The mechanism of Hsp70 chaperones: (entropic) pulling the models together.

Hsp70s are conserved molecular chaperones that can prevent protein aggregation, actively unfold, solubilize aggregates, pull translocating proteins across membranes and remodel native proteins complexes. Disparate mechanisms have been proposed for the various modes of Hsp70 action: passive prevention of aggregation by kinetic partitioning, peptide-bond isomerase, Brownian ratcheting or active power-stroke pulling. Recently, we put forward a unifying mechanism named 'entropic pulling', which proposed that Hsp70 uses the energy of ATP hydrolysis to recruit a force of entropic origin to locally unfold aggregates or pull proteins across membranes. The entropic pulling mechanism reproduces the expected phenomenology that inspired the other disparate mechanisms and is, moreover, simple.

Structure and kinematics of the Jungfrau syncline, Faflertal (Valais, Alps), and its regional significance

The formation and structural evolution of the jungrau syncline is described, based on excellent outcrops occurring in the lotschental, in the central alps of switzerland. the quality of the outcrops allows us to demonstrate that the external massifs of the swiss alps have developed due to internal folding. The jungfrau suncline, which separates the autochtonous gastern dome from the aar massif basement gneiss folds, is composed of slivers of basement rocks with their mesozoic sedimentary cover. in the inner faflertal, a side valley of the lotschental, the 200 m thick syncline cp, roses fpir imots, the gastern massif with a reduced mesozoic sedimentary cover in a normal stratigraphic succession, two units of overturned basement rocks with their mesozoic sedimentary cover, and the overturned lower limn of the tschingelhorn gneiss fold of the aar massif with lenses of its sedimentary cover. stratigraphy shows that the lower units, related to the gastern massis, are condensed and that the upper units, deposited farther away from a gastern paleo-high, form a more complete sequence, linked to the doldenhorn meso-cneozoic basin fill. the integration of these local observations with published regional data leads to the following model. on the northern margin of the doldenhorn hbasin, at the northern fringe of the alpine tethuys, the pre-triassic crystalline basement and its mesozoic sedimentary cover were folded by ductile deformation at temperatures above 300 degrees C and in the presence of high fluid pressures, as the helveti c and penninic nappes were overthrusted towards the northwest during the main alpine deformation phase, the visosity contrast between the basement gneisses and the sediments caused the formation of large basement anticlines and tight sedimentary sunclines (mullion-type structures). The edges of basement blocks bounded buy pre-cursor se-dipping normal faults at the northwestern border of the doldenhorn basin were deformed bu simple shear, creating overturned slices of crystalline rocks with their sedimentary cover in what now forms the hungfrau syncline. the localisation of ductile deformation in the vicinity of pre-existing se-dipping faults is thought to have been helped by the circulation of fluids along the faults; these fluids would have been released from the mesozoic sediments by metamorphic dehydration reactions accompanied by creep and dynamic recrystallisation of quartz at temperatures above 300 degrees C. Quantification of the deformation suggests an strain ellipsoid with a ratio (1 + e(1)/+ e(3)) of approximately 1000. The jungfrau suncline was deformed bu more brittle nw-directed shear creating well-developed shear band cleavages at a late stage, after cooling by uplift and erosion. It is suggested that the external massifs of the apls are basement gneiss folds created at temperatures of 300 degrees C by detachment through ductile deformation of the upper crust of the european plate as it was underthrusted below the adriatic plate.

Molecular crime and cellular punishment: active detoxification of misfolded and aggregated proteins in the cell by the chaperone and protease networks.

Labile or mutation-sensitised proteins may spontaneously convert into aggregation-prone conformations that may be toxic and infectious. This hazardous behavior, which can be described as a form of "molecular criminality", can be actively counteracted in the cell by a network of molecular chaperone and proteases. Similar to law enforcement agents, molecular chaperones and proteases can specifically identify, apprehend, unfold and thus neutralize "criminal" protein conformers, allowing them to subsequently refold into harmless functional proteins. Irreversibly damaged polypeptides that have lost the ability to natively refold are preferentially degraded by highly controlled ATP-consuming proteases. Damaged proteins that escape proteasomal degradation can also be "incarcerated" into dense amyloids, "evicted" from the cell, or internally "exiled" to the lysosome to be hydrolysed and recycled. Thus, remarkable parallels exist between molecular and human forms of criminality, as well as in the cellular and social responses to various forms of crime. Yet, differences also exist: whereas programmed death is the preferred solution chosen by aged and aggregation-stressed cells, collective suicide is seldom chosen by lawless societies. Significantly, there is no cellular equivalent for the role of familial care and of education in general, which is so crucial to the proper shaping of functional persons in the society. Unlike in the cell, humanism introduces a bias against radical solutions such as capital punishment, favouring crime prevention, reeducation and social reinsertion of criminals.

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.

Comparative functional analysis of two fibroblast growth factor receptor 1 (FGFR1) mutations affecting the same residue (R254W and R254Q) in isolated hypogonadotropic hypogonadism (IHH).

FGFR1 mutations have been identified in both Kallmann syndrome and normosmic HH (nIHH). To date, few mutations in the FGFR1 gene have been structurally or functionally characterized in vitro to identify molecular mechanisms that contribute to the disease pathogenesis. We attempted to define the in vitro functionality of two FGFR1 mutants (R254W and R254Q), resulting from two different amino acid substitutions of the same residue, and to correlate the in vitro findings to the patient phenotypes. Two unrelated GnRH deficient probands were found to harbor mutations in FGFR1 (R254W and R254Q). Mutant signaling activity and expression levels were evaluated in vitro and compared to a wild type (WT) receptor. Signaling activity was determined by a FGF2/FGFR1 dependent transcription reporter assay. Receptor total expression levels were assessed by Western blot and cell surface expression was measured by a radiolabeled antibody binding assay. The R254W maximal receptor signaling capacity was reduced by 45% (p<0.01) while R254Q activity was not different from WT. However, both mutants displayed diminished total protein expression levels (40 and 30% reduction relative to WT, respectively), while protein maturation was unaffected. Accordingly, cell surface expression levels of the mutant receptors were also significantly reduced (35% p<0.01 and 15% p<0.05, respectively). The p.R254W and p.R254Q are both loss-of-function mutations as demonstrated by their reduced overall and cell surface expression levels suggesting a deleterious effect on receptor folding and stability. It appears that a tryptophan substitution at R254 is more disruptive to receptor structure than the more conserved glutamine substitution. No clear correlation between the severity of in vitro loss-of-function and phenotypic presentation could be assigned.

HDLs protect the MIN6 insulinoma cell line against tunicamycin-induced apoptosis without inhibiting ER stress and without restoring ER functionality.

HDLs protect pancreatic beta cells against apoptosis induced by several endoplasmic reticulum (ER) stressors, including thapsigargin, cyclopiazonic acid, palmitate and insulin over-expression. This protection is mediated by the capacity of HDLs to maintain proper ER morphology and ER functions such as protein folding and trafficking. Here, we identified a distinct mode of protection exerted by HDLs in beta cells challenged with tunicamycin (TM), a protein glycosylation inhibitor inducing ER stress. HDLs were found to inhibit apoptosis induced by TM in the MIN6 insulinoma cell line and this correlated with the maintenance of a normal ER morphology. Surprisingly however, this protective response was neither associated with a significant ER stress reduction, nor with restoration of protein folding and trafficking in the ER. These data indicate that HDLs can use at least two mechanisms to protect beta cells against ER stressors. One that relies on the maintenance of ER function and one that operates independently of ER function modulation. The capacity of HDLs to activate several anti-apoptotic pathways in beta cells may explain their ability to efficiently protect these cells against a variety of insults.

Dissociation from BiP and Retrotranslocation of Unassembled Immunoglobulin Light Chains Are Tightly Coupled to Proteasome Activity

Unassembled immunoglobulin light chains expressed by the mouse plasmacytoma cell line NS1 (KNS1) are degraded in vivo with a half-life of 50-60 min in a way that closely resembles endoplasmic reticulum (ER)-associated degradation (Knittler et al., 1995). Here we show that the peptide aldehydes MG132 and PS1 and the specific proteasome inhibitor lactacystin effectively increased the half-life of KNS1, arguing for a proteasome-mediated degradation pathway. Subcellular fractionation and protease protection assays have indicated an ER localization of KNS1 upon proteasome inhibition. This was independently confirmed by the analysis of the folding state of KNS1and size fractionation experiments showing that the immunoglobulin light chain remained bound to the ER chaperone BiP when the activity of the proteasome was blocked. Moreover, kinetic studies performed in lactacystin-treated cells revealed a time-dependent increase in the physical stability of the BiP-KNS1complex, suggesting that additional proteins are present in the older complex. Together, our data support a model for ER-associated degradation in which both the release of a soluble nonglycosylated protein from BiP and its retrotranslocation out of the ER are tightly coupled with proteasome activity.

Targeting endoplasmic reticulum signaling pathways in cancer.

The endoplasmic reticulum (ER) orchestrates the production of membrane-bound and secreted proteins. However, its capacity to process the synthesis and folding of protein is limited. Protein overload and the accumulation of misfolded proteins in the ER trigger an adaptive response known as the ER-stress response that is mediated by specific ER-anchored signaling pathways. This response regulates cell functions aimed at restoring cellular homeostasis or at promoting apoptosis of irreparably damaged cells. Activation or deregulation of ER-signaling pathways has been associated with various diseases including cancer. Here we discuss how tumors engage ER-signaling pathways to promote tumorigenesis and how manipulation of this process by anticancer drugs may contribute to cancer treatment.

Meta-structure correlation in protein space unveils different selection rules for folded and intrinsically disordered proteins

The number of existing protein sequences spans a very small fraction of sequence space. Natural proteins have overcome a strong negative selective pressure to avoid the formation of insoluble aggregates. Stably folded globular proteins and intrinsically disordered proteins (IDP) use alternative solutions to the aggregation problem. While in globular proteins folding minimizes the access to aggregation prone regions IDPs on average display large exposed contact areas. Here, we introduce the concept of average meta-structure correlation map to analyze sequence space. Using this novel conceptual view we show that representative ensembles of folded and ID proteins show distinct characteristics and responds differently to sequence randomization. By studying the way evolutionary constraints act on IDPs to disable a negative function (aggregation) we might gain insight into the mechanisms by which function - enabling information is encoded in IDPs.

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.

Structural analysis of Turtle Mountain: Origin and influence of fractures in the development of rock slope failures

Large slope failures in fractured rocks are often controlled by the combination of pre-existing tectonic fracturing and brittle failure propagation in the intact rock mass during the pre-failure phase. This study focuses on the influence of fold-related fractures and of post-folding fractures on slope instabilities with emphasis on Turtle Mountain, located in SW Alberta (Canada). The structural features of Turtle Mountain, especially to the south of the 1903 Frank Slide, were investigated using a high-resolution digital elevation model combined with a detailed field survey. These investigations allowed the identification of six main discontinuity sets influencing the slope instability and surface morphology. According to the different deformation phases affecting the area, the potential origin of the detected fractures was assessed. Three discontinuity sets are correlated with the folding phase and the others with post-folding movements. In order to characterize the rock mass quality in the different portions of the Turtle Mountain anticline, the geological strength index (GSI) has been estimated. The GSI results show a decrease in rock mass quality approaching the fold hinge area due to higher fracture persistence and higher weathering. These observations allow us to propose a model for the potential failure mechanisms related to fold structures.

Mechanistic insights into cytosolic molecular chaperones in protein unfolding and disaggregation

Under optimal non-physiological conditions of low concentrations and low temperatures, proteins may spontaneously fold to the native state, as all the information for folding lies in the amino acid sequence of the polypeptide. However, under conditions of stress or high protein crowding as inside cells, a polypeptide may misfold and enter an aggregation pathway resulting in the formation of misfolded conformers and fibrils, which can be toxic and lead to neurodegenerative illnesses, such as Alzheimer's, Parkinson's or Huntington's diseases and aging in general. To avert and revert protein misfolding and aggregation, cells have evolved a set of proteins called molecular chaperones. Here, I focussed on the human cytosolic chaperones Hsp70 (DnaK) and HspllO, and co-chaperone Hsp40 (DnaJ), and the chaperonin CCT (GroEL). The cytosolic molecular chaperones Hsp70s/Hspll0s and the chaperonins are highly upregulated in bacterial and human cells under different stresses and are involved both in the prevention and the reversion of protein misfolding and aggregation. Hsp70 works in collaboration with Hsp40 to reactivate misfolded or aggregated proteins in a strict ATP dependent manner. Chaperonins (CCT and GroEL) also unfold and reactivate stably misfolded proteins but we found that it needed to use the energy of ATP hydrolysis in order to evict over- sticky misfolded intermediates that inhibited the unfoldase catalytic sites. Ill In this study, we initially characterized a particular type of inactive misfolded monomeric luciferase and rhodanese species that were obtained by repeated cycles of freeze-thawing (FT). These stable misfolded monomeric conformers (FT-luciferase and FT-rhodanese) had exposed hydrophobic residues and were enriched with wrong ß-sheet structures (Chapter 2). Using FT-luciferase as substrate, we found that the Hsp70 orthologs, called HspllO (Sse in yeast), acted similarly to Hsp70 as were bona fide ATP- fuelled polypeptide unfoldases and was much more than a mere nucleotide exchange factor, as generally thought. Moreover, we found that HspllO collaborated with Hsp70 in the disaggregation of stable protein aggregates in which Hsp70 and HspllO acted as equal partners that synergistically combined their individual ATP-consuming polypeptide unfoldase activities to reactivate the misfolded/aggregated proteins (Chapter 3). Using FT-rhodanese as substrate, we found that chaperonins (GroEL and CCT) could catalytically reactivate misfolded rhodanese monomers in the absence of ATP. Also, our results suggested that encaging of an unfolding polypeptide inside the GroEL cavity under a GroES cap was not an obligatory step as generally thought (Chapter 4). Further, we investigated the role of Hsp40, a J-protein co-chaperone of Hsp70, in targeting misfolded polypeptides substrates onto Hsp70 for unfolding. We found that even a large excess of monomeric unfolded a-synuclein did not inhibit DnaJ, whereas, in contrast, stable misfolded a-synuclein oligomers strongly inhibited the DnaK-mediated chaperone reaction by way of sequestering the DnaJ co-chaperone. This work revealed that DnaJ could specifically distinguish, and bind potentially toxic stably aggregated species, such as soluble a-synuclein oligomers involved in Parkinson's disease, and with the help of DnaK and ATP convert them into from harmless natively unfolded a-synuclein monomers (chapter 5). Finally, our meta-analysis of microarray data of plant and animal tissues treated with various chemicals and abiotic stresses, revealed possible co-expressions between core chaperone machineries and their co-chaperone regulators. It clearly showed that protein misfolding in the cytosol elicits a different response, consisting of upregulating the synthesis mainly of cytosolic chaperones, from protein misfolding in the endoplasmic reticulum (ER) that elicited a typical unfolded protein response (UPR), consisting of upregulating the synthesis mainly of ER chaperones. We proposed that drugs that best mimicked heat or UPR stress at increasing the chaperone load in the cytoplasm or ER respectively, may prove effective at combating protein misfolding diseases and aging (Chapter 6).  - Dans les conditions optimales de basse concentration et de basse température, les protéines vont spontanément adopter un repliement natif car toutes les informations nécessaires se trouvent dans la séquence des acides aminés du polypeptide. En revanche, dans des conditions de stress ou de forte concentration des protéines comme à l'intérieur d'une cellule, un polypeptide peu mal se replier et entrer dans un processus d'agrégation conduisant à la formation de conformères et de fibrilles qui peuvent être toxiques et causer des maladies neurodégénératives comme la maladie d'Alzheimer, la maladie de Parkinson ou la chorée de Huntington. Afin d'empêcher ou de rectifier le mauvais repliement des protéines, les cellules ont développé des protéines appelées chaperonnes. Dans ce travail, je me suis intéressé aux chaperonnes cytosoliques Hsp70 (DnaK) et HspllO, la co-chaperones Hsp40 (DnaJ), le complexe CCT/TRiC et GroEL. Chez les bactéries et les humains, les chaperonnes cytosoliques Hsp70s/Hspl 10s et les « chaperonines» sont fortement activées par différentes conditions de stress et sont toutes impliquées dans la prévention et la correction du mauvais repliement des protéines et de leur agrégation. Hsp70 collabore avec Hsp40 pour réactiver les protéines agrégées ou mal repliées et leur action nécessite de 1ATP. Les chaperonines (GroEL) déplient et réactivent aussi les protéines mal repliées de façon stable mais nous avons trouvé qu'elles utilisent l'ATP pour libérer les intermédiaires collant et mal repliés du site catalytique de dépliage. Nous avons initialement caractérisé un type particulier de formes stables de luciférase et de rhodanese monomériques mal repliées obtenues après plusieurs cycles de congélation / décongélation répétés (FT). Ces monomères exposaient des résidus hydrophobiques et étaient plus riches en feuillets ß anormaux. Ils pouvaient cependant être réactivés par les chaperonnes Hsp70+Hsp40 (DnaK+DnaJ) et de l'ATP, ou par Hsp60 (GroEL) sans ATP (Chapitre 2). En utilisant la FT-Luciferase comme substrat nous avons trouvé que HspllO (un orthologue de Hsp70) était une authentique dépliase, dépendante strictement de l'ATP. De plus, nous avons trouvé que HspllO collaborait avec Hsp70 dans la désagrégation d'agrégats stables de protéines en combinant leurs activités dépliase consommatrice d'ATP (Chapitre 3). En utilisant la FT-rhodanese, nous avons trouvé que les chaperonines (GroEL et CCT) pouvaient réactiver catalytiquement des monomères mal repliés en absence d'ATP. Nos résultats suggérèrent également que la capture d'un polypeptide en cours de dépliement dans la cavité de GroEL et sous un couvercle du complexe GroES ne serait pas une étape obligatoire du mécanisme, comme il est communément accepté dans la littérature (Chapitre 4). De plus, nous avons étudié le rôle de Hsp40, une co-chaperones de Hsp70, dans l'adressage de substrats polypeptidiques mal repliés vers Hsp70. Ce travail a révélé que DnaJ pouvait différencier et lier des polypeptide mal repliés (toxiques), comme des oligomères d'a-synucléine dans la maladie de Parkinson, et clairement les différencier des monomères inoffensifs d'a-synucléine (Chapitre 5). Finalement une méta-analyse de données de microarrays de tissus végétaux et animaux traités avec différents stress chimiques et abiotiques a révélé une possible co-expression de la machinerie des chaperonnes et des régulateurs de co- chaperonne. Cette meta-analyse montre aussi clairement que le mauvais repliement des protéines dans le cytosol entraîne la synthèse de chaperonnes principalement cytosoliques alors que le mauvais repliement de protéines dans le réticulum endoplasmique (ER) entraine une réponse typique de dépliement (UPR) qui consiste principalement en la synthèse de chaperonnes localisées dans l'ER. Nous émettons l'hypothèse que les drogues qui reproduisent le mieux les stress de chaleur ou les stress UPR pourraient se montrer efficaces dans la lutte contre le mauvais repliement des protéines et le vieillissement (Chapitre 6).

Characterisation of the putative effector interaction site of the regulatory HbpR protein from Pseudomonas azelaica by site-directed mutagenesis.

Bacterial transcription activators of the XylR/DmpR subfamily exert their expression control via σ(54)-dependent RNA polymerase upon stimulation by a chemical effector, typically an aromatic compound. Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown. Here we focus on the HbpR protein from Pseudomonas azelaica, which is a member of the XylR/DmpR subfamily and responds to biaromatic effectors such as 2-hydroxybiphenyl. We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR. A large number of site-directed HbpR mutants of residues in- and outside the predicted interaction area was created and their potential to induce reporter gene expression in Escherichia coli from the cognate P(C) promoter upon activation with 2-hydroxybiphenyl was studied. Mutant proteins were purified to study their conformation. Critical residues for effector stimulation indeed grouped near the predicted area, some of which are conserved among XylR/DmpR subfamily members in spite of displaying different effector specificities. This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition.

Chaperones and proteases: cellular fold-controlling factors of proteins in neurodegenerative diseases and aging.

The formation of toxic protein aggregates is a common denominator to many neurodegenerative diseases and aging. Accumulation of toxic, possibly infectious protein aggregates induces a cascade of events, such as excessive inflammation, the production of reactive oxygen species, apoptosis and neuronal loss. A network of highly conserved molecular chaperones and of chaperone-related proteases controls the fold-quality of proteins in the cell. Most molecular chaperones can passively prevent protein aggregation by binding misfolding intermediates. Some molecular chaperones and chaperone-related proteases, such as the proteasome, can also hydrolyse ATP to forcefully convert stable harmful protein aggregates into harmless natively refoldable, or protease-degradable, polypeptides. Molecular chaperones and chaperone-related proteases thus control the delicate balance between natively folded functional proteins and aggregation-prone misfolded proteins, which may form during the lifetime and lead to cell death. Abundant data now point at the molecular chaperones and the proteases as major clearance mechanisms to remove toxic protein aggregates from cells, delaying the onset and the outcome of protein-misfolding diseases. Therapeutic approaches include treatments and drugs that can specifically induce and sustain a strong chaperone and protease activity in cells and tissues prone to toxic protein aggregations.