EMBO
`reports
`
`review
`Getting a grip on non-native proteins
`
`review
`
`Peter C. Stirling*, Victor F. Lundin* & Michel R. Leroux+
`Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada
`
`It is an underappreciated fact that non-native polypeptides are
`prevalent in the cellular environment. Native proteins have
`the folded structure, assembled state and cellular localization
`required for activity. By contrast, non-native proteins lack func-
`tion and are particularly prone to aggregation because hydropho-
`bic residues that are normally buried are exposed on their
`surfaces. These unstable entities include polypeptides that are
`undergoing synthesis, transport to and translocation across mem-
`branes, and those that are unfolded before degradation. Non-
`native proteins are normal, biologically relevant components of a
`healthy cell, except in cases in which their misfolding results from
`disease-causing mutations or adverse extrinsic factors. Here, we
`explore the nature and occurrence of non-native proteins, and
`describe the diverse families of molecular chaperones and coor-
`dinated cellular responses that have evolved to prevent their mis-
`folding and aggregation, thereby maintaining quality control over
`these potentially damaging protein species.
`EMBO reports 4, 565–570 (2003)
`doi:10.1038/sj.embor.embor869
`
`Protein folding and the non-native protein
`All proteins are synthesized as linear polypeptide chains that are
`gradually extruded from the ribosome. To become functional, these
`nascent proteins must shield their exposed hydrophobic residues and
`adopt a precise tertiary structure. It has long been known that primary
`amino-acid sequences dictate the tertiary structures of proteins, but
`how folding into the native state occurs is still the subject of intense
`investigation. The folding process is now seen as the downward path
`that an unstructured polypeptide takes on a funnel-like free-energy
`surface representing the steadily decreasing number of conforma-
`tions available to it as it reaches its native state (Dinner et al., 2000).
`For small proteins, such as the engrailed homeodomain protein
`(7.5 kDa), folding is thought to begin with a few native-like contacts
`that, once formed, promote a rapid transition to the native state
`(Fig. 1A). In this two-step model, there are no long-lived intermedi-
`ate states, and the secondary and tertiary structures form virtually
`simultaneously (Daggett & Fersht, 2003).
`
`Department of Molecular Biology and Biochemistry, Simon Fraser University,
`8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6
`*These authors contributed equally to this work
`+Corresponding author. Tel: +1 604 268 6683; Fax: +1 604 291 5583;
`E-mail: leroux@sfu.ca
`
`Received 25 March 2003; accepted 24 April 2003
`
`The folding of larger proteins, such as lysozyme (Fig. 1B), is
`more complex, and usually involves transition states and inter-
`mediates that are represented as peaks and valleys on the energy
`landscape. One theory is that on the initiation of folding, sec-
`ondary-structure elements form autonomously and collide to
`produce the tertiary structure (Daggett & Fersht, 2003). De novo,
`co-translational protein-folding in the cell probably follows this
`type of pathway (Hartl & Hayer-Hartl, 2002). Another hypothesis
`is that the propensity of hydrophobic residues to associate stimu-
`lates the collapse of a protein into a ‘molten-globule’-like, com-
`pact state that has some non-native contacts. In this scenario, the
`folding bottleneck for proteins is the reorganization of such
`incorrect associations. A synergistic view of these two mecha-
`nisms, called nucleation-collapse, probably explains the folding
`of most proteins in vitro (Daggett & Fersht, 2003).
`Folding intermediates, and non-native protein species in general,
`are usually aggregation-prone, both in vitro and in the crowded cel-
`lular environment. Therefore, in vivo, they must be stabilized and
`ushered towards their appropriate fate, be it biogenesis (folding,
`assembly and transport), degradation, or sequestration into aggregat-
`ed forms if they cannot reach their native state or be disposed of.
`
`Cellular functions of molecular chaperones
`A diverse and ubiquitous class of proteins, known as molecular
`chaperones, has evolved to transiently stabilize exposed hydro-
`phobic residues in non-native proteins. Cellular functions for
`chaperones include assisting in biogenesis, modulation of pro-
`tein conformation and activity, disaggregation and refolding of
`proteins after cellular stress and, perhaps unexpectedly, the dis-
`assembly and unfolding of proteins for subsequent degradation
`(Leroux & Hartl, 2000a). Although they are not historically classi-
`fied as chaperones, proteins that catalyse the cis–trans isomer-
`ization of proline residues (peptidyl-prolyl isomerases) and the
`proper formation of disulphide bonds (protein disulphide iso-
`merases) often bind and stabilize non-native proteins (Leroux,
`2001). We now consider some of the best-characterized chaper-
`ones, which we classify into three broad functional categories:
`holding, folding and unfolding.
`
`Holding. Several molecular chaperones seem to have little more
`than a stabilizing effect on non-native proteins, and usually require
`the participation of other chaperones to assist with, for example,
`folding. These chaperones typically lack the ability to undergo
`ATP-dependent conformational changes.
`
`©2003 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION
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`review
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`Non-native proteins
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`
`For example, heat-shock protein 40 (Hsp40) can prevent
`protein aggregation in vitro, but requires the ATP-hydrolysing
`chaperone Hsp70 to fold substrates (Mayer et al., 2000).
`Eukaryotic prefoldin, a hetero-hexameric chaperone complex,
`interacts with nascent actin and tubulin chains and assists in their
`biogenesis by docking onto and delivering substrates to the chap-
`eronin-containing TCP1 (CCT), which is a cylindrical ‘folding
`machine’ (Martin-Benito et al., 2002). Archaeal prefoldin interacts
`indiscriminately with non-native proteins in vitro, suggesting that it
`boasts a wider range of substrates than its eukaryotic counterpart
`(Siegert et al., 2000). Hsp90 is an interesting case of a highly abun-
`dant chaperone that requires the cooperation of Hsp70 and other
`cofactors to facilitate conformational switching between active
`
`A
`
`B
`
`Unfolded
`En-HD
`
`Very fast
`
`20%
`
`70%
`
`Unfolded
`lysozyme
`
`Very fast
`
`α-intermediate
`α-intermediate
`
`Slow
`
`Very fast
`
`Very fast
`
`αβ-intermediate
`
`Native
`
`Native
`
`Fig. 1 | Folding pathways of engrailed homeodomain and egg-white lysozyme.
`(A) The engrailed homeodomain (En-HD) folds rapidly, in nanoseconds to
`microseconds (Mayor et al., 2003). (B) Lysozyme has two significantly
`populated intermediates and folds more slowly, with a timescale of milliseconds
`in vitro. The majority (70%) of the lysozyme protein population folds relatively
`quickly into the α-domain intermediate, but is slow to reach the near-native,
`short-lived αβ-intermediate. Another 20% rapidly forms the αβ-intermediate
`directly. The α-domain is shown in red, and the β-domain in yellow.
`Reproduced in part, with permission from Dinner et al., 2000.
`
`and inactive client proteins despite its ability to hydrolyse ATP (Pearl
`& Prodromou, 2002). Small heat-shock proteins (sHsps) belong to
`a diverse family of protein complexes that trap denatured proteins
`on their surfaces (van Montfort et al., 2001). It is controversial as to
`whether sHsps can use ATP to directly assist the refolding of their
`substrates, or whether they are strictly reliant on ATP-dependent
`chaperones, such as Hsp70 or chaperonins, to perform this task
`(Leroux, 2001). SecB, a bacterial chaperone, recognizes and main-
`tains newly synthesized precursor proteins in forms that are com-
`petent for translocation by SecA, a chaperone ATPase that threads
`proteins into the SecYEG translocon channel (Xu et al., 2000). Last,
`chaperones such as the periplasmic protein PapD can stabilize
`non-native proteins—in this case, unassembled pilus subunits—by
`transiently ‘donating’ a structural element that is lacking in the
`substrate’s tertiary structure, but that is present (complemented) in
`the assembled quaternary form (Sauer et al., 2002).
`
`Folding. Chaperones that assist in protein folding often couple a
`holding (or capturing) function with the ability to release the
`folded substrate in an ATP-dependent manner. Hsp70 has a mul-
`titude of functions, two of which are stabilizing nascent polypep-
`tides and promoting the folding of non-native proteins through
`rounds of ATP-dependent binding and release (Mayer et al.,
`2000). In bacteria, the function of the Hsp70 homologue, DnaK,
`partially overlaps with that of Trigger Factor (TF), a chaperone
`and prolyl isomerase that is located near the ribosomal polypep-
`tide exit tunnel. The chaperonin (Hsp60) family of chaperones
`assists the folding of newly translated proteins by sequestering
`aggregation-prone intermediates in a hydrophilic cavity and
`releasing them after ATP hydrolysis. The bacterial GroEL and
`eukaryotic cytosolic CCT chaperonins interact with a range
`of substrates, although the latter probably have specialized
`functions in folding actin and tubulin (Leroux & Hartl, 2000b).
`An emerging concept is that chaperones cooperate extensive-
`ly, sometimes forming multi-chaperone systems that work
`sequentially or simultaneously to ensure the efficient biogenesis
`of cellular proteins in their respective cellular compartments
`(Leroux & Hartl, 2000a). For example, sequential interactions
`with cytosolic Hsp70, prefoldin and CCT are probably needed to
`assist actin and tubulin folding; at least in the case of actin, pre-
`foldin and CCT also cooperate closely to help it reach its native
`state (Siegers et al., 1999; Martin-Benito et al., 2002). In mito-
`chondria, several newly imported substrates have been shown to
`interact first with Hsp70, and then with Hsp60, for productive
`folding (Manning-Krieg et al., 1991). As a final example, several
`endoplasmic reticulum (ER) chaperones, such as calnexin,
`calreticulin, Hsp70 and Hsp90 homologues, Erp57 and UDP-
`glucose:glycoprotein
`transferase
`(UGGT) work
`together or
`successively to ensure the correct biogenesis of glycosylated
`proteins (Parodi, 2000).
`Some pre-proteins contain sequences that fulfil the criteria for a
`chaperone, as their cleaved pre-domains assist folding without
`being part of the final structure. The pro-peptide of the protease
`subtilisin E encodes such an intramolecular chaperone (Shinde
`et al., 1993). An autotransporter such as BrkA provides a second
`example of an intramolecular chaperone. This bacterial protein
`contains a β-domain that forms a β-barrel channel, which is
`required for the proper biogenesis (secretion) of its α-domain and is
`subsequently removed by proteolytic cleavage (Oliver et al., 2003).
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`Non-native proteins
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`
`review
`
`Clamps
`
`L
`
`A
`
`DnaK peptide
`
`B
`
`Calnexin
`
`Cavities
`
`C
`
`CCT–actin
`
`D
`
`CCT–tubulin
`
`E
`
`HslU–HsIV
`
`F
`
`Prefoldin
`
`G
`
`Prefoldin–actin
`
`Surfaces
`
`H
`
`SecB
`
`I
`
`Cofactor A
`
`J
`
`Small HSP 16.5
`
`Structural complementation
`
`K
`
`PapD–PapK
`
`Unfolding. Chaperones of the Hsp100/Clp/AAA (ATPase associated
`with various cellular activities) ATPase family are ubiquitous ring
`structures that mediate protein unfolding and disassembly. Their
`activities depend on interactions with several regions within their
`substrates and on the conversion of the energy gained from ATP
`hydrolysis into conformational changes that exert a molecular
`‘crowbar’ effect (Horwich et al., 1999; Leroux, 2001). Most AAA
`ATPases typically cooperate with other chaperones for folding or
`with proteases for degradation. Yeast Hsp104 and bacterial ClpB,
`for example, can disassemble aggregated proteins and thus allow
`their reactivation in conjunction with an Hsp70/DnaK chaperone.
`When bound to the ends of proteases such as the eukaryotic and
`archaeal proteasomes, and the structurally related bacterial HslV,
`the respective AAA ATPase chaperones (Rpt subunits, PAN
`and HslU) promote the unfolding of their substrates and their
`subsequent threading into the proteolytic chamber (Leroux, 2001).
`
`Fig. 2 | Chaperone structures, grouped according to how they interact with their
`non-native substrates. (A) The carboxy-terminal substrate-binding domain of
`Escherichia coli DnaK, comprising a β-sheet cradle and an α-helical lid, bound to a
`peptide (red; NRLLLTG). (B) Calnexin has a globular lectin domain (yellow) and
`an extended arm domain (gold). (C,D) Cryoelectron microscopy (cryo-EM)
`reconstructions of the chaperonin CCT (blue) in complex with (C) actin and
`(D) tubulin (both in red). (E) HslU AAA ATPase (blue) shown on top of the HslV
`protease (grey). (F) Archaeal prefoldin (α-subunits shown in yellow; β-subunits
`shown in gold). (G) Cryo-EM reconstruction of eukaryotic prefoldin (gold) in
`complex with non-native actin (red). (H) SecB dimer of dimers (the different
`colours indicate monomers), with the putative substrate-binding groove shown in
`red. (I) A cofactor-A dimer (yellow and grey) with conserved putative binding
`residues (red). (J) A small heat-shock protein (Hsp16.5) from Methanococcus
`jannaschii forms a spherical oligomer; each dimer building-block is shown in a
`different colour. (K) The periplasmic chaperone PapD (grey) supplies a β-strand
`(red) to the incomplete PapK immunoglobulin fold (yellow). Structures are not
`shown to scale. The Protein Database identification numbers for these proteins are
`as follows: DnaK–peptide, 1DKX; calnexin, 1JHN; HslUV, 1G3I; prefoldin,
`1FXK; SecB, 1FX3; cofactor A, 1QSD; sHsp, 1SHS; PapD-K, 1PDK.
`
`Molecular strategies for binding non-native proteins
`The strategies used by molecular chaperones to stabilize
`non-native proteins, which have been elucidated mainly by X-ray
`crystallography and cryoelectron microscopy (cryo-EM) studies,
`can be grouped broadly into one of four classes: the use
`of clamps, cavities, specialized surfaces, and structural
`complementation.
`
`Clamps. Hsp70 contains an amino-terminal ATPase domain (45
`kDa) that has an actin-like fold and is connected by a short linker
`region to a carboxy-terminal polypeptide-binding domain (25
`kDa). The latter resembles a clamp, or jaw, which is formed by a
`β-sheet cradle and a flexible α-helical lid (Fig. 2A). The open,
`ATP-bound conformation of Hsp70 accepts a peptide segment
`that is enriched in hydrophobic residues, and ATP hydrolysis,
`which is induced by a cofactor (for example, Hsp40), locks the
`substrate in place. For DnaK, three basic features govern its inter-
`action with substrates: hydrogen bonding to the substrate back-
`bone, a hydrophobic pocket that interacts with side-chains, and
`an arch that sterically restricts bound polypeptides (Mayer et al.,
`2000). Release of the substrate is enabled by exchanging ADP for
`ATP through the action of a nucleotide-exchange cofactor, such
`as GrpE or Bag1 (Leroux & Hartl, 2000a). Like Hsp70, Hsp90
`undergoes conformational changes associated with its ATPase
`activity. In this case, the C-terminal domain mediates dimeriza-
`tion and, on ATP binding, the N-terminal domains of the two
`Hsp90 proteins also associate, forming a closed, molecular-
`clamp-like structure that probably confines the substrate (Meyer
`et al., 2003).
`Proteins that undergo maturation in the ER are stabilized by
`chaperones such as calnexin and calreticulin, which are struc-
`turally related and recognize glycosylated, non-native proteins
`through lectin-like domains. The striking tadpole-like structure of
`calnexin (Fig. 2B) suggests that it also uses a clamp strategy, sta-
`bilizing non-native proteins through its globular lectin domain
`and recruiting the disulphide-isomerase/chaperone, Erp57, with
`its extended arm (Leach et al., 2002).
`
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`review
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`Non-native proteins
`P.C. Stirling et al.
`
`Cavities. Chaperonins consist of two stacked, oligomeric rings that
`form chambers used to sequester non-native proteins and assist in
`their folding (Horwich et al., 1999). Individual subunits are composed
`of a substrate-binding (apical) domain, which lines the opening of the
`cavity and includes essential hydrophobic residues (mostly leucines,
`valines and tyrosines), an intermediate domain, and an equatorial
`ATPase domain. The multivalent binding of substrates to the homo-
`oligomeric bacterial protein, GroEL, was shown elegantly by sequen-
`tially mutating the substrate-binding sites of a single polypeptide that
`encodes a complete heptameric ring (Farr et al., 2000). Unlike GroEL,
`the eukaryotic chaperonin CCT has eight unique binding sites per
`hetero-oligomeric ring that are less hydrophobic in character. These
`probably evolved to bind a wide spectrum of substrates while retain-
`ing some specificity; consistent with these results, cryo-EM images
`show that actin and tubulin interact with two or more specific subunits
`of CCT (Fig. 2C,D; Leroux & Hartl, 2000b; Llorca et al., 2000). During
`chaperonin-assisted folding cycles, the substrates of GroEL and CCT
`are encapsulated by a cofactor (GroES) and an iris-like structure
`contained within CCT itself, respectively (Hartl & Hayer-Hartl, 2002).
`AAA ATPases form large, hexameric toroids, which probably
`also bind substrates in a multivalent manner. The exact position
`and nature of their binding sites is poorly understood, but they are
`probably adapted to the specific functions of the AAA ATPases.
`The function of these chaperones in unfolding or disassembly
`seems to involve coupling substrate interactions with large,
`nucleotide-dependent conformational changes (Horwich et al.,
`1999). As shown for the bacterial HslUV chaperone–protease
`complex (Fig. 2E), many AAA ATPases associate coaxially with
`proteases, and are poised to untangle and unfold polypeptides
`and thread them into the central proteolytic chamber.
`Prefoldin also forms a cavity and binds substrates multivalently, but
`in a unique way. The crystal structure of archaeal prefoldin shows a
`hexamer with coiled-coil ‘tentacles’ protruding from a double
`β-barrel. Truncation studies show that the combined contributions of
`its coiled coils are required to stabilize non-native proteins (Siegert
`et al., 2000; Fig. 2F). Consistent with this observation, a recent cryo-
`EM reconstruction of eukaryotic prefoldin complexed with non-native
`actin reveals several contacts near the tips of the tentacles (Martin-
`Benito et al., 2002; Fig. 2G). As the cavity of archaeal prefoldin is pre-
`dominantly polar in character, it has been suggested that the distal
`coiled-coil regions may partially unwind, exposing the inter-helical
`hydrophobic residues and creating an indiscriminate binding site for
`unfolded proteins (Siegert et al., 2000).
`
`Surfaces. To prevent inappropriate interactions between non-native
`proteins and components of the bulk cytosol, many chaperones
`use a relatively flat or corrugated surface to bind exposed, unstable
`polypeptide regions. Examples are the general chaperones TF, Hsp40
`and SecB, and cofactor A, one of several tubulin-specific chaperones.
`The crystal structure of bacterial SecB (17 kDa) shows a dimer of
`dimers that has two hydrophobic grooves on opposite faces of the
`molecule (Fig. 2H). It has been suggested that a linear polypeptide can
`wrap around a SecB tetramer, contacting both grooves simultaneously
`(Randall & Hardy, 2002). Hsp40 is a U-shaped homodimeric chaper-
`one that stabilizes non-native substrates before transferring them to
`Hsp70. It probably binds its substrates at hydrophobic depressions on
`the distal tips of its two peptide-binding domains (Lee et al., 2002). TF
`also contains a conserved hydrophobic pocket, which is the putative
`substrate-binding site of its peptidyl-prolyl isomerase domain; the
`
`preponderance of phenylalanine residues in this region confers a
`binding specificity that favours aromatic residues (Patzelt et al., 2001).
`In contrast to these other chaperones, cofactor A, which is dimeric in
`yeast (Steinbacher, 1999; Fig. 2I), but apparently monomeric in
`humans (Guasch et al., 2002), stabilizes its partially folded β-tubulin
`substrate on a primarily hydrophilic surface.
`A unique mechanism for stabilizing non-native proteins is used by
`sHsps. Methanococcus jannaschii Hsp16.5 is a spherical complex
`that is assembled from 12 dimeric building blocks (Fig. 2J; Kim et al.,
`1998). van Montfort and colleagues (2001) found that subunits of a
`wheat sHsp (Hsp16.9) form a smaller, cylindrical, dodecameric com-
`plex. They suggested, from the structure and from other studies, that
`sub-assembled species (dimers) dissociate to partition their exposed
`hydrophobic residues between substrate binding and higher-order
`assembly. On oligomerization, it is likely that non-native proteins are
`not contained within the cavity of the less ordered complexes, but are
`instead held on the outside surface, awaiting refolding by other
`chaperone systems.
`
`Structural complementation. Structural complementation is an excep-
`tional case, in which a chaperone contributes specific structural infor-
`mation to stabilize a non-native susbtrate. The interaction between
`Pap pilus subunits and the periplasmic chaperone, PapD, from
`uropathogenic Escherichia coli illustrates the use of this strategy. The
`pilus subunit contains an immunoglobulin fold that lacks a
`β-strand, which, in the assembled pilus structure, is provided by the
`neighbouring pilus subunit. Before assembly, PapD complements,
`and thus stabilizes, a pilus subunit by donating a β-strand in an analo-
`gous manner (Fig. 2K; Sauer et al., 2002).
`
`Cellular quality control
`Physical or chemical stresses that are brought about by temperature
`changes, exposure to proteotoxic agents, or other conditions that are
`conducive to protein misfolding induce a ubiquitous, protective, cel-
`lular stress response. Crucial to this response is an increase in chaper-
`one and proteolytic activities, aimed at reducing the presence of
`damaging non-native protein species. In eukaryotes, the heat-shock
`transcription factor (HSF) regulates the expression of stress-inducible
`genes, including all of the well-characterized chaperones (Leroux &
`Hartl, 2000a).
`As a first line of defence against damaging cellular insults, several
`chaperones, including sHsps and Hsp70s, can stabilize proteins
`undergoing denaturation and allow their refolding when the stress has
`subsided. Severe stresses, however, may overwhelm the ability of
`chaperones to stabilize large pools of non-native proteins, resulting in
`protein aggregation. Renaturation of these insoluble proteins by mem-
`bers of the Hsp100/Clp family, in conjunction with the Hsp70 system,
`has been shown (Glover & Lindquist, 1998).
`The ER also has its own coordinated unfolded-protein response
`(UPR). The accumulation of misfolded proteins in this compartment
`results in the upregulated transcription of numerous quality-control
`genes, including BiP and Grp94, the ER homologues of Hsp70 and
`Hsp90 (Hampton, 2000). Interestingly, the sensing mechanism of the
`UPR relies on the reversible interaction of BiP with the stress-signal-
`transducing protein IRE1. BiP interacts with and inactivates IRE1 only
`in the absence of stress, when it is not engaged with misfolded pro-
`teins. Similarly, HSF activity is modulated by at least one chaperone
`(Hsp90) that is able to detect the presence of misfolded proteins
`(Leroux & Hartl, 2000a).
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`Non-native proteins
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`Aggresome
`
`Nucleus
`
`Fig. 3 | An aggresome formed in HeLa cells by overexpression of a green-
`fluorescent-protein fusion to the polyglutamine region of huntingtin (green),
`the protein involved in the neurodegenerative disorder, Huntington’s disease.
`The nucleus is shown in blue, and the microtubules in red.
`
`Perhaps surprisingly, not all cellular strategies for maintaining pro-
`tein stability involve protein chaperones. Chemical chaperones, such
`as the disaccharide trehalose, help to stabilize non-native proteins,
`and are overproduced on heat-shock (Singer & Lindquist, 1998).
`Proteins that cannot fold properly because of mutations, errors in
`translation, faulty biogenesis or damage are normally degraded. It is
`estimated that ~30% of all nascent proteins fail to fold and are
`destroyed by the ubiquitin–proteasome system, and under stress con-
`ditions, the need for degradation increases greatly (Schubert et al.,
`2000). An emerging paradigm is that of chaperone systems cooperat-
`ing with the protein degradation machinery to ensure quality control.
`The UPR exemplifies this integration because it also regulates ER-
`associated degradation (ERAD), a process that involves the retrograde
`transport of misfolded ER proteins into the cytosol for destruction by
`the proteasome (Hampton, 2000). Mammalian cytosolic Hsp70, for
`example, uses co-chaperones such as Bag1 and CHIP to assist with the
`capture and sorting of non-native proteins that are destined for folding
`or proteasomal degradation (Höhfeld et al., 2001).
`
`Protein misfolding diseases
`Most protein misfolding diseases can be directly attributed to
`mutations in the affected proteins. As might be expected, however,
`mutations in chaperones can also lead to improper biogenesis or
`failure to stabilize particular protein substrates, and have been
`linked to these diseases. Two examples illustrate this point: the
`sHsp-encoding α-crystallin gene, when modified by a point muta-
`tion that results in an R120G amino-acid change, can lead to cata-
`racts, desmin-related myopathy or the neurodegenerative disorder,
`Alexander’s disease (Clark & Muchowski, 2000); alterations in the
`BBS6 protein, which shows weak homology to the chaperonin
`CCT, cause the symptoms seen in some patients with Bardet–Biedl
`syndrome, including blindness, obesity and kidney dysfunction
`(Katsanis et al., 2000).
`
`review
`
`In many cases, misfolded proteins associated with disease are
`sequestered in the form of fibrils or amorphous aggregates.
`Several of these conditions are collectively called amyloidoses
`because of the characteristic formation of large fibrils, known as
`amyloids. A subset of misfolding diseases that cause neurodegen-
`erative disorders, including Huntington’s disease, result from the
`expansion of CAG nucleotide repeats. The severity and onset of
`these diseases correlates with the length of the aggregation-
`prone polyglutamine regions (Taylor et al., 2002). Prion disor-
`ders, such as Creutzfeldt–Jakob disease, are unique in that the
`difference between the disease-causing (PrPSc) and normal (PrPC)
`proteins is strictly conformational; misfolded PrPSc induces PrPC
`to change its conformation and polymerize on the growing fibril
`(Taylor et al., 2002). Interestingly, studies of yeast prion-like pro-
`teins, such as Sup35, have led to the intriguing possibility that
`chaperones could influence the structural permutations of the
`prion protein (Chernoff et al., 1995).
`In protein misfolding disorders, or under severe stress, the
`machinery needed to fold or degrade a non-native protein may
`become saturated. Researchers are familiar with inclusion bod-
`ies, which are formed in response to the overexpression of some
`proteins in bacteria. In eukaryotic cells, a dynamic apparatus that
`involves microtubules and motor proteins assists the transport of
`at least some misfolded polypeptides to the centrosome, forming
`a cellular compartment known as the ‘aggresome’. This is the
`fate, for example, of a large proportion of the slow-folding cystic
`fibrosis transmembrane regulator (CFTR) protein, especially
`mutants that contain the ∆508 deletion found in most patients
`(Johnston et al., 1998). As shown in Fig. 3, a green fluorescent
`protein (GFP) fused to exon 1 of the huntingtin protein, which
`contains the long stretch of polyglutamines that is associated with
`Huntington’s disease, forms a typical perinuclear aggresome
`(Taylor et al., 2002).
`Recent findings have led to the suggestion that protein aggregates
`may sequester chaperones, proteasomes and other proteins, prevent-
`ing their normal function and thereby leading to the observed
`pathologies. Artificially increasing the levels of some chaperones has,
`in some cases, been found to provide a protective effect, and could,
`therefore, be of possible therapeutic value (Muchowski, 2002).
`
`Perspectives
`Ensuring that non-native polypeptides are directed towards their
`appropriate cellular fates, for example biogenesis or degradation,
`involves normal cellular activities that are carried out by numerous
`proteins involved in quality control. However, the number of dif-
`ferent molecular chaperones and protein-degradation-associated
`components that mediate such housekeeping functions may be
`significantly greater than is known at present. This is supported by
`the recent finding that erythroid cells have a chaperone that is
`specifically involved in stabilizing α-haemoglobin, a protein that
`has been studied for more than a century, before its assembly into a
`functional α
`β
`2-heterotetramer (Kihm et al., 2002). At a cellular
`2
`level, the possibility that unique responses tailored to deal with
`non-native proteins remain to be identified is highlighted by the
`recent discovery of a mitochondrial-specific stress response (Zhao
`et al., 2002). Clearly, the many roles of chaperones and other qual-
`ity control elements in fundamental cellular activities, as well as in
`human diseases, emphasize the need for a greater understanding
`of processes involving non-native proteins.
`
`©2003 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION
`
`EMBO reports VOL 4 | NO 6 | 2003
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`review
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`Non-native proteins
`P.C. Stirling et al.
`
`ACKNOWLEDGEMENTS
`We thank V. Daggett, A. Dinner and J. Valpuesta for providing materials to
`prepare figures, and apologize to those colleagues whose work was not cited
`due to space restrictions. We acknowledge the Canadian Institutes of Health
`Research (CIHR) and the National Cancer Institute of Canada for financial
`support. M.R.L. is the recipient of Michael Smith Foundation for Health
`Research and CIHR scholar awards, and P.C.S. holds an Natural Sciences and
`Engineering Research Council of Canada scholarship.
`
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