university of groningen dnaj proteins: more than just “co ... · pdf file144 chapter 7...

University of Groningen

DNAJ proteins: more than just “co-chaperones”Kakkar, Vaishali

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:2014

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Kakkar, V. (2014). DNAJ proteins: more than just “co-chaperones”: Implicaties voor eiwit-aggregatie ziektes[S.l.]: [S.n.]

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 13-05-2018

https://www.rug.nl/research/portal/en/publications/dnaj-proteins-more-than-just-cochaperones(5fbeb80f-7db6-41be-a34d-325181093a68).html

Chapter 7

DNAJ family shows J-domain dependent anti-aggregation

activity on RING1 mutant parkin

Vaishali Kakkar1*, Els F. E. Kuiper 1,2*, Harm H Kampinga1

* equal contribution

1University Medical Center Groningen, University of Groningen, Department of Cell Biology, A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands.

2European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, Antonius Deusinglaan 1, 9714 AV Groningen, The Netherlands.

Manuscript Submitted

144

DNAJs and ParkinChapter 7

ABSTRACT

Parkinson’s disease is one of the most common neurodegenerative disorders and several mutations in different genes have been identified to contribute to the disease. A loss of function parkin RING1 domain mutant (C289G) is associated with autosomal-recessive juvenile-onset Parkinsonism (AR-JP) and displays altered solubility and sequesters into aggregates. The heat shock protein 70 (Hsp70) chaperone machine has been shown to play a role in the prevention of aggregation of different mutant or stress unfolded proteins. For neurodegenerative CAG repeat diseases, we previously found that overexpression of members of the DNAJB co-chaperone subfamily, rather than Hsp70 itself, suppressed aggregation of the disease causing polyglutamine (polyQ) proteins. For the most potent members, DNAJB6b and DNAJB8, this activity was largely independent of the Hsp70 machine. Here, we show that upon upregulation, all cytosolic DNAJ family members are equally efficient in reducing parkin C289G aggregation. Upregulation of the DNAJs alone was sufficient to provide protection, but the action of all DNAJs tested was dependent on interaction with and activity of Hsp70s. Our data imply that different aggregation-prone, disease-causing proteins pose different challenges to the protein quality control system and require specific chaperones for their proper handling. The DNAJs can have both Hsp70 dependent and independent activities, highlighting the versatility of the DNAJs with respect to the disease causing proteins, thus making them possible interesting potential therapeutic targets.

145

7

7


INTRODUCTION

Maintenance of protein quality control (PQC) is critical for cellular and organismal health. When PQC is lost either due to stress conditions or mutations, intracellular proteins are prone to misfolding and aggregate formation. Proteins need to be folded correctly to prevent them from attaining conformations that can cause them to function improperly or aggregate into, potentially cytotoxic, complexes (1). The accumulation of misfolded and aggregated proteins is a hallmark of many ageing-related neurodegenerative diseases like Amyotrophic Lateral Sclerosis, Alzheimer’s, Parkinson’s and Huntington’s disease, also called proteinopathies. Maintenance of proteostasis is achieved by an integrated network of several hundred proteins, including the autophagy system, the ubiquitin-proteasome system (UPS), and molecular chaperones and their regulators (2, 3). Molecular chaperones play a crucial role in various ways in the prevention of misfolding and aggregation of different mutant proteins. The largest group of molecular chaperones are the heat shock proteins (HSPs). Most HSPs can recognize exposed hydrophobic regions of non-native proteins and hereby can prevent protein aggregation. In doing so, they not only assist in protein (re)folding but also in the degradation of misfolded client proteins by targeting them to the protein degradation machineries (4, 5). Due to the different characteristics of the HSPs as modifiers of proteostasis, they have been considered as a possible therapeutic target for multiple diseases involving aggregation of mutant proteins or disruption of the proteostasis network. However, there are large differences between proteinopathies with regard to which proteins form aggregates and the type of aggregates they form (6–8) and this likely will be important to determine which class of HSPs might be most suited in the prevention of aggregation of these proteins (9).

In Parkinson’s disease, progressive accumulation of stable protein aggregates in the cytoplasm, named Lewy bodies, is one of the pathological hallmarks and the cause of selective loss of dopaminergic neurons in the substantia nigra (10–13). Several dominantly inherited mutations are known, including mutations in the genes for α-synuclein (SNCA) and parkin (PARK2), which cause protein aggregations and the disease phenotype (11, 14–16). Parkin is an E3 ubiquitin ligase that plays a role in ubiquitination of several candidate substrate proteins and thereby targeting them for proteasomal degradation. Parkin is also involved in the elimination of damaged mitochondria through autophagy, called mitophagy (17, 18). Parkin contains a N-terminal ubiquitin-like (Ubl) domain, a central RING (Really Interesting New Gene) domain (RING0) and a C-terminal RING domain consisting of two RING finger motifs (RING1 and RING2) separated by an In-between-RING (IBR) domain (19). A wide variety of PARK2 mutations have been found, including exon deletions, duplications and triplications, missense, nonsense, and frameshift mutations (20). Here, we focus on one

146


of the first reported mutations in the RING1 domain of parkin, the Cys289 to Gly (C289G) mutation, that is associated with an autosomal-recessive form of juvenile parkinsonism (AR-JP) (21, 22). Besides a loss of function, the C289G mutation results in alterations in parkin solubility and sequestration in aggresome-like protein aggregates and hence might also indicate a dominant toxic-gain-of function phenomenon (23–27). Indeed, expression of parkin C289G in cells leads to impaired mitochondrial function, which is restored when this aggregation formation is prevented (18) and carrying a single allele with the C289G mutant is associated with a higher risk of parkinsonism (28–30).

Given the capacity of molecular chaperones to prevent aggregation of misfolded proteins and analogous to our previous work on polyQ aggregation (31, 32) we compared different members the Hsp70 machine for their ability to prevent aggregation of parkin C289G (6, 9, 31, 33–38). Using the same cell model as used for the polyQ screen, we now show that handling of parkin C289G requires different chaperone activities. In fact, unlike for polyQ aggregation, all cytoplasmic DNAJs were equally effective in preventing parkin C289G aggregation in an Hsp70-dependent manner. Specifically, DNAJB6 and DNAJB8, which we have previously identified as superior suppressors of polyQ aggregation in an Hsp70 independent but C-terminal SSF-SST stretch dependent manner, could prevent parkin C289G aggregation in a manner independent of its C-terminal SSF-SST stretch and only when it was able to interact with the Hsp70 machinery.

RESULTS

DNAJB2a, DNAJB6, and DNAJB8 are equally efficient in reducing Parkin C289G mediated aggregation

Previously, we have shown aggregation of polyQ proteins can be prevented most efficiently by DNAJB6b and DNAJB8 and to a lesser extent by DNAJB2a (31). To test whether these chaperones are also able to reduce parkin C289G aggregation, HEK293 cells were transiently co-transfected with V5-tagged chaperones and flag-tagged parkin wildtype (WT) or C289G mutant. Upon Triton X-100 (TX-100) lysis, most of the wild type protein distributes to the TX-100 soluble fraction (Fig. 1A, lane 1 and 6), whereas parkin C289G ends up in the TX-100 insoluble fraction (Fig. 1A, lane 2 and 7). In addition, for parkin C289G a high molecular weight (HMW) smear in the TX-100 insoluble fraction of the cell lysate can be seen (Fig. 1A, lane 7), consistent with earlier work (17) and indicative of parkin C289G aggregation. Furthermore, cells expressing parkin C289G show multiple large inclusions scattered throughout the cytoplasm and nucleus whilst parkin WT is uniformly distributed in the cells (Fig. 1B, panel 1 and 2) (18). Unlike for the inhibition of polyQ aggregation, where DNAJB6 and DNAJB8 were superior over DNAJB2a, co-transfection of all three

147

7

7


chaperones decreased parkin C289G aggregation to a similar extent (Fig. 1A, lane 8, 9 and, 10). Inclusion formation was also equally effective reduced by DNAJB2a (Fig. 1B, panel 3), DNAJB6 (not shown), and DNAJB8 (Fig. 2C, panel 1). For DNAJB2a, these data are in line with the findings of Rose et al., using different cell lines (18).

A functional J-domain is critical for activity of DNAJ proteins against parkin C289G aggregation

Since we found no differences in efficacy between DNAJB6, DNAJB8, and DNAJB2a in their ability to reduce parkin C289G aggregation, we wondered whether other members of the DNAJ family could also prevent aggregation of parkin C289G. In fact, co-expressing parkin C289G with various DNAJA (Fig. 1C) and DNAJB family members (Fig. 1D) revealed that most DNAJs are capable of preventing parkin C289G insolubilization. Exceptions are DNAJA3 which is localized to mitochondria (1, 39), DNAJA4 which is a membrane-associated protein, and DNAJB9 which is normally localized to the ER (1, 40) and was hardly expressed here. The inability of these chaperones to prevent parkin C289G aggregation can be attributed to their different localization as compared to parkin, which is predominantly cytosolic and forms perinuclear inclusions.

In case of expanded polyQ proteins, the serine rich region at the C-terminal of DNAJB6 and DNAJB8, and not the J-domain, was found to be essential for anti-aggregation activity (31). To investigate the role of the different domains of DNAJ proteins in preventing parkin C289G aggregation, different mutants of the DNAJ chaperones were co-transfected with parkin C289G (Fig. 2A). DNAJ chaperones contain a highly conserved ~70 amino acid J-domain with a histdine-proline-aspartic acid (HPD) motif which is necessary for the interaction between DNAJs and Hsp70s (41). Here, we used DNAJ mutants in which the histidine residue in the HPD motif was substituted by a glutamine (H/Q) leading to a J-domain that can no longer bind to Hsp70s. We also used a mutant of DNAJB6b in which the entire J-domain was deleted (ΔJ) (Fig. 2B). Mutations in the J-domain or deletion of the J-domain render all the DNAJs incapable of preventing aggregation of parkin C289G (Fig. 2A, lane 17, 18, 21, and 24). For DNAJB8, we confirmed this with immunohistochemistry, showing that in cells co-transfected with the H/Q mutant of DNAJB8, parkin C289G still forms large inclusions. Interestingly, unlike wild type DNAJB8, the DNAJB8 H/Q mutant was found to co-localize with parkin C289G aggregates, suggesting that it may be trapped here due to its inability to interact with Hsp70 (Fig. 2C, panel 2). Together, these data imply that Hsp70 interaction is required for all DNAJs to prevent parkin C289G aggregation.

Besides the J-domain mutants, we tested the F93L mutant of DNAJB6b with a missense mutation in the G/F-rich region. This F93L mutant was recently implicated in limb-girdle

148


Figu

re 1

. Cha

pero

nes

cont

aini

ng a

J-d

omai

n ca

n pr

even

t agg

rega

tion

of p

arki

n C

289G

. (A

) Cel

ls w

ere

trans

fect

ed w

ith fl

ag-ta

gged

par

kin

WT,

flag

-tagg

ed p

arki

n C

289G

or c

o-tra

nsfe

cted

with

flag

-tagg

ed p

arki

n C

289G

and

V5-

tagg

ed D

NAJ

B2a,

DN

AJB6

b, o

r DN

AJB8

. Exp

ress

ion

of c

hape

rone

s w

as in

duce

d w

ith te

tracy

clin

e. T

riton

X-1

00 (T

X-10

0) s

olub

le

and

inso

lubl

e fra

ctio

n w

ere

obta

ined

24

hour

s af

ter t

rans

fect

ion.

Par

kin

WT

and

C28

9G w

ere

asse

ssed

with

ant

i-par

kin

and

anti-

flag

antib

odie

s on

Wes

tern

blo

t. In

the

TX-1

00 in

solu

ble

fract

ion,

hig

h m

olec

ular

wei

ght (

HM

W)

spec

ies

of p

arki

n C

289G

are

obs

erve

d. C

o-tra

nsfe

ctio

n w

ith D

NAJ

B2a,

DN

AJB6

b, o

r D

NAJ

B8 p

reve

nts

form

atio

n of

par

kin

C28

9G H

MW

sp

ecie

s. A

nti-p

arki

n an

tibod

ies

dete

ct fu

ll-le

ngth

par

kin

and

an N

-term

inal

ly tr

unca

ted

isof

orm

(als

o re

mov

ing

the

flag-

tag)

, gen

erat

ed d

ue to

an

inte

rnal

sta

rt si

te, s

how

ing

an e

xtra

ba

nd a

roun

d 42

kD

a (6

5). T

he a

nti-fl

ag a

ntib

odie

s re

cogn

ize

only

the

full-

leng

th p

arki

n pr

otei

n. (B

) Rep

rese

ntat

ive

imm

unofl

uore

scen

ce p

ictu

res

of c

ells

co-

trans

fect

ed w

ith fl

ag-

tagg

ed p

arki

n W

T or

par

kin

C28

9G (r

ed) a

nd V

5-ta

gged

cha

pero

nes

(gre

en).

DAP

I sta

inin

g is

sho

wn

in b

lue.

Bar

repr

esen

ts 1

0 µm

. Par

kin

WT

show

s a

diffu

se p

atte

rn th

roug

hout

the

cyto

plas

m. P

arki

n C

289G

form

s ag

greg

ates

mai

nly

conc

entra

ted

into

larg

e pe

rinuc

lear

incl

usio

ns. C

o-tra

nsfe

ctio

n w

ith D

NAJ

B2a

reve

als

clea

ranc

e of

par

kin

C28

9G a

ggre

gate

s an

d co

-loca

lizat

ion

of D

NAJ

B2a

with

par

kin

C28

9G, i

ndic

ated

by

arro

whe

ads.

(C) A

ccum

ulat

ion

of p

arki

n C

289G

is p

reve

nted

whe

n co

-tran

sfec

ted

with

mem

bers

of t

he D

NAJ

A su

bfam

ily.

DN

AJA3

, whi

ch is

loca

lized

to m

itoch

ondr

ia, a

nd D

NAJ

A4, a

mem

bran

e pr

otei

n, a

re e

xcep

tions

. (D

) C

o-tra

nsfe

ctio

n w

ith D

NAJ

B ch

aper

ones

cou

ld a

lso

prev

ent p

arki

n C

289G

ag

greg

atio

n. D

NAJ

B9 s

how

s no

pre

vent

ion,

due

to lo

caliz

atio

n of

the

chap

eron

e to

the

ER. E

xpre

ssio

n of

cha

pero

nes

was

det

ecte

d w

ith a

nti-V

5 an

tibod

ies.

GAP

DH

was

use

d as

a

load

ing

cont

rol f

or th

e so

lubl

e fra

ctio

n.

149

7

7


muscular dystrophy and suggested to be slightly impaired delaying polyQ mediated aggregation (42). The F93L mutant, however, was equally effective as the wildtype DNAJB6 in preventing parkin C289G aggregation (Fig. 2A, lane 19), implying that this disease-causing mutation has no impact on the functional DNAJB6-Hsp70 interaction in dealing with parkin C298G aggregates. Next, we used a deletion mutant of DNAJB8 that lacks the serine rich SSF-SST domain (ΔSSF-SST). In contrast to the crucial role of this domain in the prevention of polyQ aggregation (31), the ΔSSF-SST mutant fully retained its anti-aggregation activity on parkin C289G as revealed by fractionation (Fig. 2A, lane 22) and immunofluorescence (Fig 2C, panel 3). These combined data further illustrate different chaperone activity requirements for dealing with either polyQ or parkin C289G aggregation.

To further consolidate that DNAJs prevent aggregation of parkin C289G via an interaction with Hsp70s, we examined whether short interfering RNA (siRNA)-mediated knockdown of HSPA1A had an effect on the DNAJ mediated clearance of parkin C289G. Interestingly, the partial RNAi-mediated knockdown of HSPA1A alone, already increases parkin C289G insolubilization considerably (Fig. 3A, lane 12), implying that constitutively expressed HSPA1A contributes to handling of parkin C289G to reduce its aggregation (see also Fig. 4A).

In the RNAi-treated cells, the overexpressed DNAJs largely have lost their ability to prevent parkin C289G aggregation. Yet, a small reduction in parkin C289G aggregation is still seen for DNAJB2a, DNAJB6b, or DNAJA1 (but not DNAJB1) (Fig. 3A, lane 14, 16, and 20 compared to lane 12). Interestingly, in the DNAJB2a, DNAJB6b, and DNAJA1 co-expressing cells, HMW smears were now detected in the TX-100 soluble fraction (Fig. 3A, lane 4, 6, 8, and 10), suggesting some Hsp70-independent actions of the DNAJs prior to parkin C289G insolubilization. When cells were treated with VER-155008, an adenosine-derived Hsp70 inhibitor that targets the ATPase binding domain of all Hsp70s (43), similar results were observed (Fig. 3B). This data demonstrates that DNAJs are capable of partially solubilising parkin C289G when Hsp70s are depleted but that Hsp70s are required for full prevention of parkin C298G aggregation.

Effects on parkin C289G aggregation upon overexpression of other components of the Hsp70 machinery

From the above results, parkin C289G appears to be a substrate for the Hsp70 machine, in which upregulation of different DNAJs can enhance the cellular capacity to prevent degradation as long as sufficient Hsp70 activity is available (Fig. 1, Fig. 2, and Fig. 3). We next asked whether upregulation of different members of the Hsp70 family might also result in reduced parkin C298G aggregation. HSPA1A and HSPA14 indeed reduced TX-100

150


Figu

re 2

. The

ant

i-agg

rega

tion

activ

ity o

f DN

AJ c

hape

rone

s is

dep

ende

nt o

n th

e J-

dom

ain,

not

on

the

SSF-

SST

dom

ain.

(A

) Ag

greg

atio

n of

par

kin

C28

9G is

pre

vent

ed w

hen

co-tr

ansf

ecte

d w

ith D

NAJ

B2a,

DN

AJB6

b, D

NAJ

B8, o

r D

NAJ

B1. M

utat

ions

in th

e J-

dom

ain

of th

e ch

aper

ones

(H

/Q)

and

dele

tion

of th

e J-

dom

ain

as a

who

le (

ΔJ),

resu

lts in

the

loss

of p

reve

ntio

n of

agg

rega

tion

of p

arki

n C

289G

. DN

AJB6

b w

ith a

mut

atio

n in

the

G/F

-rich

reg

ion

(F93

L) r

etai

ns it

s an

ti-ag

greg

atio

n ac

tivity

. Del

etio

n of

the

SSF-

SST

dom

ain

(ΔSS

F-SS

T) o

f DN

AJB8

doe

s no

t affe

ct it

s ab

ility

to p

reve

nt a

ggre

gatio

n. (B

) Sch

e mat

ic o

verv

iew

of D

NAJ

B1, D

NAJ

2a, D

NAJ

B6, a

nd D

NAJ

B8 a

nd th

e m

utan

ts. (

C) R

epre

sent

ativ

e im

mun

ofluo

resc

ence

pic

ture

s of

cel

ls c

o-tra

nsfe

cted

with

flag

-tagg

ed p

arki

n C

289G

(red

) and

V5-

tagg

ed c

hape

rone

s (g

reen

). D

API s

tain

ing

is s

how

n in

blu

e. B

ar re

pres

ents

10

µm.

Co-

trans

fect

ion

with

DN

AJB8

sho

ws

a re

duct

ion

in p

arki

n C

289G

agg

rega

tes

and

co-lo

caliz

atio

n of

DN

AJB8

with

par

kin

C28

9G, i

ndic

ated

by

arro

whe

ads.

Co-

trans

fect

ion

with

DN

AJB8

H

/Q s

till s

how

s fo

rmat

ion

of p

arki

n C

289G

agg

rega

tes

mai

nly

conc

entra

ted

into

larg

e pe

rinuc

lear

incl

usio

ns, i

ndic

ated

by

arro

whe

ads.

Par

kin

C28

9G a

nd D

NAJ

B8 H

/Q c

o-lo

caliz

e bu

t ag

greg

ate

form

atio

n is

not

pre

vent

ed. C

o-tra

nsfe

ctio

n w

ith D

NAJ

B8 Δ

SSF-

SST

reve

als

clea

ranc

e of

par

kin

C28

9G a

ggre

gate

s an

d in

occ

asio

nal s

mal

l agg

rega

tions

co-

loca

lizat

ion

of

DN

AJB8

ΔSS

F-SS

T w

ith p

arki

n, in

dica

ted

by a

rrow

head

s.

151

7

7


insolubilization to some extent (Fig. 4A, lane 10 ad 16) but without a significant increase in the amount of parkin C289G in the TX-100 soluble fraction (Fig. 4A, lanes 2 and 8). Overexpression of HSPA1L, HSPA2, HSPA6, HSPA8, and HSPA9 was ineffective (Fig. 4A, lanes 11 - 15). A minimal Hsp70 machine contains, besides DNAJs and HSPAs, also a nucleotide exchange factors (NEFs) (1). NEFs are a heterologous group of co-factors required for the dissociation of the substrate from HSPA and may also - in part - determine the fate of the substrate (1), like HSPH2, which is able to remodel the Hsp70 machine to efficiently disaggregate aggregated proteins, and BAG3 (44), which has been implied in dealing with aggregation of polyQ proteins. However, neither overexpression of the cytosolic NEFs (HSPH1, HSPH2, or HSPH3) (Fig. 4B) nor the overexpression of the BAG family members (BAG1 and BAG3) (Fig. 4C) has an effect on the aggregation of parkin C289G. So, within the background of endogenous expression levels of HSPs in HEK293 cells, overexpression of DNAJs seems the most effective way (i.e. rate limiting) to increase the efficiency of the Hsp70 machine to prevent aggregation of parkin C298G.

DISCUSSION

In the current study we show that, when upregulated in cells, all cytosolic members of the DNAJA and DNAJB subfamilies can suppress parkin C289G aggregation in an Hsp70 dependent manner. As it was already shown for DNAJB2a, prevention of parkin C289G aggregation was associated with restoring its function in mitophagy (18), implying that such anti-aggregation effects are cytoprotective. Hsp70 inhibition increased parkin C289G aggregation and only upregulation of HSPA1A and HSPA14, but none of the other Hsp70 members, resulted in reduced parkin C289G aggregation. Further, overexpression of the cytosolic nucleotide exchange factors (HSPH1, HSPH2, HSPH3, BAG1, and BAG3) did not lead to reduced parkin C289G aggregation. Comparing our current observations on parkin C298G to our previous studies (31, 32, 44) on suppression of aggregation of polyQ containing proteins, performed in the same cell model, reveals a number of intriguing features. First, different aggregation-prone, disease-causing proteins pose different challenges to the PQC systems and require specific chaperones for their handling. Second, the different HSPA members show a certain degree of functional differentiation despite their high sequence similarities and molecular activity. Third, the DNAJB6b and DNAJB8 chaperones that we previously identified as the most potent but largely Hsp70-independent suppressors of polyQ aggregation (31), can also inhibit parkin C289G aggregation but do now depend on a functional Hsp70 machine.

Why do we find such substantial differences between the chaperone requirements for these two different aggregation-prone, disease-causing proteins? First, the aggregates caused by the two mutant proteins have different biochemical properties as revealed by detergent

152


Figu

re 3

. The

ant

i-agg

rega

tion

activ

ity o

f DN

AJ c

hape

rone

s is

dep

ende

nt o

n H

sp70

s. (A

) Kno

ckdo

wn

of H

SPA

incr

ease

s le

vels

of T

X-10

0 so

lubl

e an

d in

solu

ble

park

in C

289G

. In

cells

co-

trans

fect

ed w

ith D

NAJ

s, th

e H

MW

sm

ear i

s st

ill fo

rmed

, ind

icat

ing

that

DN

AJs

lose

thei

r abi

lity

to p

reve

nt p

arki

n C

289G

agg

rega

tion.

How

ever

, a re

duct

ion

in th

e sm

ear c

an

be o

bser

ved

whe

n pa

rkin

C28

9G is

co-

trans

fect

ed w

ith D

NAJ

B2a,

DN

AJB6

b, o

r DN

AJA1

(not

DN

AJB1

) com

pare

d to

par

kin

C28

9G a

lone

. The

re is

an

incr

ease

in th

e TX

-100

sol

uble

fra

ctio

n w

hen

co-tr

ansf

ecte

d w

ith D

NAJ

s, in

dica

ting

mor

e so

lubl

e pa

rkin

C28

9G u

nder

con

ditio

ns o

f kno

ckdo

wn

of H

SPAs

and

sug

gest

ing

som

e H

sp70

-inde

pend

ent a

ctio

ns o

f the

D

NAJ

s pr

ior t

o pa

rkin

inso

lubi

lizat

ion.

(B) H

sp70

s ar

e in

hibi

ted

with

VER

-155

008,

an

aden

osin

e-de

rived

HSP

A in

hibi

tor t

hat t

arge

ts th

e AT

Pase

bin

ding

dom

ain.

Sim

ilar r

esul

ts a

re

obse

rved

as

in p

anel

D.

153

7

7


154


solubility. Whilst parkin C289G aggregates are TX-100 insoluble but largely SDS soluble (18, this report), polyQ aggregates are both TX-100 and SDS insoluble (7, 8, 31, 45–47). Further, the morphology of the aggregates is more dispersed for parkin C289G (18, this report) whilst polyQ proteins form more amyloid like inclusions (31, 45). Finally, whereas the Cys289 to Gly mutation in the RING1 domain of parkin leads to a disruption of the zinc coordination of the protein and thus to protein misfolding (48), polyQ expansions within a full length huntingtin or ataxin-3 are hardly misfolded and only initiate aggregation when fragmented by the action of proteases (49–52). So, although several neurodegenerative diseases share protein aggregation as a common feature, the characteristics of the different aggregation-prone proteins impose a substantially different challenge on the cellular PQC system (9). Also, the idea that the PQC system is challenged differently is supported by the fact that several DNAJ members, which did not suppress aggregation of polyQ proteins (31), did protect against parkin C289G aggregation (this report). Inversely, overexpression of BAG3, which resulted in prevention of polyQ aggregation (44), did not protect against parkin C289G aggregation (this report). Within the HSPB family of small HSPs, HSPB7 was the most effective in preventing polyQ aggregation and HSPB1 was ineffective (32), while HSPB1 was very effective in preventing parkin C289G aggregation (Minoia et al, submitted). Finally, the mode of action of DNAJB6 and DNAJB8, which effectively reduced both types of aggregates, was different for polyQ proteins (largely Hsp70 independent) and parkin C289G (largely Hsp70 dependent, see also below).

We have previously shown that Hsp70 members are rather ineffective on polyQ aggregates (31). In contrast, we now find that inhibition as well as upregulation of HSPA1A did substantially affect parkin C289G aggregation. Besides HSPA1A, upregulation of only HSPA14 also reduced parkin C289G aggregation, whereas overexpression of all other cytosolic Hsp70 members tested was ineffective. This not only further supports that the two investigated aggregation-prone proteins require different chaperone handling, but also suggests that besides DNAJs and NEFs (1), the diverse members of the Hsp70 family may also contribute to the functional specificity of Hsp70 machines (53) and may specifically engage in different processes. Although this may seem striking giving the large sequence identity, the similar peptide recognition motifs, and biochemical activity that these Hsp70s members are assumed to have (54, 55), work by the Frydman group already suggested that distinct Hsp70 members may act in different chaperone networks, one predominantly dealing with protein translation and one with (environmental) stress (56). Further support for functional differentiation between the Hsp70 family members was provided by the group of Jäättelä (57) and our own findings that e.g. HSPA6 was largely ineffective in refolding unfolded substrates (58).

Irrespective of the effects of Hsp70 up- and downregulation, it was striking to find that, when

155

7

7


upregulated, all cytosolic DNAJA and DNAJB members tested were suppressing parkin C289G aggregation and that they were almost all equally effective. The anti-aggregation activity of the DNAJs was largely dependent on interaction with Hsp70s, since the H/Q mutants lost most of their activity, and on the activity of endogenously expressed Hsp70 machine, because HSPA1A RNAi and an Hsp70 inhibitor largely abrogated the effects of DNAJs. For DNAJB2a, it was already shown that it generally requires interaction with Hsp70 for its action as stimulator of proteasomal degradation of clients (59) and suppressor of parkin aggregation (18). This result is rather striking as DNAJB6 and DNAJB8 suppressed polyQ aggregation in a largely Hsp70 independent manner (31, 60). In fact, purified DNAJB6 alone could suppress aggregation of purified polyQ peptides (61) suggesting that DNAJB6 acts downstream proteolytic cleavage of full length polyQ proteins to prevent polyQ peptide mediated aggregate seeding (46, 60, 61). For inhibition of polyQ aggregation, DNAJB6 required a C-terminal SSF-SST stretch rather than a functional J-domain for interaction with Hsp70 (31). This SSF-SST stretch was also required for DNAJB6 oligomerization (31) and consistently, purified DNAJB6 that was effective in vitro also formed large oligomers (61). For inhibition of parkin C289G aggregation, the SSF-SST deletion mutant which forms apparent dimers (31) like several other canonical DNAJs (1), was still effective (Fig. 3). This implies that DNAJB6 and DNAJB8 can shift from more canonical, Hsp70 dependent dimers, effective in e.g. prevention of parkin C289G aggregation (this report), to non-canonical, Hsp70 independent oligomers that are effective as peptide chaperones that can prevent aggregation of polyQ (46, 60, 61) and, as recently found, also of amyloid-beta (Månsson et al, submitted). Interestingly, the F93L mutant of DNJAB6b that is implicated in limb-girdle muscular dystrophy (42) shows no loss of anti-aggregation activity on parkin C289G. This mutant was reported to be slightly defective in suppressing polyQ aggregation (42). However, we could not detect a large defect of this mutant in polyQ aggregation in our own experimental setting (data not shown), suggesting that this mutation may have impact on yet another aspect of the function of DNAJB6.

What is the mechanism through which all the DNAJs can suppress parkin C289G aggregation? We observed lower levels of parkin C289G than of parkin WT 24 hours after transfection and co-expression of the DNAJs even further lowered parkin C289G levels (but not parkin WT levels). This effect was lost when co-expressing the various DNAJ H/Q mutants (data not shown). As revealed by our western analysis, parkin C289G is present as a high molecular weight smear in the TX-100 insoluble fraction, consistent with other studies that the mutant is polyubiquitinated (23, 62), but has aggregated before being targeted to and degraded by the proteasome. Whereas cycloheximide chase experiments did not reveal a clear effect of the DNAJs on protein degradation rates (data not shown), we hypothesize that the DNAJs can interact with the misfolded and ubiquitinated parkin C289G at a very early stage so that

156


more of it is kept soluble and can be delivered (via Hsp70) to the proteasome for degradation. It is interesting to note that DNAJs were also still slightly effective under condition of HSPA1A knockdown and that this was paralleled by an increase of high molecular weight species of parkin C289G in the TX-100 soluble fraction (Fig. 3A and 3B). Based on this, we propose a model for the handling of parkin C289G by the Hsp70 machinery (Fig. 5) in which the DNAJs primarily and transiently bind misfolding and aggregating polypeptides of parkin C289G keeping them soluble for proteasomal degradation (holding activity). Via Hsp70 (HSPA1A and/or HSPA14) the substrates are next transferred to the proteasome for degradation. The holding activity of DNAJB1 may be the weakest of the ones tested, explaining why it lacks any effect when it when Hsp70 is depleted or inhibited.

Overall, our data further strengthens the fact that DNAJ proteins may be an attractive target in prevention of aggregation of disease causing proteins. Besides that their upregulation does not seem to affect the chaperone network (data not shown), which may have adverse effects (63), they may allow for strong and specific interventions in protein aggregation diseases that are just not all the same. On the other hand, DNAJB6, which we already identified as suppressor of polyQ aggregation in vitro (31) and that recently also delayed disease onset in vivo (Kakkar et al, submitted), may also be a target in Parkinson’s disease due to its dual mode of action as canonical Hsp70 co-chaperone and non-canonical inhibitor of peptide aggregation.

Figure 5. Model for the interaction of DNAJs with HSPAs for the handling of parkin C289G. DNAJs can primarily and transiently bind with the misfolded and ubiquitinated polypeptides of parkin C289G at an early stage (holding activity). In this way parkin C289G can be kept soluble and can be delivered via the Hsp70s (HSPA1A and/or HSPA14) for proteasomal degradation. When DNAJs are not sufficiently present or when Hsp70s are depleted or inhibited, the delivery to the proteasome is prevented and parkin C289G forms aggregates.

157

7

7


MATERIALS AND METHODS

Cell line, cell culture, and transient transfectionsHEK293 stably expressing the tetracycline (tet) repressor (Flp-In T-REx HEK293, Invitrogen) were grown in DMEM (GIBCO) plus 10% FCS (Greiner Bio-one), 100 units/ml penicillin, and 100 mg/ml streptomycin (Invitrogen), and for Flp-In TREx HEK293 cells, 5 mg/ml Blasticidine (Invitrogen). For transient transfections, cells were grown to 70%–80% confluence in 35 mm diameter dishes coated with 0.001% poly-L-lysine (Sigma) and/or on coated coverslips for confocal microscopy analyses. Cells were transfected with Lipofectamine (Invitrogen) according to the manufacturer’s instructions with a 1:1 ration of parkin and molecular chaperones. When transfecting with molecular chaperones tetracyclin was added to the medium. For transfection with siRNA, Lipofectamine2000 (Invitrogen) was used according to the manufacturer’s protocol before transfection with Lipofectamine. For inhibition of Hsp70 activity, VER-155008 (Sigma) at a 40uM concentration was added to cells immediately after transfection and then cells were further incubated for 8 hours.

Gene cloning and generation of mutants Information about the (construction of) the chaperone plasmids used in this study is described in Hageman et al. (31). Information on the plasmid used for parkin is described in Ardley et al. (64).

Protein extraction to obtain soluble and insoluble fractions for Western blotting 24 hours after transfection cells were washed once in cold phosphate-buffered saline (PBS) and lysed in 200 μL 1% Triton X-100 in PBS, containing 1% complete protease inhibitor cocktail for mammalian cells (PIC) (Roche Applied Science) for 15 minutes on ice. Cell lysates were scraped and centrifuged at 14000 rpm (Eppendorf, rotor F45-30-11) for 15 minutes at 4°C. Supernatants were transferred to eppendorfs (Triton X-100 soluble fraction). Pellets were resuspended in 200 μL 1% SDS in PBS, containing 1% PIC and sonicated before centrifugation at 14000 rpm (Eppendorf, rotor F45-30-11) for 15 minutes at 4°C. Supernatants were collected (Triton X-100 insoluble fraction). Samples were diluted in 2x SDS-sample loading buffer with 20% β-mercaptoethanol (Sigma) to a final concentration of 1x and were left unboiled. Samples were used immediately or kept frozen at -20°C.

Western blot analysisFrom the unboiled samples, 10 μl of soluble fraction, 10 μl of pellet fraction and 3 μl PageRuler Prestained Protein Ladder (Thermo Scientific) were loaded on 12.5% SDS-PAGE gels. SDS-PAGE was performed using the BioRad Mini-PROTEAN 3 system. After SDS-PAGE, proteins were transferred to nitrocellulose membranes by wet electrotransfer with the BioRad Mini-

158


PROTEAN 3 system. The transfer was carried out for 1.5 hour at 100 V. To prevent non-specific protein binding, membranes were incubated in 5% (w/v) non-fat dried milk in PBS-Tween (PBS-T) for one hour at room temperature. Membranes were washed three times 5 minutes in PBS-T and incubated for at least one hour with mouse anti-parkin antibody (Cell Signaling Technology) at a 1:1000 dilution, mouse anti-flag antibody (Sigma-Aldrich) at a 1:2000 dilution, mouse anti-V5 antibody (Invitrogen) at a 1:5000 dilution, mouse anti-Hsp70 (Enzo Life Sciences) at a 1:5000 dilution, and mouse anti-GAPDH (Fitzgerald) at 1:10000 dilution. Membranes were washed three times 10 minutes and incubated for one hour in anti-mouse HRP-conjugated secondary antibody (GE Healthcare) at an 1:5000 dilution in PBS-T. After incubation, membranes were washed three times 10 minutes in PBS-T and enhanced chemiluminscence detection was performed using the ECL Western Blotting substrate kit (Thermo Scientific) using 750 μL solution 1, mixed with 750 μL solution 2 per membrane.

Immunolabeling and microscopy24 hours after transfection, indirect immunofluorescence of the flag-tag and the V5-tag was performed to detect the exogenously expressed parkin and chaperones. Cells were fixed with 3.7% formaldehyde for 15 minutes, washed three times 5 minutes with PBS, permeabilized with 0.2% Triton-X100 and blocked 10 minutes with 0.1% glycine and 30 minutes with 0.5% BSA in PBS. Incubation with rabbit anti-V5 antibody (Sigma-Aldrich) and mouse anti-flag antibody (Sigma- Aldrich) at a 1:200 dilution in PBS-T was performed overnight at 4°C. Cells were washed three times 5 minutes with PBS-T followed by a 1 hour incubation with Alexa Fluor 488 anti-rabbit (Invitrogen) and Alexa 594 anti-mouse (Invitrogen) secondary antibodies at a 1:1500 dilution in PBS-T. To visualize nuclei, cells were stained 10 minutes with 0.2 μg/ml 4’,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific). Cover slips were mounted in Citifluor (Agar Scientific). Images of flag, V5 and DAPI fluorescence were obtained using a Leica microscope (Leica DM6000 M) with a 63X oil lens. The captured images were processed using Leica Software and Adobe Photoshop CS5.

FUNDING

This work was supported by a grant from Senter Novem (IOP-IGE07004) awarded to H.H.K. and a Topmaster fellowship from the Groningen University Institute for Drug Exploration (GUIDE) awarded to E.F.E.K..

ACKNOWLEDGEMENTS

We thank Dr. Michael Cheetham (King’s College London) for providing the parkin constructs and Dr. Chris Weihl (Washington University) for the DNAJB6 F93L construct.

159

7

7


REFERENCES

1. Kampinga,H.H. and Craig,E.A. (2010) The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nat. Rev. Mol. Cell Biol., 11, 579–592.2. Hartl,F.U., Bracher,A. and Hayer-Hartl,M. (2011) Molecular chaperones in protein folding and proteostasis. Nature, 475, 324–332.3. Turturici,G., Sconzo,G. and Geraci,F. (2011) Hsp70 and its molecular role in nervous system diseases. Biochem. Res. Int., 2011, 618127.4. Powers,E.T., Morimoto,R.I., Dillin,A., Kelly,J.W. and Balch,W.E. (2009) Biological and chemical approaches to diseases of proteostasis deficiency. Annu. Rev. Biochem., 78, 959–991.5. Morimoto,R.I. (2011) The heat shock response: systems biology of proteotoxic stress in aging and disease. Cold Spring Harb. Symp. Quant. Biol., 76, 91–99.6. Bruinsma,I.B., Bruggink,K.A., Kinast,K., Versleijen,A.A.M., Segers-Nolten,I.M.J., Subramaniam,V., Bea Kuiperij,H., Boelens,W., de Waal,R.M.W. and Verbeek,M.M. (2011) Inhibition of alpha-synuclein aggregation by small heat shock proteins. Proteins Struct. Funct. Bioinforma., 79, 2956–2967.7. Lee,S.-J., Lim,H.-S., Masliah,E. and Lee,H.-J. (2011) Protein aggregate spreading in neurodegenerative diseases: Problems and perspectives. Neurosci. Res., 70, 339–348.8. Perutz,M., Johnson,T. and Suzuki,M. (1994) Glutamine repeats as polar zippers: their possible role in inherited neurodegenerative diseases. Proc. Natl. Acad. Sci. USA., 91, 5355–5358.9. Kakkar,V., Meister-Broekema,M., Minoia,M., Carra,S. and Kampinga,H.H. (2014) Barcoding heat shock proteins to human diseases: looking beyond the heat shock response. Dis. Model. Mech., 7, 421–434.10. Halliday,G., Lees,A. and Stern,M. (2011) Milestones in Parkinson’s disease--clinical and pathologic features. Mov. Disord., 26, 1015–1021.11. Hinault,M.-P., Farina-Henriquez-Cuendet,A. and Goloubinoff,P. (2011) Molecular chaperones and associated cellular clearance mechanisms against toxic protein conformers in Parkinson’s disease. Neurodegener. Dis., 8, 397–412.12. Samii,A., Nutt,J.G. and Ransom,B.R. (2004) Parkinson’s disease. Lancet, 363, 1783–1793.

13. Tsigelny,I.F., Sharikov,Y., Wrasidlo,W., Gonzalez,T., Desplats,P.A., Crews,L., Spencer,B. and Masliah,E. (2012) Role of alpha-synuclein penetration into the membrane in the mechanisms of oligomer pore formation. FEBS J., 279, 1000–1013.14. Aridon,P., Geraci,F., Turturici,G., D’Amelio,M., Savettieri,G. and Sconzo,G. (2011) Protective role of heat shock proteins in Parkinson’s disease. Neurodegener. Dis., 8, 155–168.15. Nuytemans,K., Theuns,J., Cruts,M. and Van Broeckhoven,C. (2010) Genetic etiology of Parkinson disease associated with mutations in the SNCA, PARK2, PINK1, PARK7, and LRRK2 genes: a mutation update. Hum. Mutat., 31, 763–780.16. Ruffmann,C., Zini,M., Goldwurm,S., Bramerio,M., Spinello,S., Rusconi,D., Gambacorta,M., Tagliavini,F., Pezzoli,G. and Giaccone,G. (2012) Lewy body pathology and typical Parkinson disease in a patient with a heterozygous (R275W) mutation in the Parkin gene (PARK2). Acta Neuropathol., 123, 901–903.17. Darios,F., Corti,O., Lücking,C.B., Hampe,C., Muriel,M., Abbas,N., Gu,W.-J., Hirsch,E., Rooney,T., Ruberg,M., et al. (2003) Parkin prevents mitochondrial swelling and cytochrome c release in mitochondria-dependent cell death. Hum. Mol. Gen., 12, 517–526.18. Rose,J.M., Novoselov,S.S., Robinson,P.A. and Cheetham,M.E. (2011) Molecular chaperone-mediated rescue of mitophagy by a Parkin RING1 domain mutant. Hum. Mol. Gen., 20, 16–27.19. Walden,H. and Martinez-Torres,R.J. (2012) Regulation of Parkin E3 ubiquitin ligase activity. Cell. Mol. Life Sci., 69, 3053–3067.20. Kahle,P.J. and Haass,C. (2004) How does parkin ligate ubiquitin to Parkinson’s disease? EMBO Rep., 5, 681–685.21. Kitada,T., Asakawa,S., Hattori,N., Matsumine,H., Yamamura,Y., Minoshima,S., Yokochi,M., Mizuno,Y. and Shimizu,N. (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature, 392, 605–608.22. Lücking,C., Dürr,A. and Bonifati,V. (2000) Association between early-onset Parkinson’s disease and mutations in the parkin gene. N. Engl. J. Med., 342, 1560–1567.23. Gu,W.-J., Corti,O., Araujo,F., Hampe,C., Jacquier,S.,

160


Lücking,C.B., Abbas,N., Duyckaerts,C., Rooney,T., Pradier,L., et al. (2003) The C289G and C418R missense mutations cause rapid sequestration of human Parkin into insoluble aggregates. Neurobiol. Dis., 14, 357–364.24. Hampe,C., Ardila-Osorio,H., Fournier,M., Brice,A. and Corti,O. (2006) Biochemical analysis of Parkinson’s disease-causing variants of Parkin, an E3 ubiquitin-protein ligase with monoubiquitylation capacity. Hum. Mol. Gen., 15, 2059–2075.25. Hyun,D.-H., Lee,M., Halliwell,B. and Jenner,P. (2005) Effect of overexpression of wild-type or mutant parkin on the cellular response induced by toxic insults. J. Neurosci. Res., 82, 232–244.26. Junn,E., Lee,S.S., Suhr,U.T. and Mouradian,M.M. (2002) Parkin accumulation in aggresomes due to proteasome impairment. J. Biol. Chem., 277, 47870–47877.27. Wang,C., Tan,J.M.M., Ho,M.W.L., Zaiden,N., Wong,S.H., Chew,C.L.C., Eng,P.W., Lim,T.M., Dawson,T.M. and Lim,K.L. (2005) Alterations in the solubility and intracellular localization of parkin by several familial Parkinson’s disease-linked point mutations. J. Neurochem., 93, 422–431.28. Hilker,R., Klein,C., Ghaemi,M., Kis,B., Strotmann,T., Ozelius,L.J., Lenz,O., Vieregge,P., Herholz,K., Heiss,W.D., et al. (2001) Positron emission tomographic analysis of the nigrostriatal dopaminergic system in familial parkinsonism associated with mutations in the parkin gene. Ann. Neurol., 49, 367–376.29. Khan,N.L. (2003) Parkin disease: a phenotypic study of a large case series. Brain, 126, 1279–1292.30. Khan,N.L., Brooks,D.J., Pavese,N., Sweeney,M.G., Wood,N.W., Lees,A.J. and Piccini,P. (2002) Progression of nigrostriatal dysfunction in a parkin kindred: an [18F]dopa PET and clinical study. Brain, 125, 2248–2256.31. Hageman,J., Rujano,M.A., van Waarde,M.A.W.H., Kakkar,V., Dirks,R.P., Govorukhina,N., Oosterveld-Hut,H.M.J., Lubsen,N.H. and Kampinga,H.H. (2010) A DNAJB chaperone subfamily with HDAC-dependent activities suppresses toxic protein aggregation. Mol. Cell, 37, 355–369.32. Vos,M.J., Zijlstra,M.P., Kanon,B., van Waarde-Verhagen,M.A.W.H., Brunt,E.R.P., Oosterveld-Hut,H.M.J., Carra,S., Sibon,O.C.M. and Kampinga,H.H. (2010) HSPB7 is the most potent polyQ aggregation suppressor within the HSPB family of molecular chaperones. Hum. Mol. Genet., 19, 4677–4693.

33. Carra,S., Crippa,V., Rusmini,P., Boncoraglio,A., Minoia,M., Giorgetti,E., Kampinga,H.H. and Poletti,A. (2011) Alteration of protein folding and degradation in motor neuron diseases: Implications and protective functions of small heat shock proteins. Prog. Neurobiol., 97, 83–100.34. Crippa,V., Sau,D., Rusmini,P., Boncoraglio,A., Onesto,E., Bolzoni,E., Galbiati,M., Fontana,E., Marino,M., Carra,S., et al. (2010) The small heat shock protein B8 (HspB8) promotes autophagic removal of misfolded proteins involved in amyotrophic lateral sclerosis (ALS). Hum. Mol. Genet., 19, 3440–3456.35. McLean,P.J., Kawamata,H., Shariff,S., Hewett,J., Sharma,N., Ueda,K., Breakefield,X.O. and Hyman,B.T. (2002) TorsinA and heat shock proteins act as molecular chaperones: suppression of alpha-synuclein aggregation. J. Neurochem., 83, 846–854.36. Outeiro,T.F., Klucken,J., Strathearn,K.E., Liu,F., Nguyen,P., Rochet,J.C., Hyman,B.T. and McLean,P.J. (2006) Small heat shock proteins protect against alpha-synuclein-induced toxicity and aggregation. Biochem. Biophys. Res. Commun., 351, 631–638.37. Takeuchi,H., Kobayashi,Y., Yoshihara,T., Niwa,J., Doyu,M., Ohtsuka,K. and Sobue,G. (2002) Hsp70 and Hsp40 improve neurite outgrowth and suppress intracytoplasmic aggregate formation in cultured neuronal cells expressing mutant SOD1. Brain Res., 949, 11–22.38. Uryu,K., Richter-Landsberg,C., Welch,W., Sun,E., Goldbaum,O., Norris,E.H., Pham,C.-T., Yazawa,I., Hilburger,K., Micsenyi,M., et al. (2006) Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous α-Synuclein Inclusions of α-Synucleinopathies. Am. J. Pathol., 168, 947–961.39. Elwi,A.N., Lee,B., Meijndert,H.C., Braun,J.E.A. and Kim,S.-W. (2012) Mitochondrial chaperone DnaJA3 induces Drp1-dependent mitochondrial fragmentation. Int. J. Biochem. Cell Biol., 44, 1366–1376.40. Lai,C.W., Otero,J.H., Hendershot,L.M. and Snapp,E. (2012) ERdj4 is a soluble endoplasmic reticulum DnaJ family protein that interacts with ERAD machinery. J. Biol. Chem., 287, 7969–7978.41. Cheetham,M.E. and Caplan,A.J. (1998) Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function. Cell Stress Chaperones., 8, 28–36.42. Sarparanta,J., Jonson,P.H., Golzio,C., Sandell,S.,

161

7

7


Luque,H., Screen,M., McDonald,K., Stajich,J.M., Mahjneh,I., Vihola,A., et al. (2012) Mutations affecting the cytoplasmic functions of the co-chaperone DNAJB6 cause limb-girdle muscular dystrophy. Nat. Genet., 44, 450–455.43. Williamson,D.S., Borgognoni,J., Clay,A., Daniels,Z., Dokurno,P., Drysdale,M.J., Foloppe,N., Francis,G.L., Graham,C.J., Howes,R., et al. (2009) Novel adenosine-derived inhibitors of 70 kDa heat shock protein, discovered through structure-based design. J. Med. Chem., 52, 1510–1513.44. Carra,S., Seguin,S.J., Lambert,H. and Landry,J. (2008) HspB8 chaperone activity toward poly(Q)-containing proteins depends on its association with Bag3, a stimulator of macroautophagy. J. Biol. Chem., 283, 1437–1444.45. Kubota,H., Kitamura,A. and Nagata,K. (2011) Analyzing the aggregation of polyglutamine-expansion proteins and its modulation by molecular chaperones. Methods, 53, 267–274.46. Raspe,M., Gillis,J., Krol,H., Krom,S., Bosch,K., van Veen,H. and Reits,E. (2009) Mimicking proteasomal release of polyglutamine peptides initiates aggregation and toxicity. J. Cell Sci., 122, 3262–3271.47. Ren,P.-H., Lauckner,J.E., Kachirskaia,I., Heuser,J.E., Melki,R. and Kopito,R.R. (2009) Cytoplasmic penetration and persistent infection of mammalian cells by polyglutamine aggregates. Nat. Cell Biol., 11, 219–225.48. Trempe,J.-F., Sauvé,V., Grenier,K., Seirafi,M., Tang,M.Y., Ménade,M., Al-Abdul-Wahid,S., Krett,J., Wong,K., Kozlov,G., et al. (2013) Structure of parkin reveals mechanisms for ubiquitin ligase activation. Science, 340, 1451–1455.49. Berke,S.J.S., Schmied,F.A.F., Brunt,E.R., Ellerby,L.M. and Paulson,H.L. (2004) Caspase-mediated proteolysis of the polyglutamine disease protein ataxin-3. J. Neurochem., 89, 908–918.50. Haacke,A., Broadley,S. a, Boteva,R., Tzvetkov,N., Hartl,F.U. and Breuer,P. (2006) Proteolytic cleavage of polyglutamine-expanded ataxin-3 is critical for aggregation and sequestration of non-expanded ataxin-3. Hum. Mol. Genet., 15, 555–568.51. Graham,R.K., Deng,Y., Slow,E.J., Haigh,B., Bissada,N., Lu,G., Pearson,J., Shehadeh,J., Bertram,L., Murphy,Z., et al. (2006) Cleavage at the Caspase-6 Site Is Required for Neuronal Dysfunction and Degeneration

Due to Mutant Huntingtin. Cell, 125, 1179–1191.52. Koch,P., Breuer,P., Peitz,M., Jungverdorben,J., Kesavan,J., Poppe,D., Doerr,J., Ladewig,J., Mertens,J., Tüting,T., et al. (2011) Excitation-induced ataxin-3 aggregation in neurons from patients with Machado-Joseph disease. Nature, 480, 543–546.53. Kampinga,H. and Craig,E. (2010) The Hsp70 chaperone machinery: J-proteins as drivers of functional specificity. Nat Rev Mol Cell Biol, 11, 579–592.54. Mayer,M.P. and Bukau,B. (2005) Hsp70 chaperones: cellular functions and molecular mechanism. Cell. Mol. Life Sci., 62, 670–684.55. Rüdiger,S., Germeroth,L., Schneider-Mergener,J. and Bukau,B. (1997) Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries. EMBO J., 16, 1501–1507.56. Albanèse,V., Yam,A.Y.-W., Baughman,J., Parnot,C. and Frydman,J. (2006) Systems analyses reveal two chaperone networks with distinct functions in eukaryotic cells. Cell, 124, 75–88.57. Daugaard,M., Rohde,M. and Jäättelä,M. (2007) The heat shock protein 70 family: Highly homologous proteins with overlapping and distinct functions. FEBS Lett., 581, 3702–3710.58. Heldens,L. and Genesen,S. van (2012) Protein refolding in peroxisomes is dependent upon an HSF1-regulated function. Cell Stress Chaperones, 17, 603–613.59. Westhoff,B., Chapple,J.P., van der Spuy,J., Höhfeld,J. and Cheetham,M.E. (2005) HSJ1 is a neuronal shuttling factor for the sorting of chaperone clients to the proteasome. Curr. Biol., 15, 1058–1064.60. Gillis,J., Schipper-Krom,S., Juenemann,K., Gruber,A., Coolen,S., van den Nieuwendijk,R., van Veen,H., Overkleeft,H., Goedhart,J., Kampinga,H.H., et al. (2013) The DNAJB6 and DNAJB8 protein chaperones prevent intracellular aggregation of polyglutamine peptides. J. Biol. Chem., 288, 17225–17237.61. Månsson,C., Kakkar,V., Monsellier,E., Sourigues,Y., Härmark,J., Kampinga,H.H., Melki,R. and Emanuelsson,C. (2014) DNAJB6 is a peptide-binding chaperone which can suppress amyloid fibrillation of polyglutamine peptides at substoichiometric molar ratios. Cell Stress Chaperones, 19, 227–239.62. Chaugule,V.K., Burchell,L., Barber,K.R., Sidhu,A., Leslie,S.J., Shaw,G.S. and Walden,H. (2011) Autoregulation of Parkin activity through its ubiquitin-like

162


domain. EMBO J., 30, 2853–2867.63. Dai,C., Whitesell,L., Rogers,A.B. and Lindquist,S. (2007) Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis. Cell, 130, 1005–1018.64. Ardley,H.C., Scott,G.B., Rose,S.A., Tan,N.G.S., Markham,A.F. and Robinson,P.A. (2003) Inhibition of proteasomal activity causes inclusion formation in neuronal and non-neuronal cells overexpressing Parkin.

Mol. Biol. Cell., 14, 4541–4556.65. Henn,I.H., Gostner,J.M., Lackner,P., Tatzelt,J. and Winklhofer,K.F. (2005) Pathogenic mutations inactivate parkin by distinct mechanisms. J. Neurochem., 92, 114–122.

163

7

7


university of groningen dnaj proteins: more than just “co ... · pdf file144 chapter 7...

Documents