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Review R9

Protein aggregation: folding aggregates, inclusion bodies and Anthony L Fink

Aggregation results in the formation of inclusion in vitro) are examples of ordered aggregates, whereas bodies, amyloid fibrils and folding aggregates. inclusion bodies are examples of in vivo disordered aggre- Substantial data support the hypothesis that partially gates. Corresponding disordered in vitro aggregates are folded intermediates are key precursors to aggregates, those formed during the refolding of denaturant-unfolded that aggregation involves specific intermolecular at high protein concentrations, or under weakly interactions and that most aggregates involve βb sheets. native conditions at high protein concentration; these will be referred to as folding aggregates. Address: Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA. Native, folded may aggregate under certain con- E-mail: [email protected] ditions, most notably salting out and isoelectric precipita- Folding & Design 01 February 1998, 3:R9–R23 tion (when the net charge on the protein is zero). Such http://biomednet.com/elecref/13590278003R0009 precipitates of native protein are readily distinguished from pathological aggregates by their solubility in buffer © Current Biology Ltd ISSN 1359-0278 under native-like conditions. In contrast, pathological aggregates dissociate and dissolve only in the presence of Introduction high concentrations of denaturant or detergent. In my lab- Protein aggregation can be merely a nuisance factor in oratory, it has been shown that the native conformation is many in vitro studies of proteins or it can cause major retained in salting out precipitates (Figure 1). economic and technical problems in the biotechnology and pharmaceutical industries. Its effects can be lethal in Protein aggregation has usually been assumed to involve patients who suffer from a variety of diseases involving either unfolded or native states. Inclusion body formation protein aggregation, such as the amyloidoses, dis- and other aggregates formed during protein folding have eases and other protein deposition disorders [1,2]. This been assumed to arise from hydrophobic aggregation of review focuses on the basic mechanism(s) of protein aggre- the unfolded or denatured states, whereas amyloid fibrils gation, the factors that determine whether it will occur, and other extracellular aggregates have been assumed to and the conformation of the protein molecules in the arise from native-like conformations in a process analo- aggregate. Protein aggregation is intimately tied to protein gous to the polymerization of hemoglobin S [8]. Recent folding and stability, and also, in the cell, to molecular observations suggest that aggregation is much more likely chaperones. The prevalence of protein aggregation is to arise from specific partially folded intermediates, probably much higher than generally realized — it is often however. An important consequence of this is that aggre- ignored or worked around, and in protein folding experi- gation will be favored by factors and conditions that favor ments its presence may not even be realized [3]. The population of these intermediates, and hence it is the growing recognition of the critical importance of protein properties of these intermediates that are important in aggregation has resulted in a number of reviews [4–10]. determining whether aggregation occurs. Furthermore, the characteristics and properties of the intermediates may Unless specifically noted to the contrary, in this review be significantly different from those of the native (and the term aggregation will apply to aggregated protein unfolded) conformation. involving the formation of insoluble precipitates that may be considered ‘pathological’ in nature. This is in contrast Several observations indicate that transient aggregation to the insolubility of the native state due to protein con- occurring during in vitro protein refolding may be mis- centrations exceeding the solubility limit (e.g. ‘salting taken for a transient intermediate [3]. Direct evidence for out’), or the intermolecular association involved in the for- the transient association of partially folded intermediates mation of native oligomers. It should be noted that in during refolding has been obtained in small-angle X-ray many such cases of pathological aggregation the initial scattering experiments of apomyoglobin [11,12], carbonic material formed may be soluble aggregates, but these anhydrase and phosphoglycerate kinase [13]. The experi- become insoluble when they exceed a certain size. ments show the rapid (milliseconds or less) formation of associated states that become monomeric on a slow time- It is convenient to classify protein aggregation according scale (typically seconds to minutes or longer). Another to the following categories: in vivo and in vitro, and approach indicative of transient aggregation is that involv- ordered and disordered. Amyloid fibrils (both in vivo and ing changes in the rate constants for refolding as a function R10 Folding & Design Vol 3 No 1

Figure 1 folding aggregates and amorphous deposits); how the envi- ronmental conditions affect the rate and the amount of aggregation; and how the aggregation may be prevented.

It seems likely from an evolutionary perspective that pro- teins have evolved to avoid sequences that result in a strong propensity to aggregate. It is also interesting to con- sider that many short peptide sequences containing several hydrophobic residues and a high tendency for β-sheet formation probably have a strong disposition to form aggregates and/or amyloid fibrils. It is only the flanking sequences, which are either quite polar and therefore increase the solubility limit or are sufficiently bulky to sterically prevent the required interactions, that result in

Absorbance the lack of aggregation and/or amyloid formation.

Problems due to protein aggregation Protein deposition diseases Several dozen protein deposition diseases are known. The most familiar include the amyloid diseases (amyloid- oses), such as Alzheimer’s disease, and the transmissible spongiform encephalopathies (TSEs; prion diseases such as bovine spongiform encephalopathy, BSE, or Mad Cow disease and Creutzfeldt–Jacob disease, CJD, in humans). In both amyloid and prion diseases the aggregated pro- 1700 1680 1660 1640 1620 1600 tein is usually in the form of ordered fibrils. Amyloid fibril Wavenumber (cm–1) formation has been observed to arise from both peptides and proteins. Several protein deposition diseases involve Native IL-2 non-ordered protein deposits; some examples are inclu- Ammonium sulfate precipitated IL-2 sion body myositis, light-chain deposition disease and Aggregated IL-2 Folding & Design cataracts. Many thousands of people die each year from protein deposition diseases [14]. New diseases are added Precipitates of native protein, in this case interleukin-2 (IL-2) formed by to this list every year, one of the latest being Huntington’s ‘salting out’ with ammonium sulfate, retain the native conformation. The disease [15]. figure shows the second derivatives of the FTIR spectra of the amide I region of native, ammonium sulfate precipitated IL-2 (dashed line) and, Inclusion bodies for comparison, aggregated (inclusion body) IL-2 (dotted line). IL-2 is an all-α protein, as indicated by the dominant band at 1654 cm–1 for Inclusion body formation is very common when proteins the native conformation. are overexpressed. This may facilitate their potential purification because inclusion bodies are usually highly homogeneous. The problem is that renaturation is fre- of protein concentration [3]. This study indicates that tran- quently difficult, as a result of aggregation. Several tech- sient aggregates can easily be mistaken for structured niques have been developed to help overcome the monomers and could be a general problem in time-resolved common problem of their re-aggregation during renatura- folding studies. Because aggregation is sensitive to protein tion [16] and some are discussed later. Inclusion bodies concentration, monitoring the kinetics as a function of and related insoluble non-ordered protein aggregates are concentration should reveal potential aggregation artifacts. also found in certain diseases.

Key questions relating to aggregation, many of which are Protein drugs not yet fully answered, include the following: the nature Protein aggregation is also a problem in a number of other of the species responsible for aggregation: the detailed aspects of biotechnology; for example, during storage or mechanism that leads to aggregation and the underlying delivery of protein drugs. There are several reports that kinetics scheme; the structure of the aggregates; the speci- protein aggregation can occur during lyophilization of pro- ficity of the intermolecular interaction (e.g. are the aggre- teins or during their subsequent rehydration, depending gates homogenous?); why aggregation (even of the same on the conditions (e.g. the water content of the system is protein) sometimes leads to ordered aggregates (amyloid) critical [17–22]). Because in some cases it appears that the and sometimes to disordered aggregates (inclusion bodies, dehydration of proteins, which occurs in lyophilization, Review Protein aggregation Fink R11

results in denaturation [23,24], it is probably the ensuing Figure 2 rehydration that leads to aggregation, due to the forma- tion of partially folded intermediates during the refolding Ordered (see below). Native aggregates (amyloid fibrils) Mechanisms of aggregation One of the earliest and most prescient studies of protein aggregation was that of Goldberg and coworkers [25] on I the enzyme tryptophanase, which revealed an intermedi- ate at moderate denaturant concentration that aggregated. Amorphous Evidence for the potential specificity of aggregation was Unfolded aggregates also observed in that the addition of other folded proteins (e.g. inclusion bodies) did not affect the amount of aggregated tryptophanase. Folding & Design More recently, the idea that partially folded intermediates might be responsible for aggregation has been championed The basic model for protein aggregation. The circled I represents a key by King and coworkers [4,26,27] and Wetzel [6,8,10,28]. partially folded intermediate, which can be populated either in the Recent reports supporting the involvement of partially folding direction (from unfolded) or from the native state. The folded intermediates in the aggregation of several proteins intermediate I has a strong propensity to aggregate, leading to either suggests the generality of the phenomenon [29–31]. ordered or disordered (amorphous) aggregates. It is possible that there is very local order even in apparently (macroscopically) amorphous aggregates. Intermediate I will normally be an intermediate Hypothesis on both the in vivo and in vitro folding pathways. Substantial data support the following model for the for- mation and structure of protein aggregates, in which spe- cific intermolecular interactions between hydrophobic solubility. The intermediates are more prone to aggregate surfaces of structural subunits in partially folded interme- than the unfolded state because in the unfolded state the diates are responsible for the aggregation. Key features of hydrophobic sidechains are scattered relatively randomly the model (Figure 2) are as follows. Protein folding in many small hydrophobic regions, whereas in the par- involves intermediates, each consisting of an ensemble of tially folded intermediates there will be large patches of closely related substates (the various substates of a given contiguous surface hydrophobicity, which will have a intermediate will be characterized by having common sec- much stronger propensity for aggregation (these are the ondary structure and most likely a common core of rela- surfaces that ‘normally’ interact in an intramolecular tively native-like structure, with the remainder of the manner to form the native conformation). The role of polypeptide chain disordered or in unstable structural domain (or subdomain) swapping [33] in aggregation is units). The native state is formed by the sequential inter- unclear, but it is certainly possible that in some cases it action of substructural units, the building blocks (typically may be an important factor. subdomains), which may be stable or metastable on their own, but are stabilized by the interactions with other such Aggregation often appears to be irreversible, but this is building blocks [32]. Formation of the native state usually a reflection of the very slow rates of disaggregation involves the intramolecular interaction of the hydrophobic and the fact that the equilibrium lies far in favor of the faces of structural subunits (Figure 3a). Specificity will aggregate rather than its soluble monomeric form. Under arise from a variety of features, of which the geometric certain conditions, aggregates, including in vivo amyloid shape and extent of the hydrophobic patches, the con- deposits, can be reversed [34,35]. In practice, however, straints of the polypeptide chain, and the presence of once insoluble aggregates form, the process is effectively other structural subunits are probably the most important. irreversible under native-like conditions. Aggregation occurs when these hydrophobic surfaces interact in an intermolecular manner (Figure 3b). Thus, A number of observations suggest that specific inter- the initial stages of aggregation are quite specific in the actions are involved in protein aggregation. Among the sense that they involve the interaction of specific surface clearest evidence for such specificity is the work of elements of the structural subunits of one molecule with Brems and coworkers [36–40] with bovine growth hor- ‘matching’ hydrophobic surface areas of structural sub- mone (bGH), which showed that a peptide fragment units of a neighboring molecule. Three-dimensional prop- of bGH could inhibit aggregation and that , agation of this process leads to large aggregates. Initially, which increased the hydrophobicity of the domain inter- the aggregates (e.g. dimers and tetramers) will be soluble, face, increased the propensity for aggregation. Another but eventually their size will exceed the solubility limit. strong indication of specificity in aggregation is the The fact that they may still have significant solvent- homogeneity of inclusion bodies [41], which are normally exposed hydrophobic surfaces would also minimize their highly homogeneous once any contaminating material has R12 Folding & Design Vol 3 No 1

Figure 3

Proposed mechanisms for aggregation (a) involving structural building blocks (subdomains). See text for more details. (a) The normal folding situation, in which hydrophobic surfaces (black) of the structural units interact in an intramolecular manner to form the native conformation. Each identifiable intermediate along the pathway will consist of an ensemble of substates, having in common the compact structural unit(s), with the Unfolded remainder of the chain in a disordered or Intermediates unstable structured form. (b) The situation in which aggregation occurs by intermolecular interaction of the structural building blocks, again via interaction of complementary hydrophobic surfaces. The common intermediate in folding and aggregation can be seen. Native

(b)

Unfolded

Aggregates

Folding & Design

been removed [41]. Similar arguments apply to amyloid either the native or non-native conformations of the pro- fibril formation. tein or the aggregated form), and the presence (or absence) of various molecular chaperones. In fact, a major role of Factors favoring aggregation chaperones is to prevent the potential aggregation of newly Circumstances that lead to the population of partially synthesized proteins, or those denatured through stress. folded intermediates, especially if their concentration is high, are thus likely to lead to aggregation; these circum- In most instances of aggregation, especially in vivo, there stances include mutations that lead to differential destabi- is a kinetic competition between aggregation and other lization of the native state relative to the partially folded processes, such as folding (Figure 4). The environmental intermediate, as well as environmental conditions. Conse- conditions and the protein concentration significantly affect quently, the major factors that determine whether a the degree and rate of intermolecular association. The protein will aggregate, and the extent and rate of the protein concentration enters the equation because aggre- aggregation, are: the protein amino acid sequence, the gation minimally involves a second-order kinetic process pH, the temperature and ionic strength, the concentration (although the growth of amyloid fibrils, and other aggre- of the protein, the presence of cosolutes (e.g. denaturants gates, may appear first order). In the context of physiologi- such as urea, other chaotropes or kosmotropes — includ- cal aggregation, the potential role of post-translational ing osmolytes — and ligands that interact selectively with processing may be critical. It is likely that in many cases Review Protein aggregation Fink R13

Figure 4 intermediate is responsible for both types of aggregates [26,30,43,44]. Degradation (proteolysis) In vivo aggregates: inclusion bodies Aggregates It is noteworthy that inclusion body formation is found not only in prokaryote and eukaryote cells, but also for both heterologous and homologous overexpression. This emphasizes that it is the overexpression itself that is responsible for the aggregation. For example, in vivo aggregation of β-lactamase is only observed when the rate Partially Native folded protein of expression exceeds 2.5% of the total protein synthesis intermediate(s) rate [45,46]. This mirrors the observations with in vitro refolding systems, which show that aggregation increases as the protein concentration increases [44]. Despite the Nascent fundamental and practical importance of inclusion bodies, polypeptide rather little is known about their structures and mecha- Binding to nisms of formation. No correlation has been found chaperones between inclusion body formation in recombinant pro- Folding & Design teins and a wide variety of factors, including size and hydrophobicity, although some correlation with average Aggregation usually involves kinetic competition. During in vivo protein charge and fraction of turn-forming residues has been synthesis, for example, partially folded intermediates are divided observed [47]. Inclusion bodies are frequently refractory between pathways leading to spontaneous folding and the native state, to renaturation. To obtain functional protein requires aggregation, binding to chaperones and proteolytic degradation. denaturation and solubilization, often with reducing agents, and subsequent renaturation. It is fre- when aggregation occurs from a solution of the native quently observed that the only way to renature significant protein it is the partially folded intermediates in equilib- amounts of soluble material is to use extremely low pro- rium with the native state that are the immediate precursors tein concentrations to avoid aggregation. As a result, inclu- of the aggregates. sion bodies are a major problem in biotechnology and the development of protein drugs [48]. The hypothesis that aggregation arises from partially folded intermediates explains the apparent lack of correla- Although many inclusion bodies are refractile in phase tion between protein stability and aggregation that is some- contrast microscopy, some large insoluble aggregates in times observed [42]. Thus, if the decreased stability of the Escherichia coli are not refractile, and have been called native state of a mutant form also destabilizes the partially ‘floccule-type’ inclusion bodies [49]. At least in some folded intermediate that is responsible for aggregation then cases, these appear to be less tightly packed, and more a correlation may be observed. On the other hand, if desta- easily solubilized than classical inclusion bodies. It is pos- bilization of the native conformation increases the popula- sible that these aggregates arise from native protein of tion of a partially folded intermediate that aggregates then very limited solubility, rather than partially folded inter- increased aggregation will be observed and there will be no mediates. We have noticed that there is a range in the apparent correlation with the native-state stability. denaturant solubility of in vivo insoluble proteins: classi- cal inclusion bodies are relatively resistant to solubiliza- The propensity for a given protein to aggregate, either in tion, whereas some insoluble material is much more vivo or in vitro, may well be determined in part by the readily dissolved in denaturant and is clearly morphologi- lifetime of partially folded intermediates. Longer lived cally different (S. Seshadri and A.L.F., unpublished intermediates are more likely to lead to aggregation for observations). Morphological differences have also been two reasons: first, there is a greater chance of interaction observed between cytoplasmic and periplasmic inclusion with another such partially folded intermediate, and bodies, reflected in differences in denaturant solubility second, in the in vivo situation, the molecular chaper- and protease resistance [50]. ones involved in preventing aggregation by sequestering the partially folded intermediate may become saturated, Several factors have been suggested to lead to inclusion and thus there will not be enough free chaperones avail- body formation, including: the high local concentration able to bind to additional newly synthesized protein. of protein; a reducing environment in the cytoplasm; lack Several observations indicate that in vivo and in vitro of post-translational modifications; improper interactions aggregation during folding give rise to similar aggregates, with chaperones and other enzymes involved in in vivo suggesting that it is likely that a common partially folded folding; and intermolecular crosslinking via R14 Folding & Design Vol 3 No 1

(although inclusion bodies can form from proteins intermediates rather than native states [42]. The in vivo lacking ). Three possible mechanisms for inclu- effects of sequence and growth temperature of IL-1β, as sion body formation have been proposed: aggregation of manifested by inclusion body formation, were quantita- native protein of limited solubility; aggregation of the tively reproduced in an in vitro system, indicating not unfolded state; and aggregation of partially folded inter- only that inclusion body formation is based on the intrin- mediate states. Until recently, there was no evidence for sic properties of the protein sequence, but also that inter- any regular structure in inclusion bodies and the prevail- actions with chaperones or other cellular factors were not ing view was that inclusion body formation was an aggre- significant [59]. gation process mediated by non-specific interactions in the unfolded state [10]. Consistent with the proposed model, point mutations in the hydrophobic face of the immunoglobulin light chain, In a series of elegant studies on the tailspike endorhamno- in which polar residues were introduced, significantly sidase of phage P22, King and coworkers [43,51–55] have decreased the fraction of inclusion body formed in an shown that partially folded intermediates are responsible E. coli expression system [61]. The potentially strong for the aggregation of tailspike protein as well as inclusion dependence of inclusion body formation on point muta- body formation in this unique system. Their results are tions may also be a reflection of specificity in aggregation. consistent with a model in which aggregate formation, both in vivo and in vitro, occurs by the specific association We have used attenuated total reflectance (ATR) FTIR of a partially folded intermediate that associates preferen- to examine the structure in various aggregated protein tially in an intermolecular fashion to form aggregates, forms, including inclusion bodies [30]. These included rather than intramolecular association leading to the native all-β, all-α and α+β proteins. Some common features conformation. A series of multimeric partially folded inter- were observed. First, both the inclusion bodies and the mediates, representing early stages of the aggregation folding aggregates exhibited substantial secondary struc- pathway for the P22 tailspike protein, have been trapped ture, typically 50–70% of that of the native conformation. in the cold and isolated by nondenaturing polyacrylamide We interpret this to mean that the inclusion bodies arose gel electrophoresis [26,27]. It should be noted that the P22 from the association of partially folded intermediates con- tailspike protein system is an unusual one and not typical taining substantial native-like structure. Second, the of most globular proteins, although similar ladders of mul- structure of a given protein in inclusion bodies, refolding timers were observed with the P22 coat protein and car- aggregates, and thermal aggregates (formed by heating a bonic anhydrase II [27]. Monoclonal antibodies against concentrated protein solution to a temperature just below tailspike chains that discriminate between folding inter- the beginning of the thermal denaturation transition) is mediates and native states were used to distinguish pro- the same (Figure 5). Thus, we conclude that a given ductive folding intermediates and off-pathway aggregation protein will have one partially folded intermediate that is intermediates. The aggregation intermediates displayed particularly prone to aggregate, and consequently most epitopes in common with productive folding intermedi- aggregates of that protein will arise from that intermediate ates, but were not recognized by antibodies against native and thus have similar structures. epitopes [55]. Because many other protein expression systems also show increased inclusion body formation as Furthermore, in all cases, even for all-β proteins, signifi- the temperature is raised, it is likely that ‘thermolabile’ cant new β structure, compared to that in the native confor- intermediates are common precursors to inclusion bodies mation, was observed; typically this amounted to ~20–25% at elevated temperatures. of the total secondary structure. We take this to indicate that the intermolecular interactions leading to the aggrega- There are several protein systems in which it has been tion involve β-sheet-like interactions. The exact nature of shown that point mutations may dramatically affect the the intermolecular interactions is unknown, and could be amount of aggregate (inclusion body) formation; these different in different aggregates; for example, the number include the P22 tailspike protein [53], interferon-γ [56], of strands in the β sheet may vary. What is clear, however, colicin A [57], Che Y [58], and interleukin-1β (IL-1β) is that aggregation usually leads to an increase in the [56,59]. For example, with the human IL-1β expression amount of secondary structure with IR components in the system in E. coli, the amount of inclusion body ranges 1623–1637 cm–1 region, which corresponds to the region from close to 0% for the native to close to 100% for the where β-sheet structure is observed. Interestingly, the Leu10→Thr and Lys97→Val mutants [60]. No strong amount of secondary structure in the inclusion body varies correlations were observed between the extent of inclu- from one protein to another, as does the amount of disor- sion body formation and either thermodynamic or thermal dered structure. Figure 5 shows the spectra of the native stability. This and other investigations suggest that the inclusion body and folding aggregate forms of IL-2, illus- tendency of at least some proteins to form inclusion trating the dramatic increase in β-sheet content compared bodies is related to the stability or solubility of folding to the native conformation. Review Protein aggregation Fink R15

Figure 5 the inclusion bodies were similar regardless of whether they were localized in the cytoplasm or periplasm.

In vivo aggregates: amyloid A number of human diseases involve the deposition of pro- tein aggregates; a subset of these, known as amyloidoses, are lethal diseases involving the extracellular deposition of amyloid fibrils and plaques. Amyloid plaque is usually defined by three features: characteristic birefringent stain- ing using Congo Red, fibrous morphology observed by electron microscopy, and a distinctive X-ray fiber diffrac- tion pattern similar to that of certain insect silks, consistent with high β-sheet content (the so-called cross-β structure). Amyloid fibrils are typically 7–12 nm in diameter and can be dissociated by high concentrations of denaturant.

Absorbance The molecular mechanisms leading to pathological amy- loid formation are not well understood. In most forms of β the formation of amyloid appears to be caused by a combination of factors, including high concentrations of protein, proteolysis, mutations, and other unknown factors. Certain other molecules are frequently found in association with amyloid plaques, namely serum amyloid P component [63], proteoglycans [64], apolipoprotein E [65] and metal ions. The role of these molecules, if they α have one, in amyloid deposition is unclear at present. There have been reports that these and other ligands may 1700 1680 1660 1640 1620 1600 increase or decrease aggregation (e.g. [66,67]). Wavenumber (cm–1) Evidence to support partially folded intermediates as Inclusion bodies amyloid fibril precursors has been found in at least two Folding aggregates systems. Transthyretin (TTR) is involved in two amyloid Native protein diseases, familial amyloidotic neuropathy and senile sys- Folding & Design temic amyloidosis. A partially folded monomeric interme- diate, populated at low pH, has been implicated in in vitro amyloid formation from transthyretin [68,69]. Recently, Aggregation results in a substantial increase in β structure compared to the native conformation. This figure shows the second derivatives of two naturally occurring variants of human lysozyme have the FTIR spectra of the amide I region of IL-2. The aggregated forms, been found to be amyloidogenic [70]. Both mutants are inclusion bodies and folding aggregates, show 25% more β structure significantly less stable than the wild-type protein and than the native state. The spectra of the two aggregated forms are very have populated partially folded intermediates at 37°C that similar, indicating similar underlying secondary structure. are assumed to be the precursors of amyloid. The ther- mally aggregated mutants showed an increased level of β structure compared to the native conformation. Our results, along with those of Georgiou and coworkers [44,62] on β-lactamase (see below), suggest that the gener- Some mutations leading to amyloid-fibril formation (in ation of insoluble aggregates during refolding from denat- transthyretin [71,72], lysozyme [70] and immunoglobulin urant can be considered to be an in vitro model system for variable light, VL, chain domains [73]) are also observed to inclusion body formation. result in decreased stability of the native state. Presumably, in such cases there is differential destabilization of the The secondary structure of β-lactamase (an α+β protein) native conformation compared to the aggregating intermedi- inclusion bodies in E. coli was investigated using Raman ate conformation that results in population of the aggregat- spectroscopy [62]. The amide I band spectra of the inclu- ing conformation. It has been shown, in an in vitro system, sion bodies were different from those of the native that point mutations may convert a non-amyloidogenic pro- protein: secondary structure estimates indicated significant tein into an amyloidogenic one [29,73]. In Alzheimer’s decreases in α-helical content and increases in β-sheet disease, the Aβ40 peptide is significantly less amyloidogenic content in the inclusion body samples. The structures of than the Aβ42 peptide [74]. The intermediacy of partially R16 Folding & Design Vol 3 No 1

folded intermediates in aggregation is also supported by Fiber diffraction studies have suggested that the ‘core’ the fact that amyloid fibrils are very high in β-sheet struc- structure of amyloid fibrils is a cross-β structure, involving ture, even though the corresponding native conformations β strands running perpendicular to the axis of the fibril. of some amyloid proteins and peptides are helical (e.g. The strands interact to form β sheets with their planes lysozyme [70] and the prion protein [75–78]). parallel to the axis of the fibril (e.g. [82–86]). A recently proposed model by Blake and Serpell [83] for the struc- Strong correlations between inclusion body formation and ture of amyloid fibrils from patients with familial amy- -dependent changes in residue hydrophobicity loidotic polyneuropathy (FAP), which are derived from were observed in the aggregation of immunoglobulin VL- transthyretin variants, nicely accounts for the fiber diffrac- chain domains. In this case, destabilizing mutations not tion patterns in aligned fibrils. The high resolution pattern only correlated with increased aggregation, but dramati- was in agreement with the cross-β structure and revealed a cally different morphologies were observed from different new repeat of 115 Å, which can be interpreted as a repeat- mutants, in one case leading to amyloid fibrils and in ing unit of 24 β strands that forms a complete helical turn another case to amorphous deposits, as found in light of the β sheet about an axis parallel to the fiber axis. Thus, chain deposition disease [73]. This suggests that specific the model protofilament consists of β sheets extended in amino acid residue changes can control not only the onset regular helical twists along the length of the fiber. of aggregation disease, but also aggregate morphology and Depending on the protein or peptide, four or five protofil- disease type [73]. This is consistent with a study of the aments intertwine to form a fibril, with the characteristic propensity of Bence Jones proteins to aggregate in vitro, diameter of ~10 nm. The proposed structure of the fibril which showed a very strong correlation with in vivo obser- indicates that the TTR building blocks are different from vations of deposition either as amyloid or light-chain the native tetramer conformation [83]. deposition disease [79]. Additional evidence that the same polypeptide can form different kinds of aggregate mor- Atomic force microscopy has been used to follow amyloid phology comes from the observation that the Alzheimer’s fibril formation in vitro by Aβ40 and Aβ42 [87]. Both vari- Aβ peptide forms ordered fibrils at pH 7.4, but amorphous ants first formed small ordered aggregates (protofibrils) aggregates at pH 5.8 [80]. that grew slowly and then rapidly disappeared, whereas prototypical amyloid fibrils of two discrete morphologies It has long been known that one of the structural charac- appeared. Aβ42 aggregated much more rapidly than Aβ40 teristics of amyloid fibrils is the presence of β-sheet struc- (consistent with its relation to early onset Alzheimer’s ture. This is observed not only by IR spectroscopy, but disease) and other in vitro studies. The protofibrils were by other techniques such as solid state NMR [81] and proposed as intermediates in the in vitro formation of fibrils. X-ray fiber diffraction [82–84]. For example, solid-state Numerous reports of a nonfibrillar form of Aβ aggregate in 13C NMR and isotope-edited IR spectroscopy investiga- the brains of individuals who are predisposed to Alzhei- tions of the structure of amyloid formed from a fragment mer’s disease suggest the existence of a precursor form, of the Aβ peptide were consistent with a model of an possibly the protofibril [87]. The first reports that amyloid antiparallel β sheet with specific intermolecular align- fibrils can be generated in vitro (and are indistinguishable ment [81]. The ATR FTIR spectra of amyloid fibrils are from amyloid fibrils formed in vivo), were probably those not noticeably different from those of disordered aggre- involving immunoglobulin light-chain domains [88,89]. gates, in that both show increased intensity in low fre- quency components that corresponds to β structure. This In vivo aggregates: raises the possibility that there might be local order in the The prion diseases are believed to involve a protein that apparently amorphous aggregates of inclusion bodies and undergoes conformational changes from its normal con- folding aggregates. For example, it is possible that the formation (PrPc or PrP-sens) to its pathological form amorphous aggregates actually involve local β-sheet for- (PrPsc or PrP-res). The pathological form is protease resis- mation between the associating molecules, but the tant and insoluble, usually forming amyloid fibrils [90,91]. ordered structure does not extend beyond the immediate Prions were first identified in the sheep disease scrapie vicinity. Interestingly, it has been suggested [85] that the and have now been identified in, or transmitted to, a β sheet in some amyloid fibrils may be in the form of a number of mammals, including humans, and an analogous β helix, the structure found in pectate lyase, acyltrans- phenomenon has been found in yeast involving the ferase and the P22 tailspike protein. The structures of Sup35 protein [92]. several amyloidogenic proteins and peptides can plausi- bly be modeled by β helices to form fibrils with the A very interesting model for prion-protein aggregation, observed structural properties. The IR bands of β helices involving the conversion of regions of the polypeptide are found in the same regions of the amide I band that are helical in the soluble form into β sheet in the (1623–1636 cm–1) as other forms of β sheet (R. Khurana insoluble, protease-resistant prion form, has been pro- and A.L.F., unpublished observations). posed [93]. Such a conformational change is not totally Review Protein aggregation Fink R17

without precedent, in the sense that a number of the pro- [96]. Another system in which concentration-dependent teins involved in amyloidosis diseases have substantial aggregation revealed evidence for partially folded interme- helical secondary structure in their native conformations, diates as the source of the aggregation is that of reduced- but adopt the classical cross-β structure in the amyloid denatured lysozyme [97]. Disulfide formation has been form. What is novel with prion proteins and peptides, shown to be an important factor in the aggregation occur- however, is that it is believed that the insoluble form acts ring during the renaturation of denatured-reduced lyso- as a template to catalyze the conversion of the soluble zyme: aggregation was shown to compete with productive form into the aggregate. An investigation of the conforma- folding from a partially folded intermediate [97–99]. tion of a partially folded intermediate of the scrapie prion protein at low denaturant concentration led to the sugges- In vitro aggregates: thermally gelled proteins tion that the insoluble scrapie prion particle is an aggre- It has long been known that heating solutions of many gated form of a partially folded intermediate on the proteins results in the formation of gels, due to aggrega- folding pathway [78]. tion of the protein. Biophysical studies have shown that these gels have a large component of β-sheet structure, In vitro aggregates: folding aggregates and FTIR studies indicate the preponderance of a band By folding (or refolding) aggregates we refer to precipitates in the vicinity of 1616 cm–1 (attributed to antiparallel that are formed when a (relatively) high concentration of β sheet; [18,100,101]). Evidence that these thermally protein, unfolded in high concentration of denaturant, is induced aggregates arise from partially folded intermediates refolded by dilution of the denaturant; the vast majority of has been presented (e.g. [102]). proteins will form precipitates under such experimental conditions, in many cases at micromolar concentrations. In vitro aggregates: partially folded intermediates Similar aggregates also occur for many proteins when (at We have examined the structure of partially folded inter- high concentration) they are put under relatively weak mediates (A states) of apomyoglobin [103] at low pH in native conditions (e.g. to a temperature close to that at the the monomeric and aggregated (at higher protein concen- beginning of the thermal denaturation transition, or a con- tration) states (K.A. Oberg and A.L.F., unpublished obser- centration of denaturant close to the beginning of the vations). The FTIR spectra reveal increased β structure in unfolded transition). the aggregated forms: the intensity of the β-sheet band(s) increases with increasing protein concentration (Figure 6). It is most noteworthy that the ATR FTIR spectra of refolding aggregates are essentially identical to those of Kinetics of protein aggregation the inclusion bodies in all cases we have examined (S. The kinetics of aggregation have most commonly been Seshadri, K.A. Oberg and A.L.F., unpublished observa- monitored by light scattering (e.g. [104,105]). Depending on tions). This suggests that they are formed from the same the particular system, the observed kinetics range from first partially folded intermediate as the corresponding inclu- order to complex. Typically, however, there is an initial lag, sion bodies. This is also true for aggregates formed under followed by an exponential growth phase (e.g. [7,106–109]). weakly native conditions [30]. These observations provide Such kinetic phenomena have been observed for both amor- strong support for the idea that there will be one particular phous and ordered (amyloid) aggregate formation, and are partially folded intermediate for a given protein that is usually attributed to a nucleation–polymerization process. particularly prone to aggregate, and that both in vivo and in vitro aggregates of the particular protein will thus have Several kinetic models for protein aggregation have been similar structures. put forward, including those accounting for the competi- tion between folding and aggregation [109,110], amyloid An example of the effect of protein concentration on aggre- formation [105,111,112] and prion formation [106], and gation during refolding is that of TEM β-lactamase [44]. In specific systems, such as carbonic anhydrase [95] and this case, a partially folded intermediate in the equilibrium β-lactoglobulin [113]. In amyloid formation, the kinetic denaturation was shown to correlate with aggregation: issue can be broken down into fibril formation and fibril increasing the temperature increased the amount of aggre- growth; two general models have been proposed: nuclea- gation, whereas the presence of sucrose decreased the tion-dependent polymerization [74,112] and diffusion- amount of aggregation. This in vitro data closely paralleled limited aggregation [107]. Data supporting both models in vivo observations on β-lactamase inclusion body forma- have been reported, but most are more in accord with the tion [62]. A partially folded intermediate in the refolding of nucleation-growth model [114–116]. A recent review sum- carbonic anhydrase was initially shown to form dimers and marizes the supporting evidence for a nucleation-dependent then higher oligomers before forming insoluble aggregates polymerization mechanism [108]. [94,95]. Low temperatures and low protein concentrations were shown to facilitate the refolding of rhodanese, mini- During the biosynthesis of proteins and during in vitro mizing aggregation due to a partially folded intermediate refolding, protein aggregation is in kinetic competition with R18 Folding & Design Vol 3 No 1

Figure 6 rather interesting phenomenon, namely that aggregation of a partially folded intermediate may induce substantial additional secondary structure in the protein (V.N. Uversky and A.L.F., unpublished observations). Staphylo- coccal nuclease, like many proteins [32,122], forms par- tially folded intermediates at low pH and low ionic strength [123]. Under these conditions and low protein concentrations (< 0.2 mg/ml) SNase is monomeric, and ~50% folded, based on the amount of secondary structure and the radius of gyration from small-angle X-ray scatter- ing studies. Increasing the protein concentration leads to the formation of dimers, with a significant increase in sec- ondary structure (corresponding to ∼70% of that of the native state). Further increases in protein concentration Absorbance induced a second transition leading to the formation of larger (multimeric) soluble aggregates with more sec- ondary structure. Above 4 mg/ml protein the far-UV CD spectrum is almost identical to that of the native protein. This apparently native-like conformation has a near-UV CD spectrum close to that of the unfolded molecule, however. The Kratky plot of the small-angle X-ray scatter- ing data for the monomeric intermediate is very similar to 1700 1680 1660 1640 1620 1600 that for the unfolded protein, indicating a lack of globular- –1 ity, but as the protein concentration increases the Kratky Wavenumber (cm ) Folding & Design plots show increasing amounts of globular structure. Thus, the increase in concentration of the partially folded inter- The FTIR amide I absorbance spectra of a partially folded intermediate mediate induces the appearance of new internal structure, of the helical protein apomyoglobin in the monomeric and aggregated as manifested by both increased secondary structure and states (aggregation was induced by high protein concentration). The aggregate shows a large increase in β structure (arrow) due to the the globular core. It is not clear how widespread this phe- aggregation. nomenon may be, but at least one other example exists: the formation of filamentous helical structures of a peptide consisting of the first 24 residues of barnase [124]. other processes (especially folding to the native state) and Immediately after dissolving the peptide fragment in in vivo protein aggregation is in kinetic competition with aqueous solution CD spectroscopy indicates a random coil association with molecular chaperones (Figure 4). The out- structure, but over time aggregation occurs leading to the come of this competition is highly sensitive to the particu- formation of aggregated helices. lar conditions; for example, the protein concentration and the lifetime of the intermediate. One of the first detailed Molecular chaperones and protein aggregation studies of the competition between in vitro folding and Molecular chaperones comprise several families of stress aggregation was that of the tetramer lactate dehydrogenase (heat shock) proteins that bind unfolded proteins or par- [117,118]. There have been few studies of the kinetics of tially folded intermediates. In particular, the hsp70 and partitioning between folding and inclusion body formation hsp60 families have been shown to facilitate protein in vivo. Using radiolabeling in the Che Y overexpression folding in vivo and in vitro [125]. Current understanding of system in E. coli, however, it was demonstrated that the the role of hsp70 in protein folding suggests that the chap- kinetics of aggregation were consistent with aggregation erone sequesters the unfolded or partially folded protein, arising predominantly from a partially folded intermediate thereby preventing its aggregation. The does [119]. Studies on the kinetics of formation of amyloid not, however, actively participate in the folding process: fibrils from Aβ peptide indicate that the rate of fibril for- binding of ATP (which subsequently becomes hydrolyzed) mation is very sensitive to the experimental conditions as leads to release of the substrate protein in a non-native well as sample preparation [120]. A recent report suggests conformation [126–129]. Recent studies on the E. coli that an amorphous aggregate intermediate stage might be hsp60, GroEL, and its eukaryotic homologs, suggest that important in the formation of amyloid fibrils [121]. protein folding may be facilitated by a mechanism in which the chaperone binds a partially folded intermediate (or a Aggregation can induce secondary structure partially folded domain of a large multidomain protein) in Recently, in studies on partially folded intermediates of its large hydrophobic cavity. A subsequent conformational staphylococcal nuclease (SNase) we have discovered a change induced by binding ATP leads to a much more Review Protein aggregation Fink R19

polar environment. Folding can thus occur in a situation The problem of exceeding solubility is explained by solu- where aggregation is precluded; multiple rounds of binding bility phase diagrams, which also suggest a solution. For and release may occur [129–133]. instance, a phase diagram for the solubility of apomyo- globin as a function of urea and protein concentration The longer a protein takes to fold spontaneously, the showed that the solubility was a minimum near the Cm longer it is likely to remain associated with the hsp60 and (denaturant concentration at the midpoint of the unfold- hsp70 chaperones. Some proteins fold fast and may have ing transition). Because the solubility of both polar and partially folded intermediates that have little propensity to non-polar molecules increases in the presence of low con- aggregate, thus requiring little or no chaperone assistance, centrations of urea, the observed decrease in apomyoglo- and little tendency to form inclusion bodies [134]. Evi- bin solubility is due to the population of either denatured dence to support the notion that both the conformation and or partially folded intermediate states [34]. Subsequent the lifetime of the intermediate are critical is found in the investigation of the structure of the precipitated material observation that hsp70 binds only to the initially collapsed by ATR FTIR indicated the presence of substantial partially folded intermediates of cytosolic and mitochondr- α helix, consistent with the insolubility arising from the ial aspartate aminotransferase. The intermediate of the population of partially folded intermediates. Phase dia- cytosolic enzyme has a much shorter lifetime than its mito- grams such as the one described explain the frequent chondrial homolog, which leads to decreased chaperone problems in renaturing inclusion bodies from concen- binding in the case of the cytosolic protein [135]. trated denaturant: a simple dilution of a concentrated protein solution in high denaturant crosses through the Several experiments have been conducted in which over- region where the solubility limit is exceeded, and this expression of various combinations of the DnaK and leads to precipitation. Precipitation can be avoided by GroEL chaperone systems decreases the amount of aggre- first diluting the protein concentration to a level such that gation of either a model protein (e.g. human growth as the denaturant concentration is decreased the solubil- hormone, hGH [136], or a β-galactosidase fusion protein ity limit is not exceeded. Unfortunately, in many cases [133]) or of the total cell proteins [137]. In addition, in vitro this is not a practical solution, although procedures for the experiments in which the yield of soluble or native protein large (industrial) scale renaturation of inclusion bodies for aggregating systems is increased in the presence of dissolved in high concentrations of denaturant have been various chaperones have been reported (e.g. [138–141]). developed that involve maintaining the concentration of partially folded intermediates very low in order to avoid There have been conflicting reports as to whether the their aggregation [143,144]. Additional factors also appear DnaK or GroEL systems, individually or together, yield to be involved in some cases. the optimal amount of renaturation. It appears that in some cases all the chaperones are required for maximal suppres- Several other methods have been suggested or shown to sion of aggregation whereas in other systems either only prevent or decrease aggregation, ranging from ligands that the DnaK system or the GroEL system alone was effective stabilize the native state [145,146] to the presence of [142]. It is most likely that the two systems interact at dif- competing peptides [40]. Although there are, as yet, no ferent stages of folding and thus different results may be general methods that can be applied to prevent aggrega- observed depending on intermediate lifetimes etc. [138]. tion, there are a number of strategies that are frequently successful [94,143–147]. The most popular approaches Little is known about the interplay between molecular currently involve chaperones [148,149], immobilization chaperones and amyloid formation, but it is reasonable to [16,150], low concentration, stabilizing agents such as assume that chaperones are significant in preventing arginine [143], optimization of growth conditions, and the amyloid formation in vivo by preventing aggregation of the use of fusion proteins [151]. Most of these can be readily precursor partially folded intermediates. It is possible, by rationalized based on our current understanding of the analogy with the situation we postulate for inclusion body factors involved in protein aggregation. In essence, most formation, that a lack of available chaperones is important factors that increase the partitioning of partially folded in amyloidosis. Because amyloid deposits are extracellular, intermediates towards the native conformation, rather some limitations may be placed on the accessibility of than towards aggregated species, are effective. amyloid fibrils (or their precursors) to chaperones. Immobilization of the unfolded protein (thereby prevent- Prevention of aggregation ing aggregation by allowing each molecule to fold indepen- There are probably two common reasons for the formation dently) and subsequent renaturation has been successfully of aggregates during renaturation: exceeding the solubility used as a means of preventing aggregation [150]. This limit of a partially folded intermediate, and the presence of approach may not work for oligomeric proteins, and may intramolecular misfolding due to the nature of the not work so well for some multidomain proteins. A system expressed protein — for example due to multiple domains. of artificial chaperones (detergents and cyclodextrin) has R20 Folding & Design Vol 3 No 1

been successfully used to minimize aggregation in the determining aggregation is the concentration of the protein renaturation of denatured-reduced lysozyme [152]. The (specifically the aggregating partially folded intermediate). application of molecular chaperones to facilitate the renatu- Factors that increase the concentration (population) of ration of recombinant proteins has recently been reviewed partially folded intermediates will also increase the propen- [149]. There have been a number of reports indicating sity for aggregation. In vivo, molecular chaperones are that osmolytes and other stabilizing ligands (kosmotropes very important for preventing intracellular aggregation, [153]) may decrease the amount of aggregation; this proba- especially during the folding of newly synthesized proteins. bly reflects differential stabilization of the native state [5,46]. Interestingly, osmolytes have been shown to inhibit There are still many outstanding and critical questions prion formation in the scrapie system, consistent with regarding protein aggregation. Among these are questions the involvement of protein conformation changes in the about the detailed mechanism of the aggregation process, formation of prions [154]. factors determining the kinetics of aggregation, the struc- tural nature of the intermolecular interactions, and how The observation that lower growth temperatures often aggregation may be effectively and efficiently prevented, lead to decreased inclusion body formation may be ratio- especially in vivo. Experimentally, the unambiguous deter- nalized on the basis of the slower rate of protein synthe- mination of whether specificity is shown in co-aggregating sis, changes in the level of cell stress (which could lead to systems is a very challenging problem and has not yet been changes in the amount of available chaperones), the adequately addressed. decreased rate of aggregation at lower temperature, and decreased hydrophobic interactions. These properties Note added in proof have been developed into a strategy for circumventing The reader is also directed to Adv. Protein Chem. 50 (1997), which was published after completion of the writing of this review and is devoted to aggregation using temperature jumps [155]. protein misassembly.

Several strategies for decreasing amyloid fibril formation Acknowledgements I would like to thank the present and past members of my research group have been proposed or demonstrated [156–158], and these who have made significant contributions to the work reported here. I would are summarized in a recent review [158]. Because the rate also like to thank the many colleagues with whom I have discussed the of amyloid formation is usually greatly enhanced by seeding question of protein aggregation over the years. The research in the author’s laboratory has been supported by the National Science Foundation. [108], prevention of amyloid seeds will delay the onset of amyloid formation [159]. The addition of ligands that bind References specifically to the native state of TTR inhibited the forma- 1. Sipe, J.D. (1992). Amyloidosis. Annu. Rev. Biochem. 61, 947-975. tion of TTR amyloid fibrils, presumably by stabilizing the 2. Carrell, R.W. & Lomas, D.A. (1997). Conformational disease. Lancet 350, 134-138. native state [146]. 3. Silow, M. & Oliveberg, M. (1997). Transient aggregates in protein folding are easily mistaken for folding intermediates. Proc. Natl Acad. Sci. USA 94, 6084-6086. Conclusions 4. Mitraki, A. & King, J. (1989). Protein folding intermediates and Our current understanding of protein aggregation (of the inclusion body formation. Biotechnology (N.Y.) 7, 690-697. pathological type) may be summarized as follows. It is the 5. Schein, C.H. (1990). Solubility as a function of protein structure and solvent components Biotechnology (N.Y.) 8, 308-315. amino acid sequence that ultimately determines the 6. Wetzel, R. (1992). Protein aggregation in vivo: bacterial inclusion propensity for aggregation, modulated by the environ- bodies and mammalian amyloid. In Stability of Protein mental conditions. Aggregates are most commonly Pharmaceuticals, Part B; In Vivo Pathways of Degradation and Strategies for Protein Stabilization. Vol. 3 (Ahern, T.J. and Manning, formed from the interaction of partially folded intermedi- M.C., eds), pp. 43-88, Plenum Press, New York. ates containing significant native-like structure. These 7. Deyoung, L.R., Fink, A.L. & Dill, K.A. (1993). Aggregation of globular β proteins Acc. Chem. Res. 26, 614-620. interactions involve extended chain or -sheet-like con- 8. Wetzel, R. (1994). Mutations and off-pathway aggregation of proteins. formations. Thus, both ordered and disordered aggregates Trends Biotechnol. 12, 193-198. show increased β structure relative to the native confor- 9. Jaenicke, R. (1995). Folding and association versus misfolding and aggregation of proteins. Phil. Trans. R. Soc. Lond. B 348, 97-105. mation (in the case of all-β proteins, the increased β struc- 10. Wetzel, R. (1996). For protein misassembly, it’s the “I” decade. Cell ture is distinct from that of the native protein). 86, 699-702. 11. Eliezer, D., Chiba, K., Tsuruta, H., Doniach, S., Hodgson, K.O. & Regardless of the manner formed (in vivo or in vitro), Kihara, H. (1993). Evidence of an associative intermediate on the aggregates of a given protein will probably have similar myoglobin refolding pathway. Biophys. J. 65, 912-917. structure and arise from a common intermediate. It is 12. Eliezer, D., Jennings, P.A., Wright, P.E., Doniach, S., Hodgson, K.O. & Tsuruta, H. (1995). The radius of gyration of an apomyoglobin folding likely that aggregation normally involves specific inter- intermediate. Science 270, 487-488. molecular interactions; among other consequences, this 13. Semisotnov, G.V., et al., & Kuwajima, K. (1996). Protein globularization means that aggregates are usually quite homogeneous and during folding - a study by synchrotron small-angle X-ray scattering J. Mol. Biol. 262, 559-574. that aggregation will be very sensitive to mutations in the 14. Thomas, P.J., Qu, B.H. & Pedersen, P.L. (1995). Defective protein protein. The process of aggregation itself may induce folding as a basis of human disease. Trends Biochem. Sci. 20, 456- 459. additional structure in the precursor intermediates. After 15. Scherzinger, E., et al., & Wanker, E.E. (1997). Huntingtin-encoded the amino acid sequence, the next most critical factor polyglutamine expansions form amyloid-like protein Cell 90, 549-558. Review Protein aggregation Fink R21

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