A robust assay to monitor ataxin-3 amyloid fibril assembly

Ci a ion: Figuei edo, F.; Lopes-Ma ques, M.; Almeida, B.; Ma scheko, N.; Ma ins, P.M.; Sil a, A.; Macedo-Ribei o, S. A Robus Assay o Moni o A axin-3 Amyloid Fib il Assembly. Cells 2022,11, 1969. h ps://doi.o g/10.3390/ cells11121969 Academic Edi o : Takahi o Seki Recei ed: 10 May 2022 Accep ed: 16 June 2022 Published: 19 June 2022 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 : © 2022 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/). cells A icle A Robus Assay o Moni o A axin-3 Amyloid Fib il Assembly F ancisco Figuei edo 1,2,3,4 , Mónica Lopes-Ma ques 1,2,5 , B uno Almeida 6,7 , Nena Ma scheko 8, Ped o M. Ma ins 1,2 , Alexand a Sil a 1,2,* and Sand a Macedo-Ribei o 1,2,* 1Ins i u o de In es igação e Ino ação em Saúde (i3S), Uni e sidade do Po o, 4200-135 Po o, Po ugal; [email p o ec ed] (F.F.); mma [email p o ec ed] (M.L.-M.); [email p o ec ed] (P.M.M.) 2Ins i u o de Biologia Molecula e Celula (IBMC), Uni e sidade do Po o, 4200-135 Po o, Po ugal 3In e na ional Ibe ian Nano echnology Labo a o y (INL), 4715-330 B aga, Po ugal 4Ins i u o de Ciências Biomédicas Abel Salaza (ICBAS), Uni e sidade do Po o, 4050-313 Po o, Po ugal 5Depa men o Biology, Facul y o Sciences, Uni e si y o Po o, 4169-007 Po o, Po ugal 6Li e and Heal h Sciences Resea ch Ins i u e (ICVS), School o Medicine, Uni e si y o Minho, 4710-057 B aga, Po ugal; [email p o ec ed] 7ICVS/3B’s—PT Go e nmen Associa e Labo a o y, 4710-057 B aga, Po ugal 8Dynamic Biosenso s GmbH, 82152 Ma ins ied, Ge many; [email p o ec ed] *Co espondence: [email p o ec ed] (A.S.); [email p o ec ed] (S.M.-R.) Abs ac : Spinoce ebella a axia ype 3 (SCA3) is caused by he expansion o a glu amine epea in he p o ein a axin-3, which is deposi ed as in acellula agg ega es in a ec ed b ain egions. Despi e he con o e sial ole o a axin-3 amyloid s uc u es in SCA3 pa hology, he iden i ica ion o molecules wi h he capaci y o p e en abe an sel -assembly and s abilize unc ional con o ma ion(s) o a axin-3 is a key o he de elopmen o he apeu ic solu ions. Amyloid-speci ic kine ic assays a e ou inely used o measu e a es o p o ein sel -assembly in i o and a e employed du ing sc eening o ib illa ion inhibi o s. The high endency o a axin-3 o assemble in o oligome ic s uc u es implies ha mino changes in expe imen al condi ions can modi y a axin-3 amyloid assembly kine ics. He e, we de e mine he sel -associa ion a es o a axin-3 and p esen a de ailed s udy o he agg ega ion o no mal and pa hogenic a axin-3, highligh ing he expe imen al condi ions ha should be conside ed when implemen ing and alida ing a axin-3 amyloid p og ess cu es in di e en se ings and in he p esence o a axin-3 in e ac o s. This assay p o ides a unique and obus pla o m o sc een o modula o s o he i s s eps o a axin-3 agg ega ion—a s a ing poin o u he s udies wi h cell and animal models o SCA3. Keywo ds: hio la in-T; polyglu amine expansion; ep oducibili y; ubiqui in; sel -associa ion a es; equilib ium dissocia ion cons an ; swi chSENSE 1. In oduc ion A axin-3 is a modula p o ein ha con ains a globula Josephin domain and a lexible C- e minal ail, which includes wo o h ee ubiqui in in e ac ion mo i s (UIMs) and a polyg- lu amine (polyQ) s e ch. Expansion o he polyQ ac abo e a h eshold o 45–55 esidues leads o a axin-3 sel -assembly and igge s spinoce ebella a axia ype 3 (SCA3), also known as Machado–Joseph disease, a highly incapaci a ing au osomal-dominan neu ode- gene a i e diso de . Deposi ion o amyloid-like ib illa agg ega es con aining he mu an polyQ-expanded p o ein in neu ons ep esen s a cha ac e is ic hallma k o SCA3 and o o he polyQ expansion diseases [ 1 , 2 ]. Al hough he p ecise neu o oxic culp i is widely deba ed, p o ein agg ega ion is a majo con ibu ing ac o o he unde lying neu odegen- e a ion, as pa hogenic a axin-3 has been shown o accumula e in in acellula deposi s a he a ec ed b ain egions, such as he ce ebellum, b ain s em, and spinal co d [ 3 , 4 ]. This pe cep ion is s eng hened by he obse a ion ha inc easing he leng h o he polyQ ac enhances p o ein agg ega ion p opensi y in i o , in co ela ion wi h an upsu ge in disease se e i y [ 5 – 7 ]. Despi e nea ly 30 yea s o in ensi e esea ch, which s a ed wi h he Cells 2022,11, 1969. h ps://doi.o g/10.3390/cells11121969 h ps://www.mdpi.com/jou nal/cells Cells 2022,11, 1969 2 o 21 iden i ica ion o he causa i e gene and con inued wi h he disco e y o a axin-3 unc ion(s) and i s agg ega ion mechanisms, SCA3 emains an incu able disease jus i ying he u gen need o disease-modi ying he apies [ 8 ]. Se e al s a egies ha e been employed o he a- peu ically a ge polyglu amine diso de s based on he p e en ion o he mis olding and agg ega ion o p o eins con aining expanded polyglu amine ac s ( e iewed by Minakawa and Nagai [ 9 ]). Gi en he mo phological he e ogenei y o he agg ega es and he an- sien na u e o he agg ega ion in e media es, iden i ying p o ein agg ega ion inhibi o s wi h neu op o ec i e p ope ies is a challenge and equi es a ho ough knowledge o he sel -assembly pa hways o he a ge p o eins. P e ious s udies ha e shown ha a axin-3 assembly in o amyloid ib ils ollows a mul i-s ep pa hway in ol ing mul iple domains and a leas wo well-de ined agg ega ion s eps. The i s s ep is media ed by an agg ega ion-p one egion loca ed in he Josephin domain [ 10 – 13 ]. This agg ega ion-p one egion ( 73 GFFSIQVISNALKVWGLELILFNS 96 ) leads o he o ma ion o sodium dodecyl sul a e (SDS)-soluble p o o ib ils bo h in non- expanded and expanded a axin-3. The second s ep, leading o he o ma ion o la ge, s able, and SDS- esis an amyloid-like ib ils, is polyQ-dependen and exclusi e o ex- panded a axin-3 [ 11 , 14 , 15 ]. The complexi y o he a axin-3 agg ega ion pa hway ende s he cha ac e iza ion o he sel -assembly mechanisms and i s modula ion by poin mu a- ions, in e ac ing pa ne s, o agg ega ion inhibi o s pa icula ly challenging. We ha e no iced ha small changes in bu e condi ions signi ican ly modi y a axin-3 agg ega ion kine ics in i o , al e ing bo h he oligome iza ion p ocesses and he balance be ween o - de ed ib illa s uc u es and amo phous agg ega ion. In his con ex , s udying a axin-3 agg ega ion mechanisms in i o unde con olled expe imen al condi ions can p o ide ins umen al in o ma ion o e alua e how di e en ac o s and molecules modula e i s assembly in o o de ed ib illa s uc u es—a s a ing poin o u he s udies wi h cell and animal models o SCA3. Robus and ep oducible agg ega ion assays a e necessa y o explo e a axin-3 sel - assembly and sc een o agg ega ion inhibi o s ha could lead o he de elopmen o disease-modi ying he apies o SCA3 [ 16 ]. He e, we p esen de ailed assays de eloped o moni o he agg ega ion o wild- ype (A x3 13Q) and polyQ-expanded a axin-3 (A x3 77Q) using a minia u ized hio la in-T (ThT) assay in combina ion wi h ansmission elec on mic oscopy (TEM) isualiza ion o cha ac e ize agg ega e mo phology [ 17 ]. Using his op imized assay, we e alua ed he impac o a b oad ange o condi ions on a axin-3 agg ega ion in i o , including ionic s eng h, pH, he p esence o de e gen s, and molecula c owde s. Fu he mo e, we show how a axin-3-in e ac ing pep ides and p o eins, such as polyQ binding pep ide 1 (QBP1) and linea ubiqui in chains (p e iously epo ed o modula e a axin-3 agg ega ion [ 18 – 23 ]), modula e a axin-3 amyloid assembly kine ics and ib il mo phology. 2. Ma e ials and Me hods 2.1. Exp ession Plasmids The plasmid cons uc s used in his wo k a e lis ed in Table 1. Sequence de ails a e p o ided in he Supplemen a y Ma e ials. The cDNAs coding o human no mal (13Q) and polyQ-expanded (77Q) a axin-3 (A x3) (iso o m 2, UniP o accession: P54252-2) wi h h ee ubiqui in in e ac ion mo i s (UIMs) we e cloned in o he pDEST17 Ga eway ec o (Li e Technologies, Ca lsbad, CA, USA) as p e iously desc ibed [ 10 ]. The plasmids we e u he modi ied o in oduce a obacco e ch i us (TEV) p o ease clea age si e downs eam o he hexahis idine ag [ 11 ]. To p epa e he cons uc s wi h codons op imized o imp o ed exp ession o epea ed polyQ sequences in E. coli, wo syn he ic cons uc s (Supplemen a y Table S1) we e designed and pu chased om GenSc ip (Pisca away, NJ, USA) and inse ed be ween he es ic ion si es Ppu10I and Bs GI o he o iginal ATXN3 gene sequence cloned in pDEST17. Si e-di ec ed mu agenesis (Supplemen a y Table S1) pe o med wi h he QuikChange II Si e-Di ec ed Mu agenesis Ki (Agilen Technologies, San a Cla a, CA, USA) was used o p oduce A x3 13Q I77K Q78K W87K and he A x3 77Q R388G a ian Cells 2022,11, 1969 3 o 21 (Single Nucleo ide Polymo phism Da abase locus accession code: s12895357, UniP o VAR_013689). The plasmid o he exp ession o un agged Homo sapiens polyubiqui in wi h 3 ubiqui in epea s in andem (pET23a-HsUBB) was a gi om P o . Jo ge Aze edo [ 23 ] (Addgene plasmid #69562). Table 1. Summa y o he cons uc s used in he p esen wo k and adop ed nomencla u e. Fo sequence de ails, see Supplemen a y Table S1. Cons uc Name P o ein Exp essed O iginal Re e ence Addgene ID A x3 JD A axin-3 Josephin domain [10,11] 184247 A x3 D1 A axin-3 JD + UIM1-2 [22] 184246 A x3 13Q A axin-3 (13Q) wi h a glycine esidue a e he polyQ ac This wo k 184248 A x3 13Q I77K Q78Q W87K A axin-3 (13Q) wi h a iple mu a ion on ubiqui in-binding si e 2 This wo k 185908 A x3 77Q PolyQ-expanded a axin-3 (77Q) wi h an a ginine esidue a e he polyQ ac (UniP o na u al a ian VAR_013689) [15] 184249 A x3 77Q R388G PolyQ-expanded a axin-3 (77Q) wi h a glycine esidue a e he polyQ ac This wo k 184251 HsUBB Homo sapiens linea i-ubiqui in chain [23] 69562 2.2. A axin-3 Exp ession and Pu i ica ion A axin-3 a ian s and unca ed cons uc s we e exp essed and pu i ied as p e iously desc ibed [ 10 ] wi h mino modi ica ions. B ie ly, E. coli BL21(DE3)-SI cells (Li e Technolo- gies) we e ans o med wi h pDEST17 plasmids ca ying he a ious a axin-3 a ian s and unca ed cons uc s and pla ed on LB Aga wi hou NaCl (LBON) pla es supplemen ed wi h 100 µ g mL −1 ampicillin. A p e-inoculum was p epa ed wi h 4-5 isola ed colonies in 150 mL LBON medium supplemen ed wi h 100 µ g mL −1 ampicillin, and cells we e g own o e nigh a 37 ◦ C, 180 pm. The ollowing day, 10 mL o p e-inoculum was added o 500 mL o LBON medium supplemen ed wi h 100 µ g mL −1 ampicillin and 0.4% (w/ ) glucose and g own a 37 ◦ C, 180 pm, un il he OD 600nm eached 0.8. The cul u e was hen cooled o 30 ◦C and p o ein exp ession was induced wi h 300 mM NaCl. A e 3 h o exp ession, he cells we e ha es ed by cen i uga ion and esuspended in bu e A (20 mM sodium phospha e pH 7.5, 500 mM NaCl, 2.5% ( / ) glyce ol, 20 mM imidazole) con aining 100 µg L−1lysozyme. Fo a axin-3 pu i ica ion, he cells we e dis up ed by gen le s i ing o 1 h on ice in he p esence o 0.02 mg mL −1 DNase, 0.02 mg mL −1 RNase, 1 mM MgCl 2 , and 1 mM phenyl- me hylsul onyl luo ide (PMSF— om a 1 M s ock solu ion in e hanol). The supe na an ob ained a e cen i uga ion was loaded on o a Ni 2+ -cha ged IMAC HiT ap column (GE Heal hca e Li e Sciences, Pisca away, NJ, USA) p e-equilib a ed in bu e A and elu ed wi h a se ies o imidazole s eps (50 mM, 250 mM, and 500 mM). EDTA was added o a inal concen a ion o 1 mM o elu ed ac ions con aining a axin-3 o educe p o eoly ic clea age. The 250 mM imidazole ac ion was applied o a HiP ep 26/60 Sephac yl S-300 HR column (GE Heal hca e Li e Sciences) p e-equilib a ed in p o ein s o age bu e (20 mM sodium phospha e pH 7.5, 150 mM NaCl, 5% ( / ) glyce ol, 2 mM EDTA, 1 mM DTT). A e SDS–PAGE analysis, ac ions om his column co esponding o pu e a axin-3 iso o ms we e pooled and concen a ed in an Amicon Ul a-15 cen i ugal il e uni (10 kDa, Mil- lipo e, Bu ling on, MA, USA) o 10–20 mg mL −1 , ozen in liquid ni ogen, and s o ed a − 80 ◦ C. P o ein concen a ion was de e mined by measu ing he abso bance a 280 nm using he p o ein’s ex inc ion coe icien s, as de ailed in Supplemen a y Table S2. Immedia ely be o e each agg ega ion assay, pu i ied a axin-3 aliquo s we e applied o a Supe ose 12 ® 10/300 GL column (GE Heal hca e Li e Sciences) p e-equilib a ed Cells 2022,11, 1969 4 o 21 in agg ega ion bu e (20 mM sodium phospha e pH 7.5, 150 mM NaCl, 1 mM DTT o 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT) and 0.4 mL ac ions we e collec ed. P o ein elu ion was moni o ed a 280 nm, and he p o ein concen a ion o he ac ion co esponding o monome ic p o ein was de e mined om he abso bance a 280 nm (Supplemen a y Table S2). 2.3. HsUBB Exp ession and Pu i ica ion Homo sapiens polyubiqui in con aining 3 ubiqui in uni s (HsUBB) was exp essed and pu i ied as p e iously desc ibed [ 23 ] wi h mino modi ica ions. B ie ly, E. coli BL21 (DE3) cells (Li e Technologies) we e ans o med wi h pET23a-HsUBB and pla ed on LB Aga medium supplemen ed wi h 100 µ g mL −1 ampicillin. A p e-inoculum was p epa ed om 4–5 isola ed colonies in LB medium supplemen ed wi h 100 µ g mL −1 ampicillin and g own o e nigh a 37 ◦ C, 180 pm. The p e-inoculum was added o LB medium supplemen ed wi h 100 µ g mL −1 ampicillin and g own a 37 ◦ C, 180 pm, un il he OD 600nm eached 0.8. HsUBB exp ession was induced wi h 0.5 mM IPTG o 3 h a 37 ◦ C. The cells we e ha es ed by cen i uga ion and esuspended in 50 mM T is–HCl pH 8.5, 1 mM EDTA, 0.5 mM DTT, 0.25 mg mL −1 PMSF. Cells we e lysed by sonica ion and he lysa e was cla i ied by cen i uga ion (34,957 g, 45 min, 4 ◦ C—So all ST 40R—Ro o JA25.50). The cell lysa e was applied o a 6 mL Resou ce Q ion-exchange column (GE Heal hca e Li e Sciences) and he low- h ough was concen a ed and injec ed on o a Supe dex 75 (GE Heal hca e Li e Sciences) column equilib a ed in 50 mM T is–HCl pH 8.5, 150 mM NaCl, 10% ( / ) glyce ol, 1 mM DTT. The peak co esponding o pu e HsUBB was concen a ed in an Amicon Ul a-15 cen i ugal il e uni (10 kDa, Millipo e) and s o ed a − 80 ◦ C. The p o ein concen a ion was de e mined by measu ing he abso bance a 280 nm using he ex inc ion coe icien o 5,960 M −1 cm −1 . Be o e he agg ega ion assay wi h a axin-3, HsUBB was e-pu i ied as desc ibed in Sec ion 2.2 using a Supe ose 12 ® 10/300 GL column (GE Heal hca e Li e Sciences) p e-equilib a ed wi h he a axin-3 agg ega ion bu e . 2.4. Thio la in-T Agg ega ion Assay A axin-3 amyloid o ma ion was moni o ed by ollowing he inc ease in ThT luo- escence a 480 nm (440 nm exci a ion) on a luo ime e . Fo his s udy, wo di e en luo ime e s we e used: he FluoDia T70 mic opla e luo ime e (Pho on Technology In- e na ional, Edison NJ, USA) using The mowell 96-Well Polyca bona e PCR Mic opla es (Cos a , Washing on, DC, USA) and he CHAMELEON V (HIDEX) pla e eade using 384-well mic opla es (low lange, black, la bo om, polys y ene; Co ning, Co ning, NY, USA). Samples (50 µ L) o 5 µ M a axin-3 in agg ega ion bu e con aining 30 µ M ThT and di e en concen a ions o he es ed compounds (as de ailed below), we e incuba ed a 37 ◦ C, and ThT luo escence was measu ed e e y 30 min o 60 h. To p e en e apo a ion, each well was co e ed wi h 20 µL pa a in oil. 2.5. T ansmission Elec on Mic oscopy Fo isualiza ion o p o ein ib ils/agg ega es by TEM, endpoin p o ein samples used in he ThT assay we e dilu ed i e- old in wa e and adso bed on o glow-discha ged, ca bon-coa ed ilms suppo ed on 300-mesh nickel g ids and nega i ely s ained wi h 1% (w/ ) u anyl ace a e using a p o ocol adap ed om Rames and collabo a o s [ 24 ]. G ids we e isualized using a JEM-1400 (JEOL) TEM a an accele a ing ol age o 80 kV. 2.6. Fil e Re a da ion Assay To de ec A x3 77Q ma u e SDS- esis an ib ils, samples (5 µ L) collec ed om each well a he endpoin o he ThT agg ega ion assay we e dilu ed in 200 µ L o TBS (50 mM T is–HCl pH 7.5, 150 mM NaCl) supplemen ed wi h 2% (w/ ) SDS and boiled o 5 min. Using a Bio-Do ® SF mic o il a ion appa a us (Bio-Rad, He cules, CA, USA), he samples we e il e ed h ough a cellulose ace a e memb ane (0.2 µ m, Wha man, Buckinghamshi e, UK) p e-equilib a ed in TBS, and he memb ane was washed wice wi h TBS supplemen ed Cells 2022,11, 1969 5 o 21 wi h 0.1% (w/ ) SDS. Nex , he memb ane was emo ed om he appa a us and blocked wi h TBS supplemen ed wi h 5% (w/ ) non- a d y milk o 1 h a oom empe a u e (RT). The A x3 77Q ma u e SDS- esis an ib ils e ained in he memb ane we e p obed wi h monoclonal mouse an i-A x3 clone 1H9 (Millipo e) an ibody 1:10,000 o e nigh a 4 ◦ C, incuba ed wi h an i-mouse an ibody (Sigma, Bu ling on, MA, USA) 1:10,000 o 1 h a RT, and de ec ed using Ame sham ECL P ime Wes e n blo ing chemiluminescen de ec ion eagen (Ci y a, Ma lbo ough, MA, USA). 2.7. Na i e Gel Assay Samples o 40 µ M A x3 13Q o A x3 77Q we e incuba ed wi h HsUBB a di e en mola a ios (1:0.5; 1:1; 1:2 and 1:3) in 20 mM HEPES pH 7.5, 150 mM NaCl, 5% (w/ ) glyce ol, 2 mM EDTA, 1 mM DTT, o 1 h on ice. P o eins we e loaded and sepa a ed in an 8% na i e polyac ylamide gel elec opho esis (PAGE) gel (240 mM T is–HCl pH 9.5, 8% (w/ ) ac ylamide, 0.1% (w/ ) APS, 0.1% (w/ ) TEMED), in a Mini-PROTEAN Te a cell (Bio-Rad) a 100 V, and s ained by incuba ion wi h BlueSa e (NZYTech). 2.8. A axin-3 13Q and A axin-3 JD Oligome iza ion Kine ics Measu ed ia Swi chSENSE Measu emen s we e se up in he swi chBUILD so wa e wi h “His- ag cap u e” as he immobiliza ion me hod and pe o med in s a ic measu emen mode on a DRX de ice on MPC-48-2-R1 biochips (bo h Dynamic Biosenso s GmbH (DBS), Munich, Ge many). The sys em was p imed wi h unning bu e (phospha e-bu e ed saline (PBS) pH 7.5, 1 mM TCEP) and he biochip was unc ionalized wi h 200 nM NTA3- unc ionalized cNL- B48 DNA (His- ag cap u e ki , o de numbe CK-TN-1-B48, DBS) in hyb idiza ion bu e (10 mM sodium phospha e pH 7.4, 40 mM NaCl, 0.05% ( / ) Tween 20, 50 µ M EDTA, 50 µ M EGTA) o 10 min. The NTA3 g oup was s ipped and ac i a ed by injec ion o EDTA and loading solu ion. Cap u e o 500 nM His- agged A x3 13Q o he A x3 JD domain, dilu ed in unning bu e and s o ed a 10 ◦ C in he au osample un il au oma ic pickup, was pe o med o 2 min a a low a e o 20 µ L min −1 a 37 ◦ C. Associa ion o A x3 13Q o A x3 JD domain dilu ed in unning bu e o indica ed concen a ions, as well as subsequen dissocia ion in unning bu e , was pe o med a 37 ◦ C, 100 and 50 µ L min −1 , espec i ely. Samples we e measu ed in consecu i e ounds wi h inc easing analy e concen a ions and NTA egene a ion in be ween. Da a we e analysed wi h he swi chANALYSIS so wa e kine ics ool. Da a we e no malized o he baseline o he associa ion, A x3 13Q da a we e e e enced wi h a 0 M un, and global mono-exponen ial i s we e calcula ed by leas -squa es esidual de e mina ion. 3. Resul s 3.1. De elopmen o a Minia u ized A axin-3 Agg ega ion Assay A axin-3 sel -assembly has a complex mechanism, wi h oligome ic and amyloid pa hways aking place simul aneously [ 17 , 25 ]. The high endency o a axin-3 o assemble in o high-molecula weigh oligome ic s uc u es [ 10 ], concomi an ly wi h he occu ence o wo pa allel a axin-3 oligome isa ion pa hways on- and o - ou e o he o ma ion o amyloid ib ils, implies ha a igh con ol o he mul iple a iables and agg ega ion bu e componen s is c i ical o he de elopmen o ep oducible assays o moni o p o ein agg ega ion [ 16 ]. We ha e p e iously es ablished a minia u ized assay o ollow he amyloid assembly o A x3 13Q [ 16 , 17 ]. In ha assay, eshly e-pu i ied A x3 13Q (5 µ M) was incuba ed a 37 ◦ C in phospha e bu e and amyloid o ma ion was moni o ed by ThT binding in a inal olume o 50 µ L. Unde hose p e iously es ablished condi ions, he lag phase o agg ega ion was longe han 20 h and o en a ied be ween 30 and 40 h [ 17 ]. The long lag phase o a axin-3 amyloid assembly in phospha e bu e , combined wi h he molecula s ochas ici y o he p ima y nuclea ion, accoun s o he la ge a ia ions in he lag phase du a ion ha we obse ed du ing he agg ega ion assays unde hese condi ions. In con as , he lag phase o A x3 13Q agg ega ion was dec eased by ~20 h when sodium phospha e (as in [ 16 , 17 ]) was eplaced wi h HEPES in he agg ega ion Cells 2022,11, 1969 6 o 21 solu ion (Figu e 1A). The agg ega ion assay using HEPES bu e was e y obus , as shown by he high ep oducibili y obse ed in 192 eplica es se up in a 386-well pla e using 3 µ M A x3 13Q (Supplemen a y Figu e S1), sugges ing ha his assay can be used o medium- and high- h oughpu sc eening o a axin-3 agg ega ion inhibi o s. Cells 2022, 11, x 7 o 22 Figu e 1. A axin-3 amyloid assembly assay. (A) ThT assay pe o med o moni o he o ma ion o A x3 13Q amyloid species in wo dis inc bu e s, HEPES s. phospha e bu e . Cu es ep esen he means and s anda d de ia ions o i e eplica es o each condi ion (FluoDia T70 pla e eade ). (B) Schema ic ep esen a ion o he a axin-3 cons uc s used in his wo k. (C) SDS–PAGE o eshly hawed a axin-3 cons uc s p io o e-pu i ica ion; MW Ldd– molecula weigh ma ke s. (D) Size exclusion ch oma og aphy o e-pu i ied a axin-3 iso o ms, whe e no agg ega es a e de ec able. (E) ThT assay pe o med o measu e he o ma ion o amyloid species o di e en a axin-3 cons uc s in HEPES bu e . Cu es ep esen he means and s anda d de ia ions o i e eplica es o each condi ion (CHAMELEON V pla e eade ). (F) SDS–PAGE o a axin-3 cons uc s a he end o he ThT agg ega ion assay. (G) Sel -assembly kine ics o A x3 JD and A x3 13Q de e mined using swi chSENSE echnology and de i ed a e and equilib ium cons an s. (H) TEM images a e nega- i e s aining o ThT assay endpoin samples (60 h, 37 °C) o all a axin-3 cons uc s. Scale ba s co e- spond o 200 nm. Figu e 1. A axin-3 amyloid assembly assay. ( A ) ThT assay pe o med o moni o he o ma ion o A x3 13Q amyloid species in wo dis inc bu e s, HEPES s. phospha e bu e . Cu es ep e- sen he means and s anda d de ia ions o i e eplica es o each condi ion (FluoDia T70 pla e eade ). ( B ) Schema ic ep esen a ion o he a axin-3 cons uc s used in his wo k. ( C ) SDS–PAGE o eshly hawed a axin-3 cons uc s p io o e-pu i ica ion; MW Ldd– molecula weigh ma ke s. ( D ) Size exclusion ch oma og aphy o e-pu i ied a axin-3 iso o ms, whe e no agg ega es a e de ec able. Cells 2022,11, 1969 7 o 21 ( E ) ThT assay pe o med o measu e he o ma ion o amyloid species o di e en a axin-3 cons uc s in HEPES bu e . Cu es ep esen he means and s anda d de ia ions o i e eplica es o each condi ion (CHAMELEON V pla e eade ). ( F ) SDS–PAGE o a axin-3 cons uc s a he end o he ThT agg ega ion assay. ( G ) Sel -assembly kine ics o A x3 JD and A x3 13Q de e mined using swi chSENSE echnology and de i ed a e and equilib ium cons an s. ( H ) TEM images a e nega i e s aining o ThT assay endpoin samples (60 h, 37 ◦ C) o all a axin-3 cons uc s. Scale ba s co espond o 200 nm. Nex , we e alua ed he beha iou o a axin-3 a ian s and unca ed cons uc s ( Figu e 1B ) in agg ega ion solu ions bu e ed wi h HEPES. The pu i ied p o eins (Supplemen a y Figu e S2), s o ed a − 80 ◦ C, we e hawed and e-pu i ied by size ex- clusion ch oma og aphy in he selec ed bu e be o e he agg ega ion assay (Figu e 1D). This s ep is c i ical o emo e he glyce ol used in he a axin-3 s o age bu e , which has a s ong e ec on he agg ega ion kine ics (see below), and o elimina e pu a i e ag- g ega es o med du ing eezing/ hawing, which could modi y he nuclea ion p ocess and comp omise assay ep oducibili y. A comple e p o ocol including all he s eps o a axin-3 p oduc ion and agg ega ion analysis is p esen ed in he Supplemen a y Ma e ials. One ele an ac o o ensu e high-quali y p o ein is he u iliza ion o codon-op imized nucleo ide sequences o exp ess a axin-3 wi h a ious polyQ ac sizes, pa icula ly impo - an o p oducing high-pu i y A x3 77Q. Ou esul s showed ha a axin-3 sel -assembles in o amyloid-like s uc u es wi h ib illa mo phology independen ly o he polyQ ac (Figu e 1E,H), simila o wha had been p e iously obse ed in phospha e-bu e ed ag- g ega ion solu ions [ 17 ]. In addi ion, SDS–PAGE analysis o a axin-3 a he endpoin o agg ega ion (~60 h, 37 ◦ C) showed ha no ele an p o ein deg ada ion occu ed unde he es ablished assay condi ions (Figu e 1F). P e ious da a showed ha bo h A x3 77Q and A x3 13Q agg ega e, wi h he globula JD playing a cen al ole in he i s s eps o a axin-3 sel -assembly [ 10 , 13 , 26 , 27 ]. To quan i y A x3 JD and A x3 13Q sel -associa ion kine ics, we used elec ically swi chable nanole e (swi chSENSE) echnology [ 28 , 29 ]. Swi chSENSE is a biophysical cha ac e iza ion me hod ideally sui ed o kine ic binding analysis o challenging molecules, such as agg ega ion- p one p o eins. The low su ace densi y o he immobilized p o ein o in e es allows a con olled induc ion o he oligome iza ion p ocesses upon analy e injec ion in he liquid phase. The nanole e s we e unc ionalized wi h 500 nM A x3 13Q o JD and binding o unlabeled JD o A x3 13Q o he su ace- e he ed p o eins allowed he measu emen o JD and A x3 13Q sel -associa ion kine ics. The associa ion and dissocia ion cu es we e measu ed o analy e concen a ions be ween 3.75 and 15 µ M. The equilib ium dissocia ion cons an s (K D ) o A x3 13Q and JD sel -associa ion (Figu e 1G) we e in he µ M ange, in good ag eemen wi h he K D o 4.6 µ M es ima ed om ou biophysical model o A x3 13Q agg ega ion [ 17 ]. Al hough he associa ion a e cons an s (k on ) we e simila o bo h cons uc s, he isola ed JD dissocia ed as e , showing ha he C- e minal ail o a axin-3 s abilizes i s sel -associa ion, in good ag eemen wi h he di e ences obse ed in he ThT agg ega ion kine ics (Figu e 1E). In e es ingly, he unca ed a ian lacking he polyQ segmen and he C- e minal UIM3 (A x3 D1) had he same amyloid assembly kine ics as he ull-leng h a axin-3, high- ligh ing he ele ance o he egion b idging he JD and he polyQ o sel -assembly in o amyloid-like ib ils [ 18 , 30 ]. In con as o p e iously published da a [ 15 , 31 ], he agg ega ion kine ics o he non-expanded A x3 13Q and disease- ela ed A x3 77Q ully o e lapped. This esul was ob ained consis en ly when he p o eins we e eshly e-pu i ied, imme- dia ely be o e he agg ega ion assays. This s ep emo es minu e amoun s o agg ega ed species, mos equen ly con amina ing eshly hawed A x3 77Q monome ic ac ions (Supplemen a y Figu e S2C,D), which migh ac as nuclea ion seeds and accele a e agg e- ga ion o pa hogenic a axin-3. Analysis o he ib il mo phologies by nega i e s aining ansmission elec on mic oscopy (TEM; Figu e 1H) showed ha only A x3 77Q assembled in o ma u e ib illa agglome a es [ 27 ], a p ocess ha appa en ly does no in e e e wi h Cells 2022,11, 1969 8 o 21 he maximum ThT signal (Figu e 1E). This indica es ha moni o ing a axin-3 agg ega- ion by ThT binding kine ics only e lec s he i s agg ega ion s ep ha is elian on JD sel -assembly. This assay may be used o e alua e he e ec s o mu a ions o compa e he modula o y oles o a ious bu e componen s and a axin-3-in e ac ing molecules on a axin-3 ea ly agg ega ion p ocesses, as shown below. 3.2. Analysis o he Agg ega ion Kine ics o Na u al A axin-3 Va ian s An in agenic polymo phism in he ATXN3 gene ( s12895357, UniP o a ian VAR_013689) was obse ed in he popula ion, wi h one a ian con aining a GGG codon, ansla ed o glycine downs eam o he CAG epea s, and ano he a ian con aining a CGG codon, ansla ed o a ginine. This polymo phic posi ion was used o s udy SCA3 hap- lo ype and o igins [ 32 – 34 ]. Taking in o conside a ion he da a a ailable in he 1000 Genomes P ojec , Phase 3 [ 35 ], which ga he ed genomic in o ma ion om 4973 heal hy indi iduals, he a ian con aining he glycine esidue a e he polyQ epea was p e alen in all popula- ions (Supplemen a y Figu e S3). The a ian con aining a ginine a he equi alen posi ion was also ound in all popula ions, being mo e p e alen in he Eas Asian popula ion and was o iginally linked wi h epea expansion in Japanese SCA3 pa ien s [36]. Ou A x3 13Q cons uc con ains a glycine esidue a e he polyQ sequence, while he A x3 77Q cons uc has an a ginine in he equi alen posi ion (Supplemen a y Table S2). We compa ed he agg ega ion kine ics o A x3 77Q and A x3 77Q R388G (Figu e 2) o e alua e he po en ial impac o he na u e o he esidue a his posi ion upon expanded a axin-3 agg ega ion. The esul s showed ha he p esence o a glycine o an a ginine immedia ely a e he polyQ ac did no al e he ThT luo escence cu es (Figu e 2A), as hey moni o ed he i s s ep o a axin-3 agg ega ion. Addi ionally, no di e ences we e obse ed in he A x3 77Q o A x3 77Q R388G ib il mo phologies (Figu e 2B), bo h o which ma u ed in o SDS- esis an ib illa clus e s (Figu e 2C). The e o e, we decided o use ou o iginal A x3 77Q cons uc o explo e he in luence o a ious bu e componen s on a axin-3 agg ega ion kine ics. Cells 2022, 11, x 9 o 22 Figu e 2. E ec o a axin-3 a ian s12895357 on agg ega ion kine ics. (A) ThT assay pe o med o measu e he o ma ion o amyloid-like species in A x3 77Q and A x3 77Q R388G. Cu es ep esen he means and s anda d de ia ions o i e eplica es o each condi ion (FluoDia T70 pla e eade ). (B) TEM images a e nega i e s aining o ThT assay endpoin samples (60 h, 37 °C) o bo h a axin- 3 a ian s. Scale ba s co espond o 200 nm. (C) Fil e e a da ion assay moni o ed by immunos ain- ing wi h mouse an i-a axin-3 1H9 o agg ega ion endpoin s (60 h, 37 °C) om A x3 77Q and A x3 77Q R388G; he labels A, B, and C a e eplica es. 3.3. The In luence o he Bu e Componen s on A axin-3 Agg ega ion Se e al s udies ha e shown he impac o ionic s eng h on he agg ega ion p open- si y o a ious amyloid- o ming p o eins [37–43]. To e alua e he in luence o ionic s eng h on a axin-3 sel -assembly, we moni o ed he e ec o a ying he NaCl concen- a ion be ween 50 and 300 mM on he agg ega ion kine ics. In he selec ed bu e condi- ions, a ying he NaCl concen a ion had no majo e ec on he o ma ion o ThT-posi- i e amyloid-like species o bo h expanded and non-expanded a axin-3 (Figu e 3A,B). Howe e , la ge a iabili y in he maximum ThT luo escence in he di e en eplica es was no ed o he non-expanded A x3 13Q in he p esence o 300 mM NaCl (Figu e 3A). This a iabili y possibly esul ed om he p esence o ai bubbles in his pa icula expe - imen , as eplica es o his assay did no show a educ ion o ThT signal (Supplemen a y Figu e S4). No ele an di e ences we e ound in he mo phology o he ib ils a he end- poin o he ThT agg ega ion assay (~60 h) as obse ed by TEM analysis (Figu e 3C). Sim- ila ly, no di e ences in a axin-3 agg ega ion kine ics we e obse ed when he pH o he HEPES bu e was a ied be ween 7.0 and 8.5 (Supplemen a y Figu e S5). Figu e 2. E ec o a axin-3 a ian s12895357 on agg ega ion kine ics. ( A ) ThT assay pe o med o measu e he o ma ion o amyloid-like species in A x3 77Q and A x3 77Q R388G. Cu es ep esen he means and s anda d de ia ions o i e eplica es o each condi ion (FluoDia T70 pla e eade ). ( B ) TEM images a e nega i e s aining o ThT assay endpoin samples (60 h, 37 ◦ C) o bo h a axin-3 a ian s. Scale ba s co espond o 200 nm. ( C ) Fil e e a da ion assay moni o ed by immunos aining wi h mouse an i-a axin-3 1H9 o agg ega ion endpoin s (60 h, 37 ◦ C) om A x3 77Q and A x3 77Q R388G; he labels A, B, and C a e eplica es. Cells 2022,11, 1969 9 o 21 3.3. The In luence o he Bu e Componen s on A axin-3 Agg ega ion Se e al s udies ha e shown he impac o ionic s eng h on he agg ega ion p opensi y o a ious amyloid- o ming p o eins [ 37 – 43 ]. To e alua e he in luence o ionic s eng h on a axin-3 sel -assembly, we moni o ed he e ec o a ying he NaCl concen a ion be ween 50 and 300 mM on he agg ega ion kine ics. In he selec ed bu e condi ions, a ying he NaCl concen a ion had no majo e ec on he o ma ion o ThT-posi i e amyloid- like species o bo h expanded and non-expanded a axin-3 (Figu e 3A,B). Howe e , la ge a iabili y in he maximum ThT luo escence in he di e en eplica es was no ed o he non-expanded A x3 13Q in he p esence o 300 mM NaCl (Figu e 3A). This a iabili y possibly esul ed om he p esence o ai bubbles in his pa icula expe imen , as eplica es o his assay did no show a educ ion o ThT signal (Supplemen a y Figu e S4). No ele an di e ences we e ound in he mo phology o he ib ils a he endpoin o he ThT agg ega ion assay (~60 h) as obse ed by TEM analysis (Figu e 3C). Simila ly, no di e ences in a axin-3 agg ega ion kine ics we e obse ed when he pH o he HEPES bu e was a ied be ween 7.0 and 8.5 (Supplemen a y Figu e S5). Cells 2022, 11, x 10 o 22 Figu e 3. E ec o NaCl on a axin-3 agg ega ion kine ics. ThT assay pe o med o measu e he o - ma ion o amyloid-like species in (A) A x3 13Q and (B) A x3 77Q in he p esence o 50, 75, 100, 150 (con ol), 200, and 300 mM NaCl. Cu es ep esen he means and s anda d de ia ions o i e ep- lica es o each condi ion (CHAMELEON V pla e eade ). (C) TEM images a e nega i e s aining o ThT assay endpoin samples (60 h, 37 °C) o A x3 13Q and A x3 77Q in he p esence o 150 (con- ol), 50, and 300 mM NaCl. Scale ba s co espond o 200 nm. We nex es ed he e ec o glyce ol on a axin-3 agg ega ion. Glyce ol is equen ly used o p e en p o ein agg ega ion [44,45] and was added o he a axin-3 pu i ica ion and s o age bu e s [10,16,17]. We e alua ed he e ec o a ying he glyce ol concen a- ion on a axin-3 agg ega ion (Figu e 4). In he p esence o 0.5% o 1% ( / ) glyce ol, he agg ega ion lag phase was elonga ed, bo h o polyQ-expanded and non-expanded a axin-3 (Figu e 4A,B). The p esence o 5% ( / ) glyce ol inc eased he agg ega ion lag phase and diminished he maximum ThT luo escence o bo h a axin-3 a ian s (Figu e 4A,B). Acco dingly, he mo phological analysis o he endpoin samples by TEM showed a clea dec ease in he size and numbe o A x3 13Q p o o ib ils and A x3 77Q ma u e ib ils in he p esence o 5% ( / ) glyce ol (Figu e 4C). Figu e 3. E ec o NaCl on a axin-3 agg ega ion kine ics. ThT assay pe o med o measu e he o ma ion o amyloid-like species in ( A ) A x3 13Q and ( B ) A x3 77Q in he p esence o 50, 75, 100, 150 (con ol), 200, and 300 mM NaCl. Cu es ep esen he means and s anda d de ia ions o i e eplica es o each condi ion (CHAMELEON V pla e eade ). ( C ) TEM images a e nega i e s aining o ThT assay endpoin samples (60 h, 37 ◦ C) o A x3 13Q and A x3 77Q in he p esence o 150 (con ol), 50, and 300 mM NaCl. Scale ba s co espond o 200 nm. We nex es ed he e ec o glyce ol on a axin-3 agg ega ion. Glyce ol is equen ly used o p e en p o ein agg ega ion [ 44 , 45 ] and was added o he a axin-3 pu i ica ion and s o age bu e s [ 10 , 16 , 17 ]. We e alua ed he e ec o a ying he glyce ol concen a ion on a axin-3 agg ega ion (Figu e 4). In he p esence o 0.5% o 1% ( / ) glyce ol, he agg ega ion lag phase was elonga ed, bo h o polyQ-expanded and non-expanded a axin- Cells 2022,11, 1969 16 o 21 ac o s can a ec p o ein agg ega ion in i o and assessed he agg ega ion beha iou o a axin-3 a ian s commonly ound in he human popula ion. 4.1. The Rele ance o he Bu e Sys em o A axin-3 Agg ega ion Phospha e is conside ed a biologically ele an bu e , equen ly used o s udying amyloid o ma ion in i o . Ou esul s show ha he bu e sys em used in he sel - assembly assays has an impo an impac on ThT-moni o ed a axin-3 amyloid o ma ion, as p e iously seen o A β (1–40) [ 56 ]. In pa icula , eplacing sodium phospha e wi h HEPES induced an 80% educ ion in he du a ion o he lag phase o he a axin-3 amyloid p og ess cu es and inc eased assay ep oducibili y, wi hou a ec ing he mo phology o he endpoin ib ils isualized by TEM. I is unclea why he wo bu e ions signi ican ly al e he lag phase, bu his could be he esul o weak in e ac ions wi h unique a axin-3 sequence mo i s igge ing o s abilizing he a e-limi ing assembly o he i s agg ega ion nuclei. This is an in e es ing ques ion wo h explo ing in u u e esea ch. 4.2. The Role o Addi i es on A axin-3 Agg ega ion In ou assay condi ions, a ying he NaCl concen a ion be ween 50 and 200 mM, o he pH be ween 7.0 and 8.5, did no s ongly a ec a axin-3 amyloid assembly no he mo phology o he endpoin ib ils. In con as , glyce ol, an addi i e egula ly used in a axin-3 pu i ica ion, had a majo ole in dec easing a axin-3 ib il assembly, e en a concen a ions as low as 0.5% ( / ). The e o e, he p esence o glyce ol is an impo an pa ame e o de ine when designing he agg ega ion p o ocol. In con as , EDTA (2-5 mM), PMSF (1 mM), sodium azide (0.05% (w/ )), e hanol (1% ( / ), and DMSO (0.05% ( / )) did no a ec he a axin-3 amyloid p og ess cu es. 4.3. The E ec o De e gen s on A axin-3 Agg ega ion Moni o ed by ThT The beha iou o he ThT cu es o a axin-3 amyloid assembly in he p esence o anionic and non-ionic de e gen s is wo h discussing in de ail. Al hough ThT is an amyloid- speci ic dye commonly used o moni o amyloid o ma ion kine ics in eal ime, pa icula ca e should be aken in he in e p e a ion o he expe imen al esul s [ 16 , 57 ]. When SDS, T i on X-100, and Tween 20 we e included in he agg ega ion assay, a dec ease in he luo escence signal o ThT was obse ed wi h inc easing concen a ions o he de e gen s. Howe e , a sha p dec ease in ThT luo escence, obse ed in he con ol expe imen s wi hou a axin-3, sugges ed ha hese esul s should be in e p e ed wi h cau ion and con i med by complemen a y echniques. In ac , TEM analysis showed ha he anionic de e gen SDS was able o abolish a axin-3 assembly in o amyloid ib ils, bu he non-ionic de e gen s did no a ec a axin-3 amyloid o ma ion. 4.4. Molecula C owde s and A axin-3 Agg ega ion The in acellula en i onmen is e y complex. To mimic i s complexi y and s udy how c owding could impac a axin-3 agg ega ion, we used di e en c owding agen s. I is known ha he e ec o c owde s on amyloid p o eins a ies wi h he concen a ion and ype o c owde used [ 48 , 58 – 60 ]. Recen ly, i has been shown ha hun ing in agg ega ion was a ec ed by he p esence o dex an, Ficoll, and PEG 20,000 inc easing he he e ogenei y o he hun ing in non- ib illa agg ega e species o med [ 58 ]. All he c owding agen s es ed (dex an, PEG 5000, and PEG 10,000) dec eased he ThT signal in a concen a ion-dependen manne and abolished a axin-3 ib il o ma ion, as con i med by TEM analysis. Mac o- molecula c owde s a e gene ally associa ed wi h a solubili y-dec easing e ec caused by he exclusion o p o ein molecules om he physical olume occupied by he c owding agen s [ 61 ]. Ne e heless, when mac omolecula c owde s es ablish a ac i e in e ac ions wi h he p o ein, solubili y-enhancing e ec s may p e ail o e olume exclusion [ 62 ]. Since he s udied c owding agen s he modynamically and kine ically inhibi ed a axin-3 agg ega ion, we conclude ha he en halpic e ec s p e ailed o e he en opic ones. Cells 2022,11, 1969 17 o 21 4.5. The E ec o A axin-3-In e ac ing P o eins in Amyloid Assembly A axin-3 is a deubiqui inase ha p e e en ially clea es K63-linked chains o ou o mo e ubiqui in moie ies [ 53 , 63 ]. P e ious s udies ha e shown ha he su ace a eas in- ol ed in ubiqui in binding include he agg ega ion-p one egions in JD and ha incuba ion wi h monome ic ubiqui in delayed i s agg ega ion [ 49 , 54 ]. Since i had been sugges ed ha na i e p o ein in e ac ions may de e he sel -assembly o agg ega ion-p one p o eins [ 64 ], we es ed he e ec o HsUBB, a linea i-ubiqui in chain whose s uc u e is a close mimic o he ex ended con o ma ion adop ed by K63-linked ubiqui in chains [ 65 ], on a axin-3 agg ega ion. Ou esul s showed ha in he es ed expe imen al condi ions, HsUBB does no in e e e wi h a axin-3 amyloid ib il assembly. Howe e , o A x3 77Q, a dec ease in he maximum ThT luo escence was obse ed a he highes mola a io (1:5), sugges ing ha his may be an a enue o esea ch wo h pu suing. QBP1 is an 11- esidue pep ide ha can inhibi polyQ agg ega ion bo h in i o and in i o [ 18 – 21 ]. QBP1 is e y e icien a inhibi ing he o ma ion o ma u e SDS- esis an ib ils wi hou in e e ing wi h he i s s ep o a axin-3 agg ega ion. Ou s udies co obo- a ed his and allowed us o e i y ha ThT binding cu es a e a good ool o e alua ing modula o s o he JD-media ed s ep o a axin-3 agg ega ion, al hough TEM and il e e a - da ion analysis should be complemen a ily used o moni o he expanded polyQ-dependen agg ega ion s ep. 5. Conclusions In conclusion, we ha e de eloped a s anda d op imized assay o s udy he i s s ep o a axin-3 agg ega ion. We showed ha small amoun s o addi i es, in pa icula glyce ol, can a ec he agg ega ion p ocess and ha di e en con ol expe imen s a e equi ed o iden i y wha ype o e ec is e ealed by ThT luo escence analysis. Fu he , he ele ance o complemen a y s udies o moni o ib il mo phology by nega i e s aining elec on mic oscopy o ob ain an in eg a ed iew o he e ec o di e en molecules on amyloid ib il assembly and ma u a ion has been highligh ed. Se e al independen s udies ha e shown ha indi iduals wi h he same numbe o glu amine epea s ha e di e en ages o disease onse and a ying symp oms [ 66 – 71 ]. These s udies emphasize ha polyQ expansion is no he only ac o unde lying he de elopmen o SCA3 and ha o he physiological and ex e nal ac o s play a ole in he mani es a ion o he disease. This op imized agg ega ion assay can be used o s udy he e ec o a ious modula o s o disease p og ession on a axin-3 sel -assembly and o disco e new a axin-3 agg ega ion inhibi o s o u u e he apy de elopmen . Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a : h ps:// www.mdpi.com/a icle/10.3390/cells11121969/s1, Figu e S1: Repea abili y o a axin-3 agg ega ion assay in HEPES bu e ; Figu e S2: Pu i ica ion and he mal shi assay o di e en A axin-3 cons uc s; Figu e S3: Dis ibu ion o a ian s12895357 in a heal hy popula ion; Figu e S4: E ec o 300 mM NaCl on a axin-3 agg ega ion kine ics; Figu e S5: E ec o pH on a axin-3 agg ega ion kine ics; Figu e S6: Di e en agg ega ion bu e componen s do no a ec a axin-3 agg ega ion kine ics; Figu e S7: E ec o mu a ing he second ubiqui in binding si e o he JD on a axin-3 agg ega ion kine ics; Table S1: Nucleo ide sequences o p ime s and syn he ic genes used in his wo k; Table S2: Amino acid sequences o p o eins and pep ides used in he agg ega ion assays; Table S3: Lis o eagen s used in his wo k, wi h b and name and ca alogue e e ences. Au ho Con ibu ions: Concep ualiza ion, A.S. and S.M.-R.; me hodology, F.F., A.S., B.A. and N.M.; so wa e, F.F., N.M. and A.S.; alida ion, P.M.M., A.S. and S.M.-R.; o mal analysis, F.F., N.M., P.M.M., A.S. and S.M.-R.; in es iga ion, F.F. and A.S.; esou ces, N.M. and S.M.-R.; da a cu a ion, F.F. and A.S.; w i ing—o iginal d a p epa a ion, F.F.; w i ing— e iew and edi ing, F.F., M.L.-M., B.A., N.M., P.M.M., A.S. and S.M.-R.; supe ision, A.S. and S.M.-R.; p ojec adminis a ion, S.M.-R.; unding acquisi ion, A.S. and S.M.-R. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Cells 2022,11, 1969 18 o 21 Funding: This s udy was suppo ed by FEDER unds h ough he COMPETE 2020—Ope acional P og amme o Compe i i eness and In e na ionalisa ion (POCI), Po ugal 2020; Po uguese unds h ough FCT in he amewo k o he p ojec s “PQTools: Molecula ools o Machado-Joseph Disease” (POCI-01-0145-FEDER-031173), “NAPPIT-MJD:Nuclea a axin-3 p o ein-p o ein in e ac ions as he apeu ic a ge s in Machado-Joseph disease” (POCI-01-0145-FEDER-029056), “Agg eGATE: Ta ge ing di usible oligome s o alpha-synuclein and a axin-3: a d ug epu posing oppo uni y o he ea men o neu odegene a i e diseases” (POCI-01-0145-FEDER-031323), and “Ins i u e o Resea ch and Inno a ion in Heal h Sciences” (POCI-01-0145-FEDER-007274); he Eu opean Union’s Ho izon 2020 Resea ch and Inno a ion p og amme unde g an ag eemen ID 952334 “PhasAGE”. The wo k was also suppo ed by a esea ch g an om Na ional A axia Founda ion o A.S. F.F. is he ecipien o an FCT PhD ellowship (SFRH/BD/133009/2017). The unde s had no ole in he design, collec ion, analysis o in e p e a ion o he da a, o in he w i ing o he manusc ip . Ins i u ional Re iew Boa d S a emen : No applicable. In o med Consen S a emen : No applicable. Da a A ailabili y S a emen : The da ase s gene a ed and/o analyzed du ing he cu en s udy a e a ailable om he co esponding au ho s upon eques . All plasmids used in his wo k a e deposi ed a Addgene. Acknowledgmen s: We acknowledge he suppo o he i3S Scien i ic Pla o ms HEMS (membe o he na ional in as uc u e PPBI—Po uguese Pla o m o Bioimaging (PPBI-POCI-01-0145-FEDER- 022122)) and Biochemical and Biophysical Technologies o he use o hei acili ies and equipmen . We acknowledge Bas ian G oi l and Daisylea de Souza Pai a o suppo wi h he swi chSENSE echnology, and Ma hew Me ski and Ped o J. B. Pe ei a o ca e ul e ision o he manusc ip . Con lic s o In e es : The au ho s decla e no con lic o in e es . Re e ences 1. McGowan, D.; Van Roon-Mom, W.; Holloway, H.; Ba es, G.; Mangia ini, L.; Coope , G.; Faull, R.; Snell, R. Amyloid-like inclusions in Hun ing on’s disease. Neu oscience 2000,100, 677–680. [C ossRe ] 2. Paulson, H.L.; Pe ez, M.; T o ie , Y.; T ojanowski, J.; Sub amony, S.; Das, S.; Vig, P.; Mandel, J.-L.; Fischbeck, K.; Pi man, R. In anuclea inclusions o expanded polyglu amine p o ein in spinoce ebella a axia ype 3. Neu on 1997 ,19, 333–344. [C ossRe ] 3. Seidel, K.; Meis e , M.; Dugba ey, G.J.; Zijls a, M.P.; Vine , J.; B un , E.R.; Van Leeuwen, F.W.; Rub, U.; Kampinga, H.H.; Den Dunnen, W.F. Cellula p o ein quali y con ol and he e olu ion o agg ega es in spinoce ebella a axia ype 3 (SCA3). Neu opa ho. Appl. Neu obiol. 2012,38, 548–558. [C ossRe ] [PubMed] 4. Seidel, K.; Siswan o, S.; F ed ich, M.; Bouz ou, M.; Den Dunnen, W.F.A.; Oze den, I.; Ko , H.W.; Melegh, B.; De V ies, J.J.; B un , E.R.; e al. On he dis ibu ion o in anuclea and cy oplasmic agg ega es in he b ains em o pa ien s wi h spinoce ebella a axia ype 2 and 3. B ain Pa hol. 2017,27, 345–355. [C ossRe ] [PubMed] 5. Chen, S.; Fe one, F.A.; We zel, R. Hun ing on’s disease age-o -onse linked o polyglu amine agg ega ion nuclea ion. P oc. Na l. Acad. Sci. USA 2002,99, 11884–11889. [C ossRe ] [PubMed] 6. Maciel, P.; Gaspa , C.; DeS e ano, A.L.; Sil ei a, I.; Cou inho, P.; Rad any, J.; Dawson, D.M.; Suda sky, L.; Guima ães, J.; Lou ei o, J.E. Co ela ion be ween CAG epea leng h and clinical ea u es in Machado-Joseph disease. Am. J. Hum. Gene . 1995 , 57, 54. 7. F ança, M.C., J .; Emmel, V.E.; D’Ab eu, A.; Mau e -Mo elli, C.V.; Secolin, R.; Bonadia, L.C.; Sil a, M.S.; Nucci, A.; Ja dim, L.B.; Sa ai a-Pe ei a, M.L. No mal ATXN3 allele bu no CHIP polymo phisms modula es age a onse in Machado–Joseph disease. F on . Neu ol. 2012,3, 164. [C ossRe ] 8. Cos a, M.D.C. Recen he apeu ic p ospec s o Machado-Joseph disease. Cu . Opin. Neu ol. 2020,33, 519–526. [C ossRe ] 9. Minakawa, E.N.; Nagai, Y. P o ein Agg ega ion Inhibi o s as Disease-Modi ying The apies o Polyglu amine Diseases. F on . Neu osci. 2021,15, 621996. [C ossRe ] 10. Gales, L.; Co es, L.; Almeida, C.; Melo, C.V.; Do Ca mo Cos a, M.; Maciel, P.; Cla ke, D.T.; Damas, A.M.; Macedo-Ribei o, S. Towa ds a s uc u al unde s anding o he ib illiza ion pa hway in Machado-Joseph’s disease: T apping ea ly oligome s o non-expanded a axin-3. J. Mol. Biol. 2005,353, 642–654. [C ossRe ] 11. Sca , C.A.; Sico ello, A.; Tomé, R.J.; Macedo-Ribei o, S.; Ashc o , A.E.; Rad o d, S.E. A ale o a ail: S uc u al insigh s in o he con o ma ional p ope ies o he polyglu amine p o ein a axin-3. In . J. Mass Spec om. 2013,345, 63–70. [C ossRe ] [PubMed] 12. Masino, L.; Nicas o, G.; De Simone, A.; Calde , L.; Molloy, J.; Pas o e, A. The Josephin domain de e mines he mo phological and mechanical p ope ies o a axin-3 ib ils. Biophys. J. 2011,100, 2033–2042. [C ossRe ] [PubMed] 13. Masino, L.; Nicas o, G.; Menon, R.P.; Dal Piaz, F.; Calde , L.; Pas o e, A. Cha ac e iza ion o he s uc u e and he amyloidogenic p ope ies o he Josephin domain o he polyglu amine-con aining p o ein a axin-3. J. Mol. Biol. 2004 ,344, 1021–1035. [C ossRe ] Cells 2022,11, 1969 19 o 21 14. Ellisdon, A.M.; Pea ce, M.C.; Bo omley, S.P. Mechanisms o a axin-3 mis olding and ib il o ma ion: Kine ic analysis o a disease-associa ed polyglu amine p o ein. J. Mol. Biol. 2007,368, 595–605. [C ossRe ] [PubMed] 15. Sca , C.A.; Almeida, B.; F aga, J.; Macedo-Ribei o, S.; Rad o d, S.E.; Ashc o , A.E. Examina ion o a axin-3 (a x-3) agg ega ion by s uc u al mass spec ome y echniques: A a ionale o expedi ed agg ega ion upon polyglu amine (polyQ) expansion. Mol. Cell. P o eomics 2015,14, 1241–1253. [C ossRe ] 16. Ma ins, P.M.; Na a o, S.; Sil a, A.; Pin o, M.F.; Sá kány, Z.; Figuei edo, F.; Pe ei a, P.J.B.; Pinhei o, F.; Bedna iko a, Z.; Bu dukiewicz, M. MIRRAGGE–minimum in o ma ion equi ed o ep oducible AGG ega ion expe imen s. F on . Mol. Neu osci. 2020,222, 582488. [C ossRe ] 17. Sil a, A.; Almeida, B.; F aga, J.S.; Taboada, P.; Ma ins, P.M.; Macedo-Ribei o, S. Dis ibu ion o Amyloid-Like and Oligome ic Species om P o ein Agg ega ion Kine ics. Angew. Chem. In . Ed. 2017,56, 14042–14045. [C ossRe ] 18. Knigh , P.D.; Ka amanos, T.K.; Rad o d, S.E.; Ashc o , A.E. Iden i ica ion o a no el si e o in e ac ion be ween a axin-3 and he amyloid agg ega ion inhibi o polyglu amine binding pep ide 1. Eu . J. Mass Spec om. 2018,24, 129–140. [C ossRe ] [PubMed] 19. A men, R.S.; Be na d, B.M.; Day, R.; Alonso, D.O.; Dagge , V. Cha ac e iza ion o a possible amyloidogenic p ecu so in glu amine- epea neu odegene a i e diseases. P oc. Na l. Acad. Sci. USA 2005,102, 13433–13438. [C ossRe ] 20. Nagai, Y.; Tucke , T.; Ren, H.; Kenan, D.J.; Hende son, B.S.; Keene, J.D.; S i ma e , W.J.; Bu ke, J.R. Inhibi ion o polyglu amine p o ein agg ega ion and cell dea h by no el pep ides iden i ied by phage display sc eening. J. Biol. Chem. 2000 ,275, 10437–10442. [C ossRe ] 21. Tomi a, K.; Popiel, H.A.; Nagai, Y.; Toda, T.; Yoshimi su, Y.; Ohno, H.; Oishi, S.; Fujii, N. S uc u e-ac i i y ela ionship s udy on polyglu amine binding pep ide QBP1. Bioo g. Med. Chem. 2009,17, 1259–1263. [C ossRe ] [PubMed] 22. Fe o, A.; Ca alho, A.L.; Teixei a-Cas o, A.; Almeida, C.; Tomé, R.J.; Co es, L.; Rod igues, A.-J.; Loga inho, E.; Sequei os, J.; Macedo-Ribei o, S.; e al. NEDD8: A new a axin-3 in e ac o . Biochim. Biophys. Ac a Mol. Cell Res. 2007 ,1773, 1619–1627. [C ossRe ] [PubMed] 23. G ou, C.P.; Pin o, M.P.; Mendes, A.V.; Domingues, P.; Aze edo, J.E. The de no o syn hesis o ubiqui in: Iden i ica ion o deubiqui inases ac ing on ubiqui in p ecu so s. Sci. Rep. 2015,5, 12836. [C ossRe ] [PubMed] 24. Rames, M.; Yu, Y.; Ren, G. Op imized nega i e s aining: A high- h oughpu p o ocol o examining small and asymme ic p o ein s uc u e by elec on mic oscopy. JoVE 2014,15, e51087. [C ossRe ] [PubMed] 25. Sil a, A.; De Almeida, A.V.; Macedo-Ribei o, S. Polyglu amine expansion diseases: Mo e han simple epea s. J. S uc . Biol. 2018 , 201, 139–154. [C ossRe ] [PubMed] 26. Chow, M.K.; Paulson, H.L.; Bo omley, S.P. Des abiliza ion o a non-pa hological a ian o a axin-3 esul s in ib illogenesis ia a pa ially olded in e media e: A model o mis olding in polyglu amine disease. J. Mol. Biol. 2004,335, 333–341. [C ossRe ] 27. Ellisdon, A.M.; Thomas, B.; Bo omley, S.P. The wo-s age pa hway o a axin-3 ib illogenesis in ol es a polyglu amine- independen s ep. J. Biol. Chem. 2006,281, 16888–16896. [C ossRe ] 28. Kneze ic, J.; Lange , A.; Hampel, P.A.; Kaise , W.; S asse , R.; Ran , U. Quan i a ion o a ini y, a idi y, and binding kine ics o p o ein analy es wi h a dynamically swi chable biosu ace. J. Am. Chem. Soc. 2012,134, 15225–15228. [C ossRe ] [PubMed] 29. Mülle -Landau, H.; Va ela, P.F. S anda d ope a ion p ocedu e o swi chSENSE DRX sys ems. Eu . Biophys. J. 2021 ,50, 389–400. [C ossRe ] 30. San amb ogio, C.; F ana, A.M.; Na alello, A.; Papaleo, E.; Regonesi, M.E.; Doglia, S.M.; To o a, P.; In e nizzi, G.; G ando i, R. The ole o he cen al lexible egion on he agg ega ion and con o ma ional p ope ies o human a axin-3. FEBS J. 2012 ,279, 451–463. [C ossRe ] 31. Lup on, C.J.; S ee , D.L.; Win ode, P.L.; Bo omley, S.P.; Hughes, V.A.; Ellisdon, A.M. Enhanced molecula mobili y o o dina ily s uc u ed egions d i es polyglu amine disease. J. Biol. Chem. 2015,290, 24190–24200. [C ossRe ] [PubMed] 32. Gaspa , C.; Lopes-Cendes, I.; Hayes, S.; Go o, J.; A idsson, K.; Dias, A.; Sil ei a, I.; Maciel, P.; Cou inho, P.; Lima, M. Ances al o igins o he Machado-Joseph disease mu a ion: A wo ldwide haplo ype s udy. Am. J. Hum. Gene . 2001 ,68, 523–528. [C ossRe ] [PubMed] 33. Li, T.; Ma ins, S.; Peng, Y.; Wang, P.; Hou, X.; Chen, Z.; Wang, C.; Tang, Z.; Qiu, R.; Chen, C.; e al. Is he High F equency o Machado-Joseph Disease in China Due o New Mu a ional O igins? F on . Gene . 2018,9, 740. [C ossRe ] 34. Sha ony, R.; Ma ins, S.; Cos a, I.P.D.; Zal zman, R.; Amo im, A.; Sequei os, J.; Go don, C.R. Yemeni e-Jewish amilies wi h Machado-Joseph disease (MJD/SCA3) sha e a ecen common ances o . Eu . J. Hum. Gene . 2019 ,27, 1731–1737. [C ossRe ] [PubMed] 35. Genomes P ojec , C.; Au on, A.; B ooks, L.D.; Du bin, R.M.; Ga ison, E.P.; Kang, H.M.; Ko bel, J.O.; Ma chini, J.L.; McCa hy, S.; McVean, G.A.; e al. A global e e ence o human gene ic a ia ion. Na u e 2015,526, 68–74. [C ossRe ] 36. Go o, J.; Wa anabe, M.; Ichikawa, Y.; Yee, S.-B.; Iha a, N.; Endo, K.; Iga ashi, S.; Takiyama, Y.; Gaspa , C.; Maciel, P. Machado– Joseph disease gene p oduc s ca ying di e en ca boxyl e mini. Neu osci. Res. 1997,28, 373–377. [C ossRe ] 37. Flynn, J.D.; McGlinchey, R.P.; Walke , R.L.; Lee, J.C. S uc u al ea u es o α -synuclein amyloid ib ils e ealed by Raman spec oscopy. J. Biol. Chem. 2018,293, 767–776. [C ossRe ] [PubMed] 38. Molle , J.; Sch oe , M.A.; E lkamp, M.; G obelny, S.; Paulus, M.; Tiemeye , S.; Wi ke , F.J.; Tolan, M.; Win e , R. The e ec o ionic s eng h, empe a u e, and p essu e on he in e ac ion po en ial o dense p o ein solu ions: F om nonlinea p essu e esponse o p o ein c ys alliza ion. Biophys. J. 2012,102, 2641–2648. [C ossRe ] Cells 2022,11, 1969 20 o 21 39. Munishkina, L.A.; Hen iques, J.; U e sky, V.N.; Fink, A.L. Role o p o ein − wa e in e ac ions and elec os a ics in α -synuclein ib il o ma ion. Biochemis y 2004,43, 3289–3300. [C ossRe ] 40. Ziaunys, M.; Sakalauskas, A.; Mikalauskai e, K.; Smi no as, V. Polymo phism o Alpha-Synuclein Amyloid Fib ils Depends on Ionic S eng h and P o ein Concen a ion. In . J. Mol. Sci. 2021,22, 12382. [C ossRe ] 41. Campos-Rami ez, A.; Ma quez, M.; Quin ana , L.; Rojas-Ochoa, L.F. E ec o ionic s eng h on he agg ega ion kine ics o he amida ed amyloid be a pep ide Abe a (1-40) in aqueous solu ions. Biophys. Chem. 2017,228, 98–107. [C ossRe ] [PubMed] 42. Abelein, A.; Ja e , J.; Ba h, A.; G aslund, A.; Danielsson, J. Ionic S eng h Modula ion o he F ee Ene gy Landscape o Abe a40 Pep ide Fib il Fo ma ion. J. Am. Chem. Soc. 2016,138, 6893–6902. [C ossRe ] [PubMed] 43. Ma ek, P.J.; Pa salo, V.; G een, D.F.; Raleigh, D.P. Ionic s eng h e ec s on amyloid o ma ion by amylin a e a complica ed in e play among Debye sc eening, ion selec i i y, and Ho meis e e ec s. Biochemis y 2012,51, 8478–8490. [C ossRe ] 44. Gekko, K.; Timashe , S.N. Mechanism o p o ein s abiliza ion by glyce ol: P e e en ial hyd a ion in glyce ol-wa e mix u es. Biochemis y 1981,20, 4667–4676. [C ossRe ] [PubMed] 45. Vagenende, V.; Yap, M.G.; T ou , B.L. Mechanisms o p o ein s abiliza ion and p e en ion o p o ein agg ega ion by glyce ol. Biochemis y 2009,48, 11084–11096. [C ossRe ] [PubMed] 46. Saunde s, H.M.; Hughes, V.A.; Cappai, R.; Bo omley, S.P. Con o ma ional beha io and agg ega ion o a axin-3 in SDS. PLoS ONE 2013,8, e69416. [C ossRe ] 47. Kuma , S.; Singh, A.K.; K ishnamoo hy, G.; Swamina han, R. Thio la in T displays enhanced luo escence selec i ely inside anionic micelles and mammalian cells. J. Fluo esc. 2008,18, 1199–1205. [C ossRe ] [PubMed] 48. Seelige , J.; We kmulle , A.; Win e , R. Mac omolecula c owding as a supp esso o human IAPP ib il o ma ion and cy o oxici y. PLoS ONE 2013,8, e69652. [C ossRe ] 49. Nicas o, G.; Masino, L.; Esposi o, V.; Menon, R.P.; De Simone, A.; F a e nali, F.; Pas o e, A. Josephin domain o a axin-3 con ains wo dis inc ubiqui in-binding si es. Biopolyme s 2009,91, 1203–1214. [C ossRe ] 50. Nicas o, G.; Menon, R.P.; Masino, L.; Knowles, P.P.; McDonald, N.Q.; Pas o e, A. The solu ion s uc u e o he Josephin domain o a axin-3: S uc u al de e minan s o molecula ecogni ion. P oc. Na l. Acad. Sci. USA 2005,102, 10493–10498. [C ossRe ] 51. Chai, Y.; Be ke, S.S.; Cohen, R.E.; Paulson, H.L. Poly-ubiqui in binding by he polyglu amine disease p o ein a axin-3 links i s no mal unc ion o p o ein su eillance pa hways. J. Biol. Chem. 2004,279, 3605–3611. [C ossRe ] [PubMed] 52. Nicas o, G.; Todi, S.V.; Ka aca, E.; Bon in, A.M.; Paulson, H.L.; Pas o e, A. Unde s anding he ole o he Josephin domain in he PolyUb binding and clea age p ope ies o a axin-3. PLoS ONE 2010,5, e12430. [C ossRe ] [PubMed] 53. Bu ne , B.; Li, F.; Pi man, R.N. The polyglu amine neu odegene a i e p o ein a axin-3 binds polyubiqui yla ed p o eins and has ubiqui in p o ease ac i i y. Hum. Mol. Gene . 2003,12, 3195–3205. [C ossRe ] 54. Masino, L.; Nicas o, G.; Calde , L.; Vend uscolo, M.; Pas o e, A. Func ional in e ac ions as a su i al s a egy agains abno mal agg ega ion. FASEB J. 2011,25, 45–54. [C ossRe ] [PubMed] 55. Bake , M. 1,500 scien is s li he lid on ep oducibili y. Na u e 2016,533, 452–454. [C ossRe ] [PubMed] 56. Ga ey, M.; Teppe , K.; Haup , C.; Knüp e , U.; Klemen , K.; Meinha d , J.; Ho n, U.; Balbach, J.; Fänd ich, M. Phospha e and HEPES bu e s po en ly a ec he ib illa ion and oligome iza ion mechanism o Alzheime ’s A β pep ide. Biochem. Biophys. Res. Commun. 2011,409, 385–388. [C ossRe ] 57. Gade Malmos, K.; Blancas-Mejia, L.M.; Webe , B.; Buchne , J.; Rami ez-Al a ado, M.; Naiki, H.; O zen, D. ThT 101: A p ime on he use o hio la in T o in es iga e amyloid o ma ion. Amyloid 2017,24, 1–16. [C ossRe ] 58. G oo e , S.E.; Adegbuyi o, A.; Fan, C.K.; Hodges, B.L.; Beasley, M.; Taylo , K.; S oneb ake , A.R.; Si iwa dhana, C.; Leglei e , J. Mac omolecula c owding in solu ion al e s hun ing in in e ac ion and agg ega ion a in e aces. Colloids Su . B: Bioin e aces 2021,206, 111969. [C ossRe ] 59. U e sky, V.N.; Coope , E.M.; Bowe , K.S.; Li, J.; Fink, A.L. Accele a ed α -synuclein ib illa ion in c owded milieu. FEBS Le . 2002 , 515, 99–103. [C ossRe ] 60. Fang, X.; Yousa , M.; Huang, Q.; Yang, Y.; Wang, C. Dual e ec o PEG-PE micelle o e he oligome iza ion and ib illa ion o human isle amyloid polypep ide. Sci. Rep. 2018,8, 4463. [C ossRe ] 61. Min on, A.P. The e ec o olume occupancy upon he he modynamic ac i i y o p o eins: Some biochemical consequences. Mol. Cell. Biochem. 1983,55, 119–140. [C ossRe ] [PubMed] 62. Fe ei a, C.; Pin o, M.F.; Macedo-Ribei o, S.; Pe ei a, P.J.B.; Rocha, F.A.; Ma ins, P.M. P o ein c ys als as a key o deciphe ing mac omolecula c owding e ec s on biological eac ions. Phys. Chem. Chem. Phys. 2020,22, 16143–16149. [C ossRe ] [PubMed] 63. Winbo n, B.J.; T a is, S.M.; Todi, S.V.; Scaglione, K.M.; Xu, P.; Williams, A.J.; Cohen, R.E.; Peng, J.; Paulson, H.L. The deubiqui i- na ing enzyme a axin-3, a polyglu amine disease p o ein, edi s Lys63 linkages in mixed linkage ubiqui in chains. J. Biol. Chem. 2008,283, 26436–26443. [C ossRe ] 64. Pas o e, A.; Temussi, P.A. The wo aces o Janus: Func ional in e ac ions and p o ein agg ega ion. Cu . Opin. S uc . Biol. 2012 , 22, 30–37. [C ossRe ] [PubMed] 65. Weeks, S.D.; G as y, K.C.; He nandez-Cuebas, L.; Loll, P.J. C ys al s uc u es o Lys-63-linked i-and di-ubiqui in e eal a highly ex ended chain a chi ec u e. P o eins S uc . Func . Bioin . 2009,77, 753–759. [C ossRe ] 66. Hsieh, M.; Tsai, H.-F.; Lu, T.-M.; Yang, C.-Y.; Wu, H.-M.; Li, S.-Y. S udies o he CAG epea in he Machado-Joseph disease gene in Taiwan. Hum. Gene . 1997,100, 155–162. [C ossRe ] [PubMed] Cells 2022,11, 1969 21 o 21 67. Kawaguchi, Y.; Okamo o, T.; Taniwaki, M.; Aizawa, M.; Inoue, M.; Ka ayama, S.; Kawakami, H.; Nakamu a, S.; Nishimu a, M.; Akiguchi, I. CAG expansions in a no el gene o Machado-Joseph disease a ch omosome 14q32. 1. Na . Gene . 1994 ,8, 221–228. [PubMed] 68. Gu, W.; Ma, H.; Wang, K.; Jin, M.; Zhou, Y.; Liu, X.; Wang, G.; Shen, Y. The sho es expanded allele o he MJD1 gene in a Chinese MJD kind ed wi h au onomic dys unc ion. Eu . Neu ol. 2004,52, 107–111. [C ossRe ] 69. Padia h, Q.S.; S i as a a, A.K.; Roy, S.; Jain, S.; B ahmacha i, S.K. Iden i ica ion o a no el 45 epea uns able allele associa ed wi h a disease pheno ype a he MJD1/SCA3 locus. Am. J. Med. Gene . B Neu opsychia . Gene . 2005,133, 124–126. [C ossRe ] 70. Takiyama, Y.; Sakoe, K.; Nakano, I.; Nishizawa, M. Machado-Joseph disease: Ce ebella a axia and au onomic dys unc ion in a pa ien wi h he sho es known expanded allele (56 CAG epea uni s) o he MJD1 gene. Neu ology 1997 ,49, 604–606. [C ossRe ] 71. Van Al en, N.; Sinke, R.J.; Zwa s, M.J.; Gab eëls-Fes en, A.; P aams a, P.; K eme , B.P.; Ho s ink, M.W. In e media e CAG epea leng hs (53, 54) o MJD/SCA3 a e associa ed wi h an abno mal pheno ype. Ann. Neu ol. 2001 ,49, 805–808. [C ossRe ] [PubMed]