R E V I E W S
`
`Cellular strategies for controlling
`protein aggregation
`
`Jens Tyedmers, Axel Mogk and Bernd Bukau
`
`Abstract | The aggregation of misfolded proteins is associated with the perturbation of
`cellular function, ageing and various human disorders. Mounting evidence suggests that
`protein aggregation is often part of the cellular response to an imbalanced protein
`homeostasis rather than an unspecific and uncontrolled dead-end pathway. It is a regulated
`process in cells from bacteria to humans, leading to the deposition of aggregates at specific
`sites. The sequestration of misfolded proteins in such a way is protective for cell function as it
`allows for their efficient solubilization and refolding or degradation by components of the
`protein quality-control network. The organized aggregation of misfolded proteins might also
`allow their asymmetric distribution to daughter cells during cell division.
`
`Conformer
`One of many possible
`structural states from the
`same protein species.
`
`Molecular chaperone
`One of a group of unrelated
`proteins that interact with
`non-native polypeptides to
`assist in their folding, transport
`and assembly.
`
`Heat shock protein
`A protein that shows increased
`expression in stress conditions
`through a specialized
`heat shock-response element
`in the promoter of the
`corresponding gene.
`
`Zentrum für Molekulare
`Biologie der Universität
`Heidelberg (ZMBH),
`DKFZ-ZMBH Alliance,
`Im Neuenheimer Feld 282,
`D-69,120 Heidelberg,
`Germany.
`Correspondence to
`B.B. and J.T.
`e-mails: bukau@zmbh.
`uni-heidelberg.de;
`j.tyedmers@zmbh.
`uni-heidelberg.de
`doi:10.1038/nrm2993
`Published online
`14 October 2010
`
`To be functional, most proteins must adopt a defined
`three-dimensional structure termed the native fold.
`Protein folding starts as proteins are synthesized at
`ribosomes and passes through structural intermediates
`before the native state is reached (FIG. 1). Some inter-
`mediates can be non-productive, such as misfolded
`conformers that are trapped in free energy minima. As
`the energy barriers that separate native and non-native
`conformations are usually small, even native proteins
`are at permanent risk of unfolding, especially under
`environmental stress conditions1–3. Folding intermedi-
`ates, including misfolded conformers, typically expose
`hydrophobic residues that are normally buried in the
`native structure. Such hydrophobic surfaces are prone to
`triggering the aggregation of proteins. There seems to be
`a preference for the co-aggregation of the same type of
`protein4,5. However, one aggregating protein species can
`also influence the aggregation behaviour of another one6,
`and different proteins can be found to co-aggregate, as
`was observed for aggregation-prone proteins containing
`polyglutamine stretches. This trapping of other proteins
`in aggregates was suggested to be one possible reason for
`the toxicity associated with neurodegenerative diseases
`that involves polyglutamine aggregation7–9.
`Preventing the accumulation of aggregation-prone
`misfolded proteins is the first and most effective inter-
`vention point to control protein aggregation. Cells of all
`kingdoms of life have evolved an elaborate protein quality-
`control system, which acts either to facilitate the folding
`or refolding of misfolded protein species by molecula r
`chaperones or to remove them by proteolytic degra-
`dation, thereby preventing protein aggregation10–14.
`
`The main chaperone classes that prevent the accumu-
`lation of misfolded conformers include the heat shock
`proteins (HSPs) HSP60 and HSP70, which exhibit ATP-
`dependent refolding activities11,15–17. Misfolded proteins
`that are not refolded are generally turned over by either
`cytosolic ATP-dependent AAA+ proteases (for example,
`the 26S proteasome)10 or acidic hydrolases after their
`transport into the lysosomal compartment14,18–20. A proper
`balance between these protein quality-control compo-
`nents is required for protein homeostasis, also referred
`to as proteostasis21. Protein aggregation seems to result
`from exhaustion of the above quality-control system.
`In this Review, we summarize the basic principles of
`the cellular mechanisms that control protein aggregation
`and cope with aggregates. We focus on the organization
`of aggregates in specialized intracellular deposition sites,
`mechanisms to reverse protein aggregation and strate-
`gies to eliminate aggregates or retain them in the cells
`with lower life expectancies during cell division. We do
`not cover the details of the many folding diseases that
`are related to protein aggregation and mention various
`disease-related proteins or types of aggregates only in the
`context of the general principles of protein aggregation.
`
`Conditions that result in aggregation
`The quality-control system can adapt to the sever-
`ity of protein damage through the induction of stress
`responses, which adjust the cellular levels of chaperones
`and proteases10,13,22–24. However, when the generation of
`misfolded proteins exceeds the refolding or degradative
`capacity of a cell, protein aggregates accumulate. This
`exhaustion of the cellular protein quality-control system
`
`nATuRe RevIeWS | Molecular cell Biology
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`
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`© 20 Macmillan Publishers Limited. All rights reserved10
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`

`R E V I E W S
`
`Synthesis and folding
`
`Ribosome
`
`Nascent
`polypeptide
`
`Folding intermediate
`
`Native protein
`
`Environmental
`stress, mutations or
`translational errors
`Misfolding and aggregation
`
`Refolding
`
`Quarternary
`complex
`
`Amyloid
`fibrils
`
`Prefibrillar
`aggregates
`
`Partially
`misfolded
`
`Misfolded
`
`Disordered
`aggregate
`
`Degradation
`
`Figure 1 | overview of cellular protein aggregation. A protein during and after
`Nature Reviews | Molecular Cell Biology
`its synthesis at the ribosome folds through different intermediates to its native,
`three-dimensional structure. Proteotoxic stresses, mutations in the synthesized
`protein or translational errors can cause protein misfolding. Once present, misfolded
`intermediates can be refolded to the native state or be degraded by different cellular
`proteolysis systems that prevent the accumulation of misfolded proteins. Once the
`quality-control network is overwhelmed — for example, through persisting harsh stress
`conditions, increased amounts of aberrant proteins or in aged cells — aggregates can
`form. Their formation can be guided by molecular chaperones. Forming aggregates
`can have varying degrees of structure, ranging from mostly unstructured, disordered
`aggregates to prefibrillar species and highly ordered β-sheet-rich amyloid fibrils.
`Disordered aggregates and intermediates during amyloid formation may be degraded.
`Arrows indicate a process that can include several single steps; dashed arrows indicate
`a process of minor significance.
`
`can result not only from single, severe conditions, but
`also from the combination of different moderate condi-
`tions, which do not overwhelm the system on their own4.
`various internal and external conditions have been iden-
`tified and can be categorized into the four main classes
`described below.
`The first class comprises mutations that result in the
`sustained tendency of the affected proteins to misfold
`and aggregate. Such mutations are responsible for various
`‘conformational diseases’, such as type II dia betes,
`Huntington’s disease and familial forms (which are
`in herited and have a higher probability of developing in
`the affected family) of Parkinson’s disease and Alzheimer’s
`disease21,25,26. Moreover, mutations in components of the
`protein quality-control system can provoke protein aggre-
`gation. examples are mutations in the genes en coding the
`small HSP (sHSP) α-crystallin, leading to cataract forma-
`tion27, and the E3 ubiquitin ligase Parkin, resulting in an
`early onset form of Parkinson’s disease28–30.
`The second class comprises defects in protein bio-
`genesis. These include translational errors, leading to the
`misincorporation of amino acids, and assembly defects
`of protein complexes, leading to the accumulation of
`
`AAA+ protease
`An ATP-dependent proteolytic
`machinery that acts in general
`and regulatory proteolysis.
`
`E3 ubiquitin ligase
`A component of the ubiquitin–
`proteasome system that
`delivers activated ubiquitin
`moieties to special substrates
`and thereby provides
`substrate specificity for
`ubiquitylation.
`
`non-complexed protein species that are frequently
`prone to aggregation31,32.
`The third class comprises environmental stress con-
`ditions, such as heat and oxidative stress. excessive heat
`treatment at or above the upper temperature range for
`growth of a particular cell type leads to the bulk unfold-
`ing of cellular proteins. Whereas heat-induced unfolding
`of proteins may be reversible (see below)33, oxidative
`stress can lead to several irreversible protein modifica-
`tions by reactive oxygen species (RoS), including radical-
`induced fragmentation of the polypeptide backbone and
`the replacement of side chains of specific amino acid
`residues by carbonyl groups34. Carbonyl derivates can
`be generated either by a direct oxidative modification
`of Pro, Arg, lys and Thr residues or in reactions of lys,
`Cys and His residues with reactive carbonyl compounds
`on glycoxidation products, lipids and advanced glyca-
`tion end products35. These irreversible modifications can
`then lead to misfolding and eventually aggregation. one
`possible reason for the accumulation of carbonylated
`proteins as aggregates may be that the carbonyl groups
`can further react with the α-amino group of lys residues,
`thereby leading to cross-linked derivates that are resistant
`to proteolytic degradation by the proteasome34.
`The fourth class comprises protein aggregation in
`cells during ageing, which occurs at a slower pace. For
`example, the aggregation of polyglutamine model pro-
`teins in Caenorhabditis elegans and a misfolding-prone
`mutant of human superoxide dismutase 1 (SoD1) in
`mice is exacerbated during ageing36,37. As SoD1 mutants,
`in contrast to the wild-type protein, expose more hydro-
`phobic surfaces and can be bound by chaperones, this
`example demonstrates how the quality-control system
`can become progressively exhausted during ageing37,38.
`Similarly, carbonylated proteins accumulate progress-
`ively to form visible aggregation foci in the cytoplasm
`in aged yeast cells39. These findings suggest a reduced
`capacity of ageing cells to eliminate misfolded protein
`species. Such a general decline in the capacity of cellular
`protein quality control during ageing was also suggested
`to be a reason for the late age of onset of Huntington’s dis-
`ease and many sporadic forms of Alzheimer’s disease or
`Parkinson’s disease. However, additional factors, includ-
`ing the unique combination of polymorphic variations
`in different proteins of an individual, may also influence
`its protein quality-control capacity4,23. In C. elegans, the
`general decline of protein quality control happens at an
`early stage of adulthood and thus is an early molecular
`event during cellular ageing in this organism40.
`
`Structural features of aggregates
`Protein aggregates were initially classified, based on
`electron microscopy, as either apparently amorphous
`(for example, bacterial inclusion bodies generated
`on the overproduction of recombinant proteins) or
`amyloid-like. Meanwhile, it became apparent that such
`classification is an oversimplification as, for example,
`bacterial inclusion bodies have been demonstrated
`to contain amyloid-like structures41. Intramolecular
`β-sheets have now been recognized as a common struc-
`tural element of aggregates and are shared by both
`
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`

`R E V I E W S
`
`a Bacteria
`
`Diffusion
`Misfolded protein
`Nucleoid
`
`b Yeast
`
`Aggregate
`
`Polyubiquitin
`Misfolded
`protein
`
`c Mammals
`
`Misfolded
`protein
`
`Small
`aggregates
`
`Cytoskeleton
`
`Cytoskeleton
`
`Vacuole
`
`Nucleus
`
`HDAC6
`Dynein
`
`+
`
`Microtubule
`
`–
`
`Nucleus
`
`Centriole
`Vimentin
`cage
`
`Polar aggregate
`Aggresome
`JUNQ
`IPOD
`(inclusion body)
`Figure 2 | Pathways for the cellular sequestration of protein aggregates. a | In bacteria, misfolded proteins can
`accumulate in inclusion bodies under different conditions, such as the heterogenous expression of proteins or stress.
`Inclusion bodies often form at the periphery of the cell. Nucleoid exclusion is sufficient to control the polar localization
`of aggregated proteins. Energy-driven active processes may contribute to the deposition of misfolded proteins in
`inclusion bodies. b | Yeast cells possess distinct protein quality-control compartments, the juxtanuclear quality-control
`compartment (JUNQ) and the perivacuolar insoluble protein deposit (IPOD). Soluble, misfolded, ubiquitylated proteins
`can be disposed at the JUNQ, whereas insoluble, terminally aggregated proteins can accumulate at a perivacuolar site.
`Disruption of the cytoskeleton disturbs the targeting to both compartments. c | JUNQ- and IPOD-like compartments have
`also been seen in mammals and are distinct from the perinuclear aggresomes where misfolded, ubiquitylated proteins
`accumulate. The aggresome is a vimentin-enwrapped structure located at an indentation of the nucleus surrounding
`the centriole. Aggresome formation requires the adaptor histone deacetylase 6 (HDAC6), which binds to ubiquitylated
`proteins on the one hand and the microtubule minus-end motor protein dynein on the other hand. Other sequestration
`pathways in yeast and mammals have been reported but are not shown.
`
`apparently amorphous aggregates and amyloid fibrils.
`The degree of β-sheet organization is, however, vari-
`able in the different aggregate forms, with the highest
`one present in amyloid fibrils, in which the β-sheets
`run perpendicular to the fibril axis1,25,42–44 (FIG. 1). The
`variability of protein aggregation is best illustrated by
`the finding that the morphology of protein aggregates
`generated from the same protein species can be diverse45
`and influenced by the denaturing conditions, which lead
`to different unfolding and aggregation pathways6,46. In
`an attempt to identify the underlying molecular differ-
`ences in a recent study, different amorphous and fibrillar
`aggregates of the amyloidogenic amino-terminal frag-
`ment of the protein HypF were generated and compared
`by nMR techniques. These morphologically different
`forms have shared but also distinct segments of the pro-
`tein sequence involved in the cross-β-sheet structural
`motif 46. Furthermore, subtle structural differences in
`the interphase of interacting polypeptides that result
`in differences in the morphology and toxicity of the
`particular aggregate have been shown for some prion
`proteins47–49.
`
`Cellular aggregate deposition sites
`How do cells deal with aggregated proteins? Compelling
`evidence, such as the deposition of protein aggregates
`at specific cellular sites, suggests that protein aggrega-
`tion is a much more organized process than previously
`thought (FIG. 2). The sequestration of aggregated pro-
`teins can be viewed as a second cellular response that
`occurs when the quality-control system that refolds or
`degrades misfolded proteins in their soluble state has
`been overrun.
`
`Directing aggregated proteins to specific compart-
`ments can protect the cellular environment from poten-
`tially deleterious protein species. As shown for certain
`amyloidogenic proteins, it becomes more and more
`evident that soluble oligomers themselves, rather than
`insoluble amyloids in their microscopically visible final
`state, are cytotoxic25. The formation of amyloid aggre-
`gates may even have a cytoprotective function28,50–55.
`organizing protein aggregates might also facilitate the
`efficiency of aggregate removal in a subsequent phase56–58.
`Although the spatial sequestration of misfolded proteins
`seems to be a common strategy of all cells, the specific
`localization of deposition sites differs between organisms
`and depends on the particular aggregation-prone pro-
`tein, the cellular compartment and the stress conditions
`causing protein misfolding. We do not cover the specific
`features of each aggregating species here, but we give
`some examples of more general, and better-characterized,
`deposition processes.
`
`Deposition sites for protein aggregates in bacteria.
`Aggregates of endogenous proteins can form in bacteria,
`particularly under heat or oxidative stress conditions59,60.
`Furthermore, insoluble inclusion bodies frequently form
`in bacteria and also in eukaryotic cells that overexpress
`heterologous proteins61,62. usually one or two inclusions
`form per cell, predominantly at the cell poles but also
`in mid- or quarter-cell positions, which are future sites
`for septation63–65. So far, the best quantitative analysis of
`protein aggregation has been established for heat-treated
`Escherichia coli cells. In E. coli, ~1.5–3% of total cytosolic
`proteins can aggregate and individual inclusion bodies
`contain ~2,400 –16,500 protein molecules. The number
`
`nATuRe RevIeWS | Molecular cell Biology
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`

`R E V I E W S
`
`Juxtanuclear quality-control
`compartment
`A compartment for the
`deposition of soluble,
`misfolded proteins in yeast
`and mammals. The targeting
`of substrates to this deposition
`involves ubiquitylation.
`
`Insoluble protein deposit
`A perivacuolar quality-control
`compartment for the
`deposition of terminally
`aggregated proteins observed
`in yeast and mammalian
`cells. One group of substrate
`proteins accumulating here
`are aggregates of
`amyloidogenic proteins.
`
`Ubiquitin proteasome
`system
`A cellular quality-control
`system for the ubiquitin-
`dependent degradation of
`substrate proteins by the
`proteasome.
`
`E2 ubiquitin-conjugating
`enzyme
`A component of the ubiquitin
`proteasome system that
`harbours an activated ubiquitin
`moiety and cooperates with
`E3 ligases in substrate
`ubiquitylation.
`
`Lys63-linked
`polyubiquitylation
`A polyubiquitin chain in which
`the ubiquitin molecules are
`linked by the internal Lys63
`residue of ubiquitin.
`
`of proteins that are vulnerable to thermal unfolding and
`aggregation is surprisingly high, ranging from 150–200
`individual protein species65,66.
`In E. coli, such aggregates are typically localized to the
`old cell pole for reasons that are still unclear64,65 (FIG. 2a).
`The mechanism by which aggregating proteins reach
`the cell poles is controversial. one study claims that an
`active, energy-driven process is responsible for polar
`localization67. by contrast, a second study demonstrated
`that nucleoid occlusion is necessary and sufficient
`for controlling the polar localization of aggregated pro-
`teins, indicating a passive mechanism for aggregate
`sequestration65.
`
`Deposition sites for protein aggregates in yeast. Protein
`aggregation in eukaryotic cells is often studie d in
`Saccharomyces cerevisiae68. When yeast cells are exposed
`to severe heat stress, aggregated proteins form multi-
`ple electron-dense foci of different sizes dispersed
`throughout the cytoplasm and nucleus, as visualized
`by transmission electron microscopy. no obvious spe-
`cific compartmentalization was observed for this type
`of aggregate33. notably, most of these aggregated pro-
`teins can be reactivated by chaperones during a recovery
`phase (see below).
`Proteins that form aggregates in yeast and that are
`not refolded to the native state include oxidatively dam-
`aged proteins35, proteins that are marked for degrada-
`tion by ubiquitin58, amyloidogenic proteins such as
`yeast prion s69,70 and polyglutamine model proteins (for
`example, Htt103Q — the first exon of huntingtin, with a
`stretch of 103 Gln residues)71–73. The pattern of aggrega-
`tion of these protein classes is diverse and there may be
`various ways of organizing them. For example, it was
`shown for some substrates of each class that they can
`localize, at least partially, to one of two recently identi-
`fied specialized quality-control compartments for the
`deposition of aggregated proteins58 (FIG. 2b). one com-
`partment adjacent to the nuclear membrane, termed the
`juxtanuclear quality-control compartment (JunQ), trans-
`iently accumulates misfolded proteins that are ubiquit-
`ylated and are presumably substrates for proteasomal
`degradation. Substrates at the JunQ are still mobile and
`exchange rapidly with the surrounding cytoplasm. The
`second compartment adjacent to the vacuole, termed
`the insoluble protein deposit (IPoD), harbours terminally
`aggregated, insoluble proteins, including carbonylation-
`sensitive proteins74 and amyloidogenic proteins such as
`Htt103Q or the yeast prions [RnQ] and [uRe3] (REF. 58).
`Strikingly, substrates for either compartment could be
`directed to the other compartment by experimentally
`manipulating the ubiquitin proteasome system. Impairing
`ubiquitylation of misfolded substrates — for example,
`through deletion of the E2 ubiquitin-conjugating enzyme
`pair ubc4–ubc5 — led substrates of the JunQ to be
`directed to the IPoD, whereas introducing a ubiquityla-
`tion site into otherwise typical IPoD substrates allowed
`targeting to the JunQ58. Thus, the overall picture is that
`ubiquitylated proteins that are usually substrates for
`proteasomal degradation can be stored reversibly in the
`JunQ compartment when the capacity of proteasomal
`
`degradation is limiting. by contrast, terminally misfolded
`proteins that are not usually turned over by the protea-
`some are deposited more permanently at the IPoD.
`Furthermore, the IPoD could serve as an overflow
`compartment when the ubiquitin proteasome system is
`overwhelmed. This raises several intriguing questions,
`foremost of which is the mechanism by which the mis-
`folded proteins are recognized, sorted and transported
`to the JunQ and IPoD. It is also not clear what the fate
`of the aggregated proteins in the IPoD and JunQ are.
`JunQ- and IPoD-like compartments have also been
`observed in mammalian cells58.
`
`The mammalian aggresome. A specialized form of
`inclusion bodies in the cytoplasm of mammalian cells is
`termed the aggresome75,76 (FIG. 2c). Aggresomes are not
`permanently present in the cell, but form in numerous
`disease states62,77, as a result of the expression of several
`heterologous proteins — including cystic fibrosis trans-
`membrane conductance regulator (CFTR)76 and a green
`fluorescent protein (GFP)–p115 chimaera75 — and on
`inhibition of the proteasome. Aggresomes localize to an
`indentation of the nuclear envelope at the micro tubule-
`organizing centre (MToC) and often surround the centri-
`ole. The exterior of aggresomes is sheeted by a cage-like
`shell formed by the intermediate filament vimentin.
`Their overall structure and size varies and depends on
`cell type and the aggregating substrate. Most aggresomes
`appear as a single sphere of 1–3 μm diameter or as an
`extended ribbon62,75–77.
`As shown for a GFP–p115 fusion protein, aggresome
`formation is initiated by the formation of smaller aggre-
`gates in the periphery, which then move in a dynein-
`based manner along the microtubule cytoskeleton to the
`final perinuclear site at the MToC75. Although ubiquityl-
`ation of the substrate is generally considered to be a pre-
`requisite for its recognition and transport to aggresomes,
`it could not be shown for all the substrates found in
`aggresomes, which leaves the possibility that signals other
`than ubiquitylation may also be involved62,77. The trans-
`port of aggregated proteins to aggresomes is mediated
`by histone deacetylase 6 (HDAC6), which functions as
`an adaptor that binds polyubiquitin chains of substrates
`and the microtubule motor protein dynein, thereby
`mediating the transport of polyubiquitylated cargo along
`microtubules towards the MToC during aggresome
`formation78 (FIG. 2c). The e3 ubiquitin ligase Parkin,
`which promotes the degradation of several substrates,
`has been suggested to recognize misfolded proteins and
`mark them by Lys63-linked polyubiquitylation29. This may
`provide one type of signal for HDAC6-dependent trans-
`port and sequestration of misfolded proteins to the aggre-
`some under conditions of proteasomal impairment, for
`example in pathogenic situations29,79.
`notably, HDAC6 has also been implicated in the regu-
`lation of cellular stress responses by being a component
`of a HDAC6–HSF1–HSP90 complex, which represses
`the stress response by trapping the transcription factor
`heat shock factor 1 (HSF1). The binding of HDAC6
`to ubiquitylated aggregates may cause the release and
`activ ation of HSF1. This offers the possibility of coupling
`
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`

`Table 1 | Molecular chaperones and proteases implicated in protein disaggregation
`chaperone
`organism
`Structure and
`aTP
`activity
`oligomeric state
`binding
`Hexamer
`Yes
`
`ClpB or
`Hsp104
`
`Bacteria, yeast, plants
`and mitochondria of
`animals
`
`R E V I E W S
`
`Reactivation of aggregated proteins in
`cooperation with an Hsp70 chaperone
`system
`
`Hsp70
`
`Bacteria, archaea and
`eukaryotes (cytosol,
`ER, mitochondria and
`chloroplasts)
`
`Monomer
`
`Yes
`
`sHSPs
`
`Bacteria, archaea and
`eukaryotes (cytosol)
`
`8–24-mer
`
`No
`
`Prevention of aggregation, reactivation
`of aggregated proteins in cooperation
`with ClpB or Hsp104, and folding of newly
`synthesized proteins and misfolded protein
`species
`Prevention of irreversible protein
`aggregation
`
`AAA+ proteases Bacteria and
`eukaryotes
`(mitochondria and
`chloroplasts)
`
`26S proteasome Eukaryotes (cytosol)
`
`Yes
`
`Yes
`
`Hexamer (for
`example, ClpA
`and ClpC) and
`heptamer (for
`example, ClpP)
`
`Hexamer (for
`AAA+ proteins)
`and heptamer (for
`α- and β-subunits)
`
`Degradation of misfolded or aggregated
`protein species and of native proteins
`harbouring specific degradation tags
`
`Degradation of polyubiquitylated proteins
`(including misfolded and native proteins
`harbouring specific degradation tags)
`
`VCP
`
`Eukaryotes (cytosol)
`
`Hexamer
`
`Yes
`
`Degradation of misfolded ER proteins and
`membrane fusion
`
`ER, endoplasmic reticulum; sHSP, small heat shock protein; VCP, valosin-containing protein.
`
`the sensing of aggregated proteins by HDAC6 to an
`increased chaperone expression, thereby initiating a
`cell ular response to counteract the accumulation of
`misfolded protein species80.
`
`Reversal of protein aggregation
`Aggregation is not necessarily a dead-end situation for
`a protein in vivo as disaggregation followed by refolding
`of aggregated proteins has been observed in cells from
`diverse species, from bacteria to humans. Disaggregation
`is not achieved by a single activity, but by different
`c ellular machineries, as summarized in TABLE 1.
`
`Protein disaggregation by a bi-chaperone system. The
`reversibility of protein aggregation was first demon-
`strated for S. cerevisiae33. Heat-aggregated proteins in the
`cytosol and nucleus are reactivated by the cooperative
`action of the Hsp70 system, composed of Ssa1 and the
`J protein co-chaperone yeast DnaJ protein 1 (Ydj1), and
`the oligomeric, ring-forming AAA+ chaperone Hsp104
`(REF. 81). A comparable activity exists in E. coli, provided
`by the bi-chaperone system of the corresponding ortho-
`logues of the Hsp104 (Clpb), Ssa1 (DnaK) and Ydj1
`(DnaJ) chaperones66,82. notably, each chaperone com-
`ponent on its own has only limited (the Hsp70 system)
`or no (Hsp104 and Clpb) disaggregation activity.
`
`This bi-chaperone system is also found in the cytosol
`of most other bacteria, plants and several unicellular
`eukaryotes, and in mitochondria and chloroplasts of
`unicellular and multicellular eukaryotes. Its activity
`efficiently counteracts the damaging effects of severe
`stress situations, and the induction of its expression by
`a sub-lethal heat treatment even enables cells to trans-
`iently survive a subsequent, severe heat shock treatment
`that is normally lethal, a phenomenon referred to as
`thermotolerance83–85. Further experiments showed that
`for E. coli and yeast cells the main reason for the loss of
`viability under such severe stress conditions is the mas-
`sive loss of protein activity by misfolding and aggrega-
`tion and that thermotolerance requires the bi-chaperone
`mediated reactivation, rather than the degradation, of
`aggregated proteins84,86.
`The mechanism of protein disaggregation by this
`bi-chaperone system involves an essential activity
`of the Hsp70 system during the initial phases of the
`process86,87 (FIG. 3). The binding of Hsp70 and J pro-
`teins to aggregates restricts the access of proteases to
`the aggregates and allows the transfer of aggregated
`polypeptides to the substrate-processing pore of Clpb
`or Hsp104 (REFS 88,89) (FIG. 3). These two functions
`combined provide a mechanism for pathway selection,
`in which the refolding pathway is preferred over the
`
`J protein
`A co-chaperone of HSP70
`chaperones that harbours a
`characteristic domain called a
`J domain and is responsible for
`activating the ATPase activity
`of HSP70.
`
`nATuRe RevIeWS | Molecular cell Biology
`
` voluMe 11 | noveMbeR 2010 | 781
`
`
`
`© 20 Macmillan Publishers Limited. All rights reserved10
`
`Page 5
`
`

`

`The role of sHSPs in organizing and solubilizing protein
`aggregates. The chaperone-mediated protein dis-
`aggregation process is further facilitated by sHSPs that
`directly interact with aggregating proteins92,93 (FIG. 4).
`For example, the E. coli sHSPs inclusion body-binding
`protein A (IbpA) and Ibpb were initially identified by
`their tight association with bacterial inclusion bodies94.
`sHSPs are the most widespread molecular chaper ones
`with an increasing number of family members in
`multicellular eukaryotes. They share the α-crystallin
`domain and also have variable extensions at the n and
`C termini93. Their synthesis and chaperone activity is
`often tightly controlled by temperature, allowing them
`to activate sHSP function on demand92,95,96. sHSPs bind
`tightly to misfolded protein species, resulting in the
`formation of sHSP–substrate complexes that do not
`release bound proteins spontaneously, thereby cre-
`ating a reservoir of misfolded proteins during stress
`conditions. Such complexes are still aggregates, but of
`reduced size and altered composition95,97. It is tempting
`to speculate that sHSPs might even function to seed
`the aggregation of misfolded protein species, thereby
`controlling the aggregation process in the cell and
`potentially even directing aggregates to specific
`cellular sites.
`sHSP-induced changes in aggregate architecture
`allow for more efficient disaggregation by the bi-
`chaperone system and contribute to the development
`of thermotolerance98–100. In addition, incorporation of
`sHSPs into aggregates allows for protein disaggrega-
`tion by the Hsp70 chaperone system97,101,102 (FIG. 4). The
`increased number of sHSP species in multicellular
`eukaryotes might potentially enable Hsp70 chaperones
`to work productively on aggregates even without the
`cooperation of an Hsp104-like AAA+ chaperone.
`
`Other disaggregation activities. In bacteria, several
`additional AAA+ chaperones (for example, ClpA, ClpC
`and Clpe from E. coli and Bacillus subtilis), which act
`in proteolysis by complex formation with peptidases
`(such as ClpP) (see TABLE 1), have been shown to possess
`a disaggregation activity in vitro, underlining the
`unique capacity of the AAA+ protein family to act on
`aggregates103,104. However, their contribution to protein
`disaggregation in vivo has not yet been fully clarified.
`It is remarkable that Clpb and Hsp104 homologues
`exist only in the mitochondria and chloroplasts of
`higher eukaryotes. organisms that encode cytosolic
`Clpb and Hsp104 homologues lack large-scale mobility
`and cannot escape exposure to sudden and severe
`environ mental changes such as thermal stress. The find-
`ing that the expression of Hsp104 leads to a substantial
`increase in the disaggregation activity in human cell
`lines and in C. elegans indicates that the disaggregation
`capacity is limited in such organisms105,106. nevertheless,
`various studies showed that animal cells can solubi-
`lize aggregates, demonstrating the existence of a dis-
`aggregation activity in the absence of Clpb or Hsp104
`(REFS 53,107,108). The identity of this activity is not yet
`clear, but may involve the action of Hsp70 chaperones
`and sHSPs.
`
`ATP
`ADP
`
`ATP
`ADP
`
`ATP
`ADP
`ClpB or Hsp104
`
`Loop
`segment
`
`ADP
`
`A T P
`
`R E V I E W S
`
`AAA+ protease
`
`ATP
`