Protein Misfolding Disorders Pa Tho Genesis and Intervention

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J Inherit Metab Dis (2006) 29:456–470

DOI 10.1007/s10545-006-0301-4

S S I E M S Y M P O S I U M 2 0 0 5

Protein misfolding disorders: Pathogenesis and intervention

N. Gregersen

Received: 14 December 2005 / Accepted: 2 February 2006C SSIEM and Springer 2006

Summary Newly synthesized proteins in the living cell

must go through a folding process to attain their functionalstructure. To achieve this in an efficient fashion, all organ-

isms, includinghumans, have evolved a large setof molecular

chaperones that assist the folding as well as the maintenance

of the functional structure of cellular proteins. Aberrant pro-

teins, the result of production errors, inherited or acquired

amino acid substitutions or damage, especially oxidative

modifications, can in many cases not fold correctly and will

be trapped in misfolded conformations. To rid the cell of

misfolded proteins, the living cell contains a large number of

intracellular proteases, e.g. the proteasome, which together

with the chaperones comprise the cellular protein quality

control systems. Many inherited disorders due to amino acid

substitutions exhibit loss-of-function pathogenesis because

the aberrant protein is eliminated by oneof theprotein quality

control systems. Examples are cystic fibrosis and phenylke-

tonuria. However, not all aberrant proteins can be eliminated

and the misfolded protein may accumulate and form toxic

oligomeric and/or aggregated inclusions. In this case the loss

of function maybe accompanied by a gain-of-functionpatho-

genesis, which in manycases determinesthe pathological and

clinical features. Examples are Parkinson and Huntington

diseases. Although a number of strategies have been tried to

decrease the amounts of accumulated and aggregated pro-

Communicating editor: Jean-Marie Saudubray

Competing interests: None declared

Presented at the 42nd Annual Meeting of the SSIEM, Paris, 6–9

September 2005

N. Gregersen ()

Research Unit for Molecular Medicine, Institute of Clinical

Medicine, Aarhus University Hospital, Skejby Sygehus, 8200

Aarhus N, Denmark

teins, a likely future strategy seems to be the use of chemical

or pharmacological chaperones with specific effects on themisfolded protein in question. Positive examples are enzyme

enhancement in a number of lysosomal disorders.

Introduction

The cellular functions in the body depend on proteins, the

functions of which are dependent on active conformations

that must be attained and maintained until the proteins are

turned over to degradation mechanisms. The balance between

formation and maintenance of the active conformation and

their turnover is delicate, and disturbances in the amino acidchain by inherited sequence variations or acquired amino

acid modifications may compromise the folding and/or the

conformational maintenance and result in cell dysfunction

and disease.

In this review the concept of chaperone-assisted protein

folding and organelle-specific protein quality control systems

will be introduced, and a number of misfolding (or confor-

mational) disorders will be discussed, with focus on loss-of-

function and gain-of-function pathogenic mechanisms. Also,

factors that may influence the balance between loss of func-

tion and gain of function will be discussed, to end up with

a brief discussion of strategies by which this balance can beshifted in beneficial directions.

Chaperone-assisted protein folding and quality

control systems

Nearly all cellular proteins – except 13 that are coded from

mitochondrial DNA – are nuclear-encoded and translated

in the cytosol. When the unfolded polypeptides emerge

from the ribosome, either directly into the cytosol or

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J Inherit Metab Dis (2006) 29:456–470 457

co-translationally translocated into the endoplasmic reticu-

lum, their hydrophobic domains areexposedto a complex en-

vironment with proteins and other cell components present

in high concentration and at relatively high temperatures.

These cellular conditions promote hydrophobic interactions

between emerging polypeptides and other proteins and cell

components. To overcome these unfavourable folding con-

ditions, all organisms – from bacteria to humans – haveevolved a large number of molecular systems that assist and

monitor the intracellular folding process (Frydman 2001).

Since the final structure of a protein is given by the amino

acid sequence (Anfinsen 1973), the primary function of the

molecular systems is not to catalyse the folding but rather to

minimize nonproductive interactions by shielding hydropho-

bic domains during the folding process until they are buried

inside the native structure. The majority of components in

the surveying systems are constituted by so-called molecular

chaperones, which have been defined as ‘a functional class

of unrelated proteins that assist the correct noncovalent as-

sembly of otherpolypeptide containing structures in vivo, butare not components of these assembled structures when they

are performing their biological functions’ (Ellis 1993). In

addition to protecting newly synthesized polypeptides from

nonproductive interactions, many chaperones also act in pro-

tection, refolding and elimination of damaged and denatured

proteins (Cashikar et al 2005). Indeed, the designation heat-

shock proteins (HSPs) for many chaperones stems from the

discovery in 1988 that a variety of cellular stresses, particu-

larly heat but also oxidative stress, toxic chemicals and viral

and bacterial infections, induce the production of a number

of proteins (Lindquist and Craig 1988), many of which may

act as chaperones.

The mechanism by which the chaperones act differs de-

pending on the nature and on the cellular location. How-

ever, nearly all – except the lectin-chaperones – bind to hy-

drophobic domains of the given polypeptide/protein in an

ATP-dependent fashion and cycle between binding and re-

lease in cycles driven by hydrolysis of ATP to ADP and

conformational changes. Certain proteins, especially small

ones, are easily folded, whereas larger proteins and proteins

containing amino acid substitutions or damage may require

more assistance and need to cycle through several rounds

of ATP-binding, hydrolysis and ADP-release before acquir-

ing the native conformation. If a given protein is aberrant

due to severe damage or amino acid alterations, the fold-

ing machinery may give up, and the aberrant protein will be

taken up by intracellular proteases, which then try to elimi-

nate it. Indeed, the interplay and balance between molecular

chaperones and intracellular proteases constitute the cellular

protein quality control (PQC) systems. The main functions

of the PQC systems are thus to supervise folding and pro-

tect folding intermediates from nonproductive interactions,

which may result in aggregation, and to assist the elimina-

tion of aberrant folding intermediates and unstable proteins,

which would overload the chaperone systems and damage

the cell. The longer a protein remains associated to chap-

erones, the higher is the probability that it will be captured

by the proteolytic systems. The PQC systems are thus be-

lieved to function by a competition between release to native

structure and targeting to degradation. Therefore, in addi-tion to be involved in the processing of aberrant proteins,

the components of the PQC systems are also crucial for the

normal turnover of cellular proteins, which through differ-

ent marking systems, e.g. ubiquitination, are presented to the

proteases by chaperone-mediated transfer. Although the var-

ious organelle-specific PQC systems are very complicated

and still not fully elucidated, the principle is rather simple,

as indicated in Fig. 1. The cytosol and mitochondria possess

their own PQC systems, while the endoplasmic reticulum

(ER)-processed proteins are folded in the ER and – if not

properly folded and transport-competent to be further pro-

cessed through the Golgi complex – are retrogradely translo-cated to the cytosol for degradation. Proteins that have their

function in peroxisomes and the nucleus are folded in the cy-

tosol and transported into the respective organelle as folded

proteins.

The life-saving function of the PQC systems in the young

and healthy cell has not been appreciated until a few years

ago, where it was shown that up to 30% of newly synthesised

polypeptides are degraded prematurely by the proteasome

(Schubert et al 2000). These polypeptides are called DRiPs

(defective ribosomal products), which contain errors that re-

sult in misfolding. Although the investigators did not go into

detail regarding the type of product formed, they detected

ubiquitinated proteins in the aggregates after inhibition of

the proteasome.

These experiments demonstrate that an effective degra-

dation system is important for cell-cleaning, but they also

indicate that there is a balance between degradation and ag-

gregation, which for the DRiPs depends on the degradation

capacity.

In old cells the ability to cope with misfolded protein isde-

creased (Grune et al 2004; Verbeke et al 2001), and accumu-

lated proteins that are damaged or have an inherited tendency

to misfolding, such as α-synuclein in Parkinson disease and

β-amyloid in Alzheimer disease, are found as aggregates in-

side or outside the cells. In young cells, on the other hand,

the efficiency of the PQC systems may be sufficient to rid

the cells of such protein accumulations. However, misfolded

proteins containing inherited amino acid alterations may sat-

urate and inhibit the proteases, thereby promoting forma-

tion of aggregates, such as missense variant α-synuclein,

which accumulates as aggregates in early-onset Parkinson

disease.

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458 J Inherit Metab Dis (2006) 29:456–470

losotyc

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Fig.1 Trafficking, foldingand turnover of cellularproteins. All nascentpolypeptides are protected by cytosolic chaperones, notably Hsp70,

Hsc70 and/or Hsp90. Cytosolic proteins, e.g. phenylalanine hydroxy-

lase (PAH) are turned over by the cytosolic chaperone/protease systems,

including the proteasome. Nuclear-encoded mitochondrial matrix pro-

teins, e.g. short-chain acyl-CoA dehydrogenase (SCAD), are assisted in

their import by mitochondrial Hsp70 and in their folding by the chap-

eronin Hsp60. Turnover is accomplished by proteases, mainly Lon and

ClpP.

Endoplasmic reticulum (ER)-resident proteins or proteins processed

for secretion, translocation to the lysosomes (as β-glucosidase, β-Glu)

or cell membrane insertion (as cystic fibrosis transmembrane regulator(CFTR)) are co-translationally translocated to the ER lumen assisted

by the ER Hsp70 homologue Grp78 and/or Calnexin (CAN). Turnover

and premature degradation of ER-resident or ER-processed proteins

are – after retrograde translocation to the cytosol – degraded by cytoso-

lic proteases, e.g. the proteasome. Nuclear and peroxisomal proteins

are folded in the cytosol and translocated to the organelles by specific

import proteins. Together with Hsp70, peroxins, e.g. PEX5, assist the

translocation to peroxisomes of acyl-CoA oxidase (ACOX). Many nu-

clear proteins, e.g. p53, are assisted in their transport by importin in

addition to chaperones, e.g. Hsp90

Pathogenic mechanisms

Depending on the protein and on the efficiency of the PQCsystems, the fate of a given aberrant protein may be differ-

ent, as illustrated in Fig. 2. If the misfolded protein is easily

degraded, the consequence is a loss-of-function pathogene-

sis, where a missing function and – in many cases substrate

accumulation – is responsible for the cellular pathophysiol-

ogy and clinical disease. This is the case, we still believe,

for most metabolic disorders, such as the fatty acid oxidation

defects, where the degree of energy deficiency in combina-

tion with substrate accumulation is decisive for the disease

expression and progression (Gregersen et al 2001). However,

the aberrant protein may be protease-resistant or have condi-

tionally determined resistance, for instance after heat stressthat promotes misfolding and aggregation. In such cases the

misfolded proteins may lead not only to loss of function but

also to a gain-of-function pathogenesis, either by adopting

conformations that inhibit the normal function of the corre-

sponding wild-type protein, or by forming oligomers and ag-

gregates that elicit new toxic functions in the cell or sequester

chaperones and/or other crucial cell components (see below).

Depending on the nature of the protein, the cellular compart-

ment, and the efficiency of the PQC system, the cellular con-

sequences may be quite different. First of all the misfolded

proteins will elicit a cellular stress response, including induc-

tion of PQC components (Muchowski and Walker 2005), thefunction of which is to eliminate the misfolded proteins and

protect the cell. However, if this is not possible because of the

presence of a high amount of misfolded protein and/or insuf-

ficiency of the systems, e.g. in aged cells, a whole range of

cell-damaging mechanisms and other stress responses may

be induced, including antioxidant (Winyard et al 2005) and

autophagy mechanisms (Levine and Klionsky 2004), which

may rescue the cell or lead to cell death (Friedlander 2003).

Without going into details, the cellular damage leading to

cell death may be induced by: (1) inhibition of the ubiqui-

tin – proteasome system by misfolded proteins (Bence et al

2001); (2) chaperone sequestration as well as sequestrationof transcription factors and/or other cell components by accu-

mulated proteins (Bruijn et al 2004; Muchowski and Walker

2005; Okado-Matsumoto and Fridovich 2002; Sherman and

Goldberg 2001); (3) mitochondrial dysfunction and oxida-

tive stress (Bruijn et al 2004; Butterfield and Kanski 2001;

Haynes et al 2004; Schon and Manfredi 2003); (4) chan-

nel formation and disturbances of calcium and glutamate

homeostasis (Caughey and Lansbury 2003;Dalle-Donne et al

2003; Emerit et al 2004; Stefani and Dobson 2003).

Springer





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noitalumuccA

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noitagerggA

enorepahCnoitartseuqes

noitamrofnoccixoT

noitcnuf-fo-ssoL

n o i t c n u f - f o - n i a G

egamaD

noitairaveneG

noitcnuflaudiseR

noitcnuf-fo-ssoL

nietorpdedlofnuimeS

nietorptnecsaN

Fig. 2 Pathogenetic and cellular consequences of protein misfolding.

Inherited amino acid variations and damage to proteins may result in

misfolding and decrease of functional protein. Semi-unfolded protein

(foldingintermediate or unfolded) maybe degraded andgiverise to loss-

of-function pathogenesis or accumulated protein, resulting in loss-of-function and/or gain-of-function pathogenesis. Accumulated misfolded

protein may form toxic conformations that inhibit normal function, se-

quester chaperones (and other cell components) and/or develop into

aggregates eliciting a range of cell dysfunctional mechanisms. The var-

ious steps in these processes are target points for intervention (see the

final section of the text

Although these effects provide a general framework,

which is guidedby thephysicochemical properties of themis-

folded proteins in question, the various disorders may show

quite different pathological and clinical pictures. To illustrate

this diversity in the cellular and clinical consequences, the

next section discusses a number of misfolding diseases of the

various cellular compartments. These diseases are shown in

Table 1, together with a number of other representative dis-

orders. The focus of the discussion will be on the mechanism

of misfolding and the fate of the misfolded protein, as these

are the targets for intervention.

Misfolding diseases of the nucleus

Huntington’s chorea (McKusick 143100) is an autosomal

dominant neurodegenerative disease with clinical features

comprising uncontrolled movements, cognitive changes and

dementia.The disease is dueto misfolding andaggregationin

the nucleus and cytosol of N -terminal fragments of the large

350 kDa huntingtin, which contains a polyglutamine stretch

(poly(Q)) of about 40 or more glutamine groups (Hayden and

Kremer2001; Qin andGu 2004). The accumulationof aggre-

gates is most pronounced in the striatal neurons, where the

GABA-producing cells are among the most affected, result-

ing in secondarily reduced productionof the neurotransmitter

acetylcholine. The protein is normally found in the cytosol,

and has been suggested to be involved in cytosol – nucleus

transport mechanisms (Cornett et al 2005). N -Terminal frag-

ments of huntingtin with poly(Q) stretches less than about 40

can be proteolytically degraded by the proteasome, but when

the expansion exceeds 40, the fragments are accumulated

preferentially in the nucleus, indicating a decreased nuclear

export (Cornett et al 2005). The exact link between the accu-

mulation and neuron dysfunction and death is not known, but

the composition of the aggregates, which contain chaperones

and components from the ubiquitin – proteasomal system as

well as components involved in the cell cycle and transcrip-

tion mechanisms (Suhr et al 2001), indicates that multiple

cell functions are disturbed.

The fact that the aggregation is suppressed ex vivo by

the chaperones Hsc70 and the co-chaperone Hsp40 in cells

expressing huntingtin fragments (Jana et al 2000), indicates

that the efficiency of the PQC system in the cytosol may be

a determinant of the pathogenesis.

In summary, the pathogenesis is mainly a toxic gain of

function, and there are indications that a loss-of-function

pathogenesis contributes through a decreased level of hunt-

ingtin in nerve cells.

Congenital myopathies comprise a group of diseases of the

skeletalmuscle sarcomere. The diseases canbe autosomalre-

cessive or dominant, and are characterized by the presence

of protein aggregates and rods in muscle tissue from patients

(Clarkson et al 2004). The pathogenesis may differ depend-

ing on the nature of the protein involved, i.e. α-actin, α- and

β-tropomyosin, troponin T andnebulin,as well as on the type

of mutation. The interesting cases in the present context are

the nemaline myopathies (McKusick 161800), in which the

nuclear rods contains sarcomeric α-actinin, in many cases

secondarily to gene variations in skeletalα-actin. The mech-

anisms by which α-actinin is translocated and forms rods in

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T a b l e 1

R e p r e s e n t a t i v e p r o t e i n m i s

f o l d i n g d i s o r d e r s , g e n e t i c s , t y p e o f m o l e c u l a r p a t h o g e n e s i s ( i n d i c a t i o n i n p a r e n t h e s e s i s

n o t c e r t a i n ) , a f f e c t e d c e l l u l a r c o m p a r t m e n t , m a i n c e l l u l a r p a t h o l o g y

a n d s o m e k e y r e f e r e n c e s . T h e d i s o r d e r s d i s c u s s e d i n t h i s r e v i e w a r e i n d i c a t e d b y b o l d t y p e

M o l e c u l a r

G e n e t i c s

p a t h o g e n e s i s

C o m p a r t m e n t

M a i n c e l l u l a r p a t h o l o g y

R e f e r e n c e s t o p a t h o g e n i c m e c h a n i s m

H u n t i n g t o n d i s e a s e

A u t o s o m a l d o m i n a n t

i n h e r i t a n c e

G a i n - o f - f u n c t i o n

( L o s s - o f - f u n c t i o n )

N u c l e u s a n d c y t o s o l

D y s f u

n c t i o n / d e a t h o f

G A

B A - p r o d u c i n g

b r a i n c e l l s

Q i n a n d G u ( 2 0 0 4 ) C o

r n e t t e t a l ( 2 0 0 5 )

C o n g e n i t a l m y o p a t h i e s


o r r e c e s s i v e



L o s s - o f - f u n c t i o n

N u c l e u s a n d c y t o s o l

D y s f u


m u s c l e c e l l s

C l a r k s o n e t a l ( 2 0 0 4 )

C y s t i c fi b r o s i s

A u t o s o m a l r e c e s s i v e



E n d o p l a s m i c r e t i c u l u m

D y s f u


l u n g , p a n c r e a t i c a n d

g a s t r o i n t e s t i n a l

e p i t h e l i a l c e l l s

K o p i t o ( 2 0 0 0 )

G a u c h e r d i s e a s e




( G a i n - o f - f u n c

t i o n )


D y s f u


s k e l e t a l , l i v e r , s p l e e n

a n d

b l o o d c e l l s

R o n a n d H o r o w i t z ( 2 0

0 5 )

F a m i l i a l h y p e r c h o l e s t e r o l a e m i a





D y s f u

n c t i o n o f l i v e r

c e l l

s a n d a f f e c t i o n o f

v a s c u l a r c e l l s

J o r g e n s e n e t a l ( 2 0 0 3 )

α - 1 - A n t i t r y p s i n d e fi c i e n c y






D y s f u


l i v e

r c e l l s a n d

a f f e

c t i o n o f l u n g c e l l

f u n c t i o n

C a r r e l l a n d L o m a s ( 2 0

0 2 ) P e r l m u t t e r ( 2 0 0 3 )

F a m i l i a l h y p o p h y s e a l d i a b e t e s

i n s i p i d u s






D y s f u


v a s o p r e s s i n -

p r o d u c i n g c e l l s i n

h y p

o p h y s i s

C h r i s t e n s e n e t a l ( 2 0 0 4 )

F a m i l i a l n e p h r o g e n i c d i a b e t e s

i n s i p i d u s

X - l i n k e d i n h e r i t a n c e



D y s f u

n c t i o n o f k i d n e y

c e l l

s

M o r e l l o e t a l ( 2 0 0 1 )

A s p a r t y l g l u c o s e a m i n i d a s e

d e fi c i e n c y





D y s f u

n c t i o n o f b r a i n

a n d c o n

n e c t i v e - t i s s u e

c e l l

s

S a a r e l a e t a l ( 2 0 0 1 )

P K U




C y t o s o l

D y s f u

n c t i o n o f l i v e r

c e l l

s a n d a f f e c t i o n o f

b r a i n c e l l s

P e y e t a l ( 2 0 0 3 ) W a t e r s ( 2 0 0 3 )

P a r k i n s o n d i s e a s e

A u t o s o m a l

d o m i n a n t / r e c e s s i v e

i n h e r i t a n c e a n d

a c q u i r e d


( L o s s - o f - f u n c t i o n )

C y t o s o l

D y s f u


d o p

a m i n e - p r o d u c i n g

b r a i n c e l l s

L o t h a r i u s a n d B r u n d i n

( 2 0 0 2 ) C o o k s o n ( 2 0 0 5 )

( C

o n t i n u e d o n n e x t p a g e )

Springer




T a b l e 1

( C o n t i n u e d )

M o l e c u l a r

G e n e t i c s

p a t h o g e n e s i s

C o m p a r t m e n t

M a i n c e l l u l a r p a t h o l o g y

R e f e r e n c e s t o p a t h o g e n i c m e c h a n i s m

K e r a t i n d i s e a s e s





C y t o s o l

D y s f u


s k i n

c e l l s

S o r e n s e n e t a l ( 2 0 0 3 )

F a m i l i a l c a r d i o m y o p a t h i e s





C y t o s o l ( s a r c o m e r e )

D y s f u


c a r d i a c c e l l s

B u r c h a n d B l a i r ( 1 9 9 9

)

O r n i t h i n e t r a n s c a r b a m y l a s e

( O T C ) d e fi c i e n c y

X - l i n k e d i n h e r i t a n c e



t i o n )

M i t o c h o n d r i a

D y s f u

n c t i o n o f

l i v e

r / b r a i n c e l l s

B r u s i l o w a n d H o r w i c h

( 2 0 0 1 )

S h o r t - c h a i n a c y l - C o A

d e h y d r o g e n a s e ( S C A D )

d e fi c i e n c y





t i o n )

M i t o c h o n d r i a

D y s f u


b r a i n c e l l s

G r e g e r s e n e t a l ( 2 0 0 4 )

A l t z h e i m e r d i s e a s e


i n h e r i t a n c e a n d

a c q u i r e d


C y t o s o l a n d

e x t r a c e l l u l a r

D y s f u


b r a i n c e l l s

S m i t h e t a l ( 2 0 0 2 )

thenucleus arenot known. However, although it is not known

what exactly the composition of the rods is, sarcomeric pro-

teins have been identified (Clarkson et al 2004). Accordingly,

it is not unreasonable to indicate that other proteins impor-

tant for cell function and survival may be affected, as in

Huntington disease.

How variations in the α-actin gene itself contribute to the

pathogenesis in the actin myopathies is not known and mayvary considerably, depending on the type of gene variation.

Some missense variant α-actin proteins may result in nega-

tive dominance by forming stable misfolded conformations,

which may build into the polymerized α-actin structure in

the sarcomere, but they may also be accumulated as aggre-

gates,as seen in cells expressing missense variantsof heartα-

actin identified in patients with cardiomyopathies (Vang et al

2005). Whether such a pathogenic mechanism contributes

directly to the nuclear inclusions is not known. However,

it is known that overexpression of variant skeletal α-actin

proteins in cultured fibroblasts induces the nuclear rod for-

mation (Costa et al 2004), and that overexpression of variantactins in myoblast cell lines results in increased amounts of

α-actinin (Ilkovski et al 2004) among other proteins. The rod

formation is thus not only a result of an imbalance between

interaction partners, but is probably also aggravated by com-

pensatory upregulation of the rod forming α-actinin. How

these pathogenic processes may be influenced by variations

or pharmacological manipulations of the efficiencies of the

PQC systems is not known.

In conclusion, actin myopathy may be due to a toxic gain-

of-function pathogenesis by rod formation and loss of func-

tion by the deficiency of actin fibres in the sarcomere.

Protein misfolding diseases of the ER

Cystic fibrosis (McKusick 219700) is an autosomal recessive

disorder caused by disease-causing variations in the cystic fi-

brosis transmembrane regulator (CFTR) gene. Deficiency of

the CFTR results in disturbed electrolyte transport across

epithelial membranes, and clinical symptoms comprise re-

curring lung infections, obstruction of sinuses, pancreatic

and gastrointestinal insufficiency and male infertility (Welsh

et al 2001). The CFTR protein is co-translationally translo-

cated into and processed through the ER. The trafficking in

the cytosol and translocation are assisted by the chaperones

Hsc70/Hsp70, which act as a first level of quality control,

since at this level the common delta-Phe508 variant CFTR

protein is stopped in the processing and presented to the pro-

teasome for degradation (Farinha and Amaral 2005). A sec-

ond level of conformational control is located in the ER after

the N -glycosylation has occurred and the glycosylated CFTR

is bound to calnexin, one of the lectin chaperones of the ER.

The quality control at this level is illustrated by the obser-

vation that 75% of heterologous expressed wild-type CFTR

Springer






protein is rejected here and retro-translocated to degradation

in the cytosol (Ward and Kopito 1994). Thus only a fraction

reaches the plasma membrane. Although it has subsequently

been shown that endogenously synthesized CFTR protein

in epithelial cells is more efficiently processed (Varga et al

2004), the case illustrates the fine balance between folding

and the route to degradation in the ER for wild-type and

variant CFTR proteins. Further, it is interesting to note thatoverexpression of the CFTR protein or inhibition of protea-

some activity results in accumulation of undegraded wild-

type and delta-Phe508 variant CFTR protein that may give

rise to formation of so-called aggresomes, which are per-

inuclear aggregated proteins containing ubiquitin and sur-

rounded by collapsed intermediate filament proteins (Kopito

2000). In addition to ubiquitinated proteins and filament pro-

teins, aggresomes also contain a number of chaperones, in-

cluding Hsp70 (Kopito 2000; Sherman and Goldberg 2001).

The association with chaperones and the fact that the fold-

ing and processing to arrive at the cell surface are enhanced

by low temperature (Gelman and Kopito 2003) indicate thatenhancement of the folding and transport competence is a

possible intervention strategy.

In conclusion, cystic fibroses is due to a loss-of-function

pathogenesis, butundercertain conditions, such as cell stress,

in which the aggregation tendency increases, it may also

exhibit a toxic gain-of-function pathogenesis from variant

CFTR proteins.

Gaucher disease (McKusick 230811, 230900, 231000) is

one of about 40 lysosomal storage disorders that are char-

acterized by deficiency of lysosomal enzymes (Beutler and

Grabowski 2001). Gaucher disease is inherited in an auto-

somal recessive fashion and patients are deficient in the en-

zyme acidβ-glucosidase (McKusick 606362); they accumu-

late glucosylceramide in the lysosomes, which is believed

to be the basis for a range of clinical phenotypes. Patients

with adult type I disorder show, as important features, hep-

atosplenomegaly, skeletal lesions and pancytopenia, while

infantile and juvenile patients with severe types II and III,

respectively, in addition show central nervous system dys-

function. In this context it is interesting to note that patients

carrying identical variations in the β-glucosidase gene may

show different disease severity (Ron and Horowitz 2005).

β-Glucosidase is a membrane-associated enzyme protein

that is co-translationally translocated to the ER, where it

is N -glycosylated and folded to transport competence with

the assistance of chaperones. The folded enzyme protein

is further processed in the Golgi complex, where it is tar-

geted to the lysosome. Like misfolded CFTR proteins, mis-

sense variant β-glucosidase proteins do not obtain transport

competence; they are retained in the ER and targeted for

retrograde transport to the cytosol and degraded by the pro-

teasome. However, in cells from patients carrying missense

gene variations the amount of degraded misfolded variant

β-glucosidase protein may vary considerably, resulting in

variable amounts of ER-accumulated enzyme protein (Ron

and Horowitz 2005). Indeed, even in patient cells carrying

the same genotype, the level of misfolded aggregated en-

zyme protein is not the same, indicating that other factors

related to the ER and cytosolic PQC systems are involved in

the pathogenesis. That this is the case was demonstrated by

inhibiting the proteasomal proteases and detecting increasedamounts of aggregated variant β-glucosidase protein (Ron

and Horowitz 2005). The same study showed that missense

variant enzyme proteins make complexes with the chaper-

one calnexin, indicating that manipulation of the folding and

transport competence may be a profitable strategy.

In conclusion, Gaucher disease shows a typical loss-of-

function pathogenesis. However, the neurological dysfunc-

tion indicates that the accumulated variant proteins may con-

tribute with a toxic gain of function.

Protein misfolding disorders of the cytosol

Phenylketonuria (McKusick 261600) (PKU) is an autoso-

mal recessive disorder with mental retardation as the most

prominent clinical feature (Scriver and Kaufman 2001).

The disease is due to deficiency of phenylalanine hydrox-

ylase (PAH), and results in a decreased level of tyro-

sine and accumulation of phenylalanine and phenylalanine

metabolites, which are believed to account for the clinical

symptoms.

PAH is a cytosolic resident and the turnover is accom-

plished by the cytosolic chaperone and protease system. No

detailed study of the actual folding process of the PAH en-

zyme protein has been done, but it is indicated from in vitro

studies that variant PAH enzyme proteins are dependent on

chaperones. Indeed, the yield of active variant PAH enzyme

protein was decreased at 37◦C compared to 27◦C, whereas

the total amounts of soluble protein remained the same at

high and low temperatures (Gamez et al 2000), suggesting

that the variant proteins may form complexes with chaper-

ones, as seen for protein variants of short-chain acyl-CoA

dehydrogenase (SCAD) (Pedersen et al 2003). In addition,

expression of a number of variant PAH proteins in reticulo-

cyte extracts indicated that cytosolic proteases are responsi-

ble for rapid degradation of the variant proteins (Waters et al

1999). Indeed, it has been proposed that the efficiency of

the PQC system in the cytosol in various patients and under

varying conditions, such as cell temperature, may contribute

to the lack of correlation between clinical severity and the

nature of the variation in the PAH gene (Gamez et al 2000;

Pey et al 2003; Waters 2003).

As indicated, variant PAH proteins may be associated

with chaperones. However, although variant PAH proteins

have been observed to aggregate in vitro (Gamez et al 2000;

Pey et al 2003; Waters 2003), it is not known whether

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they may do so in vivo, or whether they are degraded

rapidly.

In conclusion, PKU is predominantly subject to loss-of-

function pathogenesis. Whether there may be a contribu-

tion, perhaps in situations of cell stress, such as elevated

temperatures, from a toxic gain of function is presently not

known.

Parkinson disease (McKusick 168600) is one of the com-monest neurodegenerative disorders, with major clinical fea-

tures such as resting tremor, rigidity and slowness of move-

ments. The disease comprises both sporadic age-dependent

and earlier-onset inherited cases (Cookson 2005; Jakobsen

and Jensen 2003). The inherited forms are in most cases au-

tosomal dominant, but can also be recessive. Although the

pathogenesis has not been fully elucidated, a common fea-

ture seems to be accumulation of α-synuclein in the cytosol

of dopamine-producing neurons (Cookson 2005). This no-

tion is strengthened by the fact that early-onset Parkinson

disease may be caused by gene variations in the α-synuclein

gene as well as in the ubiquitin ligase parkin and ubiquitincarboxyl terminal hydroxylase, both of which are involved in

the turnover of α-synuclein. α-Synuclein is one of presum-

ably several hundred proteins that in their ‘resting’ location

are naturally unfolded (Uversky 2002), and which may at-

tain a structured conformation at the active location, such

as α-synuclein in the membrane of the dopamine-containing

vesicles in the dopamine-producing neurons (Lotharius and

Brundin 2002).α-Synuclein, together with an unknown num-

ber of other cellular proteins, is prone to self-aggregation

(Ellis and Pinheiro 2002). These proteins are conformation-

ally unstable, and aberrations such as inherited amino acid

alterations or oxidative modifications may promote aggrega-

tion rather than degradation of the misfolded proteins. This

is another reason – in addition to ridding the cells of DRiPs

– for cells to possess efficient PQC systems that are able to

detect and degrade such aggregation-prone proteins. In the

young and healthy cell, the PQC system can cope with the

total ‘misfolding load’. However, the efficiency of the PQC

systems declines with age and the aggregation-prone proteins

may escape degradation. Since many other factors might be

involved, such as common variations in genes coding for

factors involved in interacting mechanisms, this decline of

PQC efficiency is probably an aetiological factor in sporadic

Parkinson disease. However, in some of the inherited forms,

where either variant α-synuclein or variant components of

the degradation system are involved, the PQC systems can-

not cope, and the disease may develop at an earlier age. As

in the other aggregation diseases, the involvement of the

PQC systems in the pathogenesis of Parkinson disease is

indicated by the presence of chaperones, e.g. Hsp70, in the

α-synuclein aggregates as well as by the alleviation of α-

synuclein toxicity by overexpression of the Hsp70 in model

systems (Muchowski and Walker 2005).

In conclusion, the pathogenesis of Parkinson disease rep-

resents a classical toxic gain of function, but a loss-of-

function component caused by α-synuclein deficiency in

dopamine vesicles may also contribute to the nerve cell dys-

function and death.

Protein misfolding disorders of mitochondria

Ornithine transcarbamylase (OTC) deficiency (McKusick

311250) is a classical X-linked disorder of the urea cycle

metabolism (Brusilow and Horwich 2001). Typical clinical

symptoms arerelated to the toxicity of ammonia, i.e. lethargy

andencephalopathy, andthe pathogenesisis due to variations

in the OTC gene that may result in total lack of enzyme pro-

tein in patients with premature stop codons or misfolded pro-

tein, which – as far as we know today – are rapidly degraded

by the mitochondrial PQC system. Although accumulation

of misfolded OTC proteins due to gene variations identified

in patients has not been observed, an OTC model protein

carrying a large deletion comprising amino acid 30 to 114(delOTC) has been expressed in COS cells in order to inves-

tigate the fate and effect of this protein (Zhao et al 2002).

Because of the overexpression and the severe folding defect,

an appreciable amount of the delOTC protein apparently es-

capes the quality control and accumulates as aggregates. It

is interesting to note that the aberrant OTC protein elicited a

stress response by inducing the expression of mitochondrial

Hsp60 as well as the mitochondrial proteases Lon and ClpP.

However, the significance for continued function or dysfunc-

tion of the cell has not been investigated and is therefore not

known at present.

In conclusion, OTC deficiency shows a loss-of-function

pathogenesis. A toxic gain of function may be the case for

severe folding gene variations, if it can be shown that mito-

chondrial aggregates are cell toxic.

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency

(McKusick 201470) is a classical inherited autosomal reces-

sive disorder of the fatty acid oxidation pathway (Gregersen

et al 2004). The clinical picture in patients with enzymati-

cally and genetically verified SCAD deficiency is very di-

verse (Corydon et al 2001; Gregersen et al 2004). Most pa-

tients show unspecific neuromuscular symptoms, such as

developmental delay, hypotonia and seizures, but a minor-

ity show – additionally or alternatively – typical symptoms

seen in other fatty acid oxidation defects, such as hypogly-

caemia and vomiting. In contrast to many other inborn errors

of metabolism, including the fatty acid oxidation defects,

only a small fraction of patients investigated for SCAD defi-

ciency, characterized by elevated excretion or accumulation

of respectively ethylmalonic acid or butyrylcarnitine, carry

deactivating variations in the SCAD gene (Gregersen 2004

and unpublished). The majority of patients carry in one or

both alleles one of two common SCAD susceptibility gene

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variations, 625G>A and 511C>T (G185S and R147W),

that are present in homozygous or compound heterozygous

form in 7–14% of the general population (Gregersen et al

1998; Nagan et al 2003). According to heterologous expres-

sion studies, these common gene variations are not SCAD

deactivating but decrease the variant SCAD activity in a

temperature-dependent manner (Gregersen et al 1998). How-

ever, the amounts of variant proteins did not decrease corre-spondingly, indicating that inactive variant SCAD proteins

were present in the cells. It was proposed that the variant

SCAD proteins misfold and associate with chaperones. In

isolated mitochondria it was later shown that the G185S vari-

ant SCAD was associated with the Hsp60 chaperone more

extensively, and that the R147W variant at higher tempera-

ture formed aggregates (Pedersen et al 2003). In addition to

the investigation of these two susceptibility variant proteins,

a number of SCAD-deactivating variant proteins were also

analysed with respect to their ability to associate to Hsp60

and aggregate. In all cases (Pedersen et al 2003; Gregersen

unpublished) the variant proteins tend to complex with Hsp60and aggregate more extensively than wild-type SCAD. These

experiments have fostered the hypothesis that SCAD gene

variations, in addition to SCAD deficiency, may result in ac-

cumulation of variant SCAD proteins, which may contribute

to the pathogenesis. In the cases where the two susceptibility

gene variations are involved, it has of course also been pro-

posed that factors other than the gene variations themselves

must be involved in the pathogenesis. Since the effect in all

cases is deficiency of SCAD activity, as indicated by ele-

vated excretion of ethylmalonic acid and/or increased blood

concentration of butyrylcarnitine, it is reasonable to propose

that the(se) factor(s) may be involved in the processing of the

SCAD proteins.

In conclusion, the pathogenesis of SCAD deficiency in

patients with classical SCAD-deactivating gene variations

is characterized by loss of function, perhaps with a con-

tribution from a toxic gain of function. In patients car-

rying the SCAD susceptibility gene variations there is a

contribution from loss-of-function pathogenesis, but the

main component is proposed to come from a toxic gain of

function.

What determines the fate of misfolded proteins?

In the discussion above, a number of determinants of the

fate of a given misfolded protein have been mentioned. For

further discussion it is convenient to distinguish between

three levels of determining effects on the pathogenesis: the

nature of the protein structure, the efficiency of the fold-

ing and degradation systems, and cellular and environmental

factors.

First level: The nature of the protein structure

In their native structure, most proteins are composed of a bal-

anced mixture of α-helices,β-sheets and unstructured turns.

The ease with which these structures are attained and main-

tained is quite different for various proteins. It is known that

protein structures are flexible, and that many biological func-

tions depend on this flexibility (Zaccai 2000). The extremeis the so-called naturally unfolded proteins (Uversky 2002),

such as α-synuclein, which reside in an unstructured con-

formation until they exert their biological function. Some of

these, as well an estimated at least 20 other proteins, carry

the unfortunate ability to be stabilized in β-sheet confor-

mations, which are prone to aggregation (Ellis and Pinheiro

2002). However, since all proteins in extreme conditions,

such as low pH, high temperature, high salt and macromolec-

ular crowding, will adopt a β-sheet structure, the 20 proteins

mentioned may represent only the extreme of a continuum.

Further, not all protein aggregates consist of β-sheet fibril-

lar structures. Misfolded monomeric proteins may also formamorphous aggregates and other forms of oligomeric struc-

tures (Muchowski and Walker 2005).

One determining factor in the aggregate formation is in

all cases the amino acid composition in certain domains of

the protein. It has been shown that changes in physicochemi-

cal properties, such as hydrophobicity, charge and secondary

structure propensity, caused by amino acid alterations, corre-

late with changes in the rates of aggregation of the unfolded

peptides (Chiti et al 2003). Indeed, a change of two amino

acids in theC-terminal domainof theCFTR protein alleviates

the tendency to aggregate (Milewski et al 2002), and system-

atic substitutions in a protein domain, HypP-N, can change

the aggregation tendency dramatically in E. coli (Calloni et al

2005). Thus, even small changes in the amino acid compo-

sition and structure of a given protein may destabilize the

folding intermediates, which, at a rate faster than the PQC

systems can detect them, may initiate an aggregation process

instead of re-entering the correct folding pathway or being

targeted for degradation.

Second level: The efficiency of the folding and

degradation systems

As discussed above, the task of the chaperone components of

the PQC systems is to protect nonfolded proteins and fold-

ing intermediates from intermolecular interactions, whereas

the function of the protease components is to degrade pro-

teins that cannot fold properly as well as proteins damaged

to such degrees that they cannot refold. The amounts and

consequently the activity of the various components of the

PQC systems may therefore have a profound influence on

the balance between proper folding, degradation and aggre-

gation of variant proteins, as has been exemplified in the

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previous section. Of further relevance in this connection is

the emerging number of diseases in which a component in

a PQC system is deficient due to inherited gene variations.

In a few cases of spastic paraplegia a variation in the gene

coding for mitochondrial Hsp60 has been detected (Hansen

et al 2002). In addition, a gene variation in a potential sub-

unit of the cytosolic chaperonin TRiC has been found to be

associated with McKusick – Kaufman syndrome (Stone et al2000). Further, to illustrate the point that both chaperone and

protease components can be affected, it should be mentioned

that spastic paraplegia has also been found associated with

variations in the gene that codes for the mitochondrial mem-

brane protease, paraplegin (Casari et al 1998).

Although the pathogenic mechanisms have not been eluci-

dated fully in these diseases, thefact that the balance between

proper folding, degradation and aggregation can be easily

disturbed indicates that multiple functions may be affected

and that only mild gene variations may be allowed, simply

because severe variations may not be compatible with life.

It is relevant to the present discussion that commongene variations (single nucleotide polymorphisms (SNPs))

in genes coding for PQC components may be susceptibility

factors in protein misfolding disorders, especially in those

cases where a residual function can be rescued by manipula-

tion of the PQC systems.

Third level: Cellular and environmental factors

The PQC systems operate in cellular environments that

change according to the physiological situation. It is known

that several types of cellular stress will induce many compo-

nents of these systems. As discussedabove, themain function

of thestress response is to compensate for adverse conditions,

which may unfold and/or damage many proteins. In addi-

tion to heat, the most notably damaging agents are reactive

oxygen species (ROS) and reactive nitrogen species (NOS)

(Butterfield and Kanski 2001; Dalle-Donne et al 2003). In

the present context the question is how heat and ROS/NOS

production affect the balance between correct folding, degra-

dation and aggregation of misfolded proteins.

Since protein unfolding and the strength of hydrophobic

interactions are promoted by heat, elevation of the tempera-

ture, e.g. fevers, may decrease the yield of correctly folded

variant proteins. Fordiseases with loss-of-function pathogen-

esis the consequence may be an aggravation of the functional

deficiency. However, because adverse conditions, including

heat, may promote formation of aggregates by a combined

effect of unfolding and transition to aggregation-prone struc-

tures, such as β-sheet structures, the result may addition-

ally be a toxic gain of function. These effects are discussed

in more detail elsewhere (Gregersen et al 2005). Suffice to

say here that all proteins during their lifetime are subject to

chemical changes, such as oxidative modifications by ROS

and NOS, that decrease their functional efficiency (Dalle-

Donne et al 2003). It has been shown in model systems that

misfolded proteins, also without heat denaturation, are sus-

ceptible to oxidative changes (Dukan et al 2000). In addi-

tion to the oxidative modifications, which are generated by

ROS elicited by the cellular effects of the misfolded proteins

themselves, it is therefore very probable that constitutively

produced oxidized proteins, which may not be eliminatedby saturated/overwhelmed PQC systems, contribute to the

disease pathology and development of a variety of diseases.

In conclusion, the two examples of cellular and environ-

mental stressors, respectively ROS/NOS and heat, illustrate

that perturbation of the cellular milieu from other sources

than the misfolded protein itself may participate in the patho-

genesis of protein misfolding disorders. Indeed, these pertur-

bations and the effects of the misfolded proteins themselves

– as soluble monomers or oligomers or as aggregates – may

interplay and aggravate each other in a vicious circle, which

may start as clinically unrecognisable mild cellular dysfunc-

tion and end up by killing the cell. Further discussion of allthese possible cellular consequences is outside the scope of

this review. The interesting aspect in the present context is

that the present knowledge and techniques make it possible

to intervene very close to the root of the problem; namely in

the folding process as well as in the mechanisms involved in

the accumulation of the misfolded proteins.

Intervention strategies

It is clear from the above discussion that the most efficient

treatment of protein misfolding disorders is to enhance the

folding of the variant proteins, thus increasing the amounts

of active protein. Such treatment will at the same time de-

crease the amounts of accumulated misfolded proteins and

alleviate the pathological consequences. A large number of

such protein function enhancement strategies have been stud-

ied and are even under investigation in clinical trials. They

will be discussed in some detail below but, before that, other

treatment strategies will be mentioned briefly.

Decreased expression of the aberrant protein

The first treatment strategy aims at decreasing the amounts

of misfolded intermediates by decreasing the expression of

the aberrant protein. In case of inherited disorders, the most

radical treatment is to repair the gene defect by targeted gene

correction, but this is still at the experimental stage (Yin et al

2005). Another strategy – related to dominantly inherited

misfolding disorders with gain-of-function pathogenesis – is

to suppress the expression of the variant protein and leave the

normal/wild-type protein at heterozygous expression level.

This can be achieved by allele-specific silencing by RNAi of

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the gene carrying thedefect, as exemplified by suppression of

variant PolyQ in spinocerebellar ataxia type 3 and a missense

Tau variation in frontotemporal dementia (Miller et al 2003).

This strategy cannot be used in diseases where a PQC system

is defective or where itsefficiency hasdeclinedwith age, such

as in most cases of Parkinson and Alzheimer diseases, where

the aggregation-prone proteins (respectivelyα-synucleinand

β-amyloid) accumulate. Instead, reprogramming of the fold-ing pathway by pharmacological means may be promising.

In cases of ER accumulation it may be a useful strategy to

alleviate the load of misfolded proteins by treatment with

drugs that inhibit protein synthesis without decreasing the

stress response, as proposed by use of the dephosphorylation

inhibitor salubrinal (Wiseman and Balch 2005). However,

instead of decreasing the expression of the aberrant protein

that accumulate and aggregates, treatment strategies aiming

at reducing the amounts of aggregation-prone protein may

be more realistic in the present view.

Increased elimination of aggregation-prone proteins

Since specific amino acids in a given protein may promote

aggregation and others may alleviate this effect (Calloni et al

2005), a radical strategy to alleviate the aggregation tendency

could be to change specific amino acids in certain proteins.

Another way is to stimulate the elimination of the ac-

cumulated aberrant protein, either by induction of spe-

cific degradation pathways or intracellular proteases, or by

introducing antibodies to neutralize the gain-of-function ef-

fects. Although some target points for stimulating degra-

dation pathways or intracellular proteases are known, e.g.

components of the ERAD system (Haynes et al 2004) and

co-chaperones to Hsp70 (Morishima 2005), these strategies

has not yet been explored.

A general point in this regard, which would also apply

to induction or introduction of the known chaperones, is that

strategies aiming at nonspecific mechanisms may have many

unexpected consequences. A more feasible way is to intro-

duce specific antibodies, either specifically designed to the

particular disease and to aggregation-prone proteins (Miller

and Messer 2005) or targeted to the general β-sheeted struc-

tures, which constitute the precursor of aggregates in the

classical misfolding diseases – Alzheimer, Parkinson and

Huntington diseases (Kayed et al 2003).

These strategies may be promising because the

monomeric/oligomeric misfolded proteins, which have been

shown to be the toxic substances, are eliminated (Bucciantini

et al 2004). Although some of these strategies have been

shown to work in cell and animal models, there are many

challenges, such as means of delivery and the risk of elici-

tation of immune responses, before human treatment can be

effective and safe.

In contrast to the direct elimination of the toxic protein

conformations, many attempts to block the formation of

aggregates have been made as detailed below.

Inhibition of aggregate formation

In contrast to alleviating the misfolding load at the

monomeric/oligomeric level, it has been shown that po-

tential treatment strategies may lie in preventing aggre-

gate formation by the use of small molecules such as tre-

halose (Tanaka et al 2005), bi-functional organic molecules

(Gestwicki et al 2004) or small peptides (Zhou et al 2004), or

in disaggregating already formed aggregates by overexpres-

sion of chaperone components of the folding and degrada-

tion pathways (Cashikar et al 2005). Although some of these

strategies have shown promise in cell and animal models,

there is a potential adverse effect of dissolving the aggre-

gates, which in many cases are believed to rescue the cell

from toxic oligomeric misfolded proteins, especially in the

form of perinuclear aggresomes. However, as discussed pre-

viously, aggregates may contain a large number of vital cellu-

lar components, such as chaperones and transcription factors,

which may thus be released. Indeed, it may be speculated –

at least in some cases – that the stabilization and chaperone

association of the released misfolded proteins may confer

susceptibility to degradation, and therefore also alleviation

of the cell dysfunction caused by the accumulated misfolded

monomers and/or oligomers.

Like the two previously discussed strategies, inhibi-

tion of aggregate formation may decrease the misfolding

load, which is appreciable for disorders presenting gain-

of-function pathogenesis. However, many protein misfold-

ing diseases, including many inborn errors of metabolism,

are predominantly subject to a loss-of-function pathogene-

sis. Since the aberration – due to missense gene variations

in many cases, as discussed above for PKU and MCAD

and SCAD deficiencies – may result in folded protein with

residual function, a strategy that eliminates folding inter-

mediates may decrease the residual function and aggravate

the pathological and clinical consequences. On the other

hand, a strategy that stabilizes – in a soluble state – the

folding intermediates and targets them for further folding

instead of degradation may be beneficial for all protein

misfolding disorders, notwithstanding their pathogenic

mechanism.

Enhancement of protein function

The last intervention strategy to be discussed briefly is in-

duction of naturally occurring chaperones as well as in-

troduction of chemical and pharmacological chaperones.

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This strategy will fulfil the purpose of enhancing the pro-

tein function and alleviating the accumulation of aberrant

proteins.

As discussed previously in connection with the specific

misfolding diseases, there are many experiments showing

that overexpression of various chaperones may alleviate the

effect of misfolding and in some cases increase the protein

function. This and the general function of the chaperone net-work in the folding process and in subacute induction of

chaperones involved in damage reduction and longevity in

vitro and in vivo (Rattan 2004), together with the observa-

tion that protein aggregates contain a variety of chaperones,

have fostered the idea that enhancement of components in

the chaperone network may be a beneficial treatment strat-

egy. This can of course be achieved by gene therapy means,

as mentioned above in connection with induction of compo-

nents of the degradative pathways. However, it is probably

more promising to induce chaperone expression with small-

molecule regulators of the heat shock response (Westerheide

and Morimoto 2005). A large number of such compoundshave been identified either by candidate approaches or by

high-throughput screening, among them protein synthesis

inhibitors, proteasome inhibitors, inflammatory mediators,

sodium salicylateand flavonoids (Westerheide and Morimoto

2005).

Although the enhancement of chaperone levels by induc-

tion of the heat shock responseat a first glance seems promis-

ing, not all of the wide range of cellular factors involved in

the heat shock response (Trinklein et al 2004) may be bene-

ficial. A better approach would be to induce only the useful

chaperones or to introduce so-called chemical chaperones,

which are small-molecule compounds with general chaper-

oning effects on unfolded protein stability and prevention of

aggregate formation (Bernier et al 2004; Perlmutter 2002;

Ulloa-Aguirre et al 2004). Glycerol, dimethyl sulphoxide

(DMSO), trimethylamine N -oxide (TMAO) and deuterated

water are the best known. They have all been used in cellu-

lar systems to enhance the recovery of active proteins from

misfolding. Since phenylbutyric acid (PBA) has been shown

to stimulate the excretion of variant α-1-antitrypsin in a cell

model (Burrows et al 2000) as well as CFTR delta-Phe508

protein trafficking and expression on the cell surface of pa-

tients’ cells (Rubenstein et al 1997), it hasoften been counted

among the chemical chaperones. However, PBA is a histone

deacetylase inhibitor and – although a global analysis has not

been performed – it may influence the expression of a num-

ber of cellular factors. Indeed, in cells treated with PBA, the

chaperone Hsc70 is downregulated (Rubenstein and Zeitlin

2000), perhaps alleviating cytosolic retention of misfolded

CFTR and α-1-antitrypsin variant proteins. This compound

is therefore interesting and may represent a unique molecule

with selective effect on some cellular proteins. PBA is also

the only ‘chemical chaperone’ that has been used in clinical

trials, though without any effects in the preliminary study

(Teckman 2004).

Although it maybe worthscreening forsmall-moleculein-

ducers of specific components of the chaperone network, the

most promising intervention strategy has until now been the

in vitro and in vivo trials with the so-called pharmacological

chaperones, which are small-molecule stabilizers of specific

proteins (Bernier et al 2004; Desnick 2004; Fan 2003; Perl-mutter 2002; Sawkar et al 2002; Ulloa-Aguirre et al 2004).

The majority of pharmacological chaperones are antagonists

or agonists to receptors, membrane and secreted proteins as

well as enzymes processed through the ER. In this regard

the lysosomal enzymes are the most interesting because de-

ficiencies of these enzymes represent classical inborn errors

of metabolism, and treatment with pharmacological chaper-

ones has advanced to the level of clinical trials for some of

them (Desnick 2004; Fan 2003).

As discussed in the specific section on Gaucher disease,

the β-glucosidase related to this disease, as well as all lyso-

somal enzyme proteins, are nuclear-encoded and are co-translationally translocated into ER, where they are folded

and made transport-competent with the assistance of ER-

specific chaperones and other folding helpers before export

through the Golgi complex to the lysosomes. The crucial part

of this processing is the ER, where misfolding, for instance

due to missense gene variations, may compromise the attain-

ment of the transport-competent structure. However, since in

most cases the active enzyme centre is not affected severely

by the amino acid alterations, which compromise the folding,

the unstable folding intermediates can apparently in some

cases be stabilized by molecules that bind to the active site

(Desnick 2004; Fan 2003; Sawkar et al 2002).

Whether such an approach is possible in a broader

range of protein misfolding disorders, where the aggregation

prone/aberrant proteins are processed in the cytosol or mito-

chondria, is not known. However, a few examples may exist.

One is the cytosolically processed tumour suppressor P53,

where variant forms have been rescued by conformation- sta-

bilizing polycyclic ionizable compounds (Foster et al 1999).

No examples are known of mitochondrially processed pro-

teins, but this does not mean that the strategy is not applicable

for misfolded variants of, for example, fatty acid oxidation

enzymes and respiratory chain components.

Conclusion

The realization that many forms of inherited and acquired

diseases can be viewed as protein misfolding disorders is

slowly changing our conceptual framework regarding molec-

ular genetics, molecular pathogenesis, cellular pathology and

clinical management of many diseases. At the molecular ge-

netic level we have realized that it is important to distinguish

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between the various types of gene sequence variations asso-

ciated with disease. Many splice variations as well as most

out-of-frame deletions and insertions result in total loss-of-

function through nonsense-mediated decay of the variant

mRNA (Maquat 2005). In contrast, missense and small in-

frame deletions and insertions may lead to aberrant proteins,

which mayvaryin residual function dependenton thespecific

protein and its aberration as well as the cellular and environ-mental conditions. This leads to the molecular pathogenetic

level, which has been extensively discussed in this review.

We have realized that many inherited variant proteins and

aggregation-prone as well as damaged proteins may behave

in similar fashions in the cell. The fate of these proteins is pri-

marily guided by their physicochemical properties, such as

hydrophobicity and amino acid charges, rather than their spe-

cific functional properties. This means that the efficiency of

the cellular mechanisms, comprising molecular chaperones

and intracellular proteases, that constitute the protein quality

control systems and that rid the cell of aberrant and damaged

proteins becomes of prime importance for cellular pathology.At this level we have realized that the cellular consequences

of the pathogenesis of many diseases are a complex mix-

ture of loss of protein function, which is disease-specific,

and gain of function, which may also be specific in the sense

that it is elicited in certain cell types and cell compartments

but is general in the sense that the cell perturbations are the

result of protein accumulation and aggregation. This new

paradigm has consequences for intervention strategies, par-

ticularly exemplified by the use of pharmacological chap-

erones in current and especially in future treatment of the

lysosomal diseases.

In conclusion, it will be interesting to experience how this

emerging paradigm influences the advances in diagnostics as

well as clinical treatment of inherited and acquired protein

misfolding disorders in the coming years.

Acknowledgements The main contributors to the studies concern-

ing misfolded proteins at the Research Unit for Molecular Medicine

have been the Institute of Clinical Medicine, Aarhus University; the

Danish Medical Research Council; Aarhus University Hospital Re-

search Initiative; Karen Elise Jensen Foundation; Lundbeck Founda-

tion; Novo Nordisk Foundation; and the European Union (6th frame-

work programme).

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