J Inherit Metab Dis (2006) 29:456470 DOI 10.

1007/s10545-006-0301-4

SSIEM SYMPOSIUM 2005

Protein misfolding disorders: Pathogenesis and intervention

N. Gregersen

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

Summary Newly synthesized proteins in the living cell must go through a folding process to attain their functional structure. To achieve this in an efcient fashion, all organisms, including humans, have evolved a large set of molecular chaperones that assist the folding as well as the maintenance of the functional structure of cellular proteins. Aberrant proteins, the result of production errors, inherited or acquired amino acid substitutions or damage, especially oxidative modications, 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 one of the protein quality control systems. Examples are cystic brosis and phenylketonuria. 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 may be accompanied by a gain-of-function pathogenesis, which in many cases determines the 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-

teins, a likely future strategy seems to be the use of chemical or pharmacological chaperones with specic effects on the misfolded 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 acid chain by inherited sequence variations or acquired amino acid modications 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-specic protein quality control systems will be introduced, and a number of misfolding (or conformational) disorders will be discussed, with focus on loss-offunction and gain-of-function pathogenic mechanisms. Also, factors that may inuence the balance between loss of function and gain of function will be discussed, to end up with a brief discussion of strategies by which this balance can be shifted in benecial 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

Communicating editor: Jean-Marie Saudubray Competing interests: None declared Presented at the 42nd Annual Meeting of the SSIEM, Paris, 69 September 2005 N. Gregersen ( ) Research Unit for Molecular Medicine, Institute of Clinical Medicine, Aarhus University Hospital, Skejby Sygehus, 8200 Aarhus N, Denmark

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which may result in aggregation, and to assist the elimination of aberrant folding intermediates and unstable proteins, which would overload the chaperone systems and damage the cell. The longer a protein remains associated to chaperones, the higher is the probability that it will be captured by the proteolytic systems. The PQC systems are thus believed to function by a competition between release to native structure and targeting to degradation. Therefore, in addition 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 different marking systems, e.g. ubiquitination, are presented to the proteases by chaperone-mediated transfer. Although the various organelle-specic 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 processed through the Golgi complex are retrogradely translocated to the cytosol for degradation. Proteins that have their function in peroxisomes and the nucleus are folded in the cytosol 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 result 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 degradation system is important for cell-cleaning, but they also indicate that there is a balance between degradation and aggregation, which for the DRiPs depends on the degradation capacity. In old cells the ability to cope with misfolded protein is decreased (Grune et al 2004; Verbeke et al 2001), and accumulated proteins that are damaged or have an inherited tendency to misfolding, such as -synuclein in Parkinson disease and -amyloid in Alzheimer disease, are found as aggregates inside or outside the cells. In young cells, on the other hand, the efciency of the PQC systems may be sufcient to rid the cells of such protein accumulations. However, misfolded proteins containing inherited amino acid alterations may saturate and inhibit the proteases, thereby promoting formation of aggregates, such as missense variant -synuclein, which accumulates as aggregates in early-onset Parkinson disease.

co-translationally translocated into the endoplasmic reticulum, their hydrophobic domains are exposed to a complex environment 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 conditions, all organisms from bacteria to humans have evolved a large number of molecular systems that assist and monitor the intracellular folding process (Frydman 2001). Since the nal structure of a protein is given by the amino acid sequence (Annsen 1973), the primary function of the molecular systems is not to catalyse the folding but rather to minimize nonproductive interactions by shielding hydrophobic 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 dened as a functional class of unrelated proteins that assist the correct noncovalent assembly of other polypeptide containing structures in vivo, but are 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 protection, refolding and elimination of damaged and denatured proteins (Cashikar et al 2005). Indeed, the designation heatshock proteins (HSPs) for many chaperones stems from the discovery in 1988 that a variety of cellular stresses, particularly 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 depending on the nature and on the cellular location. However, nearly all except the lectin-chaperones bind to hydrophobic domains of the given polypeptide/protein in an ATP-dependent fashion and cycle between binding and release 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 acquiring the native conformation. If a given protein is aberrant due to severe damage or amino acid alterations, the folding machinery may give up, and the aberrant protein will be taken up by intracellular proteases, which then try to eliminate 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 protect folding intermediates from nonproductive interactions,

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mRNA
ribosome Hsp90 Importin ER nucleus CAN/Grp78 p53 Proteasome ClpP/Lon

nascent protein
cytosol Hsp70 Hsc70 Hsp90

mitochondria

Hsp70 Hsp60

PAH SCAD

-Glu CFTR

Hsp70 Lysosome golgi

PEX5

ACOX
peroxisome

cell membrane

Fig. 1 Trafcking, folding and turnover of cellular proteins. All nascent polypeptides are protected by cytosolic chaperones, notably Hsp70, Hsc70 and/or Hsp90. Cytosolic proteins, e.g. phenylalanine hydroxylase (PAH) are turned over by the cytosolic chaperone/protease systems, including the proteasome. Nuclear-encoded mitochondrial matrix proteins, e.g. short-chain acyl-CoA dehydrogenase (SCAD), are assisted in their import by mitochondrial Hsp70 and in their folding by the chaperonin 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 -glucosidase, -Glu)

or cell membrane insertion (as cystic brosis 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 cytosolic proteases, e.g. the proteasome. Nuclear and peroxisomal proteins are folded in the cytosol and translocated to the organelles by specic import proteins. Together with Hsp70, peroxins, e.g. PEX5, assist the translocation to peroxisomes of acyl-CoA oxidase (ACOX). Many nuclear 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 efciency of the PQC systems, the fate of a given aberrant protein may be different, as illustrated in Fig. 2. If the misfolded protein is easily degraded, the consequence is a loss-of-function pathogenesis, where a missing function and in many cases substrate accumulation is responsible for the cellular pathophysiology 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 deciency in combination 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 conditionally determined resistance, for instance after heat stress that 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 corresponding wild-type protein, or by forming oligomers and aggregates 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 compartment, and the efciency 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 induction of PQC components (Muchowski and Walker 2005), the function 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 insufciency 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 ubiquitin proteasome system by misfolded proteins (Bence et al 2001); (2) chaperone sequestration as well as sequestration of transcription factors and/or other cell components by accumulated proteins (Bruijn et al 2004; Muchowski and Walker 2005; Okado-Matsumoto and Fridovich 2002; Sherman and Goldberg 2001); (3) mitochondrial dysfunction and oxidative stress (Bruijn et al 2004; Buttereld and Kanski 2001; Haynes et al 2004; Schon and Manfredi 2003); (4) channel 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).

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Functional protein Residual function Toxic conformation Chaperone sequestration Accumulation Loss-of-function Semi unfolded protein Aggregation

Damage

G a i n - of - fu n c ti on

Gene variation

Degradation Loss-of-function Nascent protein

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 (folding intermediate or unfolded) may be degraded and give rise to lossof-function pathogenesis or accumulated protein, resulting in loss-offunction and/or gain-of-function pathogenesis. Accumulated misfolded

protein may form toxic conformations that inhibit normal function, sequester chaperones (and other cell components) and/or develop into aggregates eliciting a range of cell dysfunctional mechanisms. The various steps in these processes are target points for intervention (see the nal section of the text

Although these effects provide a general framework, which is guided by the physicochemical properties of the misfolded 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 disorders. 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 Huntingtons chorea (McKusick 143100) is an autosomal dominant neurodegenerative disease with clinical features comprising uncontrolled movements, cognitive changes and dementia. The disease is due to misfolding and aggregation in 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 Kremer 2001; Qin and Gu 2004). The accumulation of aggregates is most pronounced in the striatal neurons, where the GABA-producing cells are among the most affected, resulting in secondarily reduced production of 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 fragments 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 accumulation 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 transcription 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 efciency 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 huntingtin in nerve cells. Congenital myopathies comprise a group of diseases of the skeletal muscle sarcomere. The diseases can be autosomal recessive 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 depending on the nature of the protein involved, i.e. -actin, - and -tropomyosin, troponin T and nebulin, 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 -actinin, in many cases secondarily to gene variations in skeletal -actin. The mechanisms by which -actinin is translocated and forms rods in
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Table 1 Representative protein misfolding disorders, genetics, type of molecular pathogenesis (indication in parentheses is not certain), affected cellular compartment, main cellular pathology and some key references. The disorders discussed in this review are indicated by bold type Genetics Autosomal dominant inheritance Gain-of-function Loss-of-function Nucleus and cytosol Clarkson et al (2004) Loss-of-function Endoplasmic reticulum Kopito (2000) Gain-of-function (Loss-of-function) Nucleus and cytosol Molecular pathogenesis Compartment Main cellular pathology References to pathogenic mechanism Qin and Gu (2004) Cornett et al (2005)

Springer Dysfunction/death of GABA-producing brain cells Dysfunction/death of muscle cells Autosomal dominant or recessive inheritance Autosomal recessive inheritance Autosomal recessive inheritance Autosomal dominant inheritance Autosomal recessive inheritance Loss-of-function Gain-of-function Endoplasmic reticulum Loss-of-function Endoplasmic reticulum Loss-of-function (Gain-of-function) Endoplasmic reticulum Ron and Horowitz (2005) Jorgensen et al (2003) Carrell and Lomas (2002) Perlmutter (2003) Autosomal dominant inheritance Gain-of-function Loss-of-function Endoplasmic reticulum Christensen et al (2004) X-linked inheritance Autosomal recessive inheritance Loss-of-function Loss-of-function Endoplasmic reticulum Endoplasmic reticulum Morello et al (2001) Saarela et al (2001) Autosomal recessive inheritance Autosomal dominant/recessive inheritance and acquired Loss-of-function Cytosol Pey et al (2003) Waters (2003) Gain-of-function (Loss-of-function) Cytosol Dysfunction/death of lung, pancreatic and gastrointestinal epithelial cells Dysfunction/death of skeletal, liver, spleen and blood cells Dysfunction of liver cells and affection of vascular cells Dysfunction/death of liver cells and affection of lung cell function Dysfunction/death of vasopressinproducing cells in hypophysis Dysfunction of kidney cells Dysfunction of brain and connective-tissue cells Dysfunction of liver cells and affection of brain cells Dysfunction/death of dopamine-producing brain cells Lotharius and Brundin (2002) Cookson (2005) (Continued on next page)

Huntington disease

Congenital myopathies

Cystic brosis

Gaucher disease

Familial hypercholesterolaemia

-1-Antitrypsin deciency

Familial hypophyseal diabetes insipidus

Familial nephrogenic diabetes insipidus Aspartylglucoseaminidase deciency

PKU

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the nucleus are not known. However, although it is not known what exactly the composition of the rods is, sarcomeric proteins have been identied (Clarkson et al 2004). Accordingly, it is not unreasonable to indicate that other proteins important for cell function and survival may be affected, as in Huntington disease. How variations in the -actin gene itself contribute to the pathogenesis in the actin myopathies is not known and may vary considerably, depending on the type of gene variation. Some missense variant -actin proteins may result in negative dominance by forming stable misfolded conformations, which may build into the polymerized -actin structure in the sarcomere, but they may also be accumulated as aggregates, as seen in cells expressing missense variants of heart actin identied 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 -actin proteins in cultured broblasts induces the nuclear rod formation (Costa et al 2004), and that overexpression of variant actins in myoblast cell lines results in increased amounts of -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 compensatory upregulation of the rod forming -actinin. How these pathogenic processes may be inuenced by variations or pharmacological manipulations of the efciencies of the PQC systems is not known. In conclusion, actin myopathy may be due to a toxic gainof-function pathogenesis by rod formation and loss of function by the deciency of actin bres in the sarcomere. Protein misfolding diseases of the ER Cystic brosis (McKusick 219700) is an autosomal recessive disorder caused by disease-causing variations in the cystic brosis transmembrane regulator (CFTR) gene. Deciency of the CFTR results in disturbed electrolyte transport across epithelial membranes, and clinical symptoms comprise recurring lung infections, obstruction of sinuses, pancreatic and gastrointestinal insufciency and male infertility (Welsh et al 2001). The CFTR protein is co-translationally translocated into and processed through the ER. The trafcking in the cytosol and translocation are assisted by the chaperones Hsc70/Hsp70, which act as a rst level of quality control, since at this level the common delta-Phe508 variant CFTR protein is stopped in the processing and presented to the proteasome for degradation (Farinha and Amaral 2005). A second 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 observation that 75% of heterologous expressed wild-type CFTR
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References to pathogenic mechanism

Burch and Blair (1999)

Brusilow and Horwich (2001)

Sorensen et al (2003)

Gregersen et al (2004)

Main cellular pathology

Cytosol (sarcomere)

Dysfunction/death of skin cells Dysfunction/death of cardiac cells Dysfunction of liver/brain cells Dysfunction/death of brain cells Gain-of-function Loss-of-function Gain-of-function Loss-of-function Loss-of-function (Gain-of-function) Loss-of-function (Gain-of-function) Autosomal dominant inheritance Autosomal dominant inheritance X-linked inheritance Keratin diseases Familial cardiomyopathies Autosomal recessive inheritance Cytosol Mitochondria Mitochondria

Compartment

Molecular pathogenesis

Genetics

Table 1 (Continued)

Ornithine transcarbamylase (OTC) deciency Short-chain acyl-CoA dehydrogenase (SCAD) deciency Altzheimer disease

Autosomal dominant inheritance and acquired

Gain-of-function

Cytosol and extracellular

Dysfunction/death of brain cells

Smith et al (2002)

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-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 enzyme 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 increased amounts of aggregated variant -glucosidase protein (Ron and Horowitz 2005). The same study showed that missense variant enzyme proteins make complexes with the chaperone calnexin, indicating that manipulation of the folding and transport competence may be a protable strategy. In conclusion, Gaucher disease shows a typical loss-offunction pathogenesis. However, the neurological dysfunction indicates that the accumulated variant proteins may contribute with a toxic gain of function. Protein misfolding disorders of the cytosol Phenylketonuria (McKusick 261600) (PKU) is an autosomal recessive disorder with mental retardation as the most prominent clinical feature (Scriver and Kaufman 2001). The disease is due to deciency of phenylalanine hydroxylase (PAH), and results in a decreased level of tyrosine 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 accomplished by the cytosolic chaperone and protease system. No detailed study of the actual folding process of the PAH enzyme 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 chaperones, 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 reticulocyte extracts indicated that cytosolic proteases are responsible for rapid degradation of the variant proteins (Waters et al 1999). Indeed, it has been proposed that the efciency 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

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 efciently processed (Varga et al 2004), the case illustrates the ne balance between folding and the route to degradation in the ER for wild-type and variant CFTR proteins. Further, it is interesting to note that overexpression of the CFTR protein or inhibition of proteasome activity results in accumulation of undegraded wildtype and delta-Phe508 variant CFTR protein that may give rise to formation of so-called aggresomes, which are perinuclear aggregated proteins containing ubiquitin and surrounded by collapsed intermediate lament proteins (Kopito 2000). In addition to ubiquitinated proteins and lament proteins, aggresomes also contain a number of chaperones, including Hsp70 (Kopito 2000; Sherman and Goldberg 2001). The association with chaperones and the fact that the folding and processing to arrive at the cell surface are enhanced by low temperature (Gelman and Kopito 2003) indicate that enhancement of the folding and transport competence is a possible intervention strategy. In conclusion, cystic broses is due to a loss-of-function pathogenesis, but under certain 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 characterized by deciency of lysosomal enzymes (Beutler and Grabowski 2001). Gaucher disease is inherited in an autosomal recessive fashion and patients are decient in the enzyme acid -glucosidase (McKusick 606362); they accumulate 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, hepatosplenomegaly, skeletal lesions and pancytopenia, while infantile and juvenile patients with severe types II and III, respectively, in addition show central nervous system dysfunction. In this context it is interesting to note that patients carrying identical variations in the -glucosidase gene may show different disease severity (Ron and Horowitz 2005). -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 targeted to the lysosome. Like misfolded CFTR proteins, missense variant -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 proteasome. However, in cells from patients carrying missense gene variations the amount of degraded misfolded variant
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In conclusion, the pathogenesis of Parkinson disease represents a classical toxic gain of function, but a loss-offunction component caused by -synuclein deciency in dopamine vesicles may also contribute to the nerve cell dysfunction and death. Protein misfolding disorders of mitochondria Ornithine transcarbamylase (OTC) deciency (McKusick 311250) is a classical X-linked disorder of the urea cycle metabolism (Brusilow and Horwich 2001). Typical clinical symptoms are related to the toxicity of ammonia, i.e. lethargy and encephalopathy, and the pathogenesis is due to variations in the OTC gene that may result in total lack of enzyme protein in patients with premature stop codons or misfolded protein, 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 identied 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 investigate 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 escapes 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 signicance for continued function or dysfunction of the cell has not been investigated and is therefore not known at present. In conclusion, OTC deciency 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 mitochondrial aggregates are cell toxic. Short-chain acyl-CoA dehydrogenase (SCAD) deciency (McKusick 201470) is a classical inherited autosomal recessive disorder of the fatty acid oxidation pathway (Gregersen et al 2004). The clinical picture in patients with enzymatically and genetically veried SCAD deciency is very diverse (Corydon et al 2001; Gregersen et al 2004). Most patients show unspecic neuromuscular symptoms, such as developmental delay, hypotonia and seizures, but a minority show additionally or alternatively typical symptoms seen in other fatty acid oxidation defects, such as hypoglycaemia 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 deciency, 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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they may do so in vivo, or whether they are degraded rapidly. In conclusion, PKU is predominantly subject to loss-offunction pathogenesis. Whether there may be a contribution, 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 commonest neurodegenerative disorders, with major clinical features such as resting tremor, rigidity and slowness of movements. 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 autosomal dominant, but can also be recessive. Although the pathogenesis has not been fully elucidated, a common feature seems to be accumulation of -synuclein in the cytosol of dopamine-producing neurons (Cookson 2005). This notion is strengthened by the fact that early-onset Parkinson disease may be caused by gene variations in the -synuclein gene as well as in the ubiquitin ligase parkin and ubiquitin carboxyl terminal hydroxylase, both of which are involved in the turnover of -synuclein. -Synuclein is one of presumably several hundred proteins that in their resting location are naturally unfolded (Uversky 2002), and which may attain a structured conformation at the active location, such as -synuclein in the membrane of the dopamine-containing vesicles in the dopamine-producing neurons (Lotharius and Brundin 2002). -Synuclein, together with an unknown number of other cellular proteins, is prone to self-aggregation (Ellis and Pinheiro 2002). These proteins are conformationally unstable, and aberrations such as inherited amino acid alterations or oxidative modications may promote aggregation rather than degradation of the misfolded proteins. This is another reason in addition to ridding the cells of DRiPs for cells to possess efcient 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 efciency 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 efciency is probably an aetiological factor in sporadic Parkinson disease. However, in some of the inherited forms, where either variant -synuclein or variant components of the degradation system are involved, the PQC systems cannot 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 -synuclein aggregates as well as by the alleviation of synuclein toxicity by overexpression of the Hsp70 in model systems (Muchowski and Walker 2005).

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First level: The nature of the protein structure In their native structure, most proteins are composed of a balanced mixture of -helices, -sheets and unstructured turns. The ease with which these structures are attained and maintained is quite different for various proteins. It is known that protein structures are exible, and that many biological functions depend on this exibility (Zaccai 2000). The extreme is the so-called naturally unfolded proteins (Uversky 2002), such as -synuclein, which reside in an unstructured conformation 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 -sheet conformations, 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 macromolecular crowding, will adopt a -sheet structure, the 20 proteins mentioned may represent only the extreme of a continuum. Further, not all protein aggregates consist of -sheet brillar structures. Misfolded monomeric proteins may also form amorphous aggregates and other forms of oligomeric structures (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 physicochemical properties, such as hydrophobicity, charge and secondary structure propensity, caused by amino acid alterations, correlate with changes in the rates of aggregation of the unfolded peptides (Chiti et al 2003). Indeed, a change of two amino acids in the C-terminal domain of the CFTR protein alleviates the tendency to aggregate (Milewski et al 2002), and systematic 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 composition 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 efciency 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 folding intermediates from intermolecular interactions, whereas the function of the protease components is to degrade proteins 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 inuence on the balance between proper folding, degradation and aggregation of variant proteins, as has been exemplied in the

variations, 625G > A and 511C > T (G185S and R147W), that are present in homozygous or compound heterozygous form in 714% of the general population (Gregersen et al 1998; Nagan et al 2003). According to heterologous expression studies, these common gene variations are not SCAD deactivating but decrease the variant SCAD activity in a temperature-dependent manner (Gregersen et al 1998). However, the amounts of variant proteins did not decrease correspondingly, 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 variant SCAD was associated with the Hsp60 chaperone more extensively, and that the R147W variant at higher temperature 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 Hsp60 and aggregate more extensively than wild-type SCAD. These experiments have fostered the hypothesis that SCAD gene variations, in addition to SCAD deciency, may result in accumulation 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 proposed that factors other than the gene variations themselves must be involved in the pathogenesis. Since the effect in all cases is deciency of SCAD activity, as indicated by elevated 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 deciency in patients with classical SCAD-deactivating gene variations is characterized by loss of function, perhaps with a contribution from a toxic gain of function. In patients carrying 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 efciency of the folding and degradation systems, and cellular and environmental factors.
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and NOS, that decrease their functional efciency (DalleDonne et al 2003). It has been shown in model systems that misfolded proteins, also without heat denaturation, are susceptible to oxidative changes (Dukan et al 2000). In addition to the oxidative modications, 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 eliminated by saturated/overwhelmed PQC systems, contribute to the disease pathology and development of a variety of diseases. In conclusion, the two examples of cellular and environmental 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 pathogenesis of protein misfolding disorders. Indeed, these perturbations 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 dysfunction and end up by killing the cell. Further discussion of all these 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.

previous section. Of further relevance in this connection is the emerging number of diseases in which a component in a PQC system is decient 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 subunit of the cytosolic chaperonin TRiC has been found to be associated with McKusick Kaufman syndrome (Stone et al 2000). 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 membrane protease, paraplegin (Casari et al 1998). Although the pathogenic mechanisms have not been elucidated fully in these diseases, the fact 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 common gene 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 manipulation 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 components of these systems. As discussed above, the main function of the stress response is to compensate for adverse conditions, which may unfold and/or damage many proteins. In addition to heat, the most notably damaging agents are reactive oxygen species (ROS) and reactive nitrogen species (NOS) (Buttereld 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, degradation and aggregation of misfolded proteins. Since protein unfolding and the strength of hydrophobic interactions are promoted by heat, elevation of the temperature, e.g. fevers, may decrease the yield of correctly folded variant proteins. For diseases with loss-of-function pathogenesis the consequence may be an aggravation of the functional deciency. However, because adverse conditions, including heat, may promote formation of aggregates by a combined effect of unfolding and transition to aggregation-prone structures, such as -sheet structures, the result may additionally be a toxic gain of function. These effects are discussed in more detail elsewhere (Gregersen et al 2005). Sufce to say here that all proteins during their lifetime are subject to chemical changes, such as oxidative modications by ROS

Intervention strategies It is clear from the above discussion that the most efcient 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 decrease the amounts of accumulated misfolded proteins and alleviate the pathological consequences. A large number of such protein function enhancement strategies have been studied 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 briey. Decreased expression of the aberrant protein The rst 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-specic silencing by RNAi of
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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.

the gene carrying the defect, as exemplied 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 its efciency has declined with age, such as in most cases of Parkinson and Alzheimer diseases, where the aggregation-prone proteins (respectively -synuclein and -amyloid) accumulate. Instead, reprogramming of the folding 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.

Inhibition of aggregate formation In contrast to alleviating the misfolding load at the monomeric/oligomeric level, it has been shown that potential treatment strategies may lie in preventing aggregate formation by the use of small molecules such as trehalose (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 overexpression of chaperone components of the folding and degradation 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 aggregates, 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 previously, aggregates may contain a large number of vital cellular 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, inhibition of aggregate formation may decrease the misfolding load, which is appreciable for disorders presenting gainof-function pathogenesis. However, many protein misfolding diseases, including many inborn errors of metabolism, are predominantly subject to a loss-of-function pathogenesis. Since the aberration due to missense gene variations in many cases, as discussed above for PKU and MCAD and SCAD deciencies may result in folded protein with residual function, a strategy that eliminates folding intermediates 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 benecial for all protein misfolding disorders, notwithstanding their pathogenic mechanism. Enhancement of protein function The last intervention strategy to be discussed briey is induction of naturally occurring chaperones as well as introduction of chemical and pharmacological chaperones.

Increased elimination of aggregation-prone proteins Since specic 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 specic amino acids in certain proteins. Another way is to stimulate the elimination of the accumulated aberrant protein, either by induction of specic degradation pathways or intracellular proteases, or by introducing antibodies to neutralize the gain-of-function effects. Although some target points for stimulating degradation 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 nonspecic mechanisms may have many unexpected consequences. A more feasible way is to introduce specic antibodies, either specically designed to the particular disease and to aggregation-prone proteins (Miller and Messer 2005) or targeted to the general -sheeted structures, 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 elicitation of immune responses, before human treatment can be effective and safe.
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trials, though without any effects in the preliminary study (Teckman 2004). Although it may be worth screening for small-molecule inducers of specic 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 specic proteins (Bernier et al 2004; Desnick 2004; Fan 2003; Perlmutter 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 deciencies of these enzymes represent classical inborn errors of metabolism, and treatment with pharmacological chaperones has advanced to the level of clinical trials for some of them (Desnick 2004; Fan 2003). As discussed in the specic section on Gaucher disease, the -glucosidase related to this disease, as well as all lysosomal enzyme proteins, are nuclear-encoded and are cotranslationally translocated into ER, where they are folded and made transport-competent with the assistance of ERspecic 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 attainment 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 mitochondria, 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- stabilizing polycyclic ionizable compounds (Foster et al 1999). No examples are known of mitochondrially processed proteins, 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.

This strategy will full the purpose of enhancing the protein function and alleviating the accumulation of aberrant proteins. As discussed previously in connection with the specic 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 network 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 observation that protein aggregates contain a variety of chaperones, have fostered the idea that enhancement of components in the chaperone network may be a benecial treatment strategy. This can of course be achieved by gene therapy means, as mentioned above in connection with induction of components of the degradative pathways. However, it is probably more promising to induce chaperone expression with smallmolecule regulators of the heat shock response (Westerheide and Morimoto 2005). A large number of such compounds have been identied either by candidate approaches or by high-throughput screening, among them protein synthesis inhibitors, proteasome inhibitors, inammatory mediators, sodium salicylate and avonoids (Westerheide and Morimoto 2005). Although the enhancement of chaperone levels by induction of the heat shock response at a rst glance seems promising, not all of the wide range of cellular factors involved in the heat shock response (Trinklein et al 2004) may be benecial. 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 chaperoning 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 cellular systems to enhance the recovery of active proteins from misfolding. Since phenylbutyric acid (PBA) has been shown to stimulate the excretion of variant -1-antitrypsin in a cell model (Burrows et al 2000) as well as CFTR delta-Phe508 protein trafcking and expression on the cell surface of patients cells (Rubenstein et al 1997), it has often been counted among the chemical chaperones. However, PBA is a histone deacetylase inhibitor and although a global analysis has not been performed it may inuence the expression of a number 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 -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

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 molecular genetics, molecular pathogenesis, cellular pathology and clinical management of many diseases. At the molecular genetic level we have realized that it is important to distinguish
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Beutler E, Grabowski GA (2001) Gaucher disease. In: Scriver CR, Beaudet al, Sly WS, Valle D, eds; Childs B, Kinzler KW, Vogelstein B, assoc, eds. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGraw-Hill, 36353668. Bruijn LI, Miller TM, Cleveland DW (2004) Unraveling the mechanisms involved in motor neuron degeneration in ALS. Annu Rev Neurosci 27: 723749. Brusilow SW, Horwich AL (2001) Urea cycle enzymes. In: Scriver CR, Beaudet al, Sly WS, Valle D, eds; Childs B, Kinzler KW, Vogelstein B, assoc, eds. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGraw-Hill, 19091963. Bucciantini M, Calloni G, Chiti F, et al (2004) Prebrillar amyloid protein aggregates share common features of cytotoxicity. J Biol Chem 279: 3137431382. Burch M, Blair E (1999) The inheritance of hypertrophic cardiomyopathy. Pediatr Cardiol 20: 313316. Burrows JA, Willis LK, Perlmutter DH (2000) Chemical chaperones mediate increased secretion of mutant alpha 1-antitrypsin (alpha 1-AT) Z: a potential pharmacological strategy for prevention of liver injury and emphysema in alpha 1-AT deciency. Proc Natl Acad Sci USA 97: 17961801. Buttereld DA, Kanski J (2001) Brain protein oxidation in age-related neurodegenerative disorders that are associated with aggregated proteins. Mech Ageing Dev 122: 945962. Calloni G, Zoffoli S, Stefani M, Dobson CM, Chiti F (2005) Investigating the effects of mutations on protein aggregation in the cell. J Biol Chem 280: 1060710613. Carrell RW, Lomas DA (2002) Alpha1-antitrypsin deciencya model for conformational diseases. N Engl J Med 346: 4553. Casari G, De Fusco M, Ciarmatori S, et al (1998) Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin, a nuclear-encoded mitochondrial metalloprotease. Cell 93: 973 983. Cashikar AG, Duennwald M, Lindquist SL (2005) A chaperone pathway in protein disaggregation. Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hsp104. J Biol Chem 280: 2386923875. Caughey B, Lansbury PT (2003) Protobrils, pores, brils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Annu Rev Neurosci 26: 267298. Chiti F, Stefani M, Taddei N, Ramponi G, Dobson CM (2003) Rationalization of the effects of mutations on peptide and protein aggregation rates. Nature 424: 805808. Christensen JH, Siggaard C, Corydon TJ, et al (2004) Impaired trafcking of mutated AVP prohormone in cells expressing rare disease genes causing autosomal dominant familial neurohypophyseal diabetes insipidus. Clin Endocrinol (Oxf) 60: 125136. Clarkson E, Costa CF, Machesky LM (2004) Congenital myopathies: diseases of the actin cytoskeleton. J Pathol 204: 407417. Cookson MR (2005) The biochemistry of Parkinsons disease. Annu Rev Biochem 74: 2952. Cornett J, Cao F, Wang CE, et al (2005) Polyglutamine expansion of huntingtin impairs its nuclear export. Nature Genetics 37: 198 204. Corydon MJ, Vockley J, Rinaldo P, et al (2001) Role of common gene variations in the molecular pathogenesis of short-chain acyl-CoA dehydrogenase deciency. Pediatr Res 49: 1823. Costa CF, Rommelaere H, Waterschoot D, et al (2004) Myopathy mutations in alpha-skeletal-muscle actin cause a range of molecular defects. J Cell Sci 117: 33673377. Dalle-Donne I, Giustarini D, Colombo R, Rossi R, Milzani A (2003) Protein carbonylation in human diseases. Trends Mol Med 9: 169 176. Desnick RJ (2004) Enzyme replacement and enhancement therapies for lysosomal diseases. J Inherit Metab Dis 27: 385410.

between the various types of gene sequence variations associated with disease. Many splice variations as well as most out-of-frame deletions and insertions result in total loss-offunction through nonsense-mediated decay of the variant mRNA (Maquat 2005). In contrast, missense and small inframe deletions and insertions may lead to aberrant proteins, which may vary in residual function dependent on the specic protein and its aberration as well as the cellular and environmental 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 primarily guided by their physicochemical properties, such as hydrophobicity and amino acid charges, rather than their specic functional properties. This means that the efciency 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 mixture of loss of protein function, which is disease-specic, and gain of function, which may also be specic 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, particularly exemplied by the use of pharmacological chaperones in current and especially in future treatment of the lysosomal diseases. In conclusion, it will be interesting to experience how this emerging paradigm inuences 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 concerning 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 Research Initiative; Karen Elise Jensen Foundation; Lundbeck Foundation; Novo Nordisk Foundation; and the European Union (6th framework programme).

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