huntingtin interacting proteins are genetic modifiers of ... · proteins [25–28]. we reasoned...

Huntingtin Interacting Proteins AreGenetic Modifiers of NeurodegenerationLinda S. Kaltenbach

1[¤, Eliana Romero

2[, Robert R. Becklin

1, Rakesh Chettier

1, Russell Bell

1, Amit Phansalkar

1,

Andrew Strand3

, Cameron Torcassi4

, Justin Savage1

, Anthony Hurlburt1

, Guang-Ho Cha2

, Lubna Ukani2

,

Cindy Lou Chepanoske1

, Yuejun Zhen1

, Sudhir Sahasrabudhe1

, James Olson3

, Cornelia Kurschner1

, Lisa M. Ellerby4

,

John M. Peltier1

, Juan Botas2*

, Robert E. Hughes1,4*

1 Prolexys Pharmaceuticals, Salt Lake City, Utah, United States of America, 2 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas,

United States of America, 3 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 4 Buck Institute for Age

Research, Novato, California, United States of America

Huntington’s disease (HD) is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract inthe huntingtin (Htt) protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of proteininteractions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration shouldbe enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors usingtwo complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by massspectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which werefound with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encodinginteracting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD.This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, asmodification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among theinteractors is an order of magnitude higher than the 1%–4% typically observed in unbiased genetic screens. Geneticmodifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/massspectrometry methods, supporting the notion that these complementary technologies are equally useful in identifyingbiologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotyperepresent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signaltransduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration,which can be considered potential targets for therapeutic intervention. Finally, we show that seven interactingproteins from among 11 tested were able to co-immunoprecipitate with full-length Htt from mouse brain. Thesestudies demonstrate that high-throughput screening for protein interactions combined with genetic validation in amodel organism is a powerful approach for identifying novel candidate modifiers of polyglutamine toxicity.

Citation: Kaltenbach LS, Romero E, Becklin RR, Chettier R, Bell R, et al (2007) Huntingtin interacting proteins are genetic modifiers of neurodegeneration. PLoS Genet 3(5): e82.doi:10.1371/journal.pgen.0030082

Introduction

Huntington’s Disease (HD) is a member of a family ofdominantly inherited neurodegenerative diseases caused byexpansion in a glutamine-encoding CAG tract. HD occurswhen the polyglutamine (polyQ) tract in huntingtin (Htt)expands beyond ;35 glutamine (Q) repeats and manifestswith movement disorder, psychological disturbances, andcognitive dysfunction progressing over a period of about tento 15 years until death. Currently there is no effectivetreatment or cure for HD.

Mutant Htt is thought to cause cellular dysfunction,neurodegeneration, and associated clinical features primarilythrough a toxic gain of function [1]. Indeed, proteinscontaining expanded polyQ tracts are toxic when expressedin a wide range of experimental transgenic systems includingyeast, cultured mammalian cells, Caenorhabditis elegans, Droso-phila, and mouse [2–4]. Determining the precise mechanism ofpolyQ-mediated toxicity is a subject of intense inquiry, andthere is evidence supporting a role for aberrant protein-protein interactions in pathogenesis. In HD, expanded Htt isprocessed to N-terminal fragments that form inclusions

found both in the cytoplasm and nucleus [5,6]. A number ofproteins localize to expanded polyQ inclusions, includingubiquitin/proteasome components, heat shock proteins, andtranscription factors [7–12]. These findings support the ideathat mutant Htt may interfere with the functions of diversecellular proteins directly, through protein interactions. Someinteracting proteins have been shown to be functionally

Editor: Harry Orr, University of Minnesota, United States of America

Received September 21, 2006; Accepted April 6, 2007; Published May 11, 2007

Copyright: � 2007 Kaltenbach et al. This is an open-access article distributedunder the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided theoriginal author and source are credited.

Abbreviations: CCT, chaperonin-containing t-complex polypeptide 1; HD, Hun-tington’s disease; Htt, huntingtin; MS, mass spectrometry; polyQ, polyglutamine; Q,glutamine; TAP, tandem affinity purification; Y2H, yeast two hybrid

* To whom correspondence should be addressed. E-mail: [email protected] (JB);[email protected] (REH)

[ These authors contributed equally to this work.

¤ Current address: Center for Drug Discovery, Duke University, Durham, NorthCarolina, United States of America

PLoS Genetics | www.plosgenetics.org May 2007 | Volume 3 | Issue 5 | e820689

compromised when bound to mutant Htt [11,13,14]. Inaddition, some of these proteins localize to insoluble Htt-fragment-containing inclusions present in affected tissues[15,16]. Recent work, however, has suggested that inclusionsmay be benign or even protective and that other misfoldedforms of Htt may be the primary toxic species [17–19]. Sinceinteractions between cellular proteins and soluble or aggre-gated Htt may have a general role in HD pathogenesis,identification of Htt-interacting proteins will further eluci-date toxic mechanisms and therapeutic targets for thedisease.

Htt is a large, ubiquitously expressed protein comprisednearly entirely of HEAT repeats, a characteristic protein-protein interaction motif [20,21]. Nearly 50 proteins capableof interacting directly with Htt or Htt fragments have beendescribed. Most proteins have been found to interact with N-terminal polyQ containing Htt fragments and, in some cases,the strength of these interactions has been shown to besensitive to the length of the polyQ tract [22,23]. Htt-interacting proteins represent diverse cellular roles includingintracellular transport, transcription, and ubiquitin-medi-ated proteolysis. These observations suggest that the normalfunction of Htt involves multiple protein-protein interac-tions in the context of diverse multiprotein cellular com-plexes. Indeed, loss of normal Htt function is a component ofHD pathology [24]. Identifying Htt protein-protein inter-actions may help to elucidate the functions of wild-type Httas well as the novel gain of function of mutant Htt.

In this study we report a large set of novel Htt-fragment-interacting proteins using yeast two-hybrid (Y2H) and affinitypull-down/mass spectrometry (MS) protein interactionscreens. We used both approaches in parallel in an effort todefine a comprehensive set of interactors, as there is evidencethat each method explores different groups of interactingproteins [25–28]. We reasoned that if expanded Htt caninfluence the functions of its interacting proteins (and viceversa), genes encoding interacting proteins should beenriched for genetic modifiers of neurotoxicity mediated byexpression of a mutant Htt fragment. We used a Drosophilamodel of polyQ toxicity to test this idea and found that 45%of the interactors behave as high-confidence genetic modi-

fiers (i.e., interaction confirmed with more than one allele).Importantly, protein interactions validated as genetic modi-fiers in Drosophila were equally represented in the Y2H andMS derived datasets, demonstrating the complementarynature of these independent methods.A standard method for validation of large-scale interaction

datasets relies on co-affinity precipitation of samples of theprotein interaction pairs [29,30]. Whereas co-affinity precip-itation does confirm a physical interaction, it does notestablish the biological relevance of that interaction. Thehigh-content validation method used in this study (geneticinteraction in a whole organism) strongly supports theconclusion that this dataset is highly enriched for interactingproteins with functional roles in polyQ-mediated neuro-degeneration. Using co-immunoprecipitation, we show fur-ther that a number of these modifier proteins physicallyassociate with Htt in brain tissue of transgenic miceexpressing full length Htt protein.An ultimate result of this study is to provide insight into

potential therapeutic targets for HD. The 17 loss-of-functionsuppressors of Drosophila HD reported here constitute asignificant collection of novel targets (and pathways) to beconsidered as targets for therapeutic intervention.

Results

Identification of Novel Htt-Fragment-Interacting ProteinsIn a comprehensive search for novel Htt-fragment-inter-

acting proteins, we performed two large-scale screens forinteractions using MS and Y2H methods (Figure 1A). Multiplefragments of Htt, including both wild-type and mutant N-terminal fragments, were cloned and expressed for pull-downexperiments and Y2H screens (Figures 1B and S1). Wepurified five recombinant Htt-fragment baits (correspondingto amino acid residues 1-90-23 Q, 1-90-48 Q, 1-90-75 Q, 443–1,100, and 2,758–3,114) from Escherichia coli in sufficientquantities for pull-down experiments. A total of 97 pull-downexperiments were performed with these Htt-fragment baitsand mammalian tissue or cell protein lysates (Figure 1).Tandem affinity purification (TAP)-tagged Htt-fragmentcontaining protein complexes were allowed to form inprotein extracts prepared from mouse or human brain tissueor mouse muscle tissue. Complexes were copurified with theaffinity tagged Htt-fragment proteins and analyzed by MS. Ofthe five Htt-fragment bait proteins, only the 1–90 aminoterminal fragments yielded specific and reproducible proteincomplexes. The wild-type and mutant Htt-fragment baits(corresponding to exon 1 of the HD gene) (Figure 1B) werealso used to probe protein extracts prepared from culturedcells (HEK293, HeLa, and M17 neuroblastoma). Using thedatabase-searching tool MASCOT, we generated a primarydataset of 1,107 unique high-scoring peptides present in theHtt-fragment pull downs (Figure 1A; Table S1). To generate ahigh-confidence interaction list, we subjected these peptidesto a statistical test for specific association with Htt fragmentsby comparison to a database of 15,131 high-scoring peptidesidentified in pull downs performed with 88 different proteinbaits (unpublished data). This analysis was used to generate ap-value for the association of a particular peptide with Htt-fragment pull downs. A total of 410 unique peptides fromHtt-fragment pull downs met a p-value limit of �0.05, andeach of these peptides was manually validated by inspection


Huntingtin Interactome

Author Summary

Huntington’s Disease (HD) is a fatal inherited neurodegenerativedisease, which typically begins in middle age and progresses withsymptoms of severe uncontrolled movements and cognitivedysfunction. HD is uniformly fatal with death occurring ten to 15years after onset of symptoms. There is currently no effectivetreatment for HD. The genetic mutation underlying HD causes aprotein called huntingtin (Htt) to contain an abnormally long tract ofthe amino acid glutamine. This extended span of glutamineschanges the shape of the Htt protein, which can cause it to interactin abnormal ways with other cellular proteins. In this study, we haveidentified a large number of new proteins that bind to normal andmutant forms of the Htt protein. To establish a potential role forthese interacting proteins in HD, we show that changing theexpression of many of these proteins can modulate the pathologicaleffects of mutant Htt on fly neurons that deteriorate when theyexpress mutant Htt. Identifying cellular proteins that bind to Htt andmodulate its pathological activity may facilitate the discovery of aneffective treatment for HD.

of the MS spectra (see detailed methods in SupportingInformation). The data were filtered further by excluding anyproteins identified by peptides observed in control pulldowns with TAP-tag alone or proteins containing anypeptides not meeting the p-value cut-off (i.e., peptides notspecific to Htt-fragment pull downs). These methods identi-

fied 145 mouse and human proteins specific to Htt-fragmentpull downs and eliminated many proteins considered to befalse positives in other studies (Tables 1 and S1) [25]. Genesencoding orthologs of 28 of these proteins were tested forgenetic interaction with a truncated mutant N-terminalhuman HD gene in a Drosophila model of polyQ toxicity.In addition to solution-based MS protein interaction

studies, we performed Y2H searches with Htt-fragment baitsusing a high-throughput automated screening platform [31].A total of 3,749 individual Y2H searches of HD fragment baitswere performed against prey libraries prepared from 17different human tissue cDNA sources (Figure 1). Multipleoverlapping baits were searched extensively but only baitslocated near the Htt N terminus (including polyQ containingfragments) gave reproducible interactions (Figure 1B, solidlines). PolyQ containing Htt-fragment baits of amino acids 1–90, 1–450 or 1–740 were screened in both wild-type (23 Q)and mutant (.45 Q) forms. Screens were performed understringent selection conditions requiring simultaneous activa-tion of two independent auxotrophic reporter genes, HIS3and ADE2 [31,32]. Initial results identified a total of 562unique interacting prey proteins (Figure 1A; Tables S2 andS3). Because Y2H screens have been estimated to contain upto 50% false positives among the primary positives [33,34],the data were filtered using stringent criteria to eliminatefalse positives and generate a high-confidence dataset. First,only interactions that had been independently observed atleast three times in Y2H screens were included. Next,proteins were excluded if they were observed to interactwith more than 174 unique partners in a database of 110,000interacting protein pairs generated from approximately290,000 Y2H screens. These searches were performed in alarge random screen for human protein interactions (un-published data). This cut-off, designed to eliminate promis-cuous interactors, was calculated by k-means clusteringanalysis of the random dataset [31]. Finally, genes encodinginteracting preys were recovered from positive yeast colonies,sequenced twice, and reintroduced with Htt-fragment baitinto naive Y2H assay cells. Genes were excluded if theinteraction with Htt fragments could not be reproduced inthe Y2H assay (as measured by activation of two reportergenes). Previously published Htt and Htt-fragment interac-tors were included in the final list regardless of the exclusioncriteria. A total of 104 unique interacting proteins (18% ofthe primary dataset) met these conditions and were includedin the final high-confidence dataset (Table 2). A complete listof all Htt-fragment interactions found in our Y2H screens isshown in Table S2. Sequences derived from all positivecolonies used to identify the interacting proteins arepresented in Table S3. Orthologs of 35 of these genes weretested for genetic interactions with a mutant human N-terminal portion of the HD gene in a Drosophila model ofpolyQ toxicity.While more than 3,500 searches were performed with Htt

fragments, after 800 searches the rate of discovery for novelinteracting proteins approached zero, indicating that thesescreens were close to saturation (Figure S2). This resultdemonstrates that the total number of Htt-fragment-inter-acting proteins discovered in our Y2H screens represents afinite set and is not simply a function of the number ofsearches.Examining the gene ontology annotations associated with

Figure 1. Results of the Physical Interaction Screens

(A) Overview of the discovery workflow is represented. Y2H and pull-down/MS workflows are shown on the left and right, respectively. Thenumber of Htt-fragment baits used for Y2H searches or pull downsincludes wild-type (23 Q) and mutant (48 Q and 55 Q) forms. Not all Httfragments were successfully expressed in bacteria or yielded positiveinteractions in Y2H screens. For Y2H positive prey identification, the topblast score was chosen. The total number of genes found in pull down/MS includes 15 mouse and human homologs; the nonredundant setdoes not include mouse homologs (see Supporting Information).(B) A diagram of Htt baits used in Y2H and MS experiments is presented.Structural features (HEAT repeat domains and protease cleavage region)are indicated by shaded boxes on a diagram of the Htt protein. Numbersindicate reference amino acids positions (with respect to NP_002102).Lines representing Htt-fragment baits with associated amino acidpositions indicated by numbers are shown relative to the diagram ofHtt. We purified three Htt-fragments (top panel) from bacteria insufficient quantities for pull-down/MS experiments. Htt-fragment baitsused in Y2H screens are shown on the bottom panel. Some baits did notyield positive interactions (dotted lines). Htt clones that contained thepolyQ sequence were generated in wild-type (23 Q) and expanded (55 Q,75 Q, and 97 Q) forms (asterisk).(C) A functional analysis of Htt-fragment-interacting proteins ispresented. The number of proteins representing the indicated functionalcategories found in Htt-fragment Y2H screens (white bars) or pull down/MS (black bars) are shown. Proteins were assigned to categories basedon gene ontology. Only categories with more than one protein assignedare shown.doi:10.1371/journal.pgen.0030082.g001



Ta

ble

1.

Sele

cte

dP

rote

ins

Ide

nti

fie

din

Pu

llD

ow

ns

wit

hH

ttFr

agm

en

ts

Fu

nct

ion

al

Ca

teg

ory

Ge

ne

Ide

nti

fica

tio

nN

am

eR

efS

eq

GI

Nu

mb

er

Va

lid

ate

d

Pe

pti

de

s

Tis

sue

Cy

tosk

ele

ton

org

an

iza

tio

n

an

db

iog

en

esi

s;tr

aff

ick

ing

BA

P3

1_

HU

MA

NB

-ce

llre

cep

tor-

asso

ciat

ed

pro

tein

31

NP

_0

05

73

6.3

32

17

11

86

4H

EK2

93

BA

SP_

HU

MA

Nb

rain

abu

nd

ant,

me

mb

ran

eat

tach

ed

sig

nal

pro

tein

1N

P_

00

63

08

.33

07

95

23

17

,1

HB

,M

17

CLH

1_

HU

MA

Ncl

ath

rin

,h

eav

ych

ain

NP

_0

04

85

0.1

47

58

01

21

,4

HB

,H

EK2

93

DY

HC

_H

UM

AN

dyn

ein

,cy

top

lasm

ic1

,h

eav

ych

ain

1N

P_

00

13

67

.23

33

50

93

22

HEK

29

3

E41

L1_

HU

MA

Ne

ryth

rocy

tem

em

bra

ne

pro

tein

ban

d4

.1-l

ike

1N

P_

03

62

88

.23

24

90

57

22

HB

GFA

P_

HU

MA

Ng

lial

fib

rilla

ryac

idic

pro

tein

NP

_0

02

04

6.1

a1

62

65

83

62

HB

Q5

TB

35

_H

UM

AN

ery

thro

cyte

me

mb

ran

ep

rote

inb

and

4.1

NP

_9

76

21

8.1

42

71

62

89

1H

B

SEP

T7

_H

UM

AN

sep

tin

7N

P_

00

17

79

.24

50

26

95

1H

B

SPT

A2

_M

OU

SEal

ph

a-sp

ect

rin

2,

bra

inX

P_

62

28

87

.1a

23

61

84

16

1M

B

TP

M1

_M

OU

SEtr

op

om

yosi

n1

,al

ph

aN

P_

07

77

45

.22

05

22

24

01

MM

Pro

tein

turn

ov

er;

pro

tein

fold

ing

HN

RP

C_

HU

MA

Nh

ete

rog

en

eo

us

nu

cle

arri

bo

nu

cle

op

rote

inC

NP

_0

04

49

1.1

13

93

78

88

3H

EK2

93

SRP

09

_H

UM

AN

sig

nal

reco

gn

itio

np

arti

cle

9kD

aN

P_

00

31

24

.11

83

14

56

51

M1

7

STIP

1_

HU

MA

Nst

ress

-in

du

ced

-ph

osp

ho

pro

tein

1(H

sp7

0/H

sp9

0-o

rgan

izin

gp

rote

in)

NP

_0

06

81

0.1

58

03

18

12

HEK

93

TC

PQ

_H

UM

AN

chap

ero

nin

con

tain

ing

TC

P1

,su

bu

nit

8(t

he

ta)

NP

_0

06

57

6.2

60

05

72

73

HEK

29

3

TC

PZ

_H

UM

AN

chap

ero

nin

con

tain

ing

TC

P1

,su

bu

nit

6A

(ze

ta1

)N

P_

00

17

53

.14

50

26

43

1H

EK2

93

Ion

cha

nn

els

;p

um

ps

AT

1B

1_

MO

USE

AT

Pas

e,

Naþ

/Kþ

tran

spo

rtin

g,

be

ta1

po

lyp

ep

tid

eN

P_

03

38

51

.16

75

31

38

1M

B

AT

PD

_M

OU

SEA

TP

syn

thas

e,

Hþ

tran

spo

rtin

g,

mit

och

on

dri

alF1

com

ple

x,d

elt

asu

bu

nit

NP

_0

79

58

9.1

21

53

62

20

2M

M

AT

PO

_M

OU

SEA

TP

syn

thas

e,

Hþ

tran

spo

rtin

g,

mit

och

on

dri

alF1

com

ple

x,O

sub

un

itN

P_

61

30

63

.12

00

70

41

21

MM

CA

C2

D_

MO

USE

calc

ium

chan

ne

l,vo

ltag

e-d

ep

en

de

nt,

alp

ha2

/de

lta

sub

un

it1

NP

_0

33

91

4.1

67

53

23

42

MM

CM

C1

_H

UM

AN

bso

lute

carr

ier

fam

ily2

5(m

ito

cho

nd

rial

carr

ier,

Ara

lar)

,m

em

be

r1

2N

P_

00

36

96

.22

13

61

10

31

HB

CM

C1

_M

OU

SEso

lute

carr

ier

fam

ily2

5(m

ito

cho

nd

rial

carr

ier,

Ara

lar)

,m

em

be

r1

2N

P_

76

60

24

.12

73

69

58

17

MM

EAA

2_

MO

USE

solu

teca

rrie

rfa

mily

1(g

lial

hig

haf

fin

ity

glu

tam

ate

tran

spo

rte

r),

me

mb

er

2N

P_

03

55

23

.17

10

64

09

1M

B

M2

OM

_M

OU

SEso

lute

carr

ier

fam

ily2

5(m

ito

cho

nd

rial

carr

ier

oxo

glu

tara

teca

rrie

r),

me

mb

er

11

NP

_0

77

17

3.1

21

31

29

94

1M

M

MP

CP

_M

OU

SEso

lute

carr

ier

fam

ily2

5(m

ito

cho

nd

rial

carr

ier,

ph

osp

hat

eca

rrie

r),

me

mb

er

3N

P_

59

84

29

.11

95

26

81

81

MM

Q8

VD

L1_

MO

USE

citr

ate

syn

thas

eN

P_

08

07

20

.11

33

85

94

22

MM

SFX

N3

_M

OU

SEsi

de

rofl

exi

n3

NP

_4

44

42

7.1

16

71

64

99

1M

B

VD

AC

1_

HU

MA

Nb

volt

age

-de

pe

nd

en

tan

ion

chan

ne

l1

NP

_0

03

36

5.1

45

07

87

93

,3

HB

;H

EK2

93

VD

AC

1_

MO

USE

volt

age

-de

pe

nd

en

tan

ion

chan

ne

l1

NP

_0

35

82

4.1

67

55

96

37

MM

VD

AC

2_

HU

MA

Nb

volt

age

-de

pe

nd

en

tan

ion

chan

ne

l2

NP

_0

03

36

6.2

a1

17

25

54

5H

B

VD

AC

2_

MO

USE

volt

age

-de

pe

nd

en

tan

ion

chan

ne

l2

NP

_0

35

82

5.1

67

55

96

51

MM

VD

AC

3_

HU

MA

Nvo

ltag

e-d

ep

en

de

nt

anio

nch

ann

el

3N

P_

00

56

53

.3a

57

33

50

42

HB

Sig

na

ltr

an

sdu

ctio

n;

rece

pto

rs

14

33

B_

HU

MA

Nb

tyro

sin

e3

-mo

no

oxy

ge

nas

e/t

ryp

top

han

5-m

on

oo

xyg

en

ase

acti

vati

on

pro

tein

,

gam

ma

po

lyp

ep

tid

e

NP

_0

36

61

1.2

21

46

41

01

3,1

HB

,M

17

14

33

G_

MO

USE

3-m

on

oo

xyg

en

ase

/try

pto

ph

an5

-mo

no

oxy

ge

nas

eac

tiva

tio

np

rote

in,

gam

ma

po

ly-

pe

pti

de

NP

_0

61

35

9.2

92

56

64

6;

31

54

39

76

2,

1M

B,

MM

DIR

A2

_H

UM

AN

bD

IRA

Sfa

mily

,G

TP

-bin

din

gR

AS-

like

2N

P_

06

00

64

.22

17

03

36

71

HB

DIR

A2

_M

OU

SED

IRA

Sfa

mily

,G

TP

-bin

din

gR

AS-

like

2N

P_

00

10

19

64

53

24

49

87

31

MM

DP

YL2

_H

UM

AN

dih

ydro

pyr

imid

inas

e-l

ike

2N

P_

00

13

77

.12

17

50

95

61

HB

GB

B1

_H

UM

AN

gu

anin

en

ucl

eo

tid

eb

ind

ing

pro

tein

(Gp

rote

in),

be

tap

oly

pe

pti

de

1N

P_

00

20

65

.11

13

21

58

57

HB

GB

B2

_H

UM

AN

bg

uan

ine

nu

cle

oti

de

bin

din

gp

rote

in(G

pro

tein

),b

eta

po

lyp

ep

tid

e2

NP

_0

05

26

4.2

20

35

75

29

3H

B

GB

B2

_M

OU

SEg

uan

ine

nu

cle

oti

de

bin

din

gp

rote

in,

be

ta2

NP

_0

34

44

2.1

13

93

73

91

2M

B

GN

AO

1_

HU

MA

Ng

uan

ine

nu

cle

oti

de

bin

din

gp

rote

in(G

pro

tein

),al

ph

aac

tiva

tin

gac

tivi

typ

oly

pe

p-

tid

eO

NP

_0

66

26

8.1

10

56

78

16

3H

B

GN

AO

1_

MO

USE

gu

anin

en

ucl

eo

tid

eb

ind

ing

pro

tein

,al

ph

ao

NP

_0

34

43

8.1

67

54

01

23

MB

GN

AZ

_H

UM

AN

gu

anin

en

ucl

eo

tid

eb

ind

ing

pro

tein

(Gp

rote

in),

alp

ha

zp

oly

pe

pti

de

NP

_0

02

06

4.1

a4

50

40

51

3H

B

ITP

R1

_M

OU

SEin

osi

tol

1,4

,5-t

rip

ho

sph

ate

rece

pto

r1

NP

_0

34

71

5.1

67

54

39

01

MB

LAN

C1

_H

UM

AN

Lan

Cla

nti

bio

tic

syn

the

tase

com

po

ne

nt

C-l

ike

1N

P_

00

60

46

.15

17

44

45

1H

B



Ta

ble

1.

Co

nti

nu

ed

.

Fu

nct

ion

al

Ca

teg

ory

Ge

ne

Ide

nti

fica

tio

nN

am

eR

efS

eq

GI

Nu

mb

er

Va

lid

ate

d

Pe

pti

de

s

Tis

sue

Q6

NV

X7

_H

UM

AN

ne

uro

pla

stin

NP

_0

36

56

0.1

a6

91

26

46

4H

B

PH

B2

_H

UM

AN

pro

hib

itin

2N

P_

00

92

04

.16

00

58

54

1H

EK2

93

RA

C1

_H

UM

AN

ras-

rela

ted

C3

bo

tulin

um

toxi

nsu

bst

rate

1(r

ho

fam

ily,

smal

lG

TP

bin

din

gp

rote

in

Rac

1)

NP

_0

08

83

9.2

a9

84

55

11

4H

B

RA

C1

_M

OU

SER

AS-

rela

ted

C3

bo

tulin

um

sub

stra

te1

NP

_0

33

03

3.1

45

59

29

34

2M

B

Ce

lla

dh

esi

on

AO

C3

_M

OU

SEam

ine

oxi

das

e,

cop

pe

rco

nta

inin

g3

NP

_0

33

80

5.1

67

53

06

62

MM

CA

D1

3_

MO

USE

cad

he

rin

13

NP

_0

62

68

1.1

97

89

90

51

MM

CD

36

_M

OU

SEC

D3

6an

tig

en

NP

_0

31

66

9.2

31

98

24

74

1M

M

CN

TN

1_

HU

MA

Nco

nta

ctin

1N

P_

00

18

34

.2a

28

37

31

17

27

,1

HB

,M

17

GP

M6

A_

HU

MA

Ng

lyco

pro

tein

M6

AN

P_

00

52

68

.11

41

49

61

91

HB

LSA

MP

_H

UM

AN

limb

icsy

ste

m-a

sso

ciat

ed

me

mb

ran

ep

rote

inN

P_

00

23

29

.2a

23

29

19

2H

B

NT

RI_

HU

MA

Nn

eu

rotr

imin

NP

_0

57

60

6.1

a7

70

54

13

2H

B

OP

CM

_H

UM

AN

op

ioid

bin

din

gp

rote

in/c

ell

adh

esi

on

mo

lecu

le-l

ike

NP

_0

02

53

6.1

45

05

50

51

HB

FBN

2_

HU

MA

Nfi

bri

llin

2(c

on

ge

nit

alco

ntr

actu

ral

arac

hn

od

acty

ly)

NP

_0

01

99

0.1

a4

50

71

91

1H

B

SHP

S1_

HU

MA

Nsi

gn

al-r

eg

ula

tory

pro

tein

alp

ha

NP

_5

42

97

0.1

18

42

69

11

1H

B

Sy

na

pti

ctr

an

smis

sio

nC

N3

7_

HU

MA

Nb

29,

39-

cycl

icn

ucl

eo

tid

e39

ph

osp

ho

die

ste

rase

NP

_1

49

12

4.2

14

91

64

81

2H

B

CN

37

_M

OU

SEcy

clic

nu

cle

oti

de

ph

osp

ho

die

ste

rase

1N

P_

03

40

53

.16

75

34

76

5M

B

MY

PR

_H

UM

AN

bp

rote

olip

idp

rote

in1

(Pe

lizae

us-

Me

rzb

ach

er

dis

eas

e,

spas

tic

par

aple

gia

2,

un

com

-

plic

ate

d)

NP

_0

00

52

4.3

a1

90

08

52

HB

MY

PR

_M

OU

SEp

rote

olip

idp

rote

in(m

yelin

)1

NP

_0

35

25

3.1

23

95

60

58

4M

B

RA

B3

A_

MO

USE

RA

B3

D,

me

mb

er

RA

So

nco

ge

ne

fam

ilyN

P_

11

40

80

.2a

66

79

59

32

MB

SC6

A1

_H

UM

AN

solu

teca

rrie

rfa

mily

6(n

eu

rotr

ansm

itte

rtr

ansp

ort

er,

GA

BA

),m

em

be

r1

NP

_0

03

03

3.2

a2

17

07

90

81

HB

SNP

25

_H

UM

AN

syn

apto

som

al-a

sso

ciat

ed

pro

tein

,2

5kD

aN

P_

57

08

24

.1a

18

76

57

35

4H

B

SNP

25

_M

OU

SEsy

nap

toso

mal

-ass

oci

ate

dp

rote

in2

5N

P_

03

55

58

.16

75

55

88

1M

B

STX

1A

_H

UM

AN

syn

taxi

n1

A(b

rain

)N

P_

00

45

94

.14

75

91

82

3H

B

STX

1B

_H

UM

AN

bsy

nta

xin

1B

2N

P_

44

31

06

.1a

16

41

83

79

11

HB

STX

1C

_M

OU

SEsy

nta

xin

1B

2N

P_

07

77

25

.1a

13

25

93

78

7M

B

STX

B1

_H

UM

AN

bsy

nta

xin

bin

din

gp

rote

in1

NP

_0

03

15

6.1

a4

50

72

97

18

HB

STX

B1

_M

OU

SEsy

nta

xin

bin

din

gp

rote

in1

NP

_0

33

32

1.1

66

78

17

91

2M

B

SYN

1_

MO

USE

syn

apsi

nII

NP

_0

38

70

9.1

a8

56

74

10

1M

B

SYP

H_

HU

MA

Nsy

nap

top

hys

inN

P_

00

31

70

.12

77

64

86

71

HB

SYT

1_

MO

USE

syn

apto

tag

min

IN

P_

03

33

32

.16

67

81

97

1M

B

SYT

2_

HU

MA

Nsy

nap

tota

gm

inII

NP

_7

96

37

6.2

31

54

36

70

1H

B

SYT

2_

MO

USE

syn

apto

tag

min

IIN

P_

03

33

33

.23

15

43

79

72

MB

Me

tab

oli

smA

CA

DL_

HU

MA

Nac

yl-C

oe

nzy

me

Ad

eh

ydro

ge

nas

e,

lon

g-c

hai

nN

P_

03

14

07

.23

19

82

52

01

MM

ALD

OA

_M

OU

SEal

do

lase

1,

Ais

ofo

rmN

P_

03

14

64

.16

67

15

39

4M

M

ALD

OC

_H

UM

AN

ald

ola

seC

,fr

uct

ose

-bis

ph

osp

hat

eN

P_

00

51

56

.14

88

50

63

1H

B

AM

PD

1_

HU

MA

Nad

en

osi

ne

mo

no

ph

osp

hat

ed

eam

inas

e1

(iso

form

M)

iso

form

1X

P_

13

11

03

.33

80

76

93

16

MM

ASP

H_

HU

MA

Nb

asp

arta

teb

eta

-hyd

roxy

lase

NP

_0

04

30

9.2

14

58

98

66

1H

eLa

CA

H4

_H

UM

AN

carb

on

ican

hyd

rase

IVN

P_

00

07

08

.14

50

25

19

1H

B

CO

X2

_H

UM

AN

mit

och

on

dri

ally

en

cod

ed

cyto

chro

me

co

xid

ase

IIN

P_

53

68

46

.11

79

81

85

62

,1H

B,

M1

7

CO

X3

_H

UM

AN

mit

och

on

dri

ally

en

cod

ed

cyto

chro

me

co

xid

ase

IIIN

P_

53

68

49

.11

79

81

85

91

HB

CO

X5

A_

HU

MA

Nb

cyto

chro

me

co

xid

ase

sub

un

itV

aN

P_

00

42

46

.1a

18

99

93

92

1H

B

CO

X5

A_

MO

USE

cyto

chro

me

co

xid

ase

,su

bu

nit

Va

NP

_0

31

77

3.1

66

80

98

61

MM

CO

X5

B_

HU

MA

Ncy

toch

rom

ec

oxi

das

esu

bu

nit

Vb

NP

_0

01

85

3.2

17

01

79

88

4H

B

CO

X6

B_

HU

MA

Ncy

toch

rom

ec

oxi

das

esu

bu

nit

Vib

po

lyp

ep

tid

e1

(ub

iqu

ito

us)

NP

_0

01

85

4.1

45

02

98

52

HB

CO

X6

C_

HU

MA

Ncy

toch

rom

ec

oxi

das

esu

bu

nit

VIc

NP

_0

04

36

5.1

a2

14

43

67

4H

B

CP

T1

B_

MO

USE

carn

itin

ep

alm

ito

yltr

ansf

era

se1

b,

mu

scle

NP

_0

34

07

8.1

a1

42

75

78

31

MM

CX

6A

1_

HU

MA

Ncy

toch

rom

ec

oxi

das

esu

bu

nit

VIa

po

lyp

ep

tid

e1

NP

_0

04

36

4.2

17

99

95

28

1H

B



Ta

ble

1.

Co

nti

nu

ed

.

Fu

nct

ion

al

Ca

teg

ory

Ge

ne

Ide

nti

fica

tio

nN

am

eR

efS

eq

GI

Nu

mb

er

Va

lid

ate

d

Pe

pti

de

s

Tis

sue

CY

1_

HU

MA

Ncy

toch

rom

ec-

1N

P_

00

19

07

.22

13

59

86

71

HB

DLD

H_

MO

USE

dih

ydro

lipo

amid

ed

eh

ydro

ge

nas

eN

P_

03

18

87

.23

19

82

85

62

MM

ENO

A_

HU

MA

Ne

no

lase

1,

(alp

ha)

NP

_0

01

41

9.1

45

03

57

15

HEK

29

3

ENO

B_

MO

USE

en

ola

se3

,b

eta

mu

scle

NP

_0

31

95

9.1

66

79

65

14

MM

ETFB

_M

OU

SEe

lect

ron

tran

sfe

rrin

gfl

avo

pro

tein

,b

eta

po

lyp

ep

tid

eN

P_

08

09

71

.23

81

42

46

01

MM

HB

B1

_M

OU

SEh

em

og

lob

in,

be

taad

ult

maj

or

chai

nN

P_

03

22

46

.23

19

82

30

01

MM

HC

D2

_H

UM

AN

hyd

roxy

acyl

-Co

en

zym

eA

de

hyd

rog

en

ase

,ty

pe

IIN

P_

00

44

84

.11

77

83

55

1M

17

IMM

T_

MO

USE

inn

er

me

mb

ran

ep

rote

in,

mit

och

on

dri

al(m

ito

filin

)N

P_

00

68

30

.1a

29

42

76

92

5M

M

JPH

2_

MO

USE

jun

cto

ph

ilin

2N

P_

06

75

41

.11

09

47

01

01

MM

KC

RM

_M

OU

SEcr

eat

ine

kin

ase

,m

usc

leN

P_

03

17

36

.16

67

17

62

5M

M

KP

YM

_M

OU

SEp

yru

vate

kin

ase

,m

usc

leN

P_

03

52

29

.23

19

81

56

21

MM

LDH

A_

MO

USE

lact

ate

de

hyd

rog

en

ase

AN

P_

03

48

29

.16

75

45

24

2M

M

LDH

B_

HU

MA

Nla

ctat

ed

eh

ydro

ge

nas

eA

-lik

e6

AN

P_

65

94

09

.22

14

50

65

71

HEK

29

3

LDH

B_

MO

USE

lact

ate

de

hyd

rog

en

ase

BN

P_

03

25

18

.16

67

86

74

2M

M

MD

HC

_H

UM

AN

mal

ate

de

hyd

rog

en

ase

1,

NA

D(s

olu

ble

)N

P_

03

26

44

.23

19

82

17

82

MM

MT

X1

_M

OU

SEm

eta

xin

2N

P_

05

80

84

.23

15

43

27

41

MM

NB

6M

_H

UM

AN

NA

DH

de

hyd

rog

en

ase

(ub

iqu

ino

ne

)1

alp

ha

sub

com

ple

x,1

3N

P_

05

70

49

.32

13

61

82

21

HB

NI2

M_

HU

MA

NN

AD

Hd

eh

ydro

ge

nas

e(u

biq

uin

on

e)

1b

eta

sub

com

ple

x,9

,2

2kD

aN

P_

00

49

96

.16

27

45

50

2H

B

NID

M_

HU

MA

NN

AD

Hd

eh

ydro

ge

nas

e(u

biq

uin

on

e)

1b

eta

sub

com

ple

x,1

0,

22

kDa

NP

_0

04

53

9.1

47

58

77

44

HB

NIP

M_

HU

MA

NN

AD

Hd

eh

ydro

ge

nas

e(u

biq

uin

on

e)

Fe-S

pro

tein

5,

15

kDa

(NA

DH

-co

en

zym

eQ

re-

du

ctas

e)

NP

_0

04

54

3.1

47

58

79

01

HB

NU

AM

_H

UM

AN

bN

AD

Hd

eh

ydro

ge

nas

e(u

biq

uin

on

e)

Fe-S

pro

tein

1,

75

kDa

(NA

DH

-co

en

zym

eQ

re-

du

ctas

e)

NP

_0

04

99

7.4

a3

35

19

47

55

HB

NU

AM

_M

OU

SEN

AD

Hd

eh

ydro

ge

nas

e(u

biq

uin

on

e)

Fe-S

pro

tein

1N

P_

66

34

93

.12

17

04

02

01

MM

NU

BM

_H

UM

AN

NA

DH

de

hyd

rog

en

ase

(ub

iqu

ino

ne

)fl

avo

pro

tein

1,

51

kDa

NP

_0

09

03

4.2

20

14

95

68

1H

B

NU

EM_

HU

MA

Nb

NA

DH

de

hyd

rog

en

ase

(ub

iqu

ino

ne

)1

alp

ha

sub

com

ple

x,9

,3

9kD

aN

P_

00

49

93

.16

68

17

64

1H

B

NU

EM_

MO

USE

NA

DH

de

hyd

rog

en

ase

(ub

iqu

ino

ne

)1

alp

ha

sub

com

ple

x,9

NP

_0

79

63

4.1

13

38

47

20

1M

B

NU

GM

_H

UM

AN

NA

DH

de

hyd

rog

en

ase

(ub

iqu

ino

ne

)Fe

-Sp

rote

in3

,3

0kD

a(N

AD

H-c

oe

nzy

me

Qre

-

du

ctas

e)

NP

_0

04

54

2.1

47

58

78

84

,1H

B,

HEK

29

3

NU

HM

_M

OU

SEN

AD

Hd

eh

ydro

ge

nas

e(u

biq

uin

on

e)

flav

op

rote

in2

XP

_1

28

72

5.4

38

08

27

50

1M

M

NU

PM

_M

OU

SEN

AD

Hd

eh

ydro

ge

nas

e(u

biq

uin

on

e)

1al

ph

asu

bco

mp

lex,

8N

P_

08

09

79

.12

13

12

01

21

MM

OD

O2

_M

OU

SEd

ihyd

rolip

oam

ide

S-su

ccin

yltr

ansf

era

se(E

2co

mp

on

en

to

f2

-oxo

-glu

tara

teco

m-

ple

x)

NP

_0

84

50

1.1

21

31

35

36

1M

M

PP

BT

_M

OU

SEal

kalin

ep

ho

sph

atas

e2

,liv

er

NP

_0

31

45

7.1

66

71

53

31

MM

PR

IO_

HU

MA

Np

rio

np

rote

in(p

27

–3

0)

(Cre

utz

feld

t-Ja

kob

dis

eas

e,

Ge

rstm

ann

-Str

ausl

er-

Sch

ein

ker

syn

dro

me

,fa

tal

fam

ilial

inso

mn

ia)

NP

_0

00

30

2.1

45

06

11

32

HB

Q5

U5

Y5

_M

OU

SEh

ydro

xyac

yl-C

oe

nzy

me

Ad

eh

ydro

ge

nas

e/3

-ke

toac

yl-C

oe

nzy

me

Ath

iola

se/e

no

yl-

Co

en

zym

eA

hyd

rata

se(t

rifu

nct

ion

alp

rote

in),

alp

ha

sub

un

it

NP

_8

49

20

9.1

33

85

98

11

6M

M

Q8

K2

G8

_M

OU

SEd

ihyd

rolip

oam

ide

S-ac

ety

ltra

nsf

era

se(E

2co

mp

on

en

to

fp

yru

vate

de

hyd

rog

en

ase

com

ple

x)

NP

_6

63

58

9.2

31

54

25

59

1M

M

Q9

EQ6

5_

MO

USE

asp

arta

te-b

eta

-hyd

roxy

lase

NP

_0

75

55

3.1

a1

27

46

44

81

MM

TP

IS_

MO

USE

trio

sep

ho

sph

ate

iso

me

rase

1N

P_

03

34

41

.16

67

84

13

1M

M

TP

P1

_H

UM

AN

trip

ep

tid

ylp

ep

tid

ase

IN

P_

00

03

82

.35

72

97

70

1H

B

UC

RH

_H

UM

AN

ub

iqu

ino

l-cy

toch

rom

ec

red

uct

ase

hin

ge

pro

tein

NP

_0

05

99

5.1

51

74

74

51

HB

UQ

CR

1_

HU

MA

Nu

biq

uin

ol-

cyto

chro

me

cre

du

ctas

eco

rep

rote

inI

NP

_0

03

35

6.2

45

07

84

16

HB

UQ

CR

1_

MO

USE

ub

iqu

ino

l-cy

toch

rom

ec

red

uct

ase

core

pro

tein

1N

P_

07

96

83

.21

33

84

79

43

MM

UQ

CR

2_

HU

MA

Nb

ub

iqu

ino

l-cy

toch

rom

ec

red

uct

ase

core

pro

tein

IIN

P_

00

33

57

.2a

21

90

34

82

4H

B

UQ

CR

2_

MO

USE

ub

iqu

ino

lcy

toch

rom

ec

red

uct

ase

core

pro

tein

2N

P_

08

01

75

.12

22

67

44

22

MM

Nu

cle

ar

fun

ctio

nD

DB

1_

HU

MA

Nd

amag

e-s

pe

cifi

cD

NA

bin

din

gp

rote

in1

,1

27

kDa

NP

_0

01

91

4.2

13

43

53

59

3H

EK2

93



interacting proteins reveals that the two methods differ tosome degree in the type of proteins identified (Figure 1C).Y2H clearly identified more proteins involved in proteinturnover, signal transduction, and transcription, while MSidentified more proteins involved in metabolic processes.However, proteins involved in cytoskeletal or protein-trafficking processes were similarly represented among theY2H and MS data. Overall, there was little overlap of specificinteracting proteins between the two datasets. Only fourhigh-confidence proteins were found using both methods:clathrin, pyruvate kinase, GAPDH, and YWHAB (Tables S1and S2). Two of these, clathrin and GAPDH have beenpreviously reported to associate with Htt fragments [35,36].To directly address the biological relevance of the Htt-

fragment protein-interaction dataset and to assess therelative validity of results generated using the Y2H and MSmethods, we tested a sample of interacting proteins in a high-content independent method, a genetic modifier assay in a flymodel of polyQ toxicity.

Validation of Htt-Fragment-Interacting Proteins in aDrosophila Model of PolyQ ToxicityAn arbitrary sample of 60 proteins in the dataset was

tested for the ability to modify an Htt-fragment-inducedneurodegeneration phenotype in Drosophila. This polyQtoxicity model was generated using an N-terminal fragmentof the human HD cDNA, encoding the first 336 amino acidsof the protein, including a 128 Q expansion in exon 1 (seeMaterials and Methods). Directed expression of this ex-panded human HD transgene fragment in the Drosophila eyecauses a neurodegenerative phenotype evident by externalexamination and retinal histology. Of the 234 nonredundantmammalian protein interactors found in the MS and Y2Hscreens, 213 had apparent orthologs in Drosophila (unidirec-tional top hit with BLAST score less than 10�3), and 127 ofthese had available Drosophila stocks suitable for screening.We tested 60 of these, divided roughly equally between genesdiscovered using Y2H (35) and MS (28) methods (includingthree genes found in common), for possible geneticinteractions in the fly model of polyQ toxicity (Table S4). Atotal of 48 of the 60 genes in the sample (80%) eitherenhanced or suppressed the expanded Htt-fragment-inducedneurodegeneration in the Drosophila eye when tested ineither over-expressing or in partial loss-of-function strains(Tables 3 and S5). In some cases a modifier effect wasobserved, but only one background strain could be tested(Table S5). However, for 27 of these genes, modificationeither by more than one allele or in more than one geneticbackground was observed. These genes comprise a high-confidence set of genetic modifiers of mutant Htt-fragmenttoxicity (Figures S3 and S4; Table 3). The 27 high-confidencemodifiers represent a 45% validation rate among thoseinteractors tested. Since the collection of genes tested in thefly assay represented an arbitrary sample of the proteininteraction collection, this result indicates that as much ashalf of the proteins in our dataset may be modifiers ofmutant Htt toxicity. The hit rate for genetic modifiers seenamong our interactors is an order of magnitude higher thanthe expected 1%–4% typically observed in unbiased geneticscreens [37–39], including a comparable modifier screenusing a Drosophila model of the polyQ disease spinocerebellarataxia type 1 [40]. Validation rates for proteins discovered byT

ab

le1

.C

on

tin

ue

d.

Fu

nct

ion

al

Ca

teg

ory

Ge

ne

Ide

nti

fica

tio

nN

am

eR

efS

eq

GI

Nu

mb

er

Va

lid

ate

d

Pe

pti

de

s

Tis

sue

EF1

D_

HU

MA

Ne

uka

ryo

tic

tran

slat

ion

elo

ng

atio

nfa

cto

r1

de

lta

(gu

anin

en

ucl

eo

tid

ee

xch

ang

e

pro

tein

)

NP

_0

01

95

1.2

a2

54

53

47

21

HEK

29

3,

M1

7

PT

RF_

MO

USE

po

lym

era

seI

and

tran

scri

pt

rele

ase

fact

or

NP

_0

33

01

2.1

66

79

56

71

MM

Q6

PIA

5_

MO

USE

pro

line

-ric

hp

oly

pe

pti

de

6X

P_

20

33

93

.21

28

51

67

91

MB

Oth

er;

un

kn

ow

nC

D5

9_

HU

MA

NC

D5

9m

ole

cule

,co

mp

lem

en

tre

gu

lato

ryp

rote

inN

P_

00

06

02

.1a

64

02

98

4H

B

CH

CH

2_

HU

MA

Nco

iled

-co

il-h

elix

-co

iled

-co

il-h

elix

do

mai

nco

nta

inin

g2

NP

_0

57

22

3.1

77

05

85

11

HEK

29

3

CH

CH

2_

MO

USE

coile

d-c

oil-

he

lix-c

oile

d-c

oil-

he

lixd

om

ain

con

tain

ing

2N

P_

07

71

28

.21

80

79

33

42

MM

CH

CH

3_

MO

USE

coile

d-c

oil-

he

lix-c

oile

d-c

oil-

he

lixd

om

ain

con

tain

ing

3N

P_

07

96

12

.12

13

13

61

84

MM

LC7

L2_

MO

USE

LUC

7-l

ike

2(S

.ce

revi

sia

e)N

P_

61

96

21

.12

03

73

16

71

MB

MO

G_

MO

USE

mye

lino

ligo

de

nd

rocy

teg

lyco

pro

tein

NP

_0

34

94

4.1

67

54

72

01

MB

NEG

R1

_H

UM

AN

ne

uro

nal

gro

wth

reg

ula

tor

1N

P_

77

61

69

.22

77

54

17

41

HB

O8

84

92

_M

OU

SEp

lasm

am

em

bra

ne

asso

ciat

ed

pro

tein

,S3

–1

2N

P_

06

55

93

.11

01

81

20

44

MM

TH

Y1

_H

UM

AN

Th

y-1

cell

surf

ace

anti

ge

nN

P_

00

62

79

.21

99

23

36

23

HB

Hu

man

cell

line

su

sed

we

reH

EK2

93

,H

eLa

,an

dM

17

ne

uro

bla

sto

ma.

aA

dd

itio

nal

GI

nu

mb

ers

we

rem

atch

ed

(no

tsh

ow

n).

bH

um

anp

rote

inw

ho

sem

ou

sep

rote

inh

om

olo

g(d

ete

rmin

edb

yH

OM

OLO

GEN

E,N

CB

I)w

asal

sofo

un

din

pu

lld

ow

ns

(se

eSu

pp

ort

ing

Info

rmat

ion

).H

B,

hu

man

bra

in;

MB

,m

ou

seb

rain

;M

M,

mo

use

mu

scle

.d

oi:1

0.1

37

1/j

ou

rnal

.pg

en

.00

30

08

2.t

00

1



either Y2H (27/35 or 77%) or MS (21/28 or 75%) methodswere similar, indicating that these methods are comparablein their ability to uncover biologically relevant interactions(Table 3). These relative validation rates demonstrate furtherthat the MS and Y2H datasets are complementary in natureand that each dataset is similarly enriched for genes andproteins that modify mutant Htt toxicity in vivo. Further-more, the majority of these modifiers were discovered ininteraction screens performed with human brain proteinextracts or brain-derived cDNA libraries indicating that theyare expressed in tissues relevant to HD (Tables 1 and 2).

Among the 27 high-confidence modifiers, partial loss-of-function mutations were tested for 27 of them and over-expression mutations for nine. A total of 18 of the modifiersbehaved as suppressors of neurodegeneration, (14 by partialloss-of-function and four by over-expression) (Figure S3),whereas 18 behaved as enhancers (13 by partial loss-of-function and five by over-expression) (Figure S4). In all 13cases where both over-expression and loss-of-function alleleswere tested, suppression was observed in one condition andenhancement in the other. These modifiers cluster intoseveral functional groups including proteins involved incytoskeletal organization and biogenesis, signal transduction,synaptic transmission, proteolysis, and regulation of tran-scription or translation (Table 3). Histological analysis of eyephenotypes from representative enhancers and suppressorsfrom each of these groups is shown (Figure 2).

One interesting subset of modifiers is a group of proteinsinvolved in SNARE-mediated vesicle fusion [41,42]. Thisincludes STX1A, NAPA, and the voltage-gated calciumchannel delta subunit CACNA2D1. Interestingly, allelesencoding all of these proteins act both as loss-of-functionsuppressors and gain-of-function enhancers in the fly assay.Collectively, these modifier results point toward a model ofHtt toxicity involving dysregulation in synaptic function atthe level of SNARE-mediated vesicle fusion.

Additional experiments were performed to further validatea role for a SNARE component in modifying mutant Htttoxicity (Figure 3). In contrast to expression in the eye, pan-neural expression of N-terminal expanded Htt leads to ashortened lifespan in the fly model of polyQ toxicity. Pan-neural expression also results in late-onset progressive motordysfunction that can be quantified in terms of climbingperformance as a function of age. These behavioral assaysconfirm the results obtained in the eye assay: partial loss-of-function of STX1A ameliorates both the disorganization andfusion of ommatidia seen in flies expressing the gene thatencodes N-terminal expanded Htt as well as the retinaldegeneration. The shortened life-span and the late-onsetprogressive motor dysfunction phenotypes were also im-proved by a partial loss-of-function of STX1A, confirmingthat the modifier effects seen in the eye were not limited to aparticular phenotypic assay (Figure 3B and 3C). Htt is knownto interact with proteins involved in endocytosis and vesicletrafficking such as PACSIN1, HAP1, HIP1, and HIP14 [22],however, this is the first report showing that Htt interactsdirectly with the SNARE complex and that partial loss-of-function can suppress mutant Htt toxicity.

A network summarizing interactions relevant to Htt andproteins with gene ontology annotations (http://www.geneontology.org) related to vesicle traffic and/or neuro-transmission is shown in Figure 4. Included here are Y2H

interactions (rectangles and thick lines) and proteins identi-fied by MS (ovals) in pull downs using lysates prepared frommouse and/or human brain tissue (set included in dottedcircle). A total of 11 proteins in this interaction subnetwork(shown in red) are encoded by human orthologs of genesshown to act as modifiers in the Drosophila model of polyQtoxicity (Tables 3 and S3). Notably, several modifiers arepresent in a highly connected cluster of Htt-fragment-interacting proteins known to function in receptor-mediatedendocytosis: CLTC, AP2A2, AP2B1, PACSIN1, and DNM1.The observations that Htt is localized to endosomal vesiclesand associated with clathrin in fibroblasts derived from HDpatients [5,43] and that vesicle associated proteins are foundin Htt-fragment inclusions [44] makes this interconnectedcluster of modifiers particularly striking. Curated Htt-frag-ment-interacting proteins obtained from BioGRID (http://www.thebiogrid.org) [45] and/or the Human Protein Refer-ence Database (http://www.hprd.org) [46,47] are included inthe network. These bridging proteins (blue triangles) repre-sent all curated interactions contained in these databases thatconnect HD to at least one other protein in the subnetworkthough a single protein node and link some of our novel Y2Hinteractions and MS associations to known Htt-interactingproteins (e.g., HIP1, GIT1). Together, this interaction networkprovides additional proof that our dataset is enriched forproteins that are important in HD pathogenesis and under-scores the role of proteins involved in vesicle traffic as beingrelevant to HD function and pathology.

Validation of Htt-Fragment-Interacting Proteins UsingImmunoprecipitationFor further in vivo validation of Htt-fragment protein

interactions in mammalian tissue, we performed co-immu-noprecipitation experiments from brains of wild-type miceand mice expressing a 128 Q full length YAC transgene [48].Figure 5 shows the results of co-immunoprecipitation experi-ments using antibodies raised against Htt-fragment-interact-ing proteins. In all, we observed co-immunoprecipitationwith seven of 11 interacting proteins tested. These includedthe SNARE-associated proteins STX1A and CACNA2D1, bothof which are modifiers in the Drosophila assay. We alsoobserved co-immunoprecipitation with SNAP25 (anotherSNARE component). Other modifiers observed to associatewith Htt in mouse brain were the ubiquitin hydrolase USP9Xand the proteasome component PSMC2. None of theseinteracting proteins appeared to show a strong preferencefor wild-type versus CAG expanded Htt in this assay.Immunoprecipitation using antibodies directed againstGAPDH and PARP are included as positive and negativecontrols. We observed a polyQ length-dependent associationof GAPDH with Htt. The GAPDH protein has been reportedto bind Htt and act as a modifier of mutant Htt toxicity[36,49].Overall, in this sample, we observed a 60% validation rate

in this assay (seven of 11 proteins tested). Of the sevenproteins observed to co-immunoprecipitate with Htt frommouse brain, three were discovered using Y2H (CUL2,PSMC2, and USP9X), two were discovered using MS (STX1Aand SNAP25), and one by both methods (PKM2). This furtherunderscores a specific utility of both methods for discovery ofinteracting proteins.



Ta

ble

2.

Sele

cte

dP

rote

ins

Inte

ract

ing

wit

hH

ttFr

agm

en

tsin

aY

2H

Ass

ay

Fu

nct

ion

al

Ca

teg

ory

Ge

ne

Sy

mb

ol

Ge

ne

Na

me

Ge

ne

IDB

ait

aN

um

be

ro

f

Po

siti

ve

s

Po

lyQ

Pre

fere

nce

Re

lati

ve

Str

en

gth

cDN

A

Lib

rary

Ap

op

tosi

s/ce

lld

ea

thA

PI5

apo

pto

sis

inh

ibit

or

58

53

91

–4

50

(55

Q)

32

3Q

þþþþ

BN

MTU

S1m

ito

cho

nd

rial

tum

or

sup

pre

sso

r1

57

50

91

–4

50

(55

Q)

3¼

þþ

PN

OP

TNb

op

tin

eu

rin

10

13

31

–4

50

(23

Q);

1–

45

0(5

5Q

)2

7¼

þþ

BR

,B

N,

BB

,B

H,

FL,

LK

Pro

tein

fold

ing

;

pro

tein

turn

ov

er

CU

L2cu

llin

28

45

39

–1

55

a3

N/D

N/D

BR

CU

L5cu

llin

58

06

51

–4

50

(23

Q)

6¼

þP

N

DN

AJA

3D

naJ

(Hsp

40

)h

om

olo

g,

sub

fam

ilyA

,m

em

be

r3

90

93

1–

45

0(2

3Q

);1

–4

50

(55

Q)

14

¼þþ

SC,

BR

DN

AJA

5D

naJ

ho

mo

log

ysu

bfa

mily

Am

em

be

r5

13

42

18

1–

45

0(2

3Q

);1

–4

50

(55

Q)

23

¼þþ

BB

,SC

,FB

,LK

,B

R,

PR

DN

AJC

4D

naJ

(Hsp

40

)h

om

olo

g,

sub

fam

ilyC

,m

em

be

r4

33

38

1–

45

0(2

3Q

);1

–4

50

(55

Q)

20

23

Qþþ

MM

,B

R,

PN

DN

AJC

11D

naJ

(Hsp

40

)h

om

olo

g,

sub

fam

ilyC

,m

em

be

r1

15

57

35

1–

45

0(2

3Q

);1

–4

50

(55

Q)

11

23

Qþ

MM

,LK

,P

N

HIP

2a

,bh

un

tin

gti

nin

tera

ctin

gp

rote

in2

30

93

37

0–

58

0a

4¼

�B

N,

SC

HSP

D1

he

atsh

ock

60

kDa

pro

tein

1(c

hap

ero

nin

)3

32

91

–7

40

(23

Q)

3¼

þ/�

AP

MK

RN

2m

ako

rin

,ri

ng

fin

ge

rp

rote

in,

22

36

09

1–

45

0(2

3Q

);1

–4

50

(55

Q)

82

3Q

þþþþ

BB

,B

R,

LG

NY

REN

18n

eg

ativ

ere

gu

lato

ro

fu

biq

uit

in-l

ike

pro

tein

s1

51

66

71

–4

50

(23

Q);

1–

45

0(5

5Q

)1

32

3Q

þþ

MM

PSM

D4

pro

teas

om

e(p

roso

me

,m

acro

pai

n)

26

Ssu

bu

nit

,

no

n-A

TP

ase

,4

57

10

1–

45

0(2

3Q

)6

23

Qþþ

BR

,FB

RN

F20

rin

gfi

ng

er

pro

tein

20

56

25

41

–9

0(7

5Q

);1

–4

50

(23

Q)

3¼

þþþþ

FB

RN

F40

rin

gfi

ng

er

pro

tein

40

98

10

1–

45

0(2

3Q

);1

–4

50

(55

Q)

6¼

þþþþ

MM

,B

R,

PN

UB

AD

C1

ub

iqu

itin

asso

ciat

ed

do

mai

nco

nta

inin

g1

10

42

21

–4

50

(23

Q);

1–

45

0(5

5Q

)1

12

3Q

þB

R,B

H,

BB

,P

R

USP

9Xu

biq

uit

insp

eci

fic

pe

pti

das

e9

,X

-lin

ked

82

39

1–

90

(23

Q);

1–

45

0(2

3Q

);1

–7

40

(23

Q);

1–

74

0(5

5Q

);6

40

-11

00

71

¼þþ

BR

,M

M,

BH

,B

N

WB

P4

WW

do

mai

nb

ind

ing

pro

tein

4

(fo

rmin

bin

din

gp

rote

in2

1)

11

19

31

–4

50

(23

Q);

1–

45

0(5

5Q

)1

12

3Q

þC

O,

BR

,P

R

Cy

tosk

ele

tal

org

an

iza

tio

n

an

db

iog

en

esi

s

AD

D3

add

uci

n3

(gam

ma)

12

01

–4

50

(23

Q)

32

3Q

þþ

LK

CLA

SP1

cyto

pla

smic

linke

ras

soci

ate

dp

rote

in1

23

33

21

–9

0(2

3Q

);1

–9

0(7

5Q

);1

–4

50

(55

Q)

4¼

þFB

DC

TN2

dyn

acti

n2

(p5

0)

10

54

01

–4

50

(23

Q);

1–

45

0(5

5Q

)5

9¼

þþþ

BR

,LK

,B

H,

BN

,FL

,FB

DN

ALI

1b

dyn

ein

,ax

on

em

al,

ligh

tin

term

ed

iate

chai

n1

78

02

1–

45

0(5

5Q

)1

23

Qþ

BR

DN

CH

1d

yne

in,

cyto

pla

smic

1,

he

avy

chai

n1

17

78

1–

45

0(2

3Q

);1

–4

50

(55

Q);

64

0-1

10

06

¼þþþþ

PR

,M

L

DN

M2

dyn

amin

21

78

51

–4

50

(55

Q)

3¼

þC

O,

MM

HA

X1

HC

LS1

asso

ciat

ed

pro

tein

X-1

10

45

61

–4

50

(23

Q);

1–

45

0(5

5Q

)7

¼þþ

PN

,M

M

HIP

1b

hu

nti

ng

tin

inte

ract

ing

pro

tein

13

09

21

–4

50

(23

Q);

1–

45

0(5

5Q

)1

0¼

þþþþ

MM

,M

L

PP

Lp

eri

pla

kin

54

93

1–

45

0(2

3Q

);1

–4

50

(55

Q)

4¼

þB

R,

CO

TMEM

57tr

ansm

em

bra

ne

pro

tein

57

55

21

91

–4

50

(23

Q)

32

3Q

þB

R

VIL

2vi

llin

2(e

zrin

)7

43

01

–4

50

(55

Q);

1–

74

0(5

5Q

)6

23

Qþþ

MM

Intr

ace

llu

lar

tra

nsp

ort

CO

PB

coat

om

er

pro

tein

com

ple

x,su

bu

nit

be

ta1

13

15

1–

45

0(5

5Q

)3

¼þþ

CO

,P

R,

ML

PA

CSI

N1

bp

rote

inki

nas

eC

and

case

inki

nas

e

sub

stra

tein

ne

uro

ns

1

29

99

31

–9

0(4

7Q

);1

–9

0(7

5Q

)7

N/D

N/D

BH

SOR

BS1

sorb

inan

dSH

3d

om

ain

con

tain

ing

11

05

80

1–

90

(23

Q);

1–

90

(47

Q);

1–

45

0(2

3Q

);1

–4

50

(55

Q)

22

23

Qþþ

BN

,B

B,

SC,

PR

Me

tab

oli

smG

AP

DH

bg

lyce

rald

eh

yde

-3-p

ho

sph

ate

de

hyd

rog

en

ase

25

97

1–

45

0(5

5Q

)1

N/D

N/D

FB

P4H

A1

pro

colla

ge

n-p

rolin

e,

2-o

xog

luta

rate

4-d

ioxy

ge

nas

e(p

rolin

e4

-hyd

roxy

lase

),

alp

ha

po

lyp

ep

tid

eI

50

33

1–

90

(23

Q);

1–

90

(47

Q);

1–

90

(75

Q);

1–

45

0(2

3Q

);

1–

45

0(5

5Q

);1

–7

40

(55

Q);

64

0-1

10

0

25

¼N

/DB

H,

FB,

BB

,B

N,

LK

PK

M2

pyr

uva

teki

nas

e,

mu

scle

53

15

1–

45

0(2

3Q

);1

–4

50

(55

Q);

1–

74

0(5

5Q

);6

40

-11

00

25

55

Qþþ

MM

,M

L,A

P



Ta

ble

2.

Co

nti

nu

ed

.

Fu

nct

ion

al

Ca

teg

ory

Ge

ne

Sy

mb

ol

Ge

ne

Na

me

Ge

ne

IDB

ait

aN

um

be

ro

f

Po

siti

ve

s

Po

lyQ

Pre

fere

nce

Re

lati

ve

Str

en

gth

cDN

A

Lib

rary

PP

A2

pyr

op

ho

sph

atas

e(i

no

rgan

ic)

22

70

68

1–

45

0(2

3Q

);1

–4

50

(55

Q);

1–

74

0(5

5Q

)4

¼þ

MM

Sig

na

ltr

an

sdu

ctio

n;

rece

pto

rs

AP

BB

2am

ylo

idb

eta

(A4

)p

recu

rso

rp

rote

in-b

ind

ing

,

fam

ilyB

,m

em

be

r2

(Fe

65

-lik

e)

32

31

–4

50

(23

Q);

1–

45

0(5

5Q

);

1–

74

0(5

5Q

)

4¼

þþþ

BR

,P

N

AR

HG

AP

24R

ho

GT

Pas

eac

tiva

tin

gp

rote

in2

48

34

78

1–

90

(23

Q);

1–

90

(75

Q);

1–

90

(97

Q);

1–

45

0(2

3Q

);

1–

45

0(5

5Q

);1

–7

40

(23

Q);

1–

74

0(5

5Q

);6

40

-11

00

18

23

Qþþþ

LG,

PN

,M

L

AR

HG

AP

25R

ho

GT

Pas

eac

tiva

tin

gp

rote

in2

59

93

81

–4

50

(23

Q);

1–

74

0(2

3Q

)3

¼þ

AP

,C

O

BA

IAP

2B

AI1

-ass

oci

ate

dp

rote

in2

10

45

81

–4

50

(55

Q);

1–

90

(23

Q)

14

55

Qþ

MM

HR

MT1

L1p

rote

inar

gin

ine

me

thyl

tran

sfe

rase

23

27

51

–4

50

(55

Q)

4¼

þþþþ

PR

,B

H

OST

F1o

ste

ocl

ast

stim

ula

tin

gfa

cto

r1

26

57

81

–9

0(2

3Q

);1

–9

0(4

7Q

);

1–

90

(75

Q);

1–

45

0(2

3Q

);

1–

45

0(5

5Q

)

10

12

3Q

þþþ

BR

,B

N,

FL,

LK

PIK

3R1

ph

osp

ho

ino

siti

de

-3-k

inas

e,

reg

ula

tory

sub

un

it1

(p8

5al

ph

a)

52

95

1–

45

0(2

3Q

);1

–4

50

(55

Q)

12

3¼

þB

R,

BH

,B

N,

BB

,FB

,LK

PIK

3R2

Ph

osp

ho

ino

siti

de

-3-k

inas

e,

reg

ula

tory

sub

un

it2

(p8

5b

eta

)

52

96

1–

45

0(2

3Q

);1

–4

50

(55

Q)

23

¼þþþ

BR

,B

H,

BN

,B

B,

FB,

LK

PIK

3R3

ph

osp

ho

ino

siti

de

-3-k

inas

e,

reg

ula

tory

sub

un

it3

(p5

5,

gam

ma)

85

03

1–

45

0(2

3Q

);1

–4

50

(55

Q)

45

¼þþþþ

BR

,B

H,

BN

,B

B,

FB,

LK

PR

KC

BP

1p

rote

inki

nas

eC

bin

din

gp

rote

in1

23

61

31

–4

50

(23

Q)

1¼

N/D

SC

PTK

6P

TK

6p

rote

inty

rosi

ne

kin

ase

65

75

31

–4

50

(55

Q)

52

3Q

þM

M

SRG

AP

1SL

IT-R

OB

OR

ho

GT

Pas

eac

tiva

tin

gp

rote

in1

57

52

21

–9

0(2

3Q

);1

–9

0(4

7Q

);

1–

45

0(2

3Q

);1

–4

50

(55

Q);

64

0-1

10

0

18

¼þþþþ

SC,

BB

,P

N,

FB

SRG

AP

2SL

IT-R

OB

OR

ho

GT

Pas

eac

tiva

tin

gp

rote

in2

23

38

01

–4

50

(23

Q);

1–

45

0(5

5Q

)6

23

Qþþþ

BB

,B

R,

LG

SRG

AP

3SL

IT-R

OB

OR

ho

GT

Pas

eac

tiva

tin

gp

rote

in3

99

01

1–

90

(23

Q);

1–

45

0(2

3Q

);1

–4

50

(55

Q)

9¼

þþþ

MM

,B

B

TAN

KT

RA

Ffa

mily

me

mb

er-

asso

ciat

ed

NFK

Bac

tiva

tor

10

01

01

–4

50

(23

Q);

1–

45

0(5

5Q

)1

1¼

þþþþ

MM

,LK

,P

R

YW

HA

Bty

rosi

ne

3-m

on

oo

xyg

en

ase

/try

pto

ph

an

5-m

on

oo

xyg

en

ase

acti

vati

on

pro

tein

,

be

tap

oly

pe

pti

de

75

29

1–

90

(23

Q);

1–

90

(75

Q);

1–

90

(97

Q);

1–

45

0(5

5Q

)

12

¼þ

BB

,FB

Nu

cle

ocy

top

lasm

ic

tra

nsp

ort

KP

NA

3ka

ryo

ph

eri

nal

ph

a3

(im

po

rtin

alp

ha

4)

38

39

1–

90

(23

Q);

1–

45

0(2

3Q

);1

–4

50

(55

Q);

1–

74

0(2

3Q

);1

–7

40

(55

Q)

12

55

Qþþ

ML

NU

PL1

nu

cle

op

ori

nlik

e1

98

18

1–

45

0(2

3Q

);1

–4

50

(55

Q)

9¼

þþþþ

PR

,B

B,

ML

Sy

na

pti

ctr

an

smis

sio

nA

P2A

2b

Ad

apto

r-re

late

dp

rote

inco

mp

lex

2,

alp

ha

2su

bu

nit

16

11

–4

50

(55

Q)

2¼

þþþ

MM

DN

M1

dyn

amin

11

75

91

–4

50

(23

Q);

1–

45

0(5

5Q

)4

¼þ

CO

,B

H,

BN

JAK

MIP

1ja

nu

ski

nas

ean

dm

icro

tub

ule

inte

ract

ing

pro

tein

1

15

27

89

1–

45

0(2

3Q

);1

–4

50

(55

Q)

7¼

þþþþ

BR

,B

B,

BH

,B

N

NA

PB

N-e

thyl

mal

eim

ide

-se

nsi

tive

fact

or

atta

chm

en

tp

rote

in,

be

ta

63

90

81

–4

50

(23

Q)

32

3Q

þB

R,

BH

Re

gu

lati

on

of

tra

nsc

rip

tio

n/

tra

nsl

ati

on

;sp

lici

ng

;

reco

mb

ina

tio

n

BA

Z1A

bro

mo

do

mai

nad

jace

nt

tozi

nc

fin

ge

rd

om

ain

,1

A

11

17

71

–9

0(9

7Q

);1

–4

50

(23

Q);

1–

45

0(5

5Q

);1

–7

40

(23

Q)

92

3Q

þþ

PR

,SC

BZ

W2

bas

icle

uci

ne

zip

pe

ran

dW

2d

om

ain

s2

28

96

91

–4

50

(55

Q)

2¼

þþ

MM

C20

orf

178

chro

mat

inm

od

ifyi

ng

pro

tein

4B

12

88

66

1–

90

(23

Q);

1–

90

(47

Q);

1–

90

(75

Q);

1–

90

(97

Q);

64

0-1

10

0

22

¼þ

/�B

H

CB

Pb

CR

EBb

ind

ing

pro

tein

(Ru

bin

ste

in-T

ayb

i

syn

dro

me

)

13

87

1–

45

0(2

3Q

)1

N/D

N/D

LK



Ta

ble

2.

Co

nti

nu

ed

.

Fu

nct

ion

al

Ca

teg

ory

Ge

ne

Sy

mb

ol

Ge

ne

Na

me

Ge

ne

IDB

ait

aN

um

be

ro

f

Po

siti

ve

s

Po

lyQ

Pre

fere

nce

Re

lati

ve

Str

en

gth

cDN

A

Lib

rary

CTN

NB

1ca

ten

in(c

adh

eri

n-a

sso

ciat

ed

pro

tein

),

be

ta1

,8

8kD

a

14

99

1–

45

0(2

3Q

);1

–4

50

(55

Q)

11

¼þþþþ

BH

,B

B,

FB,

LK,

PR

EHM

T1e

uch

rom

atic

his

ton

e-l

ysin

e

N-m

eth

yltr

ansf

era

se1

79

81

31

–4

50

(55

Q)

15

5Q

þþþþ

BR

GTF

3C3

ge

ne

ral

tran

scri

pti

on

fact

or

IIIC

,

po

lyp

ep

tid

e3

,1

02

kDa

93

30

1–

45

0(5

5Q

)4

¼þþ

SC,

BN

,M

L

PR

PF4

0A(H

YP

A)b

PR

P4

0p

re-m

RN

Ap

roce

ssin

gfa

cto

r

40

ho

mo

log

A(y

eas

t)

55

66

01

–9

0(2

3Q

);1

–9

0(4

7Q

);

1–

90

(75

Q);

1–

90

(97

Q);

1–

45

0(2

3Q

);1

–4

50

(55

Q);

1–

74

0(2

3Q

);1

–7

40

(55

Q);

44

3-1

10

0;

64

0-1

10

0

15

47

¼a

þþþþ

PR

,FB

,B

R

HY

PB

(SET

D2)

bSE

Td

om

ain

con

tain

ing

22

90

72

1–

90

(23

Q);

1–

90

(75

Q);

1–

90

(97

Q);

1–

45

0(2

3Q

);

1–

45

0(5

5Q

)

37

¼þ

BR

,B

B,

BH

,FL

LDO

C1

leu

cin

ezi

pp

er,

do

wn

-re

gu

late

din

can

cer

12

36

41

1–

45

0(2

3Q

);1

–4

50

(55

Q)

44

¼þþ

BN

,B

B,

BH

,P

N,

PR

MEF

2DM

AD

Sb

ox

tran

scri

pti

on

en

han

cer

fact

or

2,

po

lyp

ep

tid

eD

(myo

cyte

en

han

cer

fact

or

2D

)

42

09

1–

45

0(2

3Q

);1

–4

50

(55

Q)

22

3Q

�B

R

NC

OR

1b

nu

cle

arre

cep

tor

co-r

ep

ress

or

19

61

11

–4

50

(23

Q);

1–

45

0(5

5Q

)9

¼þþ

PR

,P

N,

MM

NFk

B1

bn

ucl

ear

fact

or

of

kap

pa

ligh

tp

oly

pe

pti

de

ge

ne

en

han

cer

inB

-ce

lls1

(p1

05

)

47

90

1–

45

0(2

3Q

);1

–4

50

(55

Q);

1–

74

0(2

3Q

);4

43

-11

00

95

5Q

þþ

LK,

PR

PC

QA

P(p

osi

tive

cofa

cto

r2

,m

ult

ipro

tein

com

ple

x)

glu

tam

ine

/Q-r

ich

-ass

oci

ate

dp

rote

in

51

58

61

–4

50

(23

Q);

1–

45

0(5

5Q

)4

23

Qþþ

BR

,M

M,

PR

PP

AR

Gp

ero

xiso

me

pro

life

rato

r-ac

tiva

ted

rece

pto

rg

amm

a

54

68

1–

45

0(2

3Q

);1

–4

50

(55

Q)

26

¼þþ

MM

,LG

,FL

,C

O

SP3

Sp3

tran

scri

pti

on

fact

or

66

70

1–

45

0(5

5Q

);1

–7

40

(55

Q);

44

3-1

10

0;

64

0-1

10

0

9N

/DN

/DB

R

SREB

F2st

ero

lre

gu

lato

rye

lem

en

tb

ind

ing

tran

scri

pti

on

fact

or

2

67

21

1–

45

0(2

3Q

);1

–4

50

(55

Q);

64

0-1

10

0

19

23

Qþ

BR

,P

R

SUP

T5H

sup

pre

sso

ro

fT

y5

ho

mo

log

(S.

cere

visi

ae)

68

29

1–

45

0(5

5Q

)3

¼þ

CO

TCER

G1b

tran

scri

pti

on

elo

ng

atio

nre

gu

lato

r1

10

91

51

–9

0(2

3Q

);1

–9

0(4

7Q

);

1–

90

(97

Q);

1–

45

0(2

3Q

);

1–

45

0(5

5Q

);1

–7

40

(23

Q);

1–

74

0(5

5Q

);6

40

-11

00

16

2¼

þþþ

BR

,LK

,B

H,

BB

,FL

,FB

TIEG

2(K

LF11

)K

rup

pe

l-lik

efa

cto

r1

18

46

21

–4

50

(55

Q);

1–

74

0(5

5Q

)7

¼þ

AP

TIZ

(ZN

F675

)zi

nc

fin

ge

rp

rote

in6

75

17

13

92

1–

45

0(2

3Q

);1

–4

50

(55

Q)

31

6¼

þB

R

ZN

F655

(VIK

)zi

nc

fin

ge

rp

rote

in6

55

79

02

71

–4

50

(55

Q)

3¼

þþþ

BB

ZN

F133

zin

cfi

ng

er

pro

tein

13

37

69

21

–4

50

(23

Q);

1–

45

0(5

5Q

)4

23

Qþ

BR

ZB

TB16

zin

cfi

ng

er

and

BT

Bd

om

ain

con

tain

ing

16

77

04

1–

74

0(5

5Q

)3

¼þ

BR

ZN

F91

zin

cfi

ng

er

pro

tein

91

76

44

1–

45

0(2

3Q

);1

–4

50

(55

Q)

7¼

þþþþ

FB

Ce

llcy

cle

MR

E11A

MR

E11

me

ioti

cre

com

bin

atio

n1

1

ho

mo

log

A(S

.ce

revi

sia

e)

43

61

1–

45

0(2

3Q

);1

–4

50

(55

Q)

23

23

Qþþþþ

FB,

LK,

PR

,M

M,

ML

TAC

C1

tran

sfo

rmin

g,

acid

icco

iled

-co

il

con

tain

ing

pro

tein

1

68

67

1–

45

0(2

3Q

);1

–4

50

(55

Q)

6¼

þþþ

LK,

BH

,P

R

Oth

er

or

un

kn

ow

nA

RS2

AR

S2p

rote

in5

15

93

1–

45

0(2

3Q

);1

–4

50

(55

Q)

19

¼þþ

PR

,M

M,

ML

C13

orf

24ch

rom

oso

me

13

op

en

read

ing

fram

e2

41

04

64

1–

45

0(2

3Q

);1

–4

50

(55

Q)

4¼

þþþ

BB

,B

R,

ML

DO

CK

11d

ed

icat

or

of

cyto

kin

esi

s1

11

39

81

81

–9

0(2

3Q

);1

–9

0(7

5Q

)4

55

Qa

þC

O

DO

CK

9d

ed

icat

or

of

cyto

kin

esi

s9

23

34

81

–4

50

(55

Q);

1–

74

0(5

5Q

);

64

0-1

10

0

10

55

Qa

þþþþþ

SC,

LG



Ta

ble

2.

Co

nti

nu

ed

.

Fu

nct

ion

al

Ca

teg

ory

Ge

ne

Sy

mb

ol

Ge

ne

Na

me

Ge

ne

IDB

ait

aN

um

be

ro

f

Po

siti

ve

s

Po

lyQ

Pre

fere

nce

Re

lati

ve

Str

en

gth

cDN

A

Lib

rary

FEZ

1b

fasc

icu

lati

on

and

elo

ng

atio

n

pro

tein

zeta

1(z

ygin

I)

96

38

1–

45

0(5

5Q

)6

¼þ

BB

,B

H

FLJ1

3386

cen

tro

som

alp

rote

in6

3kD

a8

02

54

1–

45

0(2

3Q

)5

23

Qþþþ

BR

FNB

P4

form

inb

ind

ing

pro

tein

42

33

60

1–

90

(23

Q);

1–

90

(47

Q);

1–

90

(75

Q);

1–

90

(97

Q);

1–

45

0(2

3Q

);1

–4

50

(55

Q);

1–

74

0(5

5Q

);6

40

-11

00

17

5¼

þB

N,

SC,

FB,

BB

,P

N,

LK

HSP

C04

9H

SPC

04

9p

rote

in2

90

62

1–

45

0(5

5Q

)4

23

Qa

þþþþ

SC

HY

PK

bH

un

tin

gti

nin

tera

ctin

gp

rote

inK

25

76

41

–4

50

(55

Q)

1N

/DN

/DB

B

KIA

A04

23K

IAA

04

23

23

11

61

–4

50

(23

Q);

1–

45

0(5

5Q

)1

02

3Q

þþ

BR

,B

B

KIA

A12

12K

IAA

12

12

55

70

41

–4

50

(23

Q)

62

3Q

þþ

BB

KIA

A12

29(O

DF2

L)o

ute

rd

en

sefi

be

ro

fsp

erm

tails

2-l

ike

57

48

91

–4

50

(23

Q);

1–

45

0(5

5Q

)4

23

Qþ

BH

,P

R

TXN

DC

11th

iore

do

xin

do

mai

nco

nta

inin

g1

15

10

61

1–

45

0(2

3Q

)5

23

Qþ

BH

SASH

1SA

Man

dSH

3d

om

ain

con

tain

ing

12

33

28

1–

45

0(2

3Q

);1

–4

50

(55

Q)

82

3Q

þþ

PR

,LG

TRA

FD1

TR

AF-

typ

ezi

nc

fin

ge

rd

om

ain

con

tain

ing

11

09

06

1–

45

0(2

3Q

);1

–4

50

(55

Q)

7¼

þþ

BR

,LK

,P

N,

LG

WA

CW

Wd

om

ain

con

tain

ing

adap

tor

wit

hco

iled

-co

il

51

32

21

–9

0(2

3Q

);1

–9

0(4

7Q

);

1–

90

(75

Q);

1–

45

0(2

3Q

);

1–

45

0(5

5Q

);6

40

-11

00

59

23

Q�

BB

,FB

,B

R,

FB,

PR

,LK

ZFY

VE1

9zi

nc

fin

ge

r,FY

VE

do

mai

nco

nta

inin

g1

98

49

36

1–

45

0(2

3Q

);1

–4

50

(55

Q)

52

3Q

þþþþ

SC,

PR

ZN

F537

(TSH

Z3)

teas

hir

tfa

mily

zin

cfi

ng

er

35

76

16

1–

45

0(2

3Q

);1

–4

50

(55

Q)

42

3Q

þþþþ

MM

Ge

ne

sym

bo

l,g

en

en

ame

,an

dg

en

eID

are

fro

mN

CB

I.R

ela

tive

affi

nit

yw

asin

ferr

ed

by

me

asu

rem

en

to

fb

eta

-gal

acto

sid

ase

acti

vity

inM

ille

rU

nit

s(M

U)

fro

mliq

uid

cult

ure

s(s

ee

Mat

eri

als

and

Me

tho

ds)

.Are

lati

vesc

ale

was

use

das

follo

ws:þ/�,

,5

0M

Uan

dva

riab

le;þ

,0–

50

MU

;þþ,

50

–1

00

MU

;þþþ

,10

0–

20

0M

U;þþþþ,

.2

00

MU

.aH

Db

aits

are

liste

das

amin

oac

ids

coo

rdin

ate

sre

lati

veto

NP

_0

02

10

2w

ith

two

exc

ep

tio

ns:

CU

L2w

asa

bai

tth

atin

tera

cte

dw

ith

Htt

amin

oac

id9

–1

55

pre

yan

dH

IP2

was

ab

ait

that

inte

ract

ed

wit

hH

ttam

ino

acid

37

0–5

80

pre

y.b

Lite

ratu

rere

po

rte

dH

tto

rH

tt-f

rag

men

tin

tera

cto

r.N

/A,

no

tav

aila

ble

;N

/D,

no

td

on

e;¼

,p

rote

inin

tera

cte

dw

ith

bo

th2

3Q

and

55

QH

ttam

ino

acid

1–

45

0fr

agm

en

tsu

nd

er

the

rete

stco

nd

itio

ns.

cDN

Alib

rary

abb

revi

atio

ns

(hu

man

):A

P,

adip

ose

;B

B,

bra

ince

reb

ellu

m;

BH

,b

rain

hip

po

cam

pu

s;B

N,

bra

inca

ud

ate

nu

cle

us;

BR

,b

rain

wh

ole

;C

O,

colo

n;

FB,

feta

lb

rain

;FL

,fe

tal

lun

g;

LK,

leu

kocy

te;

LG,

lun

g;

ML,

me

lan

om

a;M

M,

mam

mar

yg

lan

d;

PN

,p

ancr

eas

;P

R,

pro

stat

eg

lan

d;

SC,

spin

alco

rd.

do

i:10

.13

71

/jo

urn

al.p

ge

n.0

03

00

82

.t0

02



Ta

ble

3.

Mo

dif

iers

of

the

Exp

and

ed

Htt

-Fra

gm

en

t-In

du

ced

Eye

Ph

en

oty

pe

Fu

nct

ion

al

Ca

teg

ory

Hu

ma

nG

en

e

Sy

mb

ol

So

urc

eD

roso

ph

ila

Ort

ho

log

Bio

log

ica

lP

roce

ssO

ve

r-E

xp

ress

ion

All

ele

(s)

E/S

Lo

ss-o

f-F

un

ctio

nA

lle

le(s

)E

/S

Cy

tosk

ele

tal

org

an

iza

tio

n

an

db

iog

en

esi

s

AD

D3

Y2

Hh

tsSt

ruct

ura

lco

nst

itu

en

to

fth

ecy

tosk

ele

ton

NA

NA

01

10

3,

k06

12

1,

KG

06

77

7S

CEP

36(F

LJ13

386)

Y2

Hzi

pp

erC

yto

ske

leta

lp

rote

inb

ind

ing

,st

ruct

ura

lco

nst

itu

en

t

of

the

cyto

ske

leto

n

NA

NA

02

95

7,

IIX6

2E

CTN

NB

1Y

2H

arm

ad

illo

Wn

tre

cep

tor

sig

nal

ing

pat

hw

ay,

adh

ere

ns

jun

ctio

nN

AN

AG

01

92

,G

02

34

,2

,3

S

GFA

PM

SLa

min

CC

yto

ske

leta

lo

rgan

izat

ion

NA

NA

G0

01

58

,k1

19

04

E

GP

M6

aM

SM

6C

yto

ske

leta

lo

rgan

izat

ion

,tr

ansm

issi

on

of

ne

rve

imp

uls

eEY

07

03

2E

BG

00

39

0S

KIA

A12

29Y

2H

CLI

P-1

90St

ruct

ura

lco

nst

itu

en

to

fth

ecy

tosk

ele

ton

NA

NA

KG

07

83

7,

KG

06

49

0E

KIF

5Ca

MS

Kh

cSt

ruct

ura

lco

nst

itu

en

to

fth

ecy

tosk

ele

ton

NA

NA

E02

14

1,8

,k1

33

1,4

S

PP

LY

2H

sho

rtst

op

Act

incy

tosk

ele

tal

org

aniz

atio

n,

axo

no

ge

ne

sis

NA

NA

3,

65

–2

E

SEP

T7(C

DC

10)

MS

pea

nu

tA

ctin

bin

din

g,

stru

ctu

ral

con

stit

ue

nt

of

the

cyto

ske

leto

nN

AN

AX

P,

02

50

2,

KG

00

47

8S

SOR

BS1

Y2

HC

AP

Cyt

osk

ele

tal

con

stit

ue

nt

NA

NA

BG

02

18

4,

KG

00

83

,K

G0

03

08

E

Sig

na

ltr

an

sdu

ctio

nG

NA

ZM

SG

-ia

65A

G-p

rote

inco

up

led

rece

pto

rsi

gn

alin

gp

ath

way

EY1

03

55

,EY

09

37

6b

EK

G0

19

07

S

ITP

R1

MS

Itp

-r83

AC

alci

um

ion

tran

spo

rt;

sig

nal

tran

sdu

ctio

nEY

02

52

2E

05

61

6S

NEG

R1

MS

Lach

esin

Axo

no

ge

ne

sis,

sig

nal

tran

sdu

ctio

nN

AN

AG

00

04

4,

BG

01

46

2E

PTK

6Y

2H

Src4

2AP

rote

inam

ino

acid

ph

osp

ho

ryla

tio

nU

AS-

Src4

2A

.CA

SK

G0

25

15

,k1

01

08

,E1

E

YW

HA

BM

S14

-3-3

fR

asp

rote

insi

gn

altr

ansd

uct

ion

EY0

61

47

,EY

03

32

5E

NA

NA

YW

HA

EaM

S14

-3-3

eR

asp

rote

insi

gn

altr

ansd

uct

ion

UA

S:1

4-3

-3,

UA

S:1

4-3

-3(w

eak

)

Ej2

B1

0S

Sy

na

pti

ctr

an

smis

sio

nC

LTC

MS

Ch

cIn

trac

ellu

lar

pro

tein

tran

spo

rt,

ne

uro

tran

smit

ter

secr

eti

on

NA

NA

1,

G0

43

8,

BG

02

59

3a

S

NA

PA

aM

SSN

AP

Intr

ace

llula

rp

rote

intr

ansp

ort

,n

eu

rotr

ansm

itte

rse

cre

tio

nS1

02

C#

2D

ESN

AP

G8,

SNA

PI1

,SN

AP

I65,

SNA

PM

3,

SNA

PM

4,

SNA

PP

2

S

STX

1AM

SSy

nta

xin

1At-

SNA

RE,

ne

uro

tran

smit

ter

secr

eti

on

EP3

21

5E

De

lta2

29

,0

67

37

S

Pro

teo

lysi

s/p

ep

tid

oly

sis

or

ub

iqu

itin

cycl

e

ASP

HM

SA

sph

Pro

teo

lysi

san

dp

ep

tid

oly

sis,

tyro

sin

eki

nas

esi

gn

alin

gp

ath

way

NA

NA

ZC

L16

05

,K

G0

98

81

E

DN

CH

1Y

2H

Dh

c64C

Intr

ace

llula

rp

rote

intr

ansp

ort

,p

rote

oly

sis

and

pe

pti

do

lysi

sN

AN

A4

–1

9,

KG

08

83

8E

PSM

C2a

Y2

HR

pt1

Pro

teo

lysi

san

dp

ep

tid

oly

sis

EP2

15

3E

43

Ed-1

,0

56

43

S

USP

9XY

2H

fat

face

tsP

rote

oly

sis

and

pe

pti

do

lysi

s,p

rote

ind

eu

biq

uit

inat

ion

EP3

81

SB

x4E

Re

gu

lati

on

of

tra

nsc

rip

tio

n

or

tra

nsl

ati

on

MEF

2DY

2H

Mef

2R

eg

ula

tio

no

ftr

ansc

rip

tio

nfr

om

Po

lII

pro

mo

ter

NA

NA

X1

,K

G0

12

11

aS

PP

AR

GY

2H

Eip

75B

Re

gu

lati

on

of

tran

scri

pti

on

fro

mP

ol

II;fa

tty

acid

me

tab

olis

mN

AN

AB

G0

25

76

,K

G0

01

39

,

KG

09

02

6,

07

04

1

S

ZN

F91

Y2

Hcr

oo

ked

leg

sR

NA

Po

lym

era

seII

tran

scri

pti

on

fact

or

acti

vity

EY0

89

53

S0

44

18

,k0

50

25

E

Oth

er

CA

CN

A2D

1M

SC

G12

455

Cal

ciu

mch

ann

el

acti

vity

,vo

ltag

e-g

ate

dca

lciu

mch

ann

el

acti

vity

EY0

97

50

EK

G0

02

60

S

FEZ

1Y

2H

/LIT

Un

c-76

Axo

nca

rgo

tran

spo

rtN

AN

AG

03

10

,G

03

33

,G

04

23

aE

GA

PD

HM

S/Y

2H

/LIT

Gp

dh

Car

bo

hyd

rate

me

tab

olis

mN

AN

AN

0,

n1

–4

,n

1–

5S

GP

IaM

SP

gi

Glu

con

eo

ge

ne

sis,

gly

coly

sis

nN

C1

,2E

NA

NA

ND

UFB

10M

SP

dsw

Mit

och

on

dri

ale

lect

ron

tran

spo

rtN

AN

AK

10

10

1S

VD

AC

2M

Sp

ori

nV

olt

age

-gat

ed

ion

chan

ne

lN

AN

Ak0

51

23

,f0

36

16

S

Fun

ctio

nal

clas

sifi

cati

on

asin

Tab

le2

.E,

en

han

cer;

S,su

pp

ress

or.

aG

en

este

ste

din

Dro

sop

hila

pri

or

tost

atis

tica

lfi

lte

rin

g.

do

i:10

.13

71

/jo

urn

al.p

ge

n.0

03

00

82

.t0

03



Discussion

Although the gene encoding Htt was identified over adecade ago, the normal function of this protein and theprecise mechanisms by which expanded polyQ exerts its toxiceffects remain the subjects of intense inquiry. In this study weidentified 234 potential new Htt-associated proteins usinghigh-throughput proteomic screens. The diverse functions ofHtt and Htt-fragment protein partners and modifiersreported here are consistent with the functional diversity ofpathogenic processes and targets in HD. Htt is localized to anumber of different cellular compartments, and there is alarge body of evidence showing that mutant Htt fragmentscan interfere with a diverse range of proteins and pathwaysincluding, transcriptional activation and co-activation[12,13,15], ubiquitin-mediated proteolysis [50], mitochondrialenergy metabolism [51,52], receptor-mediated signal trans-duction [53], axonal transport [54], and vesicle trafficking[43,44]. These observations suggest models of Htt-mediatedpathology that involve interference in multiple cellularpathways.

Furthermore, we have identified a novel associationbetween Htt fragment and components of the vesiclesecretion apparatus (Table 1). Stx1A, NAPA, and CACNA2D1were confirmed as modifiers in the fly polyQ toxicity model

(Table 3), and SNAP25, STX1a, and CACNA2D1 proteinswere observed to co-immunoprecipitate with full length Httfrom mouse brain (Figure 5). Protein interactions andlocalization experiments have placed Htt primarily atpostsynaptic sites (reviewed in [55]), but Htt has also beenshown to be associated with N-type calcium channels inpresynaptic cells [56]. These results suggest that modulationof SNARE-mediated neurotransmitter secretion may be anormal function for Htt and/or may be perturbed by mutantHtt.In addition to the general large-scale protein interaction

screens reported for human proteins, two screens have beenreported that focus specifically on proteins related to polyQdisease. A large-scale Y2H screen for Htt-fragment bindingproteins uncovered 15 novel interacting proteins, includingGIT1, an enhancer of polyQ aggregation [57]. A more recentscreen for protein interactions relevant to inherited ataxiasreported a large network of interaction involving 54 proteinsimplicated in human ataxia [29]. Interestingly, there was moreoverlap between high-confidence interactions in our datasetand the previously published Htt dataset [57] than the ataxiadataset [29], suggesting that protein-protein interactions maycontribute to pathogenic specificity found among the polyQdiseases. Validation of interactions in the ataxin networkstudy relied on demonstration of co-affinity precipitation of

Figure 2. Modification of the Phenotypes Caused by N-Terminal Expanded Htt in the Drosophila Eye

Retinal sections of adult Drosophila eyes show modification of the phenotypes caused by expression of different levels (B and I) of a transgene encodingan N-terminal expanded Htt fragment. Enhancers (C–G) and suppressors (J–N) include proteins involved in cytoskeletal organization (C) and (J), signaltransduction (D) and (K), neurotransmitter secretion (E) and (L), proteolysis/peptidolysis and the ubiquitin cycle (F) and (M), and transcriptional/translational regulation (G) and (N). Retinal sections of day 5 control flies cultured at 25 8C expressing the gene that encodes expanded N-terminal Httfragment (GMR-GAL4/þ; UAS:128Qhtt[M64]/þ) (B) show a degenerative phenotype when compared to controls of the same age and cultured at the sametemperature (GMR-GAL4/þ) (A). The phenotype consists of a shortening (see arrow) and detachment of the retina, as well as the presence of vacuoles inthe retina. The Htt-fragment-induced phenotype can be enhanced by (C) reduced levels of zipper (GMR-GAL4/PfPZgzip02957; UAS:128Qhtt[M64]/þ), (D)reduced levels of Src oncogene at 42A (GMR-GAL4/PflacWgSrc42Ak10108; UAS:128Qhtt[M64]/þ), (E) overexpression of soluble N-ethylmaleimide-sensitive-attachment protein (GMR-GAL4/þ; UAS:128Qhtt[M64]/UAS-S102C#2D), (F) reduced levels of fat facets (GMR-GAL4/þ; UAS:128Qhtt[M64]/fafBx4), and (G)reduced levels of crooked legs (GMR-GAL4/PfPZgcrol04418cn1; UAS:128Qhtt[M64]/þ). None of these mutations cause an abnormal eye phenotype in fliescarrying the GMR-GAL4 driver but not the UAS:128Qhtt[M64] transgene (unpublished data). However, when combined with an N-terminal expanded Httfragment, they lead to an even larger decrease in retinal thickness sometimes accompanied by an increase in retinal detachment and vacuolization.Retinal sections of day 1 control flies cultured at 27 8C expressing a gene that encodes an expanded N-terminal Htt fragment (GMR-GAL4/þ;UAS:128Qhtt[M64]/þ) (I) show a severe degenerative phenotype when compared to GMR controls of the same age and cultured at the sametemperature (H). The phenotype consists of a shortening (see arrow) and detachment of the retina, as well as the presence of vacuoles in the retina.The Htt-fragment-induced phenotype can be suppressed by (J) reduced levels of hu li tai shao (GMR-GAL4/PflacWghtsk06121; UAS:128Qhtt[M64]/þ), (K)reduced levels of G protein iasubunit 65A (GMR-GAL4/; UAS:128Qhtt[M64]/PfSUPor-PgG-ia65AKG01907ry506), (L) reduced levels of clathrin heavy chain(Chc1/þ GMR-GAL4/þ; UAS:128Qhtt[M64]/þ), (M) reduced levels of Rpt1 (GMR-GAL4/PfPZgRpt105643cn1; UAS:128Qhtt[M64]/þ), and (N) reduced levels ofmyocyte enhancing factor 2 (GMR-GAL4/Df(2R)X1,Mef2[X1]; UAS:128Qhtt[M64]/þ). These mutations decrease the vacuolization and increase the retinalthickness as well as virtually eliminating the retinal detachment.doi:10.1371/journal.pgen.0030082.g002



tagged expressed protein pairs. Here we tested the ability of agenetic model to validate protein interactions. 48 of 60 genestested in a polyQ-induced fly eye degeneration model of HDmodified the polyQ-induced toxicity, suggesting that this listcontains protein interactors that also genetically interactwith Htt. Our validation rate using the Drosophila geneticmodel (80%) is similar to that found using co-affinitypurification in the ataxia and Htt studies (80% and 65%,respectively) [29,57]. Moreover, whereas co-affinity purifica-tion gives validation of the physical interaction of proteins,the genetic modification screen provides additional informa-tion suggesting a biological role in genetic pathways relevantto HD. Overall, these observations demonstrate the utility ofcombining protein-interaction screening with genetic-inter-action screening to provide insight into disease mechanismsand identify potential targets for therapeutic intervention.

Whereas our datasets more than quadruple the potentialnumber of interactions attributed to Htt or Htt fragments,the in vitro derived interactor datasets do contain non-relevant interactions (false positives) and do not represent allbinding proteins (false negatives), an issue common to high-throughput screens. For example, despite the saturation ofthe screens we identified some, but not all, of the known Htt-fragment-interacting proteins. Our protein interactionscreens revealed 14 of the 40 interactions previouslydiscovered using Y2H methods [22,23]. Using different Y2Hmethods, a recent high-throughput screen isolated 19 Htt-fragment-interacting proteins, four of which had beenpreviously described [57]. Together, these data suggest thatdifferent Y2H methods yield overlapping but not identicaldatasets, likely due to differences in selection stringency aswell as other technical differences. Surprisingly, only Htt

Figure 3. Modification of the Expanded Htt-Fragment-Induced Phenotype by a STX1A Loss-of-Function Mutation

Modification was observed both in the eye (external phenotype and retinal sections) and in the nervous system (climbing ability and survival).(A) Retinal sections of day 5 flies raised at 25 8C (left), day 1 flies raised at 27 8C (middle), and standard error of mean of day 5 flies raised at 29 8C (right)expressing a gene that encodes expanded N-terminal Htt fragment (GMR-GAL4/þ; UAS:128Qhtt[M64]/þ).(B) Retinal sections of day 5 flies raised at 25 8C (left), day 1 flies raised at 27 8C (middle), and standard error of mean of day 5 flies raised at 29 8C (right)expressing a gene that encodes expanded N-terminal Htt fragment and carrying reduced levels of STX1A (GMR-GAL4/þ; UAS:128Qhtt[M64]/Syx1A229ry506). Note suppression of both the retinal and external eye phenotypes at all three temperatures. Overexpression of STX1A showsenhancement of the retinal degeneration and external 128 Qhtt phenotype (unpublished data).(C) Climbing assay (top) and survival assay (bottom) results confirm the suppression observed in the eye assay. Shown in red/pink are the climbingperformance and survival curve of a population of females flies expressing a gene that encodes expanded N-terminal expanded Htt fragment (elav-GAL4/þ; UAS:128Qhtt[F27B]/þ). Shown in blue/light blue are the improved climbing performance and survival curve of a population of females fliesexpressing a gene that encodes expanded N-terminal Htt fragment and carrying a heterozygous loss-of-function mutation in STX1A (elav-GAL4/þ;þ/þ;UAS:128Qhtt[F27B]/Syx1A229 ry506). (x-Axis, age of flies in days; y-axis, percent surviving or climbing flies; LOF, loss-of-function).doi:10.1371/journal.pgen.0030082.g003



fragments near the N terminus of the protein were able togenerate reproducible protein interaction in our Y2Hscreens (Table 2). This finding is consistent with a previousreport in which Y2H methods failed to detect interactionsfrom Htt-fragment baits outside the amino terminus [58] andmay be in part due to technical limitations of the Y2Hmethod. For example, C-terminal Htt fragments may not foldproperly in yeast, may require post-translational modifica-tions not found in yeast for interaction with protein partners,or may be localized away from the nucleus. Even fewer knownHtt-interacting proteins were found by pull-down/MS meth-ods. Interestingly, the cytosolic chaperonin-containing t-complex (CCT or TriC) was recently shown to physicallyinteract with Htt and modify the course of polyQ-inducedtoxicity in mammalian cells [59,60]. We found that twocomponents of the CCT complex, CCT6 and CCT8, wereassociated with Htt exon1 in pull downs. Together, these datasuggest that many potential Htt-interacting or Htt-associatedproteins remain to be discovered by other methods.

Overall, there was little overlap between interactions foundby the Y2H and pull-down methods (4/234). This low degreeof overlap is consistent with results seen in other systems-scale protein interaction datasets generated using Y2H andMS methods. For example, interaction screens of the yeastproteome using Y2H (4,476 and 915 binary protein inter-

actions) [27,28] and MS-based screens (3,767 and 3,727interactions in proteins complexes) [25,26] yielded a 2%–5% overlap. It has been suggested that this low overlapbetween interaction screening methods may arise fromseveral factors including method-specific biases [34]. Ulti-mately, the value of protein interaction data generated by anymethod needs to be evaluated through experimental vali-dation. We clearly demonstrate here that both methods aresimilarly capable of identifying Htt-fragment-interacting

Figure 4. A Network of Protein Interactions Involved in Vesicle Traffic

A network is shown that includes protein interactions described in thisstudy and interactions curated from the public domain (NCBI EntrezGene). Htt-fragment-interacting proteinss found in this study areindicated as ovals (MS) or rectangles (Y2H). Binary Y2H interactionsfound in this study are indicated as thick lines. Proteins contained in thedotted circle were identified in Htt-fragment pull downs using brainlysates. Thin lines indicate curated protein interactions. Curated bridginginteractions (blue triangles) are defined as proteins reported to interactwith HD and at least one other protein in the network. Proteins whoseDrosophila ortholog genes acted as modifiers in this study are indicatedin red.doi:10.1371/journal.pgen.0030082.g004

Figure 5. Co-Immunoprecipitation of Huntingtin-Interacting Proteins

from YAC128 Mouse Brain

Htt was immunoprecipitated with mouse monoclonal Htt antibody andprobed with rabbit polyclonal Htt BKP1 antibody (top right panel). Theinput for each protein (left panels) and resulting immunoprecipitationare shown (right panels). The lower molecular weight band in the PKM2immunoprecipitation is an immunoglobulin (IgG) band. GAPDH isincluded as a positive control. PARP is included as a negative control.doi:10.1371/journal.pgen.0030082.g005



proteins that can be validated by assays based upon geneticinteraction and physical association in mammalian tissuesrelevant to HD pathology.

Most specific molecular mechanisms proposed for Htt-mediated pathogenesis can, in principle, be attributed to adirect interaction between Htt and a protein component (orcomponents) of a given pathway. Consistent with thisassertion, we demonstrate here that a large set of Htt-interacting proteins is highly enriched for genetic modifiersof Htt-mediated neurodegeneration. Currently, there areefforts directed toward discovering genetic modifiers ofhuman HD. Since the modifiers reported here were firstdiscovered in screens performed with mammalian genes andproteins and subsequently validated in Drosophila, it would beof interest to determine whether human gene variantsencoding similar proteins and pathway act can act asmodifiers in human neurodegeneration.

Materials and Methods

Y2H screens. Automated screens were done as described inLaCount et al. [31]. Briefly, haploid yeast expressing Htt-bait fusionproteins were grown in liquid medium in 96-well plates. Aliquots ofyeast of the opposite mating type expressing prey libraries wereadded to each well and allowed to mate overnight. Matings wereplated on medium selecting for diploids, the expression of theauxotrophic markers fused to the cDNA inserts and to the activity ofthe metabolic reporter genes ADE2 and HIS3 [32,61]. cDNA preyinserts from yeast that grew under selection were PCR-amplified andsequenced. Identities of prey inserts were determined by BLASTcomparison against the National Center for Biotechnology (NCBI)RefSeq database (http://www.ncbi.nlm.nih.gov). All reported interac-tions were verified by recovering prey plasmids from positivecolonies, transforming these into yeast strains expressing Htt baitsand reconfirming the ADEþ, HISþ phenotype. Beta-galactosidasemeasurements were performed according to manufacturer’s direc-tions (Pierce, http://www.piercenet.com). Control yeast strains carry-ing Htt bait and prey plasmids without an insert were used asbaseline. The Htt 55 Q bait had slightly higher background levels thanthe corresponding Htt 23 Q bait. Y2H interactor lists were filtered toremove promiscuous proteins. Additional yeast methods can befound in Supporting Information.

MS. Htt-fragment-interacting proteins underwent TAP and wereidentified by MS [62]. Affinity-tagged Htt N-terminal fragments fusedto GST and 6 3 His were incubated with protein lysates preparedfrom mouse and human tissues and cultured cells. After TAP,proteins were digested with trypsin, desalted, and subjected to strongcation exchange (CEX). CEX fractions were further separated byreverse-phase HPLC and subjected to MS analysis by matrix-assistedlaser desorption/ionisation-time of flight (MALDI)MS/MS and electro-spray ionization MS/MS. MS/MS data were used for protein sequencedatabase searches by Mascot (Matrix Sciences, http://www.matrixscience.com) [63,64]. All searches were performed against thesubset of either human or mouse proteins in the NCBInr proteinsequence database (HumanNR or MouseNR). The minimum peptidescore was set at 10, and the minimum peptide length was set to 5;otherwise the default instrument-specific Mascot settings were used.A variable cut-off was applied to proteins, which was dependent uponthe number of peptides identified for a given protein. For anyprotein from which only one peptide was identified, a minimalpeptide score threshold of 60 was required. If two peptides wereidentified, a threshold ion score of 50 was required, and for threepeptides an ion score of 40 was required. Any peptides observed incontrol pull downs done with beads bound to TAP-tag alone wereexcluded. A statistical method, based on comparison of a wide varietyof pull downs, was used to identify nonspecific interactors, whichwere also excluded. To validate protein identification subsequent tothe automated thresholding and initial filtering, each remaining MS/MS spectrum was manually inspected to ensure that there were nospurious results matched by Mascot. Detailed MS and statisticalmethods can be found online with Supporting Information.

Drosophila polyQ toxicity model and genetic screen. A DrosophilapolyQ toxicity model was generated using an N-terminal fragment ofthe human HD cDNA that encodes the first 336 amino acids of the

protein including a 128-Q expansion in exon 1. The construct wascloned into the pUAST vector for generating transgenic lines [65].This HD Drosophila model is most similar to the expanded version (82Q) of the N171 mouse model, which shows abundant intranuclearinclusions [66] and neuronal degeneration [67]. Expression of the128-Q N-terminal Htt fragment in Drosophila leads to neurodegener-ative phenotypes. In the eye, these phenotypes are evident bothexternally and in the retina following expression using the glassmultimer reporter (GMR)-GAL4 driver (Figures 2 and 3). In thenervous system, Elav-GAL4-directed expression of the transgene leadsto progressive impaired motor ability and reduced life span (Figure3C). Also as in the N171-82Q mouse, intranuclear inclusions areobserved in Drosophila neurons expressing the 128-Q N-terminal Httfragment (unpublished data).

For the modifier screen, females of the genotype y1w118; GMR-GAL4/CyO; UAS:128QHtt[M64] were crossed to males from the mutantstrains. In cases where the mutation was on the X chromosome, thecross was reversed. Crosses were incubated at 27 8C and 29 8C toprovide two different phenotypic readouts. Strains modifying the eyephenotype were recrossed to verify the modification. Only genes thatconsistently showed modification at different temperatures or usingdifferent alleles were further analyzed. Potential modifiers behavingas enhancers were tested for possible nonspecific eye phenotypes bycrossing them to control females of the genotype y1w118; GMR-GAL4/CyO.

For scanning electron microscopy (SEM) images, flies were crossedat 29 8C and newly eclosed adults were aged for five days. Whole flieswere dehydrated in ethanol, critical-point dried, and analyzed with aJEOL JSM 6100 microscope. For paraffin sections of enhancers, flieswere crossed at 25 8C and adults were aged for five days (forsuppressors, the crosses were done at 27 8C and the flies were aged forone day). Adult heads and torsos were fixed in 4% formaldehyde/85%ethanol/5% acetic acid, dehydrated, embedded in paraffin for verticalsemi-thin sections, and then stained with Hemathox.

For the climbing and survival assays, females of the genotype Elav-GAL4; UAS:128QHtt[F27B] were crossed to males of the mutantstrains. Climbing assays were performed on 30 age-matched adultvirgin female flies raised at 27 8C as described [68]. The flies, placed ina plastic vial, were tapped to the bottom of the vial, and the numberof flies above a 5-cm line was counted after 18 seconds. A total of tentrials were performed every 48 hours. Each climbing and survivalexperiment was repeated three times.

Immunoprecipitation. Whole brains from wild-type or YAC128mice were lysed in T-PER (Pierce) with protease inhibitors (CompleteMini, Roche Applied Science, http://www.roche.com). Protein deter-mination was carried out with the BCA method (Bio-Rad, http://www.bio-rad.com). Lysate (500 lg, 0.7 ml T-PER with protease inhibitors)were precleared with mouse IgG beads (Sigma A6531, http://www.sigmaaldrich.com) and immunoprecipitated with monoclonal Httantibody (5 ll, Chemicon 2166, http://www.chemicon.com) byincubating overnight at 4 8C and then with protein G (40 ll,Amersham 17-0618-01, http://www.amersham.com). Beads werewashed 53with TBS/0.05% Tween, sample was eluted with 13 samplebuffer (Invitrogen, http://www.invitrogen.com) and then resolvedusing 4%–12% Bis-Tris precast gels (Invitrogen). Western blot waspreformed, and blots were probed with rabbit antibody to USP9X(1:200, Abcam 19879, http://www.abcam.com), Cullin 2 (1:500, Abcam1870), CACNA2D1 (1:200, Sigma CS105), Htt BKP1 (1:500), PARP(1:300, BioMol SA253, http://www.biomol.com), mouse monoclonalGAPDH (1:100, Chemicon MAB374), STX1A (1:1000, SynapticSystems, 11001, http://www.sysy.com), SNAP25 (1:1000, Santa CruzBiotechnology SC-7539, http://www.scbt.com/), goat antibody PKM2(1:500, Abcam 6191), and PSMC2 (1:1000, GeneTex 23322, http://www.genetex.com).

Supporting Information

Figure S1. Purified Htt Exon 1 Bait

Purified bait protein from the first and second purification steps wasseparated by sodium dodecyl sulfate polyacrylamide gel electro-phoresis (SDS-PAGE) and silver stained. The presence of glutathioneS-transferase (GST) and Htt in the bands was confirmed by matrix-assisted laser desorption/ionisation-time of flight MS and Westernblotting (not shown). The predicted size of the GST-Htt fusionproduct is 53 kDa. We were unable to determine the difference in thetwo GST-Htt bands by MS; they may represent expanded (48 Q) andwild-type (22 Q) Htt fragments. The band at 28 kDa represents GSTand likely occurs from cleavage of the fusion product between the



GST and the bait as we saw a band of this size with numerousheterologous purified baits.

Found at doi:10.1371/journal.pgen.0030082.sg001 (575 KB PDF).

Figure S2. Saturation of Y2H Searches with Htt Baits

Only searches with N-terminal baits (amino acid 1–90, 23 Q; aminoacid 1–90, 55 Q; amino acid 1–450, 23Q; amino acid 1–450, 55 Q) thatgave at least one positive were included in the analysis. The x-axisindicates numbers of screens performed. The y-axis shows the noveldiscovery index for prey proteins (e.g., a value of 0.3 indicates that 30%of the preys seen in a search were not seen in a prior screen). A peaknear 525 searches corresponds to introduction of new prey libraries.

Found at doi:10.1371/journal.pgen.0030082.sg002 (529 KB PDF).

Figure S3. Suppressors of Fly Eye Phenotype

Retinal sections of day-1 control flies cultured at 27 8C expressing thegene that encodes expanded N-terminal Htt (GMR-GAL4/þ;UAS:128Qhtt[M64]/þ) (B) show a severe degenerative phenotype whencompared to (A) GMR-GAL4 controls of the same age and cultured atthe same temperature. The phenotype consists of a shortening (seearrow) and detachment of the retina, as well as the presence ofvacuoles in the retina. The Htt-induced phenotype can be suppressedby (C) reduced levels of armadillo (PflacWgarmG0234; GMR-GAL4/þ;UAS:128Qhtt[M64]/þ), (D) reduced levels of hu li tai shao (GMR-GAL4/PflacWghtsk06121; UAS:128Qhtt[M64]/þ), (E) reduced levels of M6 (GMR-GAL4/þ; UAS:128Qhtt[M64]/PfGT1gM6BG00390), (F) reduced levels ofkinesin heavy chain (GMR-GAL4/b1pr1Khc8; UAS:128Qhtt[M64]/þ), (G)reduced levels of peanut (GMR-GAL4/PfSUP pr P-gpnutKG00478;UAS:128Qhtt[M64]/þ), (H) reduced levels of 14-3-3e (GMR-GAL4/þ;UAS:htt[M64]/14-3-3ej2b10), (I) reduced levels of G protein I a-subunit65A (GMR-GAL4/; UAS:128Qhtt[M64]/PfSUPor-PgG-ia65AKG01907ry506),(J) reduced levels of Itp-r83A (GMR-GAL4/þ; UAS:128Qhtt[M64]/PfPZgItp-r83A05616ry506), (K) over-expression of Src oncogene at 42A(GMR- GAL4/PfEPgy2gSrc42AEY08937; UAS:128Qhtt[M64]/þ), (L) reducedlevels of clathrin heavy chain (Chc4/þGMR-GAL4/þ; UAS:128Qhtt[M64]/þ), (M) reduced levels of soluble N-ethylmaleimide-sensitive factorattachment protein (GMR-GAL4/þ; UAS:128Qhtt[M64]/SNAPM4), (N)reduced levels of STX1A (GMR-GAL4/þ; UAS:128Qhtt[M64]/ry506PfPZgSyx1A06737), (O) reduced levels of Rpt1 (GMR-GAL4/PfPZgRpt105643cn1; UAS:128Qhtt[M64]/þ), (P) reduced levels of Eip75B(GMR-GAL4/þ; UAS:128Qhtt[M64]/PfPZgEip75B07041), (Q) reduced lev-els of myocyte enhancing factor 2 (GMR-GAL4/Df(2R)X1,Mef2X1;UAS:128Qhtt[M64]/þ), (R) reduced levels of crooked legs (GMR-GAL4/PfEPgy2gcrolEY08953), (S) reduced levels of Glycerol 3 phos-phate dehydrogenase (GMR-GAL4/Al1Gpdhn1–4; UAS:128Qhtt[M64]/þ),(T) reduced levels of Pdsw (GMR-GAL4/PfPlacZgPdswk10101;UAS:128Qhtt[M64]/þ), and (U) reduced levels of porin (GMR-GAL4/pfPlacWgporink05123; UAS:128Qhtt[M64]/þ) and reduced levels ofCG12455 (GMR-GAL4/PfSUP or-PgCG12455KG00260 ; UAS:128Qhtt[M64]/þ). These mutations decrease the vacuolization andincrease the retinal thickness as well as virtually eliminating theretinal detachment.

Found at doi:10.1371/journal.pgen.0030082.sg003 (2.7 MB PDF).

Figure S4. Enhancers of Fly Eye Phenotype

(A) Age-matched controls cultured at the same temperature (GMR-GAL4/þ).(B) Retinal sections of day 5 flies expressing N-terminal 128-Q htt(GMR-GAL4/þ; UAS:128Qhtt[M64]/þ) cultured at 25 8C show adegenerative phenotype. The phenotype consists of a shortening(arrow), vacuolization, and detachment of the retina. This phenotypecan be enhanced by (C) reduced levels of CAP (GMR-GAL4/PfSUPor-PgCAPKG00083; UAS:128Qhtt[M64]/þ), (D) reduced levels of CLIP-190(GMR-GAL4/PfSUPor-PgCLIP-190KG06490; UAS:128Qhtt[M64]/þ), (E) re-duced levels of LaminC (GMR-GAL4/PfPTTor-GBgLamCG00158;UAS:128Qhtt[M64]/þ), (F) overexpression of M6 (GMR-GAL4/þ;UAS:128Qhtt[M64]/PfEPgy2gM6EY07032), (G) reduced levels of zipper(GMR-GAL4/PfPZgzip02957; UAS:128Qhtt[M64]/þ), (H) reduced levels ofshort stop (GMR-GAL4/PfFRT(whs)gG13 shot3; UAS:128Qhtt[M64]/þ), (I)overexpression of 14-3-3e (GMR-GAL4/þ; UAS:128Qhtt[M64]/14-3-3eScerUASc.Ca), (J) overexpression of 14-3-31 (GMR-GAL4/ PfEPgy2g14-3-31EY03325; UAS:128Qhtt[M64]/þ), (K) overexpression of G-ia65A (GMR-GAL4/ þ; UAS:128Qhtt[M64]/PfEPgy2gG-ia65AEY10355), (L) overexpres-sion of Itp-r83A (GMR-GAL4/ þ; UAS:128Qhtt[M64]/PfEPgy2gItp-

r83AEY02522), (M) reduced levels of Lachesin (GMR-GAL4/PfPTT-un1gLacG00044; UAS:128Qhtt[M64]/þ), (N) reduced levels of Src oncogeneat 42A (GMR-GAL4/PflacWgSrc42Ak10108; UAS:128Qhtt[M64]/þ), (O)overexpression of soluble NSF-attachment protein (GMR-GAL4/þ;UAS:128Qhtt[M64]/UAS-S102C#2D), (P) overexpression of Syntaxin1A(GMR-GAL4/þ; UAS:128Qhtt[M64]/PfEPgSyx1AEP3215), (Q) reduced lev-els of Aspartyl b-hydroxylase (GMR-GAL4/PfSUPor-PgAsphKG09881;UAS:128Qhtt[M64]/þ), (R) reduced levels of Dynein heavy chain 64C(GMR-GAL4/þ; UAS:128Qhtt[M64]/PfSUPor-PgDhc64CKG08838), (S) re-duced levels of fat facets (GMR-GAL4/þ; UAS:128Qhtt[M64]/fafBx4), (T)overexpres s ion of Rpt1 (GMR-GAL4 /PfEPgRpt1EP 2 1 5 3 ;UAS:128Qhtt[M64]/þ), (U) reduced levels of crooked legs (GMR-GAL4/PfPZgcrol04418; UAS:128Qhtt[M64]/þ), (V) reduced levels of Phosphogluc-onate isomerase (GMR-GAL4/PginNC1; UAS:128Qhtt[M64]/þ), (W) reducedlevels of RhoGAP92B (GMR-GAL4/PfUAS-RhoGAP92B-dsRNAg2.2;UAS:128Qhtt[M64]/þ), (X) reduced levels of Unc-76 (PflacWgUnc-76G0423a; GMR-GAL4/þ; UAS:128Qhtt[M64]/þ), and (Y) overexpressionof CG12455 (GMR-GAL4/PfEPgy2gCG12455EY09750; UAS:128Qhtt[M64]/þ). These mutations do not cause an abnormal eye phenotype incontrol flies carrying the GMR-GAL4 driver without theUAS:128Qhtt[M64] transgene (unpublished data). However, whencombined with 128-Q htt, they lead to further decrease in retinalthickness and in some cases increased retinal detachment andvacuolization.

Found at doi:10.1371/journal.pgen.0030082.sg004 (2.8 MB PDF).

Table S1. Primary List of Peptides Identified in Pull Downs

Y indicates peptides that were manually validated and confirmed byinspection of the MS spectra; A refers to ambiguous peptides thatcould not be conclusively identified by manual validation of the MSspectra.

Found at doi:10.1371/journal.pgen.0030082.st001 (3.4 MB XLS).

Table S2. Primary List of Gene Sequences Identified from Y2HPositives with Htt-Fragment Baits

*The total number of unique interacting proteins refers to thenumber of unique gene sequences identified in a database of positivesfrom nearly .250,000 high-throughput random Y2H searchesperformed at Prolexys Pharmaceuticals (http://www.prolexys.com).

Found at doi:10.1371/journal.pgen.0030082.st002 (580 KB DOC).

Table S3. Sequences of Positives Identified in Y2H Searches

Search ID is an identifier given each Y2H mating event (see Materialsand Methods). Positive ID is a unique identifier given to each positivecolony picked in Y2H searches. RefSeq ID, Gene Symbol, and EntrezGene ID refer to gene designations in the NCBI database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db¼gene). E_VALUE_EXP is thenegative log of the E value produced by the highest scoring BLAST hitand has a maximum of 180 (corresponds to E value of 10E�180 or less).Amino acid coordinates of HD baits are indicated relative toNP_002102. Q-length repeats are shown in parentheses. HD baitsequences may be represented multiple times if more than one searchgenerated positives. High-throughput sequencing was performedunidirectionally for identification purposes and does not necessarilyrepresent the entirety of the clone. *Search ID 14291 (HD bait 1116–1196) was identified in a search using a complex bait library ratherthan individual bait clone.

Found at doi:10.1371/journal.pgen.0030082.st003 (3.2 MB XLS).

Table S4. Drosophila Orthologs of Human Genes Tested in the Fly HDStrain


Table S5. Drosophila Modifiers with Only One Confirmed Modifica-tion Result

*Genes tested in Drosophila prior to statistical filtering; E, Enhancer; S,Suppressor


Accession Numbers

The National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db¼Protein) accession num-bers for MS studies (RefSeq) are: NP_000302.1, NP_000382.3,



NP_000524.3, NP_000602.1, NP_000708.1, NP_001019645,NP_001367.2, NP_001377.1, NP_001419.1, NP_001753.1,NP_001779.2, NP_001834.2, NP_001853.2, NP_001854.1,NP_001907.2, NP_001914.2, NP_001951.2, NP_001990.1,NP_002046.1, NP_002064.1, NP_002065.1, NP_002102.4,NP_002329.2, NP_002536.1, NP_003033.2, NP_003124.1,NP_003156.1, NP_003170.1, NP_003356.2, NP_003357.2,NP_003365.1, NP_003366.2, NP_003696.2, NP_004246.1,NP_004309.2, NP_004364.2, NP_004365.1, NP_004484.1,NP_004491.1, NP_004539.1, NP_004542.1, NP_004543.1,NP_004594.1, NP_004850.1, NP_004993.1, NP_004996.1,NP_004997.4, NP_005156.1, NP_005264.2, NP_005268.1,NP_005653.3, NP_005736.3, NP_005995.1, NP_006046.1,NP_006279.2, NP_006308.3, NP_006576.2, NP_006810.1,NP_006830.1, NP_008839.2, NP_009034.2, NP_009204.1,NP_031407.2, NP_031457.1, NP_031464.1, NP_031669.2,NP_031736.1, NP_031773.1, NP_031887.2, NP_031959.1,NP_032246.2, NP_032518.1, NP_032644.2, NP_033012.1,NP_033033.1, NP_033321.1, NP_033332.1, NP_033333.2,NP_033441.1, NP_033805.1, NP_033851.1, NP_033914.1,NP_034053.1, NP_034078.1, NP_034438.1, NP_034442.1,NP_034715.1, NP_034829.1, NP_034944.1, NP_035229.2,NP_035253.1, NP_035523.1, NP_035558.1, NP_035824.1,NP_035825.1, NP_036288.2, NP_036560.1, NP_036611.2,NP_038709.1, NP_057049.3, NP_057223.1, NP_057606.1,NP_058084.2, NP_060064.2, NP_061359.2, NP_062681.1,NP_065593.1, NP_066268.1, NP_067541.1, NP_075553.1,NP_077128.2, NP_077173.1, NP_077725.1, NP_077745.2,NP_079589.1, NP_079612.1, NP_079634.1, NP_079683.2,NP_080175.1, NP_080720.1, NP_080971.2, NP_080979.1,NP_084501.1, NP_114080.2, NP_149124.2, NP_443106.1,NP_444427.1, NP_536846.1, NP_536849.1, NP_542970.1,NP_570824.1, NP_598429.1, NP_613063.1, NP_619621.1,NP_659409.2, NP_663493.1, NP_663589.2, NP_766024.1,NP_776169.2, NP_796376.2, NP_849209.1, NP_976218.1,XP_128725.4, XP_131103.3, XP_203393.2, and XP_622887.1.

The NCBI (GeneID) (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db¼gene) accession numbers for Y2H studies are: 120, 161, 323,1315, 1387, 1499, 1759, 1778, 1785, 2597, 3064, 3092, 3093, 3275, 3329,3338, 3839, 4209, 4361, 4790, 5033, 5295, 5296, 5315, 5468, 5493, 5710,

5753, 6670, 6721, 6829, 6867, 7430, 7529, 7644, 7692, 7704, 7802, 8065,8239, 8453, 8462, 8503, 8539, 9093, 9330, 9611, 9638, 9810, 9818, 9901,9938, 10010, 10133, 10422, 10456, 10458, 10464, 10540, 10580, 10906,10915, 11177, 11193, 23116, 23328, 23332, 23348, 23360, 23380, 23609,23613, 23641, 25764, 26578, 27068, 28969, 29062, 29072, 29993, 51061,51322, 51586, 51593, 51667, 55219, 55660, 55704, 55735, 56254, 57489,57509, 57522, 57616, 63908, 79027, 79813, 80254, 83478, 84936,128866, 134218, 139818, 152789, and 171392.

Acknowledgments

We thank our colleagues at Prolexys Pharmaceuticals, particularly JayBoniface, Paul Robbins, Mike Pyne, Peter Sheffield, John Piotrowski,Andrey Sivachenko, Crismon Garff, and Ann Gauntlett for excellenttechnical expertise throughout this project. We also thank NathaliaAllevato for excellent technical assistance with the fly studies. Wethank Hugo Bellen, Janet Fischer, and Leo Pallanck for fly strains.Michael Hayden provided YAC mice to L.M.E. Stanley Fields, AlLaSpada, and John P. Miller provided helpful comments on themanuscript.

Author contributions. LSK, ER, RRB, JS, SS, CK, LME, JMP, JB, andREH conceived and designed the experiments. LSK, ER, RRB, CT, JS,AH, GHC, LU, CLC, and YZ performed the experiments. LSK, ER,RRB, RC, RB, AP, AS, CT, JS, AH, GHC, LU, CLC, YZ, SS, JO, CK,LME, JMP, JB, and REH analyzed the data. RC, RB, AP, AS, JO, LME,JB, and REH contributed reagents/materials/analysis tools. LSK, ER,CK, LME, JMP, JB, and REH wrote the paper.

Funding. GHC was supported by the post-doctoral FellowshipProgram of Korea Science and Engineering Foundation (KOSEF).This work was supported by a grant to JB from the National Institutesof Health (NIH) (NS42179); grants to LME from the NIH (RO1NS40251) and HighQ Foundation; and grants to REH from the CureHuntington’s Disease Initiative, the Hereditary Disease Foundation,and the HighQ Foundation. SS is the CSO of Prolexys Pharmaceut-icals.

Competing interests. The authors have declared that no competinginterests exist.

References1. Landles C, Bates GP (2004) Huntingtin and the molecular pathogenesis of

Huntington’s disease. Fourth in molecular medicine review series. EMBORep 5: 958–963.

2. Hickey MA, Chesselet MF (2003) The use of transgenic and knock-in miceto study Huntington’s disease. Cytogenet Genome Res 100: 276–286.

3. Zoghbi HY, Botas J (2002) Mouse and fly models of neurodegeneration.Trends Genet 18: 463–471.

4. Driscoll M, Gerstbrein B (2003) Dying for a cause: Invertebrate geneticstakes on human neurodegeneration. Nat Rev Genet 4: 181–194.

5. DiFiglia M, Sapp E, Chase K, Schwarz C, Meloni A, et al. (1995) Huntingtinis a cytoplasmic protein associated with vesicles in human and rat brainneurons. Neuron 14: 1075–1081.

6. Davies SW, Turmaine M, Cozens BA, DiFiglia M, Sharp AH, et al. (1997)Formation of neuronal intranuclear inclusions underlies the neurologicaldysfunction in mice transgenic for the HD mutation. Cell 90: 537–548.

7. Cummings CJ, Mancini MA, Antalffy B, DeFranco DB, Orr HT, et al. (1998)Chaperone suppression of aggregation and altered subcellular proteasomelocalization imply protein misfolding in SCA1. Nat Genet 19: 148–154.

8. Perez MK, Paulson HL, Pendse SJ, Saionz SJ, Bonini NM, et al. (1998)Recruitment and the role of nuclear localization in polyglutamine-mediated aggregation. J Cell Biol 143: 1457–1470.

9. Kazantsev A, Preisinger E, Dranovsky A, Goldgaber D, Housman D (1999)Insoluble detergent-resistant aggregates form between pathological andnonpathological lengths of polyglutamine in mammalian cells. Proc NatlAcad Sci U S A 96: 11404–11409.

10. Suhr ST, Senut MC, Whitelegge JP, Faull KF, Cuizon DB, et al. (2001)Identities of sequestered proteins in aggregates from cells with inducedpolyglutamine expression. J Cell Biol 153: 283–294.

11. Nucifora FC Jr., Sasaki M, Peters MF, Huang H, Cooper JK, et al. (2001)Interference by huntingtin and atrophin-1 with cbp-mediated transcrip-tion leading to cellular toxicity. Science 291: 2423–2428.

12. Boutell JM, Thomas P, Neal JW, Weston VJ, Duce J, et al. (1999) Aberrantinteractions of transcriptional repressor proteins with the Huntington’sdisease gene product, huntingtin. Hum Mol Genet 8: 1647–1655.

13. Steffan JS, Kazantsev A, Spasic-Boskovic O, Greenwald M, Zhu YZ, et al.(2000) The Huntington’s disease protein interacts with p53 and CREB-binding protein and represses transcription. Proc Natl Acad Sci U S A 97:6763–6768.

14. Schaffar G, Breuer P, Boteva R, Behrends C, Tzvetkov N, et al. (2004)

Cellular toxicity of polyglutamine expansion proteins: Mechanism oftranscription factor deactivation. Mol Cell 15: 95–105.

15. Dunah AW, Jeong H, Griffin A, Kim YM, Standaert DG, et al. (2002) Sp1 andTAFII130 transcriptional activity disrupted in early Huntington’s disease.Science 296: 2238–2243.

16. Steffan JS, Bodai L, Pallos J, Poelman M, McCampbell A, et al. (2001)Histone deacetylase inhibitors arrest polyglutamine-dependent neuro-degeneration in Drosophila. Nature 413: 739–743.

17. Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S (2004) Inclusionbody formation reduces levels of mutant huntingtin and the risk ofneuronal death. Nature 431: 805–810.

18. Slow EJ, Graham RK, Osmand AP, Devon RS, Lu G, et al. (2005) Absence ofbehavioral abnormalities and neurodegeneration in vivo despite wide-spread neuronal huntingtin inclusions. Proc Natl Acad Sci U S A 102:11402–11407.

19. Zala D, Benchoua A, Brouillet E, Perrin V, Gaillard MC, et al. (2005)Progressive and selective striatal degeneration in primary neuronalcultures using lentiviral vector coding for a mutant huntingtin fragment.Neurobiol Dis 20: 785–798.

20. MacDonald ME (2003) Huntingtin: Alive and well and working in middlemanagement. Sci STKE 2003: pe48.

21. Andrade MA, Bork P (1995) HEAT repeats in the Huntington’s diseaseprotein. Nat Genet 11: 115–116.

22. Harjes P, Wanker EE (2003) The hunt for huntingtin function: Interactionpartners tell many different stories. Trends Biochem Sci 28: 425–433.

23. Li SH, Li XJ (2004) Huntingtin-protein interactions and the pathogenesisof Huntington’s disease. Trends Genet 20: 146–154.

24. Cattaneo E, Zuccato C, Tartari M (2005) Normal huntingtin function: Analternative approach to Huntington’s disease. Nat Rev Neurosci 6: 919–930.

25. Gavin AC, Bosche M, Krause R, Grandi P, Marzioch M, et al. (2002)Functional organization of the yeast proteome by systematic analysis ofprotein complexes. Nature 415: 141–147.

26. Ho Y, Gruhler A, Heilbut A, Bader GD, Moore L, et al. (2002) Systematicidentification of protein complexes in Saccharomyces cerevisiae by massspectrometry. Nature 415: 180–183.

27. Ito T, Chiba T, Ozawa R, Yoshida M, Hattori M, et al. (2001) Acomprehensive two-hybrid analysis to explore the yeast protein inter-actome. Proc Natl Acad Sci U S A 98: 4569–4574.

28. Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, et al. (2000) A



comprehensive analysis of protein-protein interactions in Saccharomycescerevisiae. Nature 403: 623–627.

29. Lim J, Hao T, Shaw C, Patel AJ, Szabo G, et al. (2006) A protein-proteininteraction network for human inherited ataxias and disorders of Purkinjecell degeneration. Cell 125: 801–814.

30. Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, et al. (2005)Towards a proteome-scale map of the human protein-protein interactionnetwork. Nature 437: 1173–1178.

31. Lacount DJ, Vignali M, Chettier R, Phansalkar A, Bell R, et al. (2005) Aprotein interaction network of the malaria parasite Plasmodium falciparum.Nature 438: 103–107.

32. James P, Halladay J, Craig EA (1996) Genomic libraries and a host straindesigned for highly efficient two-hybrid selection in yeast. Genetics 144:1425–1436.

33. Sprinzak E, Sattath S, Margalit H (2003) How reliable are experimentalprotein-protein interaction data? J Mol Biol 327: 919–923.

34. von Mering C, Krause R, Snel B, Cornell M, Oliver SG, et al. (2002)Comparative assessment of large-scale data sets of protein-proteininteractions. Nature 417: 399–403.

35. Waelter S, Scherzinger E, Hasenbank R, Nordhoff E, Lurz R, et al. (2001)The huntingtin interacting protein HIP1 is a clathrin and alpha-adaptin-binding protein involved in receptor-mediated endocytosis. Hum MolGenet 10: 1807–1817.

36. Burke JR, Enghild JJ, Martin ME, Jou YS, Myers RM, et al. (1996) Huntingtinand DRPLA proteins selectively interact with the enzyme GAPDH. Nat Med2: 347–350.

37. Laviolette MJ, Nunes P, Peyre JB, Aigaki T, Stewart BA (2005) A geneticscreen for suppressors of Drosophila NSF2 neuromuscular junction over-growth. Genetics 170: 779–792.

38. Singh A, Chan J, Chern JJ, Choi KW (2005) Genetic interaction of Lobe withits modifiers in dorsoventral patterning and growth of the Drosophila eye.Genetics 171: 169–183.

39. Kazemi-Esfarjani P, Benzer S (2000) Genetic suppression of polyglutaminetoxicity in Drosophila. Science 287: 1837–1840.

40. Fernandez-Funez P, Nino-Rosales ML, de Gouyon B, She WC, Luchak JM, etal. (2000) Identification of genes that modify ataxin-1-induced neuro-degeneration. Nature 408: 101–106.

41. Brunger AT (2005) Structure and function of SNARE and SNARE-interacting proteins. Q Rev Biophys 38: 1–47.

42. Sudhof TC (2004) The synaptic vesicle cycle. Annu Rev Neurosci 27: 509–547.

43. Velier J, Kim M, Schwarz C, Kim TW, Sapp E, et al. (1998) Wild-type andmutant huntingtins function in vesicle trafficking in the secretory andendocytic pathways. Exp Neurol 152: 34–40.

44. Qin ZH, Wang Y, Sapp E, Cuiffo B, Wanker E, et al. (2004) Huntingtinbodies sequester vesicle-associated proteins by a polyproline-dependentinteraction. J Neurosci 24: 269–281.

45. Stark C, Breitkreutz BJ, Reguly T, Boucher L, Breitkreutz A, et al. (2006)BioGRID: A general repository for interaction datasets. Nucleic Acids Res34: D535–D539.

46. Mishra GR, Suresh M, Kumaran K, Kannabiran N, Suresh S, et al. (2006)Human protein reference database–2006 update. Nucleic Acids Res 34:D411–D414.

47. Peri S, Navarro JD, Amanchy R, Kristiansen TZ, Jonnalagadda CK, et al.(2003) Development of human protein reference database as an initialplatform for approaching systems biology in humans. Genome Res 13:2363–2371.

48. Slow EJ, van Raamsdonk J, Rogers D, Coleman SH, Graham RK, et al. (2003)Selective striatal neuronal loss in a YAC128 mouse model of Huntingtondisease. Hum Mol Genet 12: 1555–1567.

49. Bae BI, Hara MR, Cascio MB, Wellington CL, Hayden MR, et al. (2006)

Mutant huntingtin: Nuclear translocation and cytotoxicity mediated byGAPDH. Proc Natl Acad Sci U S A 103: 3405–3409.

50. Venkatraman P, Wetzel R, Tanaka M, Nukina N, Goldberg AL (2004)Eukaryotic proteasomes cannot digest polyglutamine sequences and releasethem during degradation of polyglutamine-containing proteins. Mol Cell14: 95–104.

51. Browne SE, Bowling AC, MacGarvey U, Baik MJ, Berger SC, et al. (1997)Oxidative damage and metabolic dysfunction in Huntington’s disease:Selective vulnerability of the basal ganglia. Ann Neurol 41: 646–653.

52. Panov AV, Gutekunst CA, Leavitt BR, Hayden MR, Burke JR, et al. (2002)Early mitochondrial calcium defects in Huntington’s disease are a directeffect of polyglutamines. Nat Neurosci 5: 731–736.

53. Tang TS, Tu H, Chan EY, Maximov A, Wang Z, et al. (2003) Huntingtin andhuntingtin-associated protein 1 influence neuronal calcium signalingmediated by inositol-(1,4,5) triphosphate receptor type 1. Neuron 39:227–239.

54. Gunawardena S, Her LS, Brusch RG, Laymon RA, Niesman IR, et al. (2003)Disruption of axonal transport by loss of huntingtin or expression ofpathogenic polyQ proteins in Drosophila. Neuron 40: 25–40.

55. Li JY, Plomann M, Brundin P (2003) Huntington’s disease: A synaptopathy?Trends Mol Med 9: 414–420.

56. Swayne LA, Chen L, Hameed S, Barr W, Charlesworth E, et al. (2005)Crosstalk between huntingtin and syntaxin 1A regulates N-type calciumchannels. Mol Cell Neurosci 30: 339–351.

57. Goehler H, Lalowski M, Stelzl U, Waelter S, Stroedicke M, et al. (2004) Aprotein interaction network links GIT1, an enhancer of huntingtinaggregation, to Huntington’s disease. Mol Cell 15: 853–865.

58. Faber PW, Barnes GT, Srinidhi J, Chen J, Gusella JF, et al. (1998) Huntingtininteracts with a family of WW domain proteins. Hum Mol Genet 7: 1463–1474.

59. Kitamura A, Kubota H, Pack CG, Matsumoto G, Hirayama S, et al. (2006)Cytosolic chaperonin prevents polyglutamine toxicity with altering theaggregation state. Nat Cell Biol 8: 1163–1170.

60. Tam S, Geller R, Spiess C, Frydman J (2006) The chaperonin TRiC controlspolyglutamine aggregation and toxicity through subunit-specific interac-tions. Nat Cell Biol 8: 1155–1162.

61. Hudson JR Jr., Dawson EP, Rushing KL, Jackson CH, Lockshon D, et al.(1997) The complete set of predicted genes from Saccharomyces cerevisiae in areadily usable form. Genome Res 7: 1169–1173.

62. Peltier JM, Askovic S, Becklin RR, Chepanoske CL, Ho Y-SJ, et al. (2004) Anintegrated strategy for the discovery of drug targets by the analysis ofprotein-protein interactions. Int J Mass Spectrom 238: 119–130.

63. Pappin DJ, Hojrup P, Bleasby AJ (1993) Rapid identification of proteins bypeptide-mass fingerprinting. Curr Biol 3: 327–332.

64. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-basedprotein identification by searching sequence databases using massspectrometry data. Electrophoresis 20: 3551–3567.

65. Brand AH, Perrimon N (1993) Targeted gene expression as a means ofaltering cell fates and generating dominant phenotypes. Development 118:401–415.

66. Schilling G, Becher MW, Sharp AH, Jinnah HA, Duan K, et al. (1999)Intranuclear inclusions and neuritic aggregates in transgenic miceexpressing a mutant N-terminal fragment of huntingtin. Hum Mol Genet8: 397–407.

67. Yu ZX, Li SH, Evans J, Pillarisetti A, Li H, et al. (2003) Mutant huntingtincauses context-dependent neurodegeneration in mice with Huntington’sdisease. J Neurosci 23: 2193–2202.

68. Ganetzky B, Flanagan JR (1978) On the relationship between senescenceand age-related changes in two wild-type strains of Drosophila melanogaster.Exp Gerontol 13: 189–196.



huntingtin interacting proteins are genetic modifiers of ... · proteins [25–28]. we reasoned...

Documents