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
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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 .
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