H-15, H-16, and H-17), h ee me hyl g oups, and p o ons H-8, H-11, and H-4 we e obse ed o in e ac wi h In . J. Mol. Sci. 2022,23, 391 7 o 17 TTRw amyloid ib ils (Figu e 3B, pH 2.0). Howe e , addi ional signals be ween 2.5 and 3 ppm could no be unambiguously assigned due o he high densi y o esonances in his egion. Mo eo e , he b oadening o he ligand signals in he p esence o ib ils is due o al e a ion o he elaxa ion ime, and he e o e is e idence o binding [ 53 ]. When he pH o he ib ils o med a pH 2.0 was adjus ed o 7.4, he signals om he a oma ic p o ons disappea ed, excep he p o on H-4 and p o ons om he h ee me hyl g oups, as can be seen in he co esponding STD spec um (Figu e 3B, pH 2.0 o 7.4). Doxycycline p o on signals we e absen in he 1 H STD NMR spec um o doxycycline in he p esence o TTRw amyloid s uc u es a pH 4.4 (Figu e 3B, pH 4.4). Con a y o wha occu ed wi h he o he agg ega ed samples p epa ed a pH 2.0 wi h 0.1 M NaCl and pH 7.4 a 60 ◦ C, he sample wi h TTRw agg ega es and ib ils assembled a pH 4.4 was ex emely u bid, and he amyloid con en seemed o quickly p ecipi a e in he NMR ube du ing he assay. A pH 7.4, he doxycycline p o ons in ol ed in he in e ac ion wi h he hea -induced ib ils we e simila o hose a pH 2.0 (Figu e 3B, pH 7.4). 2.3. E ec o Doxycycline on TTRw Fib il Disagg ega ion The e ec o doxycycline on ib il dis up ion was assessed by DLS, TEM, and in some o he agg ega ion p o ocols by u bidi y. P e o med TTRw agg ega es and ib ils we e incuba ed bo h in he absence and p esence o a 50 × mola excess o doxycycline ela i e o e ame ic TTRw . DLS measu emen s we e ca ied ou e e y day un il he appa en size s abiliza ion o he species in solu ion was achie ed. TEM images we e acqui ed p io o doxycycline addi ion and a e ea men wi h doxycycline when he sizes o he pa icle popula ions (assessed by DLS) had s abilized. Doxycycline, a 50 × he mola concen a ion o TTRw , did no ha e an immedia e pe sis en e ec on he agg ega es/ ib ils p epa ed p e iously wi h he di e en p o ocols, aking om 11 o 17 days o each a maximum obse able dis up i e e ec . A pH 2.0, a e 11 days o incuba ion wi h doxycycline, ib ils showed a maximum leng h o 130 nm and we e 4 o 7 nm in diame e , which was a clea dec ease o he ib il leng h and diame e obse ed by TEM, indica ing ib il dis up ion (Figu e 2A, pH 2.0). Mo eo e , DLS esul s also suppo ed his obse a ion, which was isible as an inc ease in pa icle popula ions be ween 100 and 1000 nm (Figu e 2B, pH 2.0). Tu bidi y assays we e also ca ied ou o ollow TTRw amyloid ib il agg ega ion and disagg ega ion in he p esence o doxycycline. Howe e , no signal in u bidi y in ensi y was obse ed, bo h in ea ed and un ea ed samples wi h doxycycline (da a no shown). This esul may be explained by he o ma ion o a clea and comple ely anspa en solu ion, hus implica ing no ligh sca e ing signal de ec ion in he pH 2.0 samples, especially when compa ed o u bid solu ions as is he case o pH 4.4 samples. When he ib ils assembled a pH 2.0 unde wen pH adjus men o 7.4 and we e hen ea ed wi h doxycycline, he dis up i e e ec was isible a e 17 days bo h by TEM, wi h a dec ease in he size o he non- ib illa s uc u es wi h diame e s o 4 nm (Figu e 2A, pH 2.0 o 7.4), and DLS, wi h he disappea ance o pa icle popula ions equal o o la ge han 1000 nm (Figu e 2B, pH 2.0 o 7.4). The dis up i e e ec o doxycycline was also obse ed in he p e-assembled agg e- ga es/ ib ils a pH 4.4 a e 17 days o incuba ion. A dec ease in he size o ib illa s uc u es ollowing ea men wi h doxycycline was obse ed by TEM (maximum o 15 nm in leng h and diame e be ween 3 and 4 nm) (Figu e 2A, pH 4.4). DLS esul s showed ha he con ol sample had sizes anging be ween 1000 and 10,000 nm, whe eas in he doxycycline- ea ed sample, a dec ease in he la ge size pa icle popula ions and an inc ease in he smalle size popula ions be ween 100 and 1000 nm was no iceable (Figu e 2B, pH 4.4). Addi ionally, a u bidi y assay was also pe o med o u he cha ac e ize he dis up i e e ec o doxycycline o e ime in p e o med amyloid ma e ial (Figu e 4). A mo e p onounced dec ease in he u bidi y o doxycycline- ea ed samples was obse ed a day 11, demons a ing ha he agg ega ed species in solu ion we e smalle o /and in In . J. Mol. Sci. 2022,23, 391 8 o 17 smalle amoun . This e ec inc eased in he ollowing days, eaching a maximum a day 17 wi h an agg ega ion es ima e o app oxima ely 50% o he con ol. In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 o 17 The dis up i e e ec o doxycycline was also obse ed in he p e-assembled agg e- ga es/ ib ils a pH 4.4 a e 17 days o incuba ion. A dec ease in he size o ib illa s uc- u es ollowing ea men wi h doxycycline was obse ed by TEM (maximum o 15 nm in leng h and diame e be ween 3 and 4 nm) (Figu e 2A, pH 4.4). DLS esul s showed ha he con ol sample had sizes anging be ween 1000 and 10,000 nm, whe eas in he doxycy- cline- ea ed sample, a dec ease in he la ge size pa icle popula ions and an inc ease in he smalle size popula ions be ween 100 and 1000 nm was no iceable (Figu e 2B, pH 4.4). Addi ionally, a u bidi y assay was also pe o med o u he cha ac e ize he dis up i e e ec o doxycycline o e ime in p e o med amyloid ma e ial (Figu e 4). A mo e p o- nounced dec ease in he u bidi y o doxycycline- ea ed samples was obse ed a day 11, demons a ing ha he agg ega ed species in solu ion we e smalle o /and in smalle amoun . This e ec inc eased in he ollowing days, eaching a maximum a day 17 wi h an agg ega ion es ima e o app oxima ely 50% o he con ol. Figu e 4. E ec o doxycycline on p e o med TTRw ib ils a pH 4.4 moni o ed by u bidi y. Sam- ples o p e o med TTRw ib illa species (3.6 μM) we e incuba ed bo h in he absence (con ol) and p esence o 50× mola excess o doxycycline (180 μM) o he indica ed ime, a oom empe a u e. When he p e-assembled agg ega es and ib ils a pH 4.4 unde wen pH adjus men o 7.4 and we e ea ed wi h doxycycline, he dis up i e e ec was also obse ed. A e 17 days o incuba ion wi h doxycycline, he agg ega es we e 4 o 5 nm in diame e , as ob- se ed by TEM images. Mo eo e , al hough he ib illa s uc u es main ained hei ap- p oxima e diame e (4 nm) and leng h (maximum o 25 nm), hey had a lowe equency o occu ence when compa ed o he un ea ed samples (Figu e 2A, pH 4.4 o 7.4). The DLS esul s showed ha , a his ime, he pa icle popula ions o doxycycline- ea ed sam- ples we e comp ised o pa icles wi h sizes be ween 100 and 1000 nm, whe eas in he con- ol samples he majo i y o he pa icle popula ions we e highe han 1000 nm, which suppo s a ib il disagg ega ion mechanism (Figu e 2B, pH 4.4 o 7.4). The dis up i e e ec o doxycycline was also obse ed in he p e o med hea -in- duced TTRw ib ils a pH 7.4. The TEM images acqui ed a e 17 days o ea men showed ib illa s uc u es exhibi ing diame e s be ween 3 o 4 nm and leng hs be ween 10 o 25 nm, and a change in mo phology o mo e amo phous s uc u es in some cases (Figu e 2A, pH 7.4). A his incuba ion ime, he DLS esul s showed he doxycycline- ea ed samples we e comp ised o pa icles on a e age smalle , wi h sizes be ween 100 and well below 10,000 nm, whe eas in con ol samples wi h no doxycycline he majo pa icle popula ions we e abo e 1000 nm (Figu e 2B, pH 7.4). Thus, doxycycline also in- duces ib il dis up ion o hea -induced TTRw ib ils. Tu bidi y assays we e also pe - o med in o de o ollow TTRw amyloid ib il disagg ega ion in he p esence o doxycy- cline. Howe e , he esul s showed a sligh inc ease in he u bidi y in ensi y, when com- pa ed o con ol samples (da a no shown), which migh be explained by he la ge amoun o smalle species o med du ing he disagg ega ion induced by doxycycline, in pa icula amo phous species, as men ioned p e iously, leading o highe ligh sca e ing in ensi y Agg ega ion (%) Figu e 4. E ec o doxycycline on p e o med TTRw ib ils a pH 4.4 moni o ed by u bidi y. Samples o p e o med TTRw ib illa species (3.6 µ M) we e incuba ed bo h in he absence (con ol) and p esence o 50×mola excess o doxycycline (180 µM) o he indica ed ime, a oom empe a u e. When he p e-assembled agg ega es and ib ils a pH 4.4 unde wen pH adjus men o 7.4 and we e ea ed wi h doxycycline, he dis up i e e ec was also obse ed. A e 17 days o incuba ion wi h doxycycline, he agg ega es we e 4 o 5 nm in diame e , as obse ed by TEM images. Mo eo e , al hough he ib illa s uc u es main ained hei app oxima e diame e (4 nm) and leng h (maximum o 25 nm), hey had a lowe equency o occu ence when compa ed o he un ea ed samples (Figu e 2A, pH 4.4 o 7.4). The DLS esul s showed ha , a his ime, he pa icle popula ions o doxycycline- ea ed samples we e comp ised o pa icles wi h sizes be ween 100 and 1000 nm, whe eas in he con ol samples he majo i y o he pa icle popula ions we e highe han 1000 nm, which suppo s a ib il disagg ega ion mechanism (Figu e 2B, pH 4.4 o 7.4). The dis up i e e ec o doxycycline was also obse ed in he p e o med hea -induced TTRw ib ils a pH 7.4. The TEM images acqui ed a e 17 days o ea men showed ib illa s uc u es exhibi ing diame e s be ween 3 o 4 nm and leng hs be ween 10 o 25 nm, and a change in mo phology o mo e amo phous s uc u es in some cases (Figu e 2A, pH 7.4). A his incuba ion ime, he DLS esul s showed he doxycycline- ea ed samples we e comp ised o pa icles on a e age smalle , wi h sizes be ween 100 and well below 10,000 nm, whe eas in con ol samples wi h no doxycycline he majo pa icle popula ions we e abo e 1000 nm (Figu e 2B, pH 7.4). Thus, doxycycline also induces ib il dis up ion o hea -induced TTRw ib ils. Tu bidi y assays we e also pe o med in o de o ollow TTRw amyloid ib il disagg ega ion in he p esence o doxycycline. Howe e , he esul s showed a sligh inc ease in he u bidi y in ensi y, when compa ed o con ol samples (da a no shown), which migh be explained by he la ge amoun o smalle species o med du ing he disagg ega ion induced by doxycycline, in pa icula amo phous species, as men ioned p e iously, leading o highe ligh sca e ing in ensi y when compa ed o he sho and unb anched ib illa s uc u es obse ed in he con ol sample (Figu e 2A, pH 7.4). 3. Discussion Wi h he goal o inding an e ec i e and ela i ely as sc eening p o ocol o sea ch o TTR amyloid ib il dis up o s, we ha e e iewed, cha ac e ized and selec ed he mos app op ia e TTR agg ega ion p o ocol among hose p e iously desc ibed. A e a ca e ul analysis o he a ailable li e a u e, h ee TTRw agg ega ion p o ocols we e selec ed o compa ison, pe o med a di e en pH alues: pH 7.4, pH 4.4, and pH 2.0 (Tables 1and 2). The p o ocol in ol ing hea induc ion a pH 7.4 was selec ed since i is pe o med a he pH o physiological condi ions [ 51 ]. Acidi ica ion is he mos widely used s a egy o induce TTR agg ega ion. A pH 4.4 he TTR e ame ic s uc u e dissocia es in o monome ic species ha unde go pa ial un olding and, consequen ly, associa e o o m amyloid s uc u es [ 21 ]. In . J. Mol. Sci. 2022,23, 391 9 o 17 Acidi ica ion a pH 2.0 p omo es e ame dissocia ion ollowed by dena u a ion, and he addi ion o 0.1 M NaCl induces agg ega ion, since chlo ide ions shield he posi i e cha ges o he p o ein, allowing o con o ma ional changes and he assembly o monome s in o oligome s and amyloid ib ils [ 54 ]. Thus, agg ega ion p ocesses a e ex emely dependen no only on pH changes, bu also on p o ein concen a ion, ionic s eng h, empe a u e, and agi a ion. The agg ega ion p o ocol o TTRw a pH 2.0 wi h 0.1 M NaCl has been p e iously well cha ac e ized and i s kine ics s udied by ou esea ch g oup [ 20 ]. This is he mos ime-consuming o he h ee p o ocols, and i equi es a ela i ely high concen a ion o e ame ic TTRw o be e ec i e in an accep able ime pe iod. Agg ega ion a his pH, wi h a physiological plasma p o ein concen a ion (3.6 µ M), was also pe o med and he p ocess was ollowed by DLS o app oxima ely 30 days. Howe e , no signi ican changes in pa icle popula ions we e obse ed du ing he expe imen (da a no shown), which indica es ha DLS could no de ec ib il o ma ion in his pe iod o ime a 3.6 µ M TTR. The ib ils o med a pH 2.0 a e unb anched, long, well- o med and ma u e (Figu e 2A, pH 2.0), and bind ThT (Table 2), a good indica ion o p esence o amyloid s uc u es. Adjus men o pH o 7.4 o TTRw ib ils p e o med a pH 2.0 was an a emp o mimic he in i o condi ions o TTRw ib ils and o be able o s udy he ac ion and e icacy o po en ial dis up o s in close o physiological condi ions. TTRw amyloid deposi s a e mos ly ex acellula [ 55 ] whe e pH is gene ally close o neu al. The e ec o pH modi ica ion on he size, s uc u e, and mo phology o p e o med TTRw amyloid was s udied by DLS, CD, and TEM expe imen s, espec i ely. The DLS esul s showed ha incuba ion in phospha e bu e ed saline (PBS), a pH 7.4, o TTRw amyloid ib ils p e o med a pH 2.0 led o a dec ease in he a e age size o he pa icles, wi h popula ions o pa icles smalle han 1000 nm being mo e common (Figu e S1B). The CD esul s e ealed a loss o β -shee seconda y s uc u e (Figu e 1A and Table 3), and he TEM images co obo a ed he p e ious obse a ions showing ha ib ils lose hei mo phology, appea ing as smalle ib ils and amo phous agg ega es (Figu e 2A, pH 2.0 o pH 7.4); howe e , hey s ill emained ThT posi i e (Table 2). The ib il o ma ion p o ocol a pH 4.4 is widely used o in i o TTRw amyloid ib- illogenesis inhibi o sc eening, as i is easily implemen ed in medium o high h oughpu sc eening o ma s in ubes o mic owell pla es, usually ollowed by u bidi y measu e- men s [ 56 – 58 ]. Thus, his p o ocol seems o be a e y good candida e o use in TTRw disagg ega ion assays. This me hod is less ime-consuming han he p e ious me hod a pH 2.0, does no equi e high concen a ions o na i e TTRw , and amyloid species can be o med a physiological concen a ions (3.6 µ M) a e only 3 days (Figu e S4). The o ma ion o amyloid agg ega es and ib ils using his p o ocol can be moni o ed by bo h DLS and u bidi y. In an a emp o ollow agg ega ion, CD was also pe o med e e y day o 3 days; howe e , he spec a emained unal e ed and simila o Figu e 1B. A pH 4.4, he species o med a e composed by small ib ils and agg ega es, while samples emained ThT posi i e (Table 2). pH adjus men o 7.4 was also pe o med o hese amyloid s uc u es, wi h CD (Figu e 1B), DLS (Figu e 2B, pH 4.4 o pH 7.4), and TEM (Figu e 2A, pH 4.4 o pH 7.4) assays demons a ing ha he pH change sligh ly al e ed he s uc u e, size, and mo phology o hese agg ega es; howe e , hey emained ThT posi i e (Table 2). The hi d agg ega ion p o ocol is he leas s udied p ocess o he h ee. Hea -induced ib il o ma ion a pH 7.4 seems o be a e y good candida e o use in TTRw ib il dis up o sc eening assays because a oids he in luence o low pH on he mechanism o ac ion o he dis up o . Ou expe imen s showed ha his p o ocol is no e y ime-consuming, i occu s a physiological concen a ions, and he unb anched ib ils a e o med and main ained a physiological pH o a leas 6 days (Figu e S5). The ib il o ma ion p ocess was ollowed by DLS (Figu e 2B, pH 7.4), u bidi y (Figu e 4), and CD (Figu e 1C) assays. The inc ease in u bidi y obse ed o e he 6 days o incuba ion a 60 ◦ C (Figu e S3), he inc ease in pa icle popula ions wi h sizes be ween 1000 and 10,000 nm, and he dec ease in pa icle popula ions o smalle sizes obse ed by DLS demons a e agg ega ion (Figu e S5). This p ocess was no e e ed by empe a u e adjus men o 37 ◦ C, as e idenced by absence In . J. Mol. Sci. 2022,23, 391 16 o 17 23. 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