A Rare Missense Mutation in GJB3 (Cx31g45e) Is Associated with a Unique Cellular Phenotype Resulting in Necrotic Cell Death Easton, J

University of Dundee

A rare missense mutation in GJB3 (Cx31G45E) is associated with a unique cellular phenotype resulting in necrotic cell death Easton, J. A.; Alboulshi, A. K.; Kamps, M. A. F.; Brouns, G. H.; Broers, M. R.; Coull, B. J.; Oji, V.; van Geel, M.; van Steensel, M. A. M.; Martin, P. E. Published in: Experimental Dermatology

DOI: 10.1111/exd.13542

Publication date: 2018

Document Version Peer reviewed version

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Citation for published version (APA): Easton, J. A., Alboulshi, A. K., Kamps, M. A. F., Brouns, G. H., Broers, M. R., Coull, B. J., ... Martin, P. E. (2018). A rare missense mutation in GJB3 (Cx31G45E) is associated with a unique cellular phenotype resulting in necrotic cell death. Experimental Dermatology. https://doi.org/10.1111/exd.13542

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• Users may download and print one copy of any publication from Discovery Research Portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • You may freely distribute the URL identifying the publication in the public portal. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. This article isprotected rights by All copyright. reserved. 10.1111/exd.13542 doi: lead todifferences version between this andtheof Pleasec Version Record. been and throughtypesetting, proofreadingwhich may thecopyediting, process, pagination This article undergone has for and buthas accepted been not review publication fullpeer Key words: Tel: +44 141 331 3726 Glasgow Caledonian University, Glasgow,G4 0BA, Scotland, UK. Postal 3 Correspondence to # Medical Biology,Singapore Immunos, Medicine, of School Research, Cancer 5 Du of University Sciences, Life of UK; College Discovery, Glasgow, Drug and Chemistry University, Biological Caledonian Glasgow Sciences, Life and Health Netherlands, The Maastricht, University, Maastricht 1 van Geel # resultingin phenotype necroticcell death A Articletype : Regular Article DR. PATRICIA E MARTIN (Orcid ID 0000: J.A. Easton J.A. Patricia EPatricia Martin: JAE and AKAcontributed equally to this workand sharefirst Department Dermatology, of Department

Accepted Articlerare n dee, UK dee, address: missense mutation in mutation in missense 1,2, 5

, 1,2 Connexin, EKV M.A.M. Steensel van , ,

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This article isprotected rights by All copyright. reserved. Accepted the EKV phenotype. skin data Cx31G45E rescue completely necrotic. was Cx31G45E expressing cells in death cell in increase the where proteins Cx43 expressing stably cells HeLa and cells HaCaT both in membrane plasma contact cell to cell of points at plaques W connexons. heteromeric form to phenotype interaction, myc network microtubular the (ER) reticulum Articlemembrane, line cell keratinocyte human model a cells, HaCaT Cx31G45E of Expression c.134G GJB3 (EKV progressiva et variabilis Erythrokeratodermia Abstract iodide; junction; KID:ichthyosiskeratitisdeafness syndrome;polymerisechainPCR:reaction;PI: propidium compartment; intermediate golgi reticulum endoplasmic EKV Abbreviations: - - : rtrkrtdri vraii e progressiva; et variabilis Erythrokeratodermia P: suggests 6xHis

SERCA2b: ( C3) or (Cx31) A (p.G45E) >A

togy co strongly .

Cx31 and Cx31G45E both co Cx31G45Eboth and Cx31 but GFP but

n Cx31G45E and

that that 18 , Blocking

expression of Cx31G45E of expression co the mutant protein mutant the

 sarcoplasmic reticulum calcium damages GA: entrapment of Cx43 and Cx43 of entrapment - GJB4 necrosis expression with Cx31WT with expression - , localised

n w urltd patients unrelated two in 18



connexin connexin - (Cx30.3). gene

GFP

Glycyrrhetinic occurred h clua mmrn idpnet f t canl function. channel its of independent membrane cellular the or

prevent -

ih h vcluie ER vacolourised the with GFP caused caused accumulate hne function channel .

T N propidium iodide propidium co - , previously undescribed undescribed previously

E srs rsoss ee evoked were responses stress ER o x1 and Cx31 Cx31G45E restricted the ability of Cx43 to reach the reach to Cx43 of ability the restricted Cx31G45E acid; - - - muorcpttd idctv o h of indicative immunoprecipitated, GFP immunoprecipatedCx43 with n e dniid a identified We ecrotic cell death cell ecrotic - Cx: Connexin Cx:

mCherry

ATPase d caused

within the ER membrane ER the within n investigated and Cx . Cx31WT . - -

; F: re Furset Protein Fluorescent Green GFP: P GFP 3 sebe in assembled 43 ih 18α with UPR: unfolded protein response. vacuolar , Cx26 or Cx30.3 or Cx26 , ) is caused by mutations in either the the either in mutations by caused is ) uptake, suggesting that expression of expression that suggesting uptake, R edpamc eiuu; ERGIC: reticulum; endoplasmic ER:

. ; DAPI:; 4',6

, which FACS which , Cell viability assays identified identified assays viability Cell

rare in the epidermis the in changes within HeLacells within changes

- expan - GFP lcrhtnc acid Glycyrrhetinic its GJB3

diamidino s localised to the plasma the to localised cellular ion to ,

indicating ability the

analysis the mitigate not did

missense and disassembly of of disassembly and ag gp junction gap large of the endoplasmic the of Cx31WT .

characteristics - could 2 - phenylindole;

determined eteromeric mutation

; GJ :ga GJ ;

underlie did not not did - myc

Our and and an an p - . ,

This article isprotected rights by All copyright. reserved. reported previously as Netherlands) (WT) type Wild 2.1 2. human disease mechanism. ER the on effect EKV rare 28 Cx31 mutations Cx30.3 by 1) red permanent to erythemas moving fast temporary, Cx30.3 or Cx31 either in mutations progressiva et variabilis different in mutations by caused disorders various layersupper the in cellsdifferentiated ( by (encoded [ channel neighbouring a with dock and align to membrane plasma the under open to induced hemichannel reticulum endoplasmic the va one with EL2) and (EL1 loops extracellular conserved highly two by linked domains transmembrane four on basednomenclature (Cxs) connexins communication intercellular which A 1. encoded by encoded 1

. With the possible exception of an erythema gyratum repens gyratum erythema an of exception possible the With . , healthy skin barrier skin healthy Accepted] Article . 3 Construction of tagged Cx I Several studies indicatethatstudiesSeveral NTRODUCTION ] M

. mutations

riable intracellular loop loop intracellular riable ATERIALS AND METHODS Within normal epidermis normal Within nlds dees ucin, ih junctions tight junctions, adherens includes - a ssociated Cx31 mutant (G45E) mutant Cx31 ssociated ) GJA1 GJB2 , ht s rfikd n coe sae o the to state closed a in trafficked is that , , when expressed in HeLa cells, HeLa in expressed when ,

with and entrapment of Cx43 of entrapment and ) ) x1 a isre it te etr pE vector the into inserted was Cx31 , Cx30 , and Cx31 (encoded by Cx31(encodedand

, a genotype a , 21 subtypes identified in humans in identifiedsubtypes 21 requires

pathological (EKV their

( encoded by encoded

- n pa a e role key a play and P) (ER) mass molecular - WT and mutant constructs a highly ordered and tightly regulatedtightlyand orderedhighly a and carboxyl tail carboxyl and (Mendes da Costa, EKV Costa, da (Mendes ,

ER - , at least at phenotype correlation is not present not is correlation phenotype

hr they where stress conditions and a range of mutations (Cx31G45E, Cx31delG45, Cx31G45A, Cx31delG45, (Cx31G45E, mutations of range a and e.g. [ GJB 4 -

8 . We suggest that this may be a novel a be may this that suggest We . GJB3

[ is ] 6 six Cx subtypes are d are subtypes Cx six [ 2 . 17 )

0 The importance of connexins in the skin is illustrated byillustrated is skinthe connexinsin importance of The , Cx30.3 , involved , ,

- e.g. , but normally but , ) are ) 25 22

result in both apoptotic both in result

[ oligomer 3 ] , in , .

] 24 Cx31 is a 31kDa protein 31kDaa is Cx31

. expressed throughoutexpressedthe h ciia apaac o EKV of appearance clinical The

epidermal Cxs

beta

( ] in encoded by encoded

- n gp junctions gap and , cau - brown

P rang this process this follow a classic biosynthetic pathway biosynthetic classic a follow , MIM #133200) MIM ,

- plasma ise Cx e ncoi cl dah hog a unique a through death cell necrotic ses ing

GFP

accrete subtypes o om hxmrc connexon hexameric a form to hyperkeratosis

integrity in size from 25 to 62 kDa 62 to 25 from size in - - ifferentially expressed ifferentially like manifestation of EKV of manifestation like 1 B Bocecs Bea T Breda, Biosciences, (BD N1 membran GJB4

[

intercellular laterally 27 and

) , [

[

9 and necrotic cell death cell necrotic and [ 29 20 tend to be associated with associated be to tend

1 -

16 ] form a functional a form (GJ) , , [

e . caused by heterozygousby caused 2

30 26 Hmcanl cn be can Hemichannels . ] They

] .

in a closed state closed a in . Cxs . ( epidermis supplementary . ] ] E . . rythrokeratodermia GJ connexin We describe how a howdescribe We Disease adhesion network adhesion are s are

- P eit rapid mediate

. Connexin43 . of composed composed ofcomposed ags from ranges , - , with theirwith , while associated - - mediated P

GJ caused

along Table Cx26 from

unit [ 27 ( he or , ,

This article isprotected rights by All copyright. reserved. Accepted Photoshop. and Airyscan Mannheim, DMRBE, (Leica a using out carried was localisa determined autoflourescence GFP or mCherry antibodiesof (see supplementaryinformation) requirements)processedand immunocytochemical for analysisaspreviously described ice either in fixed were Cells 2.3 and protein extraction to inducestressER durationforexperiment. theThapsigargin the Sigma) of (1 Articletr were Cells assessedGFP by or mCherry h. 24 for DNA plasmid relevant with transfected were and Cx26 or Cx43 expressing l Cell 2.2 construct. 12377 # plasmid (Addgene Weimbs two the into inserted sup directed site by introduced Cx31G45W) and Cx31G45Q Cx31G45S, Cx31G45K, lmnay ehd ad upeetr Table supplementary and methods plementary Immunofluorescence

- Cell transfection Culture and were processed with ImageJ software, version 1.43 ( 1.43 version software, ImageJ with processed were step process replacing the GFP tag with a myc ines , confocal system confocal

ea Ohio HeLa

eated as required with the Cx channel blocker channel Cx the with required as eated pmCherry

AT) n HCT el ( cells HaCaT and (ATTC) [

32 . Images were acquired under similar conditions and magnifications magnifications and conditions similar under acquired Imageswere . ec TS P cnoa lsr cnig ursec microscope ﬂuorescence scanning laser confocal SPE TCS Leica - ] auto fluorescence 1 etr Cotc, i Wsbr B, ese, h Netherlands) The Leusden, BV, Westburg via (Clontech, vector N1

were cultured were - Germany) od ehnl r 4% or methanol cold

[ 31 ]

) whereappropriate

a ue t gnrt a Cx31WT a generate to used was or a Zeiss a or

under optimal conditions (see supplementary methods)supplementary (see conditions optimal under .

[ 33 - ] CLS, myc .

2 )

- Axiovert microscope linke microscope Axiovert [ pehi, Germany Eppelheim, 6xHis tagplasmid from a gifted from Thomas 12 ( w/v  ] . M, Sigma) was added for 8h prior to RNApriortoSigma) added 8h wasM, for in f Cx of tion 18 . x1T C2W a Cx26WT Cx31WT, ) http://rsbweb.nih.gov/ij/

 aaomleye a pr antibody per (as paraformaldehyde rnfcin fiiny a 30 was efficiency Transfection

glycyrrhetinic - agd rtis I proteins. tagged - ) myc

or acid ( acid d x03T were Cx30.3WT nd - myc mutagenesis ea el stably cells HeLa d 18αGA

up to a to up - ) and Adobe and ) xi tagged 6xHis using a panelusinga ) maging maging

(25µM, n -

50%, (see 800 . A

This article isprotected rights by All copyright. reserved. and s 15 for 95°C of cycles Vii inperformed wasreaction The design).(Primerreactions TaqmanPCR in used Southhampton,(Promega, Primersagainst UK). to according Kit Mini RNA II manufacturers’synthesisedinstructionscDNA the M with and(Bioline) ISOLATE using cells HeLa transfected from isolated was RNA Total 2.6 information) procedures standard Protei 2.5 Laem non any remove to washed 500 with incubated previously X Triton ( buffer RIPA in harvested were cells HeLa43 mM Imidazol and 0.1% elut by followed NP40), proteinboundremoved was washingbyNaCl, (250 Tris250 mM mM Non agitation. with overnight 4°C at incubated was mix protein and bead The added. was protein L Healthcare (GE beads Ni Sepharose Mag His 50µl To cells wereharvested in NP40 lysis buffer (150mM NaCl, 250mM Tris expression tagged myc to tagged Cx31G45E or Cx31WT HeLa 2.4

AcceptedA Article Quantitative Reverse Western blotting Protein interactions

TM μ

m cells selected to selected cells 7 n (30 n g of total prote total of g li buffer Real timeRealPCR - 100

 . ). g) was subject was g)

Twenty following manufactures instructions

vector were transfected were vector

also

and blots were probed with appropriate antibodies (please see supplementary see (please antibodiesappropriate with probed were blots and systemfollowingtheundercycling min,followed40conditions:forby 95°C15 in - stably ( five ion of bound protein in elution buffer (500 mM NaCl, 250 mM Tris mM 250 NaCl, mM (500 buffer elution in protein bound of ion

v/v

and - rnfce wt ete C3W o C3G5 tge t GFP to tagged Cx31G45E or Cx31WT either with transfected 125 transcriptase PCR (qRT polyclonal )

- 60°C for 30 s. Gene expression levels were obtained from the value of value the from obtained were levels expression Gene s. 30 for 60°C  NP40). specifically bound proteins and interacting proteins finally eluted in eluted finally proteins interacting and proteins bound specifically incubated at 4°C overnight with agitation with overnight 4°C at incubated

express o sep anti sheep of l mM NaCl, 5 NaCl, mM

to SDS to

- - myc rabbit Cx31WT PAGE, and Western blot analysis was performed according to according performed was analysis blot Western and PAGE, -

6xHis. mM EDTA, 1% EDTA, mM

W the for control positive a as -

- GFP antibody (1:100, Abcam, UK) Abcam, (1:100, antibody GFP - abt g Dnbas (T Dynabeads IgG rabbit GFP CHOP,GADD34

- The expression vector eGFP vector expression The PCR) . (HeLa31

f Sine, igm Blim 50g total 500µg Belgium) Diegem, Sciences, ife

(w/v) - GFP cells) GFP

Sodium Deoxycholate Sodium and - the referencegene the HCl (pH 7.3)and 1% - HCl, 5 mMImidazol,HCl,0.1 5 %

- . The beads were extensively were beads The . were transfected with either with transfected were MLV reversetranscriptionMLV kit emfse sinii, UK) scientific, hermofisher estern blots. estern - N1 and a contro a and N1 a ,

Applied BiosystemsApplied was then added to added then was

and 0.5% and GAPDH

( Transfected v/v

n were and - HCl, 500 HCl, )

NP40). l myc l were (v/v) ( v/v 5x - ) - ,

This article isprotected rights by All copyright. reserved. 1 (Figure ER the network from microtubule formed are vacuoles the that suggesting protein mutant the with 1 Figure supplementary cel transfected ( SERCA2b cytoplasm structures Cx31WT intracellular components 3.1 RESULTS multiple comparison testas appropriate. ** P<0.05; t unpaired using performed analysis statistical and S.E.M ± mean as expressed Experimentswereperformedin triplic 2.8 ( 575/26 a using freeze by detected was PI analysis. to prior ice, 300V at set fluorochrome on 1h, for PI 40µg/ml with incubated respect 450V and 340Vat detected were scatter sideand Forward 400V. at setfluorochrome 530/30 a usingdetected was Fluorescence laser. 488nm a using FITC to gated were cells counted, were cells expressing) (GFP transfected only eveeachpopulationFACSCantoII. 20,000 For analysisusinga BD for PBS in pre were cells Transfected 2.7 [ (∆∆C GAPDH of Ct the against normalised and gene specific each for (Ct) cycle threshold Accepted Articlehttp://www.flowingsoftware.com 34

] Statistical Analysis Propidium Iodide (PI) uptake assays and FACS analysis Expressiontheof mutation Cx31G45E unique causesa cellular morphology .

GFP - thawing cells three times prior to analysis. Analysis was carried out usingcarried was analysis. out Analysis priorto timescells three thawing

acpamc eiuu clim ATPase) calcium reticulum sarcoplasmic in n ea ho cells Ohio HeLa in trafficked to the plasmatraffickedthe tomembrane andinto assembledtypical gap junction pronounced s n i cls rnfce t epes Cx31WT express to transfected cells in and ls

) . By contrast, in Cx31G45E in contrast, By .

, in the 488nM gated cells gated 488nM the in , - vacuol treated with 18 with treated D (Figure e ws irpe i cls xrsig x14E wt eiec of evidence with Cx31G45E, expressing cells in disrupted was ) , v2.5.1). - ie structures like ively. For PI uptake assays the cells were prepared as above then above asprepared were cells the assaysuptake PI For ively. ate andate compiled usingGraphPadPrism6 software. are Results 1A  )

I cnrs, Cx31G45E contrast, In . GA as required prior to trypsinisation and resuspension and trypsinisation to prior required as GA

Fgr 1B) (Figure -

. A control necrotic cell population was prepare was populationcell necrotic control A . GFP expressing HeLa cells HeLa expressing GFP ***P<0.001.

hwd dfue R itiuin n l non all in distribution ER diffuse a showed

Co .

- tiig iha atbd against antibody an with staining - - GFP F accumulated GFP

nts were gated. To ensuregated. ntswere Fgr 1 (Figure ,

SERCA2b co SERCA2b - in vitroin test, and Dunnett’s and test,

F (Figure lowing and A

andimpacts plaque ihn the within T -

S 1B localised method) oftware B )

. - and The like d -

This article isprotected rights by All copyright. reserved. reduced. markedly was membrane co and structures keratinocytes in phenotype of collapse vac 2C (Figure perinu and B and the assessed also was Cx43 cytoplasm the structures plaque like (Figure Cx30.3 epidermis on protein Cx31 mutant the of dominant be to reported are EKV with associatedmutations Cx31 3. and gap clear junction plaqueswere visible Cx31G45E expressing cells Supplementary Supplementary (G45A,alanine data supplementary different of number determine To T and apparatus Golgi Cx31G45E. of expression (Figure in network cell the of periphery the in bundling and collapse microtubule ) 2 .

Accepted Article

uolat Cx31G45E plasma ) . In HeLa26 cells transfected with Cx31G45E the mutant protein mutant the Cx31G45E with transfected cells HeLa26 In .

1 edER E and F and E . C . )

clear regions clear

B contrast, By . Cx31 membrane in gap junction plaques junction gap in membrane the o - , with,

impact transfect 2B whether (Figure 2A and B) and 2A (Figure Supplementary Figure WT ) and nuclear envelope ( envelope nuclear and ) microtubul Figure )

resulted in resulted - -

oaie wt C4, utemr, h lclsto o C4 a te plasma the at Cx43 of localisation the furthermore, Cx43, with localised ) limited GFP cells(F GFP proteasomes . s , however, , Glycine was either deleted (delG45, deleted either was Glycine

on the spatial localisation of Cx43 3 ion , however punctate staining punctate however , mutations in HeLa cells stably ex stably cells HeLa in H h osre C3G5 phenotype Cx31G45E observed the 3 F . Each ). n HeLa in ; te itaellr raels ee lo irpe icuig h ERGIC, the including disrupted also were organelles intracellular Other

, eie (G45S, serine ),

e of HeLa cells HeLa of however, localisationat

network co hr i ws rpe i prncer ein ad n vacuole in and regions perinuclear in trapped was it where

Figure igure 1 igure the -

. localisation of of localisation the wild the uain caused mutation , all key waystations in the Cx life cycle life Cx the in waystations key all , 43

T intracellular localisation of localisation intracellular ee nrdcd o eiu 45 residue to introduced were he impact of Cx31G45E on the spatial localisation of of localisation spatial the on Cx31G45E of impact he Fgr 2 (Figure

el Cx43 cells 3 in the case of G45A of case the in

C n x14E xrsig cells expressing Cx31G45E in D

and 1 and ), Supplementary with Cx31G45E with

- the plasmathe membrane lysine(G45K, . type protein was also held in vesicular structures within structures vesicular in held also was protein type

Supplementary ad F and E D (red cells (red D pressing Cx43 or Cx26 or Cx43 pressing the wild type protein type wild the with co

- oaie extensively localised h sm mrhlgcl hne s bevd in observed as change morphological same the both Cx43 and Cx26 and Cx43 both of of ) Wild . Supplementary Cx26 - Figure

GFP and GFP

and is adominant mutation only) and G45K and Supplementary -

ye x1F co Cx31GFP type at the plasma membrane plasma the at was

Figure ). However, the nuclear pore complexnuclear pore the However, ).

wild in contrast to the stable microtubule stable the to contrast in 2

U (Figure2D

- - Cx31WT negative mutation X and Y and X - type connexins type

. . the effect was less pronounce less was effect the

and Cx31G45E in Cx31G45E and Wild x14E rdcd similar a produced Cx31G45E 3

still accumulated in vacuoles in accumulated still Figure G

(

, r rpohn (G45W, tryptophan or ), with

umrsd n Table in summarised ( ( Supplementary - - - Supplementary Figure 4 Figure Supplementary . Figure BB type Cx31 co Cx31 type mCherry (Figure mCherry ) - . We assessed t assessed We

T r residue or ) 3 hiscoincided withthe

the - were not affected by affected not were E oaie extensively localised ),

glutamine(G45Q,

Cx31G45E C expressed in the in expressed ), replaced with replaced ),

gap junction gap was evident was - - localised at localised

specific Figure he impact he Cx26 and Cx26 2A

n the in )

- and like 2 ,

E 1 A d a - - ,

This article isprotected rights by All copyright. reserved. in stress ER and UPR with associated genes of expression the in increase significant no was there that determined PCR Cx3suggestreports Previous 3.4 Cx31G45E expression is associated with necrotic cell death butdoes notinduce ER stress and Cx31 immunoprecip detected also was ~57kD of band eGFP express to transfected cells tag connexins with coated epidermal with were experiments interactions Cx31 immunoprecipitation probe further To Cx31WT. +31 GFP Cx31WT with interacted myc H31 and con+GFP+31myc while 57kD, of c HeLa protei To 3.3 evident for Cx43. wild the by plaques cytoplasm the within structures vesicular in Cx31G45E the end with

Accepted Articleobservedco the that confirm ged connexin ged

Formation of heteromeric cell and cell n ells transfected ells - rti interactions protein -

ogenously expressed Cx43, both in intracellular stores, typically around typically intracellularstores, in both Cx43, expressedogenously B contrast By . - y ad H31 and myc probed

G45E

in gap junction plaques. junction gap in a GFP antibodyGFP it , representative of the Cx31 the of representative , ated with the Cx31 constructs Cx31 the with ated can co .

for Cx43. In the case the In Cx43. for Western blot analysis of the co the of analysis blot Western

performed in HeLa cells stably expressing Cx43 (Figure 3B). (Figure Cx43 expressing stably cells HeLa in performed - type proteins, suggesting that Cx31G45E that suggesting proteins, type

assays - to express Cx31WT express to , - oligomerise

F + GFP - - o HeLa43 for myc myc - GFP input lanes input GFP

enabling coenabling , 1

- - o td te neato o C3 ad x14E ih Cx43. with Cx31G45E and Cx31 of interaction the study to myc - 6xHis ikl pull nickel Connexons EKV associatedmutation EKV G45E - localisationof . In . - 6xHis, as well as with Cx31G45E with as well as 6xHis,

- with Cx43 agd pr tagged -

Thus y lns respectively lanes myc HeLa43 el tasetd ih wild with transfected cells

- of the of immunoprecipitation with proteins associating with the GFP the proteinsassociatingimmunoprecipitationwith with - down down

- t

). Nickel pull Nickel ). - he wild he and Cx31G45E and GFP protein was detected was protein GFP HeLa cells expressing either pEGFP either expressing cells HeLa . cells transfected cells

(Figure 3B, IP: Cx43) IP: 3B, (Figure

HeLa cells HeLa Cx31G45E oteins were were oteins n immunoprecipitation and - IP assays IP - type proteins did proteins type s induce ER stress ER induce s - down assays determined that Cx31WT that determined assays down ,

transfected to express eGFP express to transfected - with wild with n the and

GFP detected eGFP (28kD eGFP detected ).

has hs x14E om canl with channels forms Cx31G45E Thus to express the express to - ~ tagged proteins expressed a protein a expressed proteins tagged 3 - 3 a dominant a ye r uat Cx31 mutant or type re . D (iue 3A (Figure kDa - - typ myc These results indicate results These

not rescue not was (Figure 3 3 (Figure e Cx31 and Cx43 represented andCx43 Cx31 e - myc

[ 27 reduced sas ee performed. were assays - , e lo performed also we CHOP/BiP 6xHis (Figure 3A (Figure 6xHis Cx31 29 effect - T B, the accumulation of accumulation the N1, Cx31W N1, ) the , he

30 from control from - IP GFP constructs a constructs GFP

omto o GJ of formation ] IgG . : GFP : ERGIC region of region ERGIC . Real . con+31myc, , This was most was This no Cx43 no

or or beads were beads , ). – that x3 co Cx43 GADD34,

T time RT time The - GFP or GFP

, H31 , T

HeLa Cx31

- hese

GFP blot was - - - This article isprotected rights by All copyright. reserved. ‘leaky function channel of loss F137L andG12D G12R, mutations Firstly mutations Cx and function present The 3. channels’ and that 18 with PI Cx31G45E expressing PI a cells expressing GFP gated, In population. control the in cells non Under to compared profile necrotic (p<0.05) Cx31. wildtype expressing significant statistically a showed cells expressing co by ~ express to transfected established induced conditions Suppl 18 of absence or presence the were t Finally, driving the observed changesER and localisation the theof protein.mutant Cx31G45E of expression that observed suggest were GADD34 or BiP of level the in changes no cells analysis of expression induced Cx31G45E 2 (bright) Accepted 5% Article Discussion

ementary associated withassociated , - of hemichannels’

ramn with treatment peak was marginally, but not significantly reduced in the Cx31G45E the in reduced significantly not but marginally, was peak mutations  o determine if determine o re ertc population necrotic cells expressing Cx31 expressing cells A Ti sget ta te bevd emaiiy o I s o de o lay connexin ‘leaky to due not is PI to permeability observed the that suggests This GA. - , vealed - F (iue 4 (Figure GFP

transfected conditions, cells subject to necrosis exhibited a PI a exhibited necrosis to subject cells conditions, transfected ~

work details the details work conditions control in 4% of the population the of 4% suggests that altered interaction with Cx43 with interaction altered that suggests soitd ih kn disease skin with associated

Figure 5 Figure no change in the level of expression of BiP of expression of level the in change no modify the Cx31G45E cells have a compromised membrane ( commonly considered as gain as considered commonly - apopto F, u ws bet rm cell from absent was but GFP, CHOP Cx31WT the gross changes occurring in cellular integrity in cells expressing cells in integrity cellular in occurring changes gross the )

18 . [

A 35

C trafficking to the plasma membrane plasma the to trafficking  . y otat tasgri, kon R stre ER known a thapsigargin, contrast, By ). Integrity of the cell membrane was membrane cell the of Integrity ells were subject were ells - GA

sis 37 by ( importance - Figure 4D 4D Figure G45E -

]  GFP or necro or 4.7 . o lc hmcanl activity hemichannel block to GA Secondly, cause the connexins to be connexinsto the cause , exhibited a necrotic profile compared to compared profile necrotic a exhibited

± - a onxn hne blocker, channel connexin a , were detected were GFP ~

0.8 6 - F de nt nue R stress ER induce not does GFP o cells of % sis

n splmnay Figure supplementary and

fold and fold were necrotic were of of ,

mu FACS analysis and analysis FACS a atr th alter can the amino acid position G45 in G45 position acid amino the ed tant proteins tant

to FACS analysis FACS to GADD34

- in the necrotic quadrant necrotic the in transfected of epesn Cx31WT expressing s - f

unction mutations unction and the effect was not significantly reduced significantly not was effect the and cnei lf cycle life connexin e is

by associated sequestered within the cytoplasmthe withinsequestered target the plasma membrane but membrane plasma the target

in HeLa cells HeLa in propidium iodide( propidium (bright) 7.2

also

with F and u and Fgr 4E) (Figure

r example or ±

Fgr 4C) (Figure

ee performed were

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This article isprotected rights by All copyright. reserved. subtype connexins of loop extracellular first the on located G45, acid other T speculations require further analysis. and signalling cellular differ and proteins Cx43/Cx31 of G45E associated co Cx43 mutation Cx31G45E HeLa both In proteins. two these of interaction cells. the HaCaT confirmed and cells HeLa43 both in contact cell to cell of regions protein two Cx43 the to capacities non were mice homozygous closure wound of rates mice deficient Cx43 of skin channels these of ability hetero functional form not do Cx43 and Cx31 that suggested Cx43 of that to profile similar a with skin the inexpressed is 31 Connexin connexins, that indicate studies loop extracellular first of part first the and domain terminal stat functional the in A keratodermapalmoplantar with associated H73R and syndrome Vohwinkel with associated are ER the 36 protein mutant the with trafficking ER the within M Keratitis disease skin inflammatory the Ichthyosis with associated Cx26G45E, including mutations, Cx26 he effect of Cx31G45E expression Cx31G45E of effect he recent report suggests that the H73R mutation may interact with Cx43 channelsCx43 with interact maymutation H73R the thatsuggestsreport recent utations introduced to introduced utations Accepted Article,

38 also non -

40 and microtubule network microtubule and

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This article isprotected rights by All copyright. reserved. Interestingly death. cell Accepteddeath cell promoted promote with associated Our event following expression theof proteinmutant network microtubule keratinization altered to obs alsoendpoint an death, cell necrotic stress ER of evidence a and and stress ER interact with t network trafficking re profound delivery membrane efficient for required consortin as such partners and trafficking Connexin maintaining C Articlelevels however reported, KID of form lethal a with associated h 32 and Cxs43 in mutation G45 of deletion including to hydrophilic a from switch a causes i while acids charge amino positive neutral to results glutamine negative and a positive induced to mutation charge G45K in The change respectively. a in result mutations G45S and G45K introduce calcium which in mutation G45E functionchannel for criticalis acidaminothis suggesting that funnel, bevtos ugs ta diverse that suggest observations re

[ could be rescued by incubating cells in >5 in cells incubating by rescued be could 48

key event key sensing d and further studies are now required to determine how heteromeric mutant channels may channels mutant heteromeric how determine to required now are studies further and d subtle d ,

. It is well established that Cx43 trafficking is highly dependent on an intact microtubular intact an on dependent highly is trafficking Cx43 that established well is It . pro 49 - localisation x31 structure ] ransport networks duction of reactive oxygen species (ROS), which (ROS), species oxygen reactive of duction a .

n unfolded protein response protein unfolded n the EKV the hs u dt cery lutae te motne f the of importance the illustrates clearly data our Thus centre

Cxs30, 32 and 43 and 32 Cxs30, s n cag i te ie f h aio aci amino the of size the in change a in changes to to changes

in inducing necrotic cell necrotic inducing in ek hemichannels leaky relies [ , 27 ,

eurd o mitiig R structure ER maintaining for required

AS nlss n P utk asy dtrie ta te uain induced mutation the that determined assays uptake PI and analysis FACS , -

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P phenotype. P the cell death phenotype was promoted when the cells were grown at low at grown were cells the when promoted was phenotype death cell the . ,

f x3 ih Cx with Cx43 of [ H deregulated on an intact microtubule network microtubule intact an on 52 . emichannel blockers or high extracellular calcium decreased the level of level the decreased calcium extracellular high or blockers emichannel

ad no effect o effect no ad ] . properties T

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Cx31 transport. Cx31 mutation , n cells expressing the mutation Cx31R42P, induced ER stress ER induced Cx31R42P, mutation the expressing cells n

] Each a erved for the other EKV otherthe for erved

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l tee mn ai changes, acid amino these All [ 46 ] lead - within 24 within . oiat fet n Cx43 on effect dominant th

Zhang et al et

lcn at glycine hemichannel activity hemichannel [ several EKV several e 50

our studies show studies our that and highly likely linkedlikely highly and o ertc el death cell necrotic to mutation , . showed that showed . i bekon f the of breakdown a te rsn study present the n 51 et al. et G45

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ed 45 G45E

and

no in is , This article isprotected rights by All copyright. reserved. differentkeratinocyte subpopulations including connexin 31. 4. 3. Chem Sohl, G.,Structural and functional diversity connexinof genesin the mouseand human genome. 2. integrity that aretherapeutic targets. 1. References versionfinal of manuscript.the JAE antibody.43 Connexin the scholarship PhD byBiology and Acknowledgements: Conflictof Interest: mechanisms of triggering thedisease state mutati the to attributed mechanism underlying the that clear EKV the suggest trans diseaseprogressionthat is associatedwithnecroticcell death. o conclusion, In assemble atplasma the membrane. trans a had and lysosomes in accumulated non with associated V174M, mutation authors mutations of function (26 temperature ur Accepted Article report

and AAand - dominant effect on the trafficking of of trafficking the on effect dominant 2002,

- Willecke,K.; Eiberger,J.; Degen, J.;Eckardt, D.; Romualdi, A.; Guldenagel, M.; Deutsc Martin, E.;P. Easton, J. A.; Hodgins, B.; M. Wright,C. S., Connexins: sensors of epidermal Di, W.Di, L.; Rugg, E. L. Laird, D. W., Life cycle connexinsof inhealth and disease. altered interactionindicativealteredsevereCxCx43be of with may P also phenotype is the firstisthe

383, (5), 725. oiat fet n h tafcig f x6 u dd o atr h aiiy f Tx1 to WTCx31 of ability the alter not did but Cx26 of trafficking the on effect dominant

wrote draft wrote report

four clinical reports clinical four the Dutch Cancer Society (KWF),grantDutch Cancer 2009 the o to AA (PEM) AA to C).

ed None to declare

ag n coll and Tang to detailto associated with this mutation is similar to other EKV other to similar is mutation this with associated hs ok a funded was work This

Cx31 that s

of the manuscript contributions with the of Author contribution: JAE contribution: Author ; ; Leigh, I. M.; Kelsell, D. P., Multipleepidermal connexins are expressed in - BiP G12D, R42P and F137L was recove was F137L and R42P G12D,

the impactthethisof mutationcellular on integrityandprovides evidence

. /Hs the ER the

We thank Dr Edward Leithe (University of Oslo) for the supply of supply the for Oslo) of (University Leithe Edward Dr thank We p eagues indicate that indicate 70

FEBS Lett

[ and 54 - ydoi dans bt ih o kn ahlg reported, pathology skin no with but deafness syndromic

]

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Cx43 28 cFos

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which, G45E J RW Sho fr nooy n Developmental and Oncology for School GROW, un , GB , B

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ahas r cnrl n EKV in central are pathways is associated with EKV with associated is s

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4352

from We alsoWeshow red by culturing cells at 27 at cells culturing by red n s ifrn, ugsig variable suggesting different, is on

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and PEM. and - and a and P mutations reported it is it reported mutations P - 2006, functional channels and channels functional that thethathasa mutation . -

P. 2001, Kuwait GovernmentKuwait To our knowledge, our To

394, (Pt3), 527.

PEM collated thePEM .

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These , Biol 43

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This article isprotected rights by All copyright. reserved. Dermatol Richard, G.,Anew,recurrent mutation of GJB3 (Cx31) inerythrokeratodermia variabilis. 21. 2011, mutation intheGJB3 gene resulting insevereerythrokeratodermia variabilis. 20. (4), 406. Erythrokeratoderma variabilis caused byrecessive a mutation inGJB3. 19. causing erythrokeratoderma variabilis. 18. erythrokeratoderma variabilis. Grabczynska, S.; Peris, Z.; Kansky, A.; Kelsell, D. P., 17. (KID) syndromewith the GJB2 mutation G45E. Denoyelle,F.; Bodemer,C.; Marlin, S.; Hadj 16. 1338. hyperactivehemichannels ichthyosis Olivero, P.; Perez 15. 721. erythrokeratodermia variabilis islethalfor HeLa cells. 14. Genet Fryns, J. P., Novel GJA1 mutations inpatients with oculo 13. focalcauses palmoplantar keratoderma with deafness. van Steensel, A.,M. A novel missense mutation in GJB2 disturbs gap junction protein transportand 12. CommunRes studies human of skin disease 11. connexin disorders. 26(D66H) mimics true Vohwinkel syndrome and providesa model for the pathogenesis of Finbow, Greenhalgh, M.; D.; Hodgins, M., Targeted epidermal expression of mutantConnexin 10. differential effects of two mutations inconnexin 32. Bennett, V.; M. Chen, L.; Sahenk, Z., Pathogenesis of X 9. differentiation. keratinocytes as an organotypic model for examining therole of Cx43 and Cx26 in skin 8. connexins during stratification of human keratinocytes. 7. analyzed with transgenic mouse mut 6. 1039. cell imaging: analysis theof roles of connexins in the epidermis. 5.

Accepted Article

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This article isprotected rights by All copyright. reserved. Accepted Article