protein solubility and protein homeostasis: a generic view...

Protein Solubility and Protein Homeostasis: AGeneric View of Protein Misfolding Disorders

Michele Vendruscolo, Tuomas P.J. Knowles, and Christopher M. Dobson

Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom

Correspondence: [email protected]

According to the “generic view” of protein aggregation, the ability to self-assemble intostable and highly organized structures such as amyloid fibrils is not an unusual featureexhibited by a small group of peptides and proteins with special sequence or structuralproperties, but rather a property shared by most proteins. At the same time, through a widevariety of techniques, many of which were originally devised for applications in otherdisciplines, it has also been established that the maintenance of proteins in a soluble stateis a fundamental aspect of protein homeostasis. Taken together, these advances offer aunified framework for understanding the molecular basis of protein aggregation and forthe rational development of therapeutic strategies based on the biological and chemicalregulation of protein solubility.

Virtually every complex biochemical processtaking place in living cells depends on the

ability of the molecules involved to self-assemble into functional structures (Dobson2003; Robinson et al. 2007; Russel et al. 2009),and a sophisticated quality control system isresponsible for regulating the reactions leadingto this organization within the cellular environ-ment (Dobson 2003; Balch et al. 2008; Hartland Hayer-Hartl 2009; Powers et al. 2009; Ven-druscolo and Dobson 2009). Proteins are themolecules that are essential for enabling, regulat-ing, and controlling almost all the tasks necessaryto maintain such a balance. To function, themajority of our proteins need to fold into specificthree-dimensional structures following theirbiosynthesis in the ribosome (Hartl and Hayer-Hartl 2002). The wide variety of highly specific

structures that results from protein folding, andwhich serve to bring key functional groups intoclose proximity, has enabled living systems todevelop an astonishing diversity and selectivityin their underlying chemical processes by usinga common set of just 20 basic molecular com-ponents, the amino acids (Dobson 2003). Giventhe central importance of protein folding, it is notsurprising that the failure of proteins to fold cor-rectly, or to remain correctly folded, is at the ori-gin of a wide variety of pathological conditions,including late-onset diabetes, cystic fibrosis, andAlzheimer’s and Parkinson’s diseases (Dobson2003; Chiti and Dobson 2006; Haass and Selkoe2007). In many of these disorders proteins self-assemble in an aberrant manner into large mo-lecular aggregates, notably amyloid fibrils (Chitiand Dobson 2006; Ramirez-Alvarado et al. 2010).

Editors: Richard I. Morimoto, Dennis Selkoe, and Jeff Kelly

Additional Perspectives on Protein Homeostasis available at www.cshperspectives.org

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PROTEIN FOLDING AND MISFOLDING

In the late 1990s, experimental data started tobecome available, suggesting that the amyloidstate is not simply associated with a few dis-ease-related proteins, but it is a “generic” stateaccessible in principle to essentially all proteins(Guijarro et al. 1998; Dobson 1999). In addi-tion, because the types of structural interactionswithin the amyloid state and the native state aresimilar, their thermodynamic stabilities arelikely to be comparable under many circum-stances (Dobson 2003; Knowles et al. 2007a).On this assumption, there will be a competitionbetween these two states that results in normalor aberrant biological behavior depending onwhether the native or the aggregated state ispopulated under given circumstances. Moregenerally, the maintenance of the correct bal-ance in the populations of different states ofproteins is of great significance, as even mar-ginal alterations in such populations can resultin disease in the long term (Dobson 2003; Balchet al. 2008; Geiler-Samerotte et al. 2011).Indeed, it has been recently shown that the limitto the “safe” concentration of proteins in livingsystems is reached when the stability of the alter-native aggregated state becomes comparable tothat of the native state (Tartaglia et al. 2007;Powers et al. 2009). It is therefore of greatimportance to complement the well-establishedcharacterization of the structure, folding, andstability of native states with a similar analysisof the structure, assembly, and stability of otherstates—ranging from unfolded and partiallyfolded species, including natively unfoldedstates, to aggregated species such as amyloidfibrils (Fig. 1). One particularly effective ap-proach has been to incorporate data fromnuclear magnetic resonance (NMR) spectro-scopy and other experiments in moleculardynamics simulations (Vendruscolo et al. 2001;Lindorff-Larsen et al. 2005; Vendruscolo andDobson 2005; Gianni et al. 2010).

There is an increasing interest in develop-ing an understanding of how proteins foldcorrectly to generate normal biological func-tion, or misfold to generate disease. To achievethis objective, it is necessary to create highly

interdisciplinary research environments that usewide ranges of advanced procedures, both ex-perimental and theoretical (Morimoto 2008;Hartl and Hayer-Hartl 2009; Powers et al.2009). In our own group we initially used thesemethods in the fields of biophysics and struc-tural biology (Dobson 2003; Jaroniec et al.2004; Carulla et al. 2005, 2009; Smith et al.2006; Knowles et al. 2007a, 2009; Orte et al.2008); they have now been extended into bio-chemistry and cell biology (Chiti et al. 1999;Fandrich et al. 2001; de la Paz et al. 2002;Dumoulin et al. 2003; Dedmon et al. 2005;Wright et al. 2005; Baglioni et al. 2006), andthe results have led to fundamentally new ideasabout the nature of protein structures, how theyhave evolved to be as they are, and why they arenow increasingly associated with emerging dis-eases in the modern world (Chiti and Dobson2006). Most recently, these various ideas havebeen tested in living systems by using modelorganisms such as Caenorhabditis elegans(Roodveldt et al. 2009) and Drosophila mela-nogaster (Luheshi et al. 2007, 2010). Thesedevelopments, as well as many others (Balchet al. 2008; Hartl and Hayer-Hartl 2009; Powerset al. 2009), have made it possible to begin torelate in detail the mechanisms of protein fold-ing and misfolding, uncovered from in vitrostudies, with the origins and progression invivo of the most common and debilitatingneurodegenerative diseases. In addition to pro-moting a deeper understanding of these rapidlyproliferating disorders, such an understandingwill form a novel and robust basis for newapproaches to their prevention and therapy(Dobson 2004).

PROTEIN AGGREGATION AND DISEASE

Strategies based on biophysical approaches arelikely to play an increasingly important role inthe study of protein homeostasis (Fig. 1). Thisview has been prompted by the realization,made through initial observations of proteinaggregation by us and by others, that the ki-netics of this phenomenon are of critical impor-tance but have great complexity (Harper andLansbury 1997; Kelly 1998; Serio et al. 2000;

M. Vendruscolo et al.

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Dobson 2003; Haass and Selkoe 2007; Knowleset al. 2009). The experience we gained in ourstudies of protein folding, in which biophys-ical and theoretical methods have togetherbeen crucial to establish a firm understandingof this process (Wolynes et al. 1995; Dobsonet al. 1998), suggested that to make advancesin the field of protein aggregation, similar strat-egies were needed. It is now important to bringtogether methods of biochemistry and cellbiology with those of physical chemistry andbiophysics, to develop approaches for observ-ing the fundamental molecular events in the

process of aggregation, and thus be in a positionto carry out a quantitative analysis of the cellularand organismal responses associated with thisphenomenon.

A particularly clear example of the greatpotential of this approach has been the studyof the microscopic processes underlying pro-tein aggregation. Here, the analytical solutionof the equations describing the time evolutionof the fibrillar assemblies identified the threeprincipal events that play a key role in deter-mining this phenomenon—nucleation, elonga-tion, and fragmentation (Knowles et al. 2009).

Disorderedaggregate

Disorderedaggregate

Oligomer

Fiber

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Figure 1. Protein behavior in the cell. Protein homeostasis refers to the ability of cells to generate and regulatethe levels of their constituent proteins in terms of conformations, interactions, concentrations, and cellularlocalization. Failure to fold, or to remain correctly folded, can result in misfolding and aggregation, whichare processes associated with a wide range of human disorders, including Alzheimer’s and Parkinson’s diseases(Dobson 2003).

Protein Solubility and Protein Homeostasis

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Specific biophysical methods for measuringeach of them in a quantitative manner weredeveloped, including the quartz crystal micro-balance (QCM) approach for quantifying theelongation process (Knowles et al. 2007b) andatomic force microscopy methods for measur-ing fragmentation (Smith et al. 2006; Knowleset al. 2007a). These developments are enablingthe quantification not only of directly observ-able macroscopic parameters, such as lag timesand growth rates, but also of the microscopicrates characterizing the fundamental molec-ular processes underlying aggregation. By build-ing further on these achievements, it will nowbe possible to extend this approach to includeprocesses that are expected to play a centralrole in vivo.

THE KEY ROLE OF PROTEIN SOLUBILITYIN PROTEIN HOMEOSTASIS

The “generic view” of protein aggregation(Dobson 1999) offers a unified framework forunderstanding the fundamental inherent mo-lecular basis of protein aggregation and foridentifying possible ways to regulate this phe-nomenon (Fig. 2). This goal can be achievedby linking theory and experiment to develop acomprehensive conceptual basis of misfoldingdiseases to suggest rational means for theiravoidance and therapy. This approach is basedon the realization that major advances can bemade by exploiting the opportunities offeredby bringing together a series of technical andconceptual developments in this area of science:

1. new ideas about protein aggregation, includ-ing the finding that the ability to assembleinto stable and highly organized structures(e.g., amyloid fibrils) is not an unusual fea-ture exhibited by a small group of peptidesand proteins with special sequence or struc-tural properties, but rather a property likelyto be shared by most proteins (Dobson 1999)

2. the discovery that specific aspects of thebehavior of proteins, including their pro-pensities for aggregation (Chiti et al. 2003;Tartaglia et al. 2008) and for interactingwith molecular chaperones (Tartaglia et al.

2010), as well as the degree of toxicity associ-ated with the aggregation (Luheshi et al.2007), can be predicted with a remarkabledegree of accuracy from the knowledge oftheir amino acid sequences

3. the realization that a wide variety of techni-ques, originally devised for applications inother disciplines, can be used to probe thenature of protein aggregation and assembly,and of the structures that emerge from theseevents (Knowles et al. 2007a,b, 2009)

4. the observation that proteins are present atconcentrations at which they are only mar-ginally soluble (Tartaglia et al. 2007) andthat stress or aging can lead to widespreadaggregation in living organisms (Gidalevitzet al. 2006; Ben-Zvi et al. 2009; Narayanas-wamy et al. 2009; David et al. 2010; Geiler-Samerotte et al. 2011; Olzscha et al. 2011)

5. the recognition that a fundamental under-standing of these issues offers unique oppor-tunities for the rational development oftherapeutic strategies (Dumoulin et al. 2003;Dobson 2004)

In the light of these advances, it is becom-ing increasingly clear that protein homeostasisis crucially associated with the maintenance ofproteins in their soluble state, and that even rel-atively small impairments in the quality controlmechanisms that regulate the concentration ofproteins in living systems will eventually leadto disease. If this view is correct, an effectivetype of therapeutic intervention will involvethe biological and chemical regulation of pro-tein solubility.

KINETICS OF PROTEIN AGGREGATION

A major challenge in the study of protein ag-gregation is to define the microscopic processesunderlying this phenomenon. There have beengreat advances recently in methods for analyti-cally solving the system of coupled differentialequations that describe the conversion of nor-mally soluble peptides and proteins into amy-loid structures (Knowles et al. 2009). Thisapproach is very accurate for the intermediate





stages of protein aggregation, when there is aclose interplay between nucleation, elongation,and fragmentation (Fig. 3). The mathematicalmethod used to achieve this result is basedon the power of fixed-point analysis to treatsystems of differential equations having greatcomplexity, and appears to be of very generalapplicability. Because this approach allowssolutions to be derived to highly nonlinearequations, which form the majority of casesencountered in biology, it is now possible toexplore a series of fundamental problems toclarify the microscopic processes underlyingprotein aggregation in increasingly complexsituations, ranging from in vitro to in vivoenvironments. For instance we have recentlyproposed a master equation for the early stagesof protein aggregation describing the primary

nucleation processes and the dynamics of oligo-meric and prefibrillar aggregate populations,which are likely to be directly associated withtoxic effects (N Cremades, SIA Cohen, AYChen, et al. in prep.).

This approach is uniquely suited for estab-lishing a clear connection between the micro-scopic processes that determine the molecularmechanisms of protein aggregation, and thecorresponding macroscopic processes, whichare those that are directly measurable (Knowleset al. 2009). This theoretical analysis can becombined with a range of biophysical experi-ments that allow key properties governingprotein aggregation to be defined in vitro andin vivo in a highly quantitative manner by usinga variety of sophisticated fluorescence methods(van Ham et al. 2010), as well as label-free

Generic viewof protein aggregation

Generic view ofmisfolding diseases

Biological regulation ofprotein aggregation

Chemical regulation ofprotein aggregation

Thermodynamics ofprotein aggregation

Kinetics ofprotein aggregation

Key role of solubility

Metastability of thehuman proteome

Figure 2. Schematic illustration of the link between solubility and homeostasis of proteins. One of the majorconsequences of the generic view of protein aggregation is that protein solubility is expected to play a crucialrole in protein homeostasis. By carrying out accurate measurements of the kinetics and thermodynamics of pro-tein aggregation, it will be possible to define the key microscopic processes underlying this phenomenon and toidentify those proteins that are particularly prone to aggregate under stress conditions. These advances will openavenues by which the behavior of such proteins could be potentially regulated, both by enhancing the activity ofnatural cellular defenses and by pharmacological intervention.





techniques such as atomic force microscopy(Smith et al. 2006; Knowles et al. 2007b),dynamic light scattering, and QCM measure-ments (Knowles et al. 2007b).

One of the key challenges in elucidating themolecular mechanisms that underlie proteinaggregation is the difficulty in quantifying therates of the individual steps that are involvedin the overall process. Specific biophysicalmethods for measuring each of them in a quan-titative manner were developed, including theQCM approach for quantifying the elongationprocess (Knowles et al. 2007b), and atomic forcemicroscopy methods for measuring fragmenta-tion (Smith 2006; Knowles et al. 2007a).

THERMODYNAMICS OF PROTEINAGGREGATION

One of the most powerful implications of thegeneric view of protein aggregation is that thebehavior of proteins, in vitro as well as in vivo,should depend in an essential manner on thenature and relative stabilities of their differentconformational states. On this perspective, thesuggestion that most peptides and proteins arecapable of converting into the amyloid stateimplies that this state can be thermodynami-cally stable under a wide range of conditions.If this is true, then the conversion in vivo of

proteins from their native states into fibrilscould well be limited by kinetic factors, a con-clusion that differs from many current viewsbut is consistent with much that is known aboutmisfolding diseases, including the fact that suchdisorders tend to be age related (Dobson 2003;Haass and Selkoe 2007; Balch et al. 2008; Powerset al. 2009).

The challenge now is to gather quantitativeevidence to probe this overall concept, and toestablish how proteins have evolved (or rathercoevolved with their cellular environments) toremain soluble on the timescales required forthe successful functioning of living organisms(Tartaglia et al. 2007). At high concentrations,proteins will be thermodynamically unstablein their native states and prone to undergo rapidaggregation. Therefore, there will be strong evo-lutionary pressure on their amino acid sequenceto maintain their solubility. In contrast, at lowconcentrations, proteins will be highly solubleas their native states are thermodynamicallyvery stable. There is consequently no evolution-ary pressure acting against a natural drift, result-ing, e.g., from random mutations, toward lesssoluble sequences. This drift, however, muststop when proteins reach a regime in whichtheir native states are still kinetically, eventhough not thermodynamically stable. Indeed,when a regime of kinetic instability is reached,

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Figure 3. (A) Illustration of the nucleation-elongation-fragmentation model of fibril formation, for which (B) ananalytical solution has recently been obtained (Knowles et al. 2009).





protein solubility is no longer guaranteed,and the functioning of a biological system iscompromised.

An essential requirement to establish theprinciple of metastability of native states againstaggregation (Tartaglia et al. 2007; Olzscha et al.2011) is the ability to quantify the relativestabilities of the native, unfolded, oligomeric,and fibrillar states. Experimental measurementsof these stabilities can be obtained by usingdenaturant-induced disaggregation experiments(O’Nuallain et al. 2005). Our initial studieshave already indicated that it is possible toexploit this strategy successfully to probe thethermodynamic characteristics of a wide varietyof proteins (Baldwin et al. 2011). These experi-ments establish a close connection between ear-lier biophysical studies that allowed the natureof protein folding to be elucidated (Wolyneset al. 1995; Dobson et al. 1998), and the phe-nomenon of amyloid growth, whose generalmechanistic principles are still emerging.

These investigations have been greatly sti-mulated by our observation of a close relation-ship between the messenger RNA (mRNA)expression levels and the aggregation rates ofcorresponding series of human proteins (Tarta-glia et al. 2007), which further suggests thatthe concentration of proteins in the cell shouldbe closely related to the critical concentrationunder the corresponding conditions. We havereferred to this relationship as the “life on theedge” hypothesis, because according to thisview, proteins are just soluble enough to remainin their active states at the concentrationsrequired for their optimal function (Tartagliaet al. 2007).

THE METASTABILITY OF THE HUMANPROTEOME

A key implication of the life on the edge hypoth-esis (Tartaglia et al. 2007) is that the human pro-teome might include proteins that are highlymetastable, as their solubility is significantlylower than would be expected on the basisof their cellular concentration. This view isreceiving support from a series of recent studiesthat have shown how protein aggregation is a

widespread phenomenon, involving, in par-ticular, metastable proteins (Gidalevitz et al.2006; Narayanaswamy et al. 2009; David et al.2010; Geiler-Samerotte et al. 2011; Olzschaet al. 2011).

These results provide new insight into theobservation that our biochemical pathways arehighly susceptible to the formation of misfoldedor aggregated proteins as a consequence of agingor stress conditions (Haass and Selkoe 2007;Ben-Zvi et al. 2009). Because misfolding andits consequences put the cellular quality controlsystems under further pressure, these initialfindings suggest that misfolding diseases canresult from a “misfolding cascade,” giving riseto a variety of unregulated events that can leadto biochemical dysfunction and cell death.This view provides a novel perspective of neuro-degenerative conditions, according to whichevents cause the accumulation of the peptideor protein aggregates that initiate the disease(e.g., amyloid b and tau in the case of Alz-heimer’s disease) activate a set of downstreamcellular signaling and metabolic events that ulti-mately damage or destroy nerve cells (Hardyand Selkoe 2002).

BIOLOGICAL REGULATION OF PROTEINAGGREGATION

A series of quantitative techniques has beenrecently developed to study the structures ofthe states involved in the interactions betweenmisfolded proteins and chaperones and otherprotective mechanisms, as well as to probe theireffects on the aggregation process (Dumoulinet al. 2003; Knowles et al. 2007b; Balch et al.2008; Hartl and Hayer-Hartl 2009; Powerset al. 2009; Roodveldt et al. 2009). One of thekey difficulties in studying the effects of cellularcomponents on the aggregation of proteins isthe interfering effects that these species canexercise on the assays that are used to monitorprotein aggregation itself. In particular, chaper-ones can compete for the binding of dye labelsand molecular crowding can influence the affin-ity of fluorescent labels to amyloid structures(White et al. 2010; Buell et al. 2011). We haverecently proposed the application of a variety





of biophysical techniques to overcome thesedifficulties. Using an alternative readout to theprevalent optical signals used in traditionalaggregation studies, namely, direct measure-ments of the changes in mass of an ensembleof aggregates by means of a QCM, we havebeen able to develop aggregation assays thatfunction reliably even in a complex environ-ment (Knowles et al. 2007b). The success ofthis concept has been shown in early experi-ments, in which we showed that the inhibitionof filament growth by the heat shock proteinaB-crystallin occurs through interactions withthe aggregated species and primarily with theirmonomeric precursors as had commonly beenassumed (Knowles et al. 2007b). In addition,by using this biosensor-based approach, wehave been able to probe directly the effect thatmolecular crowding has on protein assembly(White et al. 2010). In combination with atomicforce microscopy studies, which allow individ-ual aggregates to be resolved prior, during,and after growth, and enable a correlation tobe established between kinetic signals and thechanges in the morphology of the aggregatesthat are formed, these biosensor-based ap-proaches provide a robust platform for under-standing the effects of biological modulatorsof protein aggregation (Fig. 4).

These quantitative approaches have openedup the possibility of probing more generallythe effects of specific cellular components onthe factors that modulate protein aggregation.Biosensor-based assays have the potential todissect the aggregation pathway and elucidatethe steps with which given components inter-fere. Following the development of these meth-ods and their shown success in quantifyingprotein aggregation in our earlier studies, ithas become possible to use them to obtaininformation on the interactions of a range ofcellular components, including clusterin, heatshock proteins, ATP-dependent chaperonesystems, with aggregates formed from variouspolypeptide chains, including short peptides,protein fragments, protein domains, and full-length proteins. With this approach it willbecome possible to elucidate the general physi-cal principles that govern biological regulation

of protein aggregation and understand hownature has developed mechanisms able to coun-ter protein aggregation in a highly effective way.

Furthermore, it is becoming crucial toacquire a better understanding of the natureof protein stability and solubility. One of theexpectations inherent in the life on the edgehypothesis (Tartaglia et al. 2007) is that the

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Figure 4. Biosensor assays allow the mechanisms ofamyloid growth inhibition to be investigated (Whiteet al. 2009). (A) Growth inhibition by a chemicalchaperone (trimethylamine oxide [TMAO]). Insulinfibrils are first grown in the presence of soluble pro-tein (1), then in the presence of both protein andchaperone (2), and finally, in the presence of proteinalone (3). In the absence of chaperone, the growthresumes to the initial level, indicating no permanentinteractions between the fibrils and the chaperone.(B) The growth inhibition assay is repeated foraB-crystallin. The growth rate (3) after exposure tochaperone (2) is significantly lower than that mea-sured for pristine fibrils (1), revealing a strong inter-action between the fibrils and aB-crystallin as thebasis of the growth inhibition (Knowles et al. 2007b).





presence of chaperones should enhance theeffective solubility of proteins in the cellularenvironment (Gidalevitz et al. 2006; Olzschaet al. 2011). In this sense, chaperones act toprotect the cell from the pathological effectsof misfolded proteins and aberrant aggregates,which are particularly abundant in the contextof specific pathways (Dobson 2003; Hartland Hayer-Hartl 2009; Powers et al. 2009).The presence of a general relationship betweenprotein solubility and abundance (Fig. 5) pro-vides further insight by understanding how thespecific cellular conditions in which proteinscarry out their functions can modulate thisrelationship.

CHEMICAL REGULATION OF PROTEINAGGREGATION

A powerful idea that has recently emerged isthat fundamental physicochemical propertiesof peptides and proteins are important indetermining the toxicity of aberrant assembliesby studying in vivo models (Bucciantini et al.2002; Dobson 2004; Luheshi et al. 2007,2010). In this context, we anticipate that pro-cesses that activate particular pathways, suchas oxidative phosphorylation or proteasomaldegradation, without affecting the supply ofproteins into those pathways, will not be fullyeffective by themselves in treating misfolding

diseases. Our view is that early interventionsmay be targeted and specific, but that later strat-egies must restore health to the proteome as awhole. Recent studies performed by JeffreyKelly, Richard Morimoto, and coworkers haveindeed provided initial evidence that favoringthe return to normal homeostasis may representa powerful strategy in combating neurodegen-erative diseases (Balch et al. 2008; Morimoto2008; Powers et al. 2009).

The palette of biophysical techniques thatwe have developed and applied to the phenom-enon of protein aggregation allows a uniquelydetailed understanding to be established ofthe chemical factors that regulate this processin vivo and in vitro. A key requirement to restorebiological function is the solubilization orelimination of aggregates that have formed inan aberrant way. Quantitative assays of both ofthese processes are therefore of paramountimportance in this context. We have alreadyshown that biosensor approaches allow theeffect on protein aggregation of small mole-cules to be defined in a highly quantitativeway (Knowles et al. 2007b). This methodologyshould also be highly suited for probing thedisaggregation of aberrant assemblies underthe action of a variety of components, and henceforms an ideal platform for studying the mech-anism of action of antiaggregation compounds,such as b breakers (Soto et al. 1998).

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Figure 5. Correlation between in vivo mRNA expression levels and in vitro protein aggregation rates for a set of 11human proteins, either related (red) or unrelated (blue) to disease (Tartaglia et al. 2007).





More generally, a central question in devis-ing strategies against protein aggregation isabout the relative effectiveness of interventionat the different specific stages involved in theoverall process. Studies of the inhibition of ag-gregation in bulk solution assays, even thoughthey frequently show evidence for the inhibitoryaction of various compounds, do not in generalallow precise information to be obtained on thespecific molecular events on the aggregationpathway that have been affected, leading to dif-ficulties for a rational design of strategies to pre-vent loss of protein solubility. A combination ofinnovative biophysical approaches, includingbiosensing and atomic force microscopy techni-ques, is becoming available to dissect the effectsof antiaggregation compounds at the level ofindividual microscopic rates and processes.Biosensing approaches allow for the growthand potential degradation of aggregates to beprobed in the absence of nucleation events,whereas atomic force microscopy provides aconnection to structural and morphologicalchanges that accompany aggregation or disag-gregation under the effect of chemical agentsfavoring solubilization.

Finally, the information obtained fromthese core biophysical and theoretical investiga-tions can be used in the context of treatmentsfor misfolding diseases based on targetingamyloidogenic proteins or on improving thecapacity of the cell to protect unfolded proteinsagainst a range of aggregation pathways. Thefundamental concepts emerging from biophys-ical studies can be applied to in vivo model sys-tems including C. elegans. By using this type oftechnique, it will become possible to enhanceprotein homeostasis by antibodies (Dumoulinet al. 2003; Luheshi et al. 2010) or by small mol-ecule inhibitors of aggregation (Dobson 2004;Lendel et al. 2009).

A UNIFIED VIEW OF PROTEIN MISFOLDINGAND ITS ASSOCIATED DISORDERS

The studies that we have discussed are creatinga general framework for understanding pro-tein misfolding disorders in terms of the prop-erties of the molecules involved in them. This

approach is inspired by one of the major conse-quences of the generic view of amyloid aggre-gation (Dobson 1999)—that the study of thefactors that influence the stability of the variousstates of proteins in the cell is capable not only ofproviding an understanding of the molecularbasis of misfolding disorders, but also of sug-gesting general strategies for their treatment.This approach is based on the advances thathave been recently made in developing a rangeof novel biophysical, theoretical, and computa-tional techniques.

The approach that we have presented,instead of being targeted to the specific proc-esses involved in a particular disease, is aimedat establishing a unified theory of protein mis-folding disorders. This theory is inspired bythe general association of misfolding diseaseswith the problem of protein solubility and themetastability of the human proteome. It isalso based on the premise that comparison ofthe molecular events associated with relateddiseases will enable us to understand the keyfeatures that define the pathogenesis at themolecular level. These advances are clarifyinghow misfolding diseases are caused by the fail-ure of the natural cellular protective mecha-nisms to provide sufficient defenses againstthe naturally limited solubility of our proteins.

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published online August 8, 2011Cold Spring Harb Perspect Biol Michele Vendruscolo, Tuomas P.J. Knowles and Christopher M. Dobson Misfolding DisordersProtein Solubility and Protein Homeostasis: A Generic View of Protein

Subject Collection Protein Homeostasis

Proteostasis in Living CellsProteome-Scale Mapping of Perturbed

Isabel Lam, Erinc Hallacli and Vikram KhuranaBiology and MedicineThe Amyloid Phenomenon and Its Significance in

Michele VendruscoloChristopher M. Dobson, Tuomas P.J. Knowles and

AdaptabilityThe Proteasome and Its Network: Engineering for

Daniel Finley and Miguel A. Prado

Functional Modules of the Proteostasis NetworkGopal G. Jayaraj, Mark S. Hipp and F. Ulrich Hartl

Functional AmyloidsDaniel Otzen and Roland Riek Method in the Study of Protein Homeostasis

Protein Solubility Predictions Using the CamSol

Pietro Sormanni and Michele VendruscoloChaperone Interactions at the Ribosome

KreftElke Deuerling, Martin Gamerdinger and Stefan G. Proteins in Health and Disease

Recognition and Degradation of Mislocalized

Ramanujan S. Hegde and Eszter ZavodszkyMechanisms of Small Heat Shock Proteins

Christopher N. Woods, et al.Maria K. Janowska, Hannah E.R. Baughman, Chaperone Network

The Nuclear and DNA-Associated Molecular

FreemanZlata Gvozdenov, Janhavi Kolhe and Brian C.

MachineryStructure, Function, and Regulation of the Hsp90

Maximilian M. Biebl and Johannes Buchner Capacity of the Endoplasmic ReticulumProtein-FoldingResponding to Fluctuations in the

The Unfolded Protein Response: Detecting and

WalterG. Elif Karagöz, Diego Acosta-Alvear and Peter

of the Hsp104 DisaggregaseSpiraling in Control: Structures and Mechanisms

James Shorter and Daniel R. Southworth DiseasesDegradation (ERAD) and Protein Conformational

Associated−Chaperoning Endoplasmic Reticulum

Jeffrey L. BrodskyPatrick G. Needham, Christopher J. Guerriero and

ChaperonesModulation of Amyloid States by Molecular

Bernd BukauAnne Wentink, Carmen Nussbaum-Krammer and

Mitochondrial Proteolysis and Metabolic ControlSofia Ahola, Thomas Langer and Thomas MacVicar

http://cshperspectives.cshlp.org/cgi/collection/ For additional articles in this collection, see

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