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).

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.

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.

Hsp70 and the Cochaperone StiA (Hop) Orchestrate Hsp90-Mediated Caspofungin Tolerance in Aspergillus fumigatus.

Aspergillus fumigatus is the primary etiologic agent of invasive aspergillosis (IA), a major cause of death among immunosuppressed patients. Echinocandins (e.g., caspofungin) are increasingly used as second-line therapy for IA, but their activity is only fungistatic. Heat shock protein 90 (Hsp90) was previously shown to trigger tolerance to caspofungin and the paradoxical effect (i.e., decreased efficacy of caspofungin at higher concentrations). Here, we demonstrate the key role of another molecular chaperone, Hsp70, in governing the stress response to caspofungin via Hsp90 and their cochaperone Hop/Sti1 (StiA in A. fumigatus). Mutation of the StiA-interacting domain of Hsp70 (C-terminal EELD motif) impaired thermal adaptation and caspofungin tolerance with loss of the caspofungin paradoxical effect. Impaired Hsp90 function and increased susceptibility to caspofungin were also observed following pharmacologic inhibition of the C-terminal domain of Hsp70 by pifithrin-μ or after stiA deletion, further supporting the links among Hsp70, StiA, and Hsp90 in governing caspofungin tolerance. StiA was not required for the physical interaction between Hsp70 and Hsp90 but had distinct roles in the regulation of their function in caspofungin and heat stress responses. In conclusion, this study deciphering the physical and functional interactions of the Hsp70-StiA-Hsp90 complex provided new insights into the mechanisms of tolerance to caspofungin in A. fumigatus and revealed a key C-terminal motif of Hsp70, which can be targeted by specific inhibitors, such as pifithrin-μ, to enhance the antifungal activity of caspofungin against A. fumigatus.

Sponge Mass Mortalities in a Warming Mediterranean Sea: Are Cyanobacteria-Harboring Species Worse Off?

Mass mortality events are increasing dramatically in all coastal marine environments. Determining the underlying causes of mass mortality events has proven difficult in the past because of the lack of prior quantitative data on populations and environmental variables. Four-year surveys of two shallow-water sponge species, Ircinia fasciculata and Sarcotragus spinosulum, were carried out in the western Mediterranean Sea. These surveys provided evidence of two severe sponge die-offs (total mortality ranging from 80 to 95% of specimens) occurring in the summers of 2008 and 2009. These events primarily affected I. fasciculata, which hosts both phototrophic and heterotrophic microsymbionts, while they did not affect S. spinosulum, which harbors only heterotrophic bacteria. We observed a significant positive correlation between the percentage of injured I. fasciculata specimens and exposure time to elevated temperature conditions in all populations, suggesting a key role of temperature in triggering mortality events. A comparative ultrastructural study of injured and healthy I. fasciculata specimens showed that cyanobacteria disappeared from injured specimens, which suggests that cyanobacterial decay could be involved in I. fasciculata mortality. A laboratory experiment confirmed that the cyanobacteria harbored by I. fasciculata displayed a significant reduction in photosynthetic efficiency in the highest temperature treatment. The sponge disease reported here led to a severe decrease in the abundance of the surveyed populations. It represents one of the most dramatic mass mortality events to date in the Mediterranean Sea

Cyanobacteria from the Baltic Sea and Finnish lakes as an energy source and modulators of bioenergetic pathways

Cyanobacteria are a diverse group of oxygenic photosynthetic bacteria that inhabit in a wide range of environments. They are versatile and multifaceted organisms with great possibilities for different biotechnological applications. For example, cyanobacteria produce molecular hydrogen (H2), which is one of the most important alternatives for clean and sustainable energy. Apart from being beneficial, cyanobacteria also possess harmful characteristics and may become a source of threat to human health and other living organisms, as they are able to form surface blooms that are producing a variety of toxic or bioactive compounds. The University of Helsinki Culture Collection (UHCC) maintains around 1,000 cyanobacterial strains representing a large number of genera and species isolated from the Baltic Sea and Finnish lakes. The culture collection covers different life forms such as unicellular and filamentous, N2-fixing and non-N2-fixing strains, and planktonic and benthic cyanobacteria. In this thesis, the UHCC has been screened to identify potential strains for sustainable biohydrogen production and also for strains that produce compounds modifying the bioenergetic pathways of other cyanobacteria or terrestrial plants. Among the 400 cyanobacterial strains screened so far, ten were identified as high H2-producing strains. The enzyme systems involved in H2 metabolism of cyanobacteria were analyzed using the Southern hybridization approach. This revealed the presence of the enzyme nitrogenase in all strains tested, while none of them are likely to have contained alternative nitrogenases. All the strains tested, except for two Calothrix strains, XSPORK 36C and XSPORK 11A, were suggested to contain both uptake and bidirectional hydrogenases. Moreover, 55 methanol extracts of various cyanobacterial strains were screened to identify potent bioactive compounds affecting the photosynthetic apparatus of the model cyanobacterium, Synechocystis PCC 6803. The extract from Nostoc XPORK 14A was the only one that modified the photosynthetic machinery and dark respiration. The compound responsible for this effect was identified, purified, and named M22. M22 demonstrated a dual-action mechanism: production of reactive oxygen species (ROS) under illumination and an unknown mechanism that also prevailed in the dark. During summer, the Baltic Sea is occupied by toxic blooms of Nodularia spumigena (hereafter referred to as N. spumigena), which produces a hepatotoxin called nodularin. Long-term exposure of the terrestrial plant spinach to nodularin was studied. Such treatment resulted in inhibition of growth and chlorosis of the leaves. Moreover, the activity and amount of mitochondrial electron transfer complexes increased in the leaves exposed to nodularin-containing extract, indicating upregulation of respiratory reactions, whereas no marked changes were detected in the structure or function of the photosynthetic machinery. Nodularin-exposed plants suffered from oxidative stress, evidenced by oxidative modifications of various proteins. Plants initiated strategies to combat the stress by increasing the levels of alpha-tocopherol, mitochondrial alternative oxidase (AOX), and mitochondrial ascorbate peroxidase (mAPX).

Algas planctônicas do reservatório da Pampulha (MG): Euglenophyta, Chrysophyta, Pyrrophyta, Cyanobacteria

Neste trabalho foram descritos 28 táxons de algas planctônicas pertencentes às divisões Euglenophyta, Chrysophyta, Pyrrophyta e Cyanobacteria, encontradas no reservatório da Pampulha, estado de Minas Gerais. Foram analisadas amostras no período de maio/1992 a fevereiro/1997. Em número de espécies, Euglenophyta foi a que apresentou maior número (13 espécies em uma família), seguidas de Cyanobacteria (oito espécies), Pyrrophyta (cinco espécies) e Chrysophyta (duas espécies). Vinte e três táxons são primeiras citações para o estado de Minas Gerais.

Cylindrospermopsis raciborskii (Woloszynska) Seenayya & Subba Raju(Cyanobacteria): variação semanal e relações com fatores ambientais em um reservatório eutrófico, São Paulo, SP, Brasil

Florações de Cylindrospermopsis raciborskii têm sido cada vez mais freqüentes em reservatórios brasileiros em virtude da sua alta competitividade em ambientes tropicais eutrofizados. Esta espécie é produtora de toxinas extremamente agressivas, as quais podem causar problemas de saúde pública e danos ao meio ambiente. Nosso objetivo foi acompanhar a variação temporal da densidade desta população relacionando-a com variáveis ambientais e detectando os possíveis fatores que interferem no seu desenvolvimento. O lago estudado localiza-se na zona sul do Município de São Paulo (23º39' S e 46º37' W). É um corpo d'água eutrofizado e vem apresentando florações freqüentes de C. raciborskii. Amostras de água foram coletadas semanalmente, na parte mais profunda do lago, durante o período de setembro/97 a setembro/98. Foram analisados o perfil térmico, zona de mistura, transparência, zona eufótica, turbidez, pH, alcalinidade, condutividade, oxigênio dissolvido, nutrientes, clorofila a, densidade da população e densidade total da comunidade fitoplanctônica. A ordenação dos dados abióticos e bióticos foi feita através da análise de componentes principais (PCA) e análise de correspondência canônica (CCA). Durante os meses de novembro e dezembro/97 foram registrados os mais altos valores de densidade de C. raciborskii. Entre 10/12 e 17/12, a espécie contribuiu com 50% para a densidade total da comunidade. Nesse período foram registradas estratificações térmicas, elevados valores de temperatura da água e de turbidez e baixos valores de transparência da água. Os meses de outono e inverno de 1998 foram caracterizados pela ausência da espécie estudada, desestratificação térmica da coluna d'água, baixas temperaturas da água, altos valores de transparência (os maiores já registrados para o ambiente) e pelo aumento considerável dos bancos da macrófita aquática flutuante Eichhornia crassipes (Mart.) Solms. Tais interrelações são discutidas no presente trabalho.

Planktic Cyanobacteria from São Paulo State, Brazil: Chroococcales

The biodiversity studies of planktic cyanobacteria started in 1997 with intensive collecting in different water bodies of São Paulo State. Due to the problems brougth by eutrophication and cyanobacterial blooms, the samplings were concentrated in reservoirs of Alto Tietê region that supply drinking water to millions of people. The samples were collected with 20 µm plankton net or Van Dorn's bottle. Part of each one was preserved in formaldehyde or lugol solution and part was isolated. The culture strains were kept in BG11 and/or AMS1 media. Based on natural and culture material, 26 species were identified belonging to the families Chroococcaceae (2 taxa), Merismopediaceae (12), Microcystaceae (7) and Synechococcaceae (5). Among these species, six are potentialy toxic: Aphanocapsa incerta (Lemmerm.) Cronberg & Komárek, Microcystis aeruginosa (Kütz.) Kütz., M. botrys Teiling, M. panniformis Komárek et al., M. wesenbergii (Komárek) Komárek, and Radiocystis fernandoi Komárek & Komár.-Legn. Bacularia and Coelosphaeriopsis are genera reported for the first time in Brazil.

Effect of nitrate and ammonium on the growth and protein concentration of Microcystis viridis Lemmermann (Cyanobacteria)

Cyanobacteria are a very important group in aquatic systems, particularly in eutrophic waters. Therefore studies about their success in the environment are essential. Many hypotheses have tried to explain the dominance of Cyanobacteria, and several emphasized the importance of various nitrogen sources for the success of the group. In this study, we measured the effect of ammonium and nitrate on the growth and protein concentration of Microcystis viridis (Cyanobacteria). This species is well-known because bloom formation in eutrophic waters. The study was carried out, in experimental batch cultures, using the WC medium with different nitrogen sources: ammonium, nitrate, ammonium + nitrate (50% ammonium + 50% nitrate) and ammonium at different concentrations (to test for possible NH4+ toxicity). Protein, ammonium and nitrate concentrations were measured at end of each experiment, whereas samples for cell counts were taken daily. Results showed that Microcystis viridis grew faster with ammonium (µ = 0.393 day-1) than with nitrate (µ = 0.263 day-1) and ammonium + nitrate (µ = 0.325 day-1). This pattern is explained by the metabolism of ammonium that presents higher uptake and assimilation rates than nitrate. Maximum cell concentration, however, was higher in the ammonium + nitrate treatment, followed by nitrate treatment. Higher protein concentration were observed in the treatment with nitrate. In the ammonium toxicity test, no difference between the control and NH4+ at 50% was found. Thus, the ammonium concentrations used in these experiments were not toxic. Our results suggest that Cyanobacteria is able to grow on both nitrogen sources even if ammonium may allow faster growth and bloom development.

Occurrence of the rare genus Microcrocis P. Richter (Chroococcales, Cyanobacteria) in a coastal lagoon from southern Brazil

The paper records the first occurrence of the genus Microcrocis P. Richter (Chroococcales, Cyanobacteria), represented by M. pulchella (Buell) Geitler, in Brazil. The species was found in two zones, one with freshwater and the other with brackish water, of a coastal lagoon of Rio Grande do Sul State (31°15’-31°30’ S and 50°54’-51°09’ W). Comparison between M. pulchella and its most closely related species is presented. Up to now this species had occurrence records limited to freshwater systems in temperate regions. Its presence in a subtropical coastal lagoon from southernmost Brazil, either in fresh or in brackish water, broadened the knowledge of the distribution area of M. pulchella.

Planktic Cyanobacteria from upper Tietê basin reservoirs, SP, Brazil

Considering the great ecological and sanitary importance of the cyanobacteria and the need of detailed information about these organisms in Brazilian water bodies, the present study aims at contributing towards the knowledge of the cyanobacterial flora of five reservoirs belonging to the upper Tietê Basin, São Paulo: Billings, Guarapiranga, Jundiaí, Pirapora, Ponte Nova and Taiaçupeba. In the past several years, these reservoirs have been submitted to severe environmental deterioration and have repeatedly presented cyanobacterial blooms, including those of toxic species. The samples were collected between 1997 and 2003 either with plankton net (20 µm mesh) or van Dorn's bottle, and preserved with lugol solution or formaldehyde. Some species were isolated and maintained in culture. Forty-eight species of cyanobacteria were identified, with predominance of the order Chroococcales (58%), followed by the orders Oscillatoriales (21%) and Nostocales (21%). Among the 48 studied species, 17 (35%) were considered potentially toxic. The occurrence and biodiversity of the cyanobacteria in each reservoir depend on the environmental conditions. Among the five water bodies, Billings Reservoir presented the most adequate situation for the development of a greater number of species (34), probably due to its high pH values (around 8). Pirapora Reservoir on the other hand, with highest conductivity (445.0 µS cm-1) and lowest Secchi depth values (0.2 m), presented the lowest cyanobacterial biodiversity (14 species).

Cyanoaggregatum brasiliense gen. et sp. nov., a new chroococcal Cyanobacteria from Southern Brazil

The paper proposed Cyanoaggregatum brasiliense, a new genus and species from the plankton of a subtropical brackish coastal lagoon from Rio Grande do Sul State, South Brazil. It differs from all other members of Chroococcales by its characteristic arrangement of cells in irregular groups distributed in a single, flat or slightly curved layer, on irregular rows, slightly distant from each other, forming a mosaic-like pattern slightly below the surface of the mucilaginous colonial envelope. The cell division in one plane perpendicular to the long axis and the lack of pseudo-filaments indicate its classification in the family Synechococcaceae, sub-family Aphanothecoideae. The general characteristics, the diagnostic criteria and the taxonomic position are discussed, and a comparison between Cyanoaggregatum and its most closely related genera is presented. Physical and chemical data on the studied lagoon and geographical distribution are presented.