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Protein destabilization by mutations or external stresses may lead to misfolding and aggregation in the cell. Often, damage is not limited to a simple loss of function, but the hydrophobic exposure of aggregate surfaces may impair membrane functions and promote the aggregation of other proteins. Such a "proteinacious infectious" behavior is not limited to prion diseases. It is associated to most protein-misfolding neurodegenerative diseases and to aging in general. With the molecular chaperones and proteases, cells have evolved powerful tools that can specifically recognize and act upon misfolded and aggregated proteins. Whereas some chaperones passively prevent aggregate formation and propagation, others actively unfold and solubilize stable aggregates. In particular, ATPase chaperones and proteases serve as an intracellular defense network that can specifically identify and actively remove by refolding or degradation potentially infectious cytotoxic aggregates. Here we discuss two types of molecular mechanisms by which ATPase chaperones may actively solubilize stable aggregates: (1) unfolding by power strokes, using the Hsp100 ring chaperones, and (2) unfolding by random movements of individual Hsp70 molecules. In bacteria, fungi, and plants, the two mechanisms are key for reducing protein damages from abiotic stresses. In animals devoid of Hsp100, Hsp70 appears as the core element of the chaperone network, preventing the formation and actively removing disease-causing protein aggregates.

Stable alpha-synuclein oligomers strongly inhibit chaperone activity of the Hsp70 system by weak interactions with J-domain co-chaperones.

α-Synuclein aggregation and accumulation in Lewy bodies are implicated in progressive loss of dopaminergic neurons in Parkinson disease and related disorders. In neurons, the Hsp70s and their Hsp40-like J-domain co-chaperones are the only known components of chaperone network that can use ATP to convert cytotoxic protein aggregates into harmless natively refolded polypeptides. Here we developed a protocol for preparing a homogeneous population of highly stable β-sheet enriched toroid-shaped α-Syn oligomers with a diameter typical of toxic pore-forming oligomers. These oligomers were partially resistant to in vitro unfolding by the bacterial Hsp70 chaperone system (DnaK, DnaJ, GrpE). Moreover, both bacterial and human Hsp70/Hsp40 unfolding/refolding activities of model chaperone substrates were strongly inhibited by the oligomers but, remarkably, not by unstructured α-Syn monomers even in large excess. The oligomers acted as a specific competitive inhibitor of the J-domain co-chaperones, indicating that J-domain co-chaperones may preferably bind to exposed bulky misfolded structures in misfolded proteins and, thus, complement Hsp70s that bind to extended segments. Together, our findings suggest that inhibition of the Hsp70/Hsp40 chaperone system by α-Syn oligomers may contribute to the disruption of protein homeostasis in dopaminergic neurons, leading to apoptosis and tissue loss in Parkinson disease and related neurodegenerative diseases.

Hsp70 chaperones accelerate protein translocation and the unfolding of stable protein aggregates by entropic pulling.

Hsp70s are highly conserved ATPase molecular chaperones mediating the correct folding of de novo synthesized proteins, the translocation of proteins across membranes, the disassembly of some native protein oligomers, and the active unfolding and disassembly of stress-induced protein aggregates. Here, we bring thermodynamic arguments and biochemical evidences for a unifying mechanism named entropic pulling, based on entropy loss due to excluded-volume effects, by which Hsp70 molecules can convert the energy of ATP hydrolysis into a force capable of accelerating the local unfolding of various protein substrates and, thus, perform disparate cellular functions. By means of entropic pulling, individual Hsp70 molecules can accelerate unfolding and pulling of translocating polypeptides into mitochondria in the absence of a molecular fulcrum, thus settling former contradictions between the power-stroke and the Brownian ratchet models for Hsp70-mediated protein translocation across membranes. Moreover, in a very different context devoid of membrane and components of the import pore, the same physical principles apply to the forceful unfolding, solubilization, and assisted native refolding of stable protein aggregates by individual Hsp70 molecules, thus providing a mechanism for Hsp70-mediated protein disaggregation.

Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis.

In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here "the chaperome," which can prevent formation of potentially harmful misfolded protein conformers and use the energy of adenosine triphosphate (ATP) to rehabilitate already formed toxic aggregates into native functional proteins. In an attempt to extend knowledge of chaperome mechanisms in cellular proteostasis, we performed a meta-analysis of human chaperome using high-throughput proteomic data from 11 immortalized human cell lines. Chaperome polypeptides were about 10 % of total protein mass of human cells, half of which were Hsp90s and Hsp70s. Knowledge of cellular concentrations and ratios among chaperome polypeptides provided a novel basis to understand mechanisms by which the Hsp60, Hsp70, Hsp90, and small heat shock proteins (HSPs), in collaboration with cochaperones and folding enzymes, assist de novo protein folding, import polypeptides into organelles, unfold stress-destabilized toxic conformers, and control the conformal activity of native proteins in the crowded environment of the cell. Proteomic data also provided means to distinguish between stable components of chaperone core machineries and dynamic regulatory cochaperones.

Biophysical characterization of two different stable misfolded monomeric polypeptides that are chaperone-amenable substrates.

Misfolded polypeptide monomers may be regarded as the initial species of many protein aggregation pathways, which could accordingly serve as primary targets for molecular chaperones. It is therefore of paramount importance to study the cellular mechanisms that can prevent misfolded monomers from entering the toxic aggregation pathway and moreover rehabilitate them into active proteins. Here, we produced two stable misfolded monomers of luciferase and rhodanese, which we found to be differently processed by the Hsp70 chaperone machinery and whose conformational properties were investigated by biophysical approaches. In spite of their monomeric nature, they displayed enhanced thioflavin T fluorescence, non-native β-sheets, and tertiary structures with surface-accessible hydrophobic patches, but differed in their conformational stability and aggregation propensity. Interestingly, minor structural differences between the two misfolded species could account for their markedly different behavior in chaperone-mediated unfolding/refolding assays. Indeed, only a single DnaK molecule was sufficient to unfold by direct clamping a misfolded luciferase monomer, while, by contrast, several DnaK molecules were necessary to unfold the more resistant misfolded rhodanese monomer by a combination of direct clamping and cooperative entropic pulling.

Soluble major histocompatibility complex-peptide octamers with impaired CD8 binding selectively induce Fas-dependent apoptosis.

Fluorescence-labeled soluble major histocompatibility complex class I-peptide "tetramers" constitute a powerful tool to detect and isolate antigen-specific CD8(+) T cells by flow cytometry. Conventional "tetramers" are prepared by refolding of heavy and light chains with a specific peptide, enzymatic biotinylation at an added C-terminal biotinylation sequence, and "tetramerization" by reaction with phycoerythrin- or allophycocyanin-labeled avidin derivatives. We show here that such preparations are heterogeneous and describe a new procedure that allows the preparation of homogeneous tetra- or octameric major histocompatibility complex-peptide complexes. These compounds were tested on T1 cytotoxic T lymphocytes (CTLs), which recognize the Plasmodium berghei circumsporzoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid on Lys(259) in the context of H-2K(d). We report that mutation of the CD8 binding site of K(d) greatly impairs the binding of tetrameric but not octameric or multimeric K(d)-PbCS(ABA) complexes to CTLs. This mutation abolishes the ability of the octamer to elicit significant phosphorylation of CD3, intracellular calcium mobilization, and CTL degranulation. Remarkably, however, this octamer efficiently activates CTLs for Fas (CD95)-dependent apoptosis.

Molecular chaperones as enzymes that catalytically unfold misfolded polypeptides.

Stress-denatured or de novo synthesized and translocated unfolded polypeptides can spontaneously reach their native state without assistance of other proteins. Yet, the pathway to native folding is complex, stress-sensitive and prone to errors. Toxic misfolded and aggregated conformers may accumulate in cells and lead to degenerative diseases. Members of the canonical conserved families of molecular chaperones, Hsp100s, Hsp70/110/40s, Hsp60/CCTs, the small Hsps and probably also Hsp90s, can recognize and bind with high affinity, abnormally exposed hydrophobic surfaces on misfolded and aggregated polypeptides. Binding to Hsp100, Hsp70, Hsp110, Hsp40, Hsp60, CCTs and Trigger factor may cause partial unfolding of the misfolded polypeptide substrates, and ATP hydrolysis can induce further unfolding and release from the chaperone, leading to spontaneous refolding into native proteins with low-affinity for the chaperones. Hence, specific chaperones act as catalytic polypeptide unfolding isomerases, rerouting cytotoxic misfolded and aggregated polypeptides back onto their physiological native refolding pathway, thus averting the onset of protein conformational diseases.

L'agrégation toxique des protéines: une forme de "delinquance moléculaire" activement combattue dans la cellule par les chaperones moléculaires et les protéases [The toxic aggregation of proteins: a kind of "molecular delinquency" actively fought in the cell by molecular chaperones and proteases]

Under various stresses, mutation-sensitised proteins may spontaneously convert into inactive, aggregation-prone structures, which may be cytotoxic and infectious. In the cell, this new kind of "molecular criminality" is actively fought against by a network of molecular chaperones that can specifically identify, isolate and unfold damaged (delinquent) proteins and favour their subsequent native refolding. Irreversibly damaged molecules unable to natively refold are preferentially "executed" and recycled by proteases. Failing that, they are "imprisoned" within compact amyloids, or "evicted" from the cell. Thus, striking parallels, although of questionable ethical value, exist between protein and human criminality, and between the cellular and social responses to these different types of criminality. Fundamental differences also exist. Whereas programmed death (apoptosis) is the preferred solution chosen by aged and aggregation-stressed cells, collective suicide is seldom an option chosen by lawless human societies. More significantly, there is no clear cellular equivalent for the role of the family and the education system, which are so essential to the proper shaping of functional individuals in the society, and give rise to humanism, that favours crime prevention, reeducation and reinsertion programs over capital punishment. To the cardiologist and transplantation surgeon, the interest of molecular chaperones, in particular of Hsp70, Hsp90 and Hsp27, lays in their ability to inhibit the signalling pathway of programmed cell death. Their induction before and during ischemia, by various treatments and drugs could significantly reduce damages from the post ischemic reperfusion of organs.

Heavy metal ions are potent inhibitors of protein folding.

Environmental and occupational exposure to heavy metals such as cadmium, mercury and lead results in severe health hazards including prenatal and developmental defects. The deleterious effects of heavy metal ions have hitherto been attributed to their interactions with specific, particularly susceptible native proteins. Here, we report an as yet undescribed mode of heavy metal toxicity. Cd2+, Hg2+ and Pb2+ proved to inhibit very efficiently the spontaneous refolding of chemically denatured proteins by forming high-affinity multidentate complexes with thiol and other functional groups (IC(50) in the nanomolar range). With similar efficacy, the heavy metal ions inhibited the chaperone-assisted refolding of chemically denatured and heat-denatured proteins. Thus, the toxic effects of heavy metal ions may result as well from their interaction with the more readily accessible functional groups of proteins in nascent and other non-native form. The toxic scope of heavy metals seems to be substantially larger than assumed so far.

The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase.

Hsp70-Hsp40-NEF and possibly Hsp100 are the only known molecular chaperones that can use the energy of ATP to convert stably pre-aggregated polypeptides into natively refolded proteins. However, the kinetic parameters and ATP costs have remained elusive because refolding reactions have only been successful with a molar excess of chaperones over their polypeptide substrates. Here we describe a stable, misfolded luciferase species that can be efficiently renatured by substoichiometric amounts of bacterial Hsp70-Hsp40-NEF. The reactivation rates increased with substrate concentration and followed saturation kinetics, thus allowing the determination of apparent V(max)' and K(m)' values for a chaperone-mediated renaturation reaction for the first time. Under the in vitro conditions used, one Hsp70 molecule consumed five ATPs to effectively unfold a single misfolded protein into an intermediate that, upon chaperone dissociation, spontaneously refolded to the native state, a process with an ATP cost a thousand times lower than expected for protein degradation and resynthesis.

Physical interaction between bacterial heat shock protein (Hsp) 90 and Hsp70 chaperones mediates their cooperative action to refold denatured proteins.

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.

Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses.

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.

Study of biomacromolecule conformational changes by Scanning Force Microscopy

L?objectif de ce travail de thèse est l?étude des changements conformationels des biomacromolecules à l?échelle d?une molécule unique. Pour cela on a utilisé la Microscopie à Force Atomique (AFM) appliqué à l?étude des protéines et des acides nucléiques déposés sur une surface. Dans ce type de microscopie, une pointe très fine attachée à l?extrémité d?un levier est balayée au dessus d?une surface. L?interaction de la pointe avec la surface de l?échantillon induit la déflection du levier et ce phénomène permet de reconstruire la topographie de l?échantillon. Très importante dans cette technique est la possibilité de travailler en liquide. Cela permet de étudier les biomolécules en conditions quasi-physiologiques sans qu?elles perdent leur activité. On a étudié GroEL, la chaperonin de E.coli, qui est un homo oligomère avec une structure à double anneau qui joue un rôle très important dans le repliement des protéines dénaturées et celles qui viennent d?être synthétisées. En particulier on a focalisé notre attention sur la stabilité mécanique et sur les changements conformationels qui ont lieu pendant l?activité de GroEL. Une analyse détaillée des changements dans la stabilité mécanique et des effets produits par la liaison et l?hydrolyse de l?ATP est présentée dans ce travail. On a montré que le point le plus faible dans la structure de GroEL est l?interface entre les deux anneaux et que l?étape critique dans l?affaiblissement de la structure est l?hydrolyse de l?ATP. En ce qui concerne le changement conformationel, le passage d?une surface hydrophobe à hydrophile, induit par l?hydrolyse de l?ATP, a été montré. Ensuite on a étudié le changement dans la conformation et dans la topologie de l?ADN résultant de l?interaction avec des molécules spécifiques et en réponse à l?exposition des cellules de E.coli à des conditions de stress. Le niveau de surenroulement est un paramètre très sensible, de façon variée, à tous ces facteurs. Les cellules qui ont crus à de températures plus élevées que leur température optimale ont la tendance à diminuer le nombre de surenroulements négatif pour augmenter la stabilité thermique de leur plasmides. L?interaction avec des agents intercalant induit une transition d?un surenroulement négatif à un surenroulement positif d?une façon dépendante de la température. Finalement, l?effet de l?interaction de l?ADN avec des surfaces différentes a été étudié et une application pratique sur les noeuds d?ADN est présentée.<br/><br/>The aim of the present thesis work is to study the conformational changes of biomacromolecules at the single molecule level. To that end, Atomic Force Microcopy (AFM) imaging was performed on proteins and nucleic acids adsorbed onto a surface. In this microcopy technique a very sharp tip attached at the end of a soft cantilever is scanned over a surface, the interaction of the tip with the sample?s surface will induce the deflection of the cantilever and thus it will make possible to reconstruct the topography. A very important feature of AFM is the possibility to operate in liquid, it means with the sample immersed in a buffer solution. This allows one to study biomolecules in quasi-physiological conditions without loosing their activity. We have studied GroEL, the chaperonin of E.coli, which is a double-ring homooligomer which pays a very important role in the refolding of unfolded and newly synthetized polypeptides. In particular we focus our attention on its mechanical stability and on the conformational change that it undergoes during its activity cycle. A detailed analysis of the change in mechanical stability and how it is affected by the binding and hydrolysis of nucleotides is presented. It has been shown that the weak point of the chaperonin complex is the interface between the two rings and that the critical step to weaken the structure is the hydrolysis of ATP. Concerning the conformational change we have directly measured, with a nanometer scale resolution, the switching from a hydrophobic surface to a hydrophilic one taking place inside its cavity induced by the ATP hydrolysis. We have further studied the change in the DNA conformation and topology as a consequence of the interaction with specific DNA-binding molecules and the exposition of the E.coli cells to stress conditions. The level of supercoiling has been shown to be a very sensitive parameter, even if at different extents, to all these factors. Cells grown at temperatures higher than their optimum one tend to decrease the number of the negative superhelical turns in their plasmids in order to increase their thermal stability. The interaction with intercalating molecules induced a transition from positive to negative supercoiling in a temperature dependent way. The effect of the interaction of the DNA with different surfaces has been investigated and a practical application to DNA complex knots is reported.<br/><br/>Observer les objets biologiques en le touchant Schématiquement le Microscope a Force Atomique (AFM) consiste en une pointe très fine fixée a l?extrémité d?un levier Lors de l?imagerie, la pointe de l?AFM gratte la surface de l?échantillon, la topographie de celui-ci induit des déflections du levier qui sont enregistrées au moyen d?un rayon laser réfléchi par le levier. Ces donnés sont ensuit utilisés par un ordinateur pour reconstituer en 3D la surface de l?échantillon. La résolution de l?instrument est fonction entre autre de la dureté, de la rugosité de l?échantillon et de la forme de la pointe. Selon l?échantillon et la pointe utilisée la résolution de l?AFM peut aller de 0.1 A (sur des cristaux) a quelque dizaine de nanomètres (sur des cellules). Cet instrument est particulierment intéressant en biologie en raison de sa capacité à imager des échantillons immergés dans un liquide, c?est à dire dans des conditions quasiphysiologiques. Dans le cadre de ce travail nous avons étudié les changements conformationels de molécules biologiques soumises à des stimulations externes. Nous avons essentielment concentré notre attention sur des complexes protéiques nommé Chaperons Moléculaires et sur des molécules d?ADN circulaire (plasmides). Les Chaperons sont impliqués entre autre dans la résistance des organismes vivants aux stress thermiques et osmotiques. Leur activité consiste essentielment à aider les autres protéines à être bien pliés dans leur conformation finale et, en conséquence, à eviter que ils soient dénaturées et que ils puissent s?agréger. L?ADN, quant à lui est la molécule qui conserve, dans sa séquence, l?information génétique de tous les organismes vivants. Ce travail a spécifiquement concerné l?étude des changements conformationels des chaperonins suit a leur activation par l?ATP. Ces travaux ont montrés a l?échelle de molécule unique la capacité de ces protéines de changer leur surface de hydrophobique a hydrophilique. Nous avons également utilisé l?AFM pour étudier le changement du nombre des surenroulements des molécules d?ADN circulaire lors d?une exposition à un changement de température et de force ionique. Ces travaux ont permis de montrer comment la cellule regle le nombre de surenroulements dans ces molécules pour répondre et contrôler l?expression génétique même dans de conditions extrêmes. Pour les deux molécules en général, c?était très important d?avoir la possibilité de observer leur transitions d?une conformation a l?autre directement a l?échelle d?une seul molécule et, surtout, avec une résolution largement au dessous des la longueur d?onde de la lumière visible que représente le limite pour l?imagerie optique.

The Sottunga-Jurmo shear zone : structure and deformation history of a crustal-scale ductile shear zone in SW Finland

The aim of this study is to gain a better understanding of the structure and the deformation history of a NW-SE trending regional, crustal-scale shear structure in the Åland archipelago, SW Finland, called the Sottunga-Jurmo shear zone (SJSZ). Approaches involving e.g. structural geology, geochronology, geochemistry and metamorphic petrology were utilised in order to reconstruct the overall deformation history of the study area. The study therefore describes several features of the shear zone including structures, kinematics and lithologies within the study area, the ages of the different deformation phases (ductile to brittle) within the shear zone, as well as some geothermobarometric results. The results indicate that the SJSZ outlines a major crustal discontinuity between the extensively migmatized rocks NE of the shear zone and the unmigmatised, amphibolite facies rocks SW of the zone. The main SJSZ shows overall dextral lateral kinematics with a SW-side up vertical component and deformation partitioning into pure shear and simple shear dominated deformation styles that was intensified toward later stages of the deformation history. The deformation partitioning resulted in complex folding and refolding against the SW margin of the SJSZ, including conical and sheath folds, and in a formation of several minor strike-slip shear zones both parallel and conjugate to the main SJSZ in order to accommodate the regional transpressive stresses. Different deformation phases within the study area were dated by SIMS (zircon U-Pb), ID-TIMS (titanite U-Pb) and 40Ar/39Ar (pseudotachylyte wholerock) methods. The first deformation phase within the ca. 1.88 Ga rocks of the study area is dated at ca. 1.85 Ga, and the shear zone was reactivated twice within the ductile regime (at ca. 1.83 Ga and 1.79 Ga), during which the strain was successively increasingly partitioned into the main SJSZ and the minor shear zones. The age determinations suggest that the orogenic processes within the study area did not occur in a temporal continuum; instead, the metamorphic zircon rims and titanites show distinct, 10-20 Ma long breaks in deformation between phases of active deformation. The results of this study further imply slow cooling of the rocks through 600-700ºC so that at 1.79 Ga, 2 the temperature was still at least 600ºC. The highest recorded metamorphic pressures are 6.4-7.1 kbar. At the late stages or soon after the last ductile phase (ca. 1.79 Ga), relatively high-T mylonites and ultramylonites were formed, witnessing extreme deformation partitioning and high strain rates. After the rocks reached lower amphibolite facies to amphibolite-greenschist facies transitional conditions (ca. 500-550ºC), they cooled rapidly, probably due to crustal uplift and exhumation. The shear zone was reactivated at least once within the semi-brittle to brittle regime between ca. 1.79 Ga and 1.58 Ga, as evidenced by cataclasites and pseudotachylytes. In summary, the results of this study suggest that the Sottunga-Jurmo shear zone (and the South Finland shear zone) defines a major crustal discontinuity, and played a central role in accommodating the regional stresses during and after the Svecofennian orogeny.

Synergism between a foldase and an unfoldase: reciprocal dependence between the thioredoxin-like activity of DnaJ and the polypeptide-unfolding activity of DnaK.

The role of bacterial Hsp40, DnaJ, is to co-chaperone the binding of misfolded or alternatively folded proteins to bacterial Hsp70, DnaK, which is an ATP-fuelled unfolding chaperone. In addition to its DnaK targeting activity, DnaJ has a weak thiol-reductase activity. In between the substrate-binding domain and the J-domain anchor to DnaK, DnaJ has a unique domain with four conserved CXXC motives that bind two Zn(2+) and partly contribute to polypeptide binding. Here, we deleted in DnaJ this Zn-binding domain, which is characteristic to type I but not of type II or III J-proteins. This caused a loss of the thiol-reductase activity and strongly reduced the ability of DnaJ to mediate the ATP- and DnaK-dependent unfolding/refolding of mildly oxidized misfolded polypeptides, an inhibition that was alleviated in the presence of thioredoxin or DTT. We suggest that in addition to their general ability to target misfolded polypeptide substrates to the Hsp70/Hsp110 chaperone machinery, Type I J-proteins carry an ancillary protein dithiol-isomerase function that can synergize the unfolding action of the chaperone, in the particular case of substrates that are further stabilized by non-native disulfide bonds. Whereas the unfoldase can remain ineffective without the transient untying of disulfide bonds by the foldase, the foldase can remain ineffective without the transient ATP-fuelled unfolding of wrong local structures by the unfoldase.