Engineering a commercial cold-adapted xylanase for improved low pH stability

Dissertação de mestrado em Genética Molecular

Recombinant " Physcomitrella patens " as a sensitive tool to study heat sensing and heat-shock signal transduction in plants

SUMMARY When exposed to heat stress, plants display a particular set of cellular and molecular responses, such as chaperones expression, which are highly conserved in all organisms. In chapter 1, I studied the ability of heat shock genes to become transiently and abundantly induced under various temperature regimes. To this aim, I designed a highly sensitive heat-shock dependent conditional gene expression system in the moss Physcomitrella patens, using the soybean heatinducible promoter (hsp17.3B). Heat-induced expression of various reporter genes was over three orders of magnitude, in tight correlation with the intensity and duration of the heat treatments. By performing repeated heating/cooling cycles, a massive accumulation of recombinant proteins was obtained. Interestingly, the hsp17.3B promoter was also activated by specific organic chemicals. Thus, in chapter 2, I took advantage of the extreme sensitivity of this promoter to small temperature variations to further address the role of various natural and organic chemicals and develop a plant based-bioassay that can serve as an early warning indicator of toxicity by pollutants and heavy metals. A screen of several organic pollutants from textile and paper industry showed that chlorophenols as well as sulfonated anthraquinones elicited a heat shock like response at noninducing temperatures. Their effects were synergistically amplified by mild elevated temperatures. In contrast to standard methods of pollutant detection, this plant-based biosensor allowed to monitor early stress-responses, in correlation with long-term toxic effect, and to attribute effective toxicity thresholds for pollutants, in a context of varying environmental cues. In chapter 3, I deepened the study of the primary mechanism by which plants sense mild temperature variations and trigger a cellular signal leading to the heat shock response. In addition to the above described heat-inducible reporter line, I generated a P. patens transgenic line to measure, in vivo, variations of cytosolic calcium during heat treatment, and another line to monitor the role of protein unfolding in heat-shock sensing and signalling. The heat shock signalling pathway was found to be triggered by the plasma membrane, where temperature up shift specifically induced the transient opening of a putative high afimity calcium channel. The calcium influx triggered a signalling cascade leading to the activation of the heat shock genes, independently on the presence of misfolded proteins in the cytoplasm. These results strongly suggest that changes in the fluidity of the plasma membrane are the primary trigger of the heatshocksignalling pathway in plants. The present thesis contributes to the understanding of the basic mechanism by which plants perceive and respond to heat and chemical stresses. This may contribute to developing appropriate better strategies to enhance plant productivity under the increasingly stressful environment of global warming. RÉSUME Les plantes exposées à des températures élevées déclenchent rapidement des réponses cellulaires qui conduisent à l'induction de gènes codant pour les heat shock proteins (HSPs). En fonction de la durée d'exposition et de la vitesse à laquelle la température augmente, les HSPs sont fortement et transitoirement induites. Dans le premier chapitre, cette caractéristique aété utilisée pour développer un système inductible d'expression de gènes dans la mousse Physcomitrella patens. En utilisant plusieurs gènes rapporteurs, j'ai montré que le promoteur du gène hsp17.3B du Soja est activé d'une manière. homogène dans tous les tissus de la mousse proportionnellement à l'intensité du heat shock physiologique appliqué. Un très fort taux de protéines recombinantes peut ainsi être produit en réalisant plusieurs cycles induction/recovery. De plus, ce promoteur peut également être activé par des composés organiques, tels que les composés anti-inflammatoires, ce qui constitue une bonne alternative à l'induction par la chaleur. Les HSPs sont induites pour remédier aux dommages cellulaires qui surviennent. Étant donné que le promoteur hsp17.3B est très sensible à des petites augmentations de température ainsi qu'à des composés chimiques, j'ai utilisé les lignées développées dans le chapitre 1 pour identifier des polluants qui déclenchent une réaction de défense impliquant les HSPs. Après un criblage de plusieurs composés, les chlorophénols et les antraquinones sulfonés ont été identifiés comme étant activateurs du promoteur de stress. La détection de leurs effets a été réalisée seulement après quelques heures d'exposition et corrèle parfaitement avec les effets toxiques détectés après de longues périodes d'exposition. Les produits identifiés montrent aussi un effet synergique avec la température, ce qui fait du biosensor développé dans ce chapitre un bon outil pour révéler les effets réels des polluants dans un environnement où les stress chimiques sont combinés aux stress abiotiques. Le troisième chapitre est consacré à l'étude des mécanismes précoces qui permettent aux plantes de percevoir la chaleur et ainsi de déclencher une cascade de signalisation spécifique qui aboutit à l'induction des gènes HSPs. J'ai généré deux nouvelles lignées afin de mesurer en temps réel les changements de concentrations du calcium cytosolique ainsi que l'état de dénaturation des protéines au cours du heat shock. Quand la fluidité de la membrane augmente après élévation de la température, elle semble induire l'ouverture d'un canal qui permet de faire entrer le calcium dans les cellules. Ce dernier initie une cascade de signalisation qui finit par activer la transcription des gènes HSPs indépendamment de la dénaturation de protéines cytoplasmiques. Les résultats présentés dans ce chapitre montrent que la perception de la chaleur se fait essentiellement au niveau de la membrane plasmique qui joue un rôle majeur dans la régulation des gènes HSPs. L'élucidation des mécanismes par lesquels les plantes perçoivent les signaux environnementaux est d'une grande utilité pour le développement de nouvelles stratégies afin d'améliorer la productivité des plantes soumises à des conditions extrêmes. La présente thèse contribue à décortiquer la voie de signalisation impliquée dans la réponse à la chaleur.

The heat shock response in moss plants is regulated by specific calcium-permeable channels in the plasma membrane.

Land plants are prone to strong thermal variations and must therefore sense early moderate temperature increments to induce appropriate cellular defenses, such as molecular chaperones, in anticipation of upcoming noxious temperatures. To investigate how plants perceive mild changes in ambient temperature, we monitored in recombinant lines of the moss Physcomitrella patens the activation of a heat-inducible promoter, the integrity of a thermolabile enzyme, and the fluctuations of cytoplasmic calcium. Mild temperature increments, or isothermal treatments with membrane fluidizers or Hsp90 inhibitors, induced a heat shock response (HSR) that critically depended on a preceding Ca(2+) transient through the plasma membrane. Electrophysiological experiments revealed the presence of a Ca(2+)-permeable channel in the plasma membrane that is transiently activated by mild temperature increments or chemical perturbations of membrane fluidity. The amplitude of the Ca(2+) influx during the first minutes of a temperature stress modulated the intensity of the HSR, and Ca(2+) channel blockers prevented HSR and the onset of thermotolerance. Our data suggest that early sensing of mild temperature increments occurs at the plasma membrane of plant cells independently from cytosolic protein unfolding. The heat signal is translated into an effective HSR by way of a specific membrane-regulated Ca(2+) influx, leading to thermotolerance.

The membrane-associated transient receptor potential vanilloid channel is the central heat shock receptor controlling the cellular heat shock response in epithelial cells.

The heat shock response (HSR) is a highly conserved molecular response to various types of stresses, including heat shock, during which heat-shock proteins (Hsps) are produced to prevent and repair damages in labile proteins and membranes. In cells, protein unfolding in the cytoplasm is thought to directly enable the activation of the heat shock factor 1 (HSF-1), however, recent work supports the activation of the HSR via an increase in the fluidity of specific membrane domains, leading to activation of heat-shock genes. Our findings support the existence of a plasma membrane-dependent mechanism of HSF-1 activation in animal cells, which is initiated by a membrane-associated transient receptor potential vanilloid receptor (TRPV). We found in various non-cancerous and cancerous mammalian epithelial cells that the TRPV1 agonists, capsaicin and resiniferatoxin (RTX), upregulated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70 and Hsp90 respectively, while the TRPV1 antagonists, capsazepine and AMG-9810, attenuated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70, Hsp90, respectively. Capsaicin was also shown to activate HSF-1. These findings suggest that heat-sensing and signaling in mammalian cells is dependent on TRPV channels in the plasma membrane. Thus, TRPV channels may be important drug targets to inhibit or restore the cellular stress response in diseases with defective cellular proteins, such as cancer, inflammation and aging.

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.

Regulation of innate immunity by signaling pathways emerging from the endoplasmic reticulum.

The innate immune system has evolved the capacity to detect specific pathogens and to interrogate cell and tissue integrity in order to mount an appropriate immune response. Loss of homeostasis in the endoplasmic reticulum (ER) triggers the ER-stress response, a hallmark of many inflammatory and infectious diseases. The IRE1/XBP1 branch of the ER-stress signaling pathway has been recently shown to regulate and be regulated by innate immune signaling pathways in both the presence and absence of ER-stress. By contrast, innate immune pathways negatively affect the activation of two other branches of the ER-stress response as evidenced by reduced expression of the pro-apoptotic transcription factor CHOP. Here we will discuss how innate immune pathways and ER-signaling intersect to regulate the intensity and duration of innate immune responses.

Structural study of BSA/poly(ethylene glycol) lipid conjugate complexes

In this work we report the structural characteristics of bovine serum albumin/poly(ethylene glycol) lipid conjugate (BSA/PEG(2000)-PE) complexes under physiological conditions (37 degrees C and pH 7.4) for particular fractions of BSA to PEG-lipid concentration, CBSA/C-PEG2000-PE. Ultraviolet fluorescence spectroscopy (UV) results shown that PEG(2000)-PE is associated to BSA, leading to;protein unfolding for fixed C-BSA = 0.01 wt % and variable C-PEG2000-PE = 0.0015-0.6 wt %. Tryptophan groups on the BSA surface are in contact with the PEG-lipid at C-PEG2000-PE = 0.0015 wt %, while they are exposed to water at C-PEG2000-PE (>)0.0015 wt %. Dynamic and static light scattering (DLS and SLS) and small-angle neutron scattering (SANS) point out the existence of individual BSAIPEG-lipid complexes in the system for fixed C-BSA = 1 wt % and variable C-PEG2000-PE = 0.15-2 wt %. DLS shows that there is only one BSA molecule per protein/PEG-lipid complex, while SLS shows that the PEG-lipid associates to the BSA without promoting aggregation between adjacent protein/ polymer-lipid conjugate complexes. SANS was used to show that BSA/PEG(2000)-PE complexes adopt an oblate ellipsoidal shape. Partially unfolded BSA is contained in the core of the oblate ellipsoid, which is surrounded by an external shell containing the PEG(2000)-PE.

Pressure-jump X-ray studies of liquid crystal transitions in lipids

In this paper, we give an overview of our studies by static and time-resolved X-ray diffraction of inverse cubic phases and phase transitions in lipids. In 1, we briefly discuss the lyotropic phase behaviour of lipids, focusing attention on non-lamellar structures, and their geometric/topological relationship to fusion processes in lipid membranes. Possible pathways for transitions between different cubic phases are also outlined. In 2, we discuss the effects of hydrostatic pressure on lipid membranes and lipid phase transitions, and describe how the parameters required to predict the pressure dependence of lipid phase transition temperatures can be conveniently measured. We review some earlier results of inverse bicontinuous cubic phases from our laboratory, showing effects such as pressure-induced formation and swelling. In 3, we describe the technique of pressure-jump synchrotron X-ray diffraction. We present results that have been obtained from the lipid system 1:2 dilauroylphosphatidylcholine/lauric acid for cubic-inverse hexagonal, cubic-cubic and lamellar-cubic transitions. The rate of transition was found to increase with the amplitude of the pressure-jump and with increasing temperature. Evidence for intermediate structures occurring transiently during the transitions was also obtained. In 4, we describe an IDL-based 'AXCESS' software package being developed in our laboratory to permit batch processing and analysis of the large X-ray datasets produced by pressure-jump synchrotron experiments. In 5, we present some recent results on the fluid lamellar-Pn3m cubic phase transition of the single-chain lipid 1-monoelaidin, which we have studied both by pressure-jump and temperature-jump X-ray diffraction. Finally, in 6, we give a few indicators of future directions of this research. We anticipate that the most useful technical advance will be the development of pressure-jump apparatus on the microsecond time-scale, which will involve the use of a stack of piezoelectric pressure actuators. The pressure-jump technique is not restricted to lipid phase transitions, but can be used to study a wide range of soft matter transitions, ranging from protein unfolding and DNA unwinding and transitions, to phase transitions in thermotropic liquid crystals, surfactants and block copolymers.

Diphtheria toxoid conformation in the context of its nanoencapsulation within liposomal particles sandwiched by chitosan

Chitosan (alpha alpha-(1-4)-amino-2-deoxy-beta beta-D-glucan) is a deacetylated form of chitin, a polysaccharide from crustacean shells. Its unique characteristics, such as positive charge, biodegradability, biocompatibility, nontoxicity, and rigid structure, make this macromolecule ideal for an oral vaccine delivery system. We prepared reverse-phase evaporation vesicles (REVs) sandwiched by chitosan (Chi) and polyvinylic alcohol (PVA). However, in this method, there are still some problems to be circumvented related to protein stabilization. During the inverted micelle phase of protein nanoencapsulation, hydrophobic interfaces are expanded, leading to interfacial adsorption, followed by protein unfolding and aggregation. Here, spectroscopic and immunological techniques were used to ascertain the effects of the Hoffmeister series ions on diphtheria toxoid (Dtxd) stability during the inverted micelle phase. A correlation was established between the salts used in aqueous solutions and the changes in Dtxd solubility and conformation. Dtxd alpha alpha-helical content was quite stable, which led us to conclude that encapsulation occurred without protein aggregation or without exposition of hydrophobic residues. Dtxd aggregation was 98% avoided by the kosmotropic, POprotein use.

Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A(2) from Bothrops pirajai venom

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Sodium dodecyl sulfate (SDS) effect on the thermal stability of oxy-HbGp: Dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) studies

Glossoscolex paulistus (HbGp) hemoglobin is an oligomeric protein, presenting a quaternary structure constituted by 144 globin and 36 non-globin chains (named linkers) with a total molecular mass of 3.6MDa. SDS effects on the oxy-HbGp thermal stability were studied, by DLS and SAXS, at pH 5.0, 7.0 and 9.0. DLS and SAXS data show that the SDS-oxy-HbGp interactions induce a significant decrease of the protein thermal stability, with the formation of larger aggregates, at pH 5.0. At pH 7.0, oxy-HbGp undergoes complete oligomeric dissociation, with increase of temperature, in the presence of SDS. Besides, oxy-HbGp 3.0mg/mL, pH 7.0, in the presence of SDS, has the oligomeric dissociation process reduced as compared to 0.5mg/mL of protein. At pH 9.0, oxy-HbGp starts to dissociate at 20°C, and the protein is totally dissociated at 50°C. The thermal dissociation kinetic data show that oxy-HbGp oligomeric dissociation at pH 7.0, in the presence of SDS, is strongly dependent on the protein concentration. At 0.5mg/mL of protein, the oligomeric dissociation is complete and fast at 40 and 42°C, with kinetic constants of (2.1±0.2)×10-4 and (5.5±0.4)×10-4s-1, respectively, at 0.6mmol/L SDS. However, at 3.0mg/mL, the oligomeric dissociation process starts at 46°C, and only partial dissociation, accompanied by aggregates formation is observed. Moreover, our data show, for the first time, that, for 3.0mg/mL of protein, the oligomeric dissociation, denaturation and aggregation phenomena occur simultaneously, in the presence of SDS. Our present results on the surfactant-HbGp interactions and the protein thermal unfolding process correspond to a step forward in the understanding of SDS effects. © 2013 Elsevier B.V.

Effect of Chlorogenic Acid (5-Caffeoylquinic Acid) Isolated from Baccharis oxyodonta on the Structure and Pharmacological Activities of Secretory Phospholipase A2 from Crotalus durissus terrificus

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La spettroscopia di forza basata sull'AFM nello studio dello spazio conformazionale e dei processi aggregativi di proteine prioniche

Le malattie neurodegenerative sono caratterizzate da aggregazione proteica, dipendente dalla perdita della usuale struttura fisiologica funzionale delle proteine coinvolte, a favore di conformazioni tossiche (patologiche). Il modello corrente descrive questi cambiamenti conformazionali come eventi rari e ritiene che non esista una sola conformazione patogena, ma che tali possibili conformazioni siano piuttosto eterogenee. La caratterizzazione di queste strutture è, di conseguenza, difficile con le tradizionali tecniche in bulk che permettono di studiare solo la conformazione media e non rendono possibile il riconoscimento delle caratteristiche dei conformeri individuali. Lo sviluppo delle tecniche di singola molecola ha permesso di studiare in modo approfondito le conformazioni possibili. In questo lavoro la spettroscopia di forza di singola molecola basata sull'AFM viene applicata a PrP (proteina responsabile delle encefalopatie spongiformi trasmissibili). Si studiano gli equilibri conformazionali del monomero e quelli di costrutti oligomerici, allo scopo di caratterizzare gli step iniziali dei processi aggregativi. Nel corso di questo lavoro di tesi è stato, in particolare, sviluppato un sistema di analisi dati, al fine di studiare in modo quantitativo le distribuzioni di eventi ottenute. Grazie a tale strumento è stato possibile riconoscere i segnali di unfolding della conformazione nativa del monomero e notare come essa sia presente anche in costrutti oligomerici, ad indicare come questo ripiegamento sia stabile anche in presenza di più monomeri ravvicinati. Si è osservato l'effetto del pH sulla stabilità di tale struttura, notando come pH acidi destabilizzino il ripiegamento nativo. Inoltre si è studiato il ruolo dell'orientazione dei monomeri nella formazione di strutture dimeriche. Monomeri e oligomeri di PrP sono stati descritti come proteine parzialmente strutturate il cui panorama energetico contiene molti minimi locali, dando origine a parecchie conformazioni transienti.

Phloem sap proteins from Cucurbita maxima and Ricinus communis have the capacity to traffic cell to cell through plasmodesmata

In angiosperms, the functional enucleate sieve tube system of the phloem appears to be maintained by the surrounding companion cells. In this study, we tested the hypothesis that polypeptides present within the phloem sap traffic cell to cell from the companion cells, where they are synthesized, into the sieve tube via plasmodesmata. Coinjection of fluorescently labeled dextrans along with size-fractionated Cucurbita maxima phloem proteins, ranging in size from 10 to 200 kDa, as well as injection of individual fluorescently labeled phloem proteins, provided unambiguous evidence that these proteins have the capacity to interact with mesophyll plasmodesmata in cucurbit cotyledons to induce an increase in size exclusion limit and traffic cell to cell. Plasmodesmal size exclusion limit increased to greater than 20 kDa, but less than 40 kDa, irrespective of the size of the injected protein, indicating that partial protein unfolding may be a requirement for transport. A threshold concentration in the 20–100 nM range was required for cell-to-cell transport indicating that phloem proteins have a high affinity for the mesophyll plasmodesmal binding site(s). Parallel experiments with glutaredoxin and cystatin, phloem sap proteins from Ricinus communis, established that these proteins can also traffic through cucurbit mesophyll plasmodesmata. These results are discussed in terms of the requirements for regulated protein trafficking between companion cells and the sieve tube system. As the threshold value for plasmodesmal transport of phloem sap proteins falls within the same range as many plant hormones, the possibility is discussed that some of these proteins may act as long-distance signaling molecules.

Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A(2) from Bothrops pirajai venom

Background: Harpalycin 2 (HP-2) is an isoflavone isolated from the leaves of Harpalyce brasiliana Benth., a snakeroot found in northeast region of Brazil and used in folk medicine to treat snakebite. Its leaves are said to be anti-inflammatory. Secretory phospholipases A(2) are important toxins found in snake venom and are structurally related to those found in inflammatory conditions in mammals, as in arthritis and atherosclerosis, and for this reason can be valuable tools for searching new anti-phospholipase A(2) drugs.Methods: HP-2 and piratoxin-III (PrTX-III) were purified through chromatographic techniques. The effect of HP-2 in the enzymatic activity of PrTX-III was carried out using 4-nitro-3-octanoyloxy-benzoic acid as the substrate. PrTX-III induced platelet aggregation was inhibited by HP-2 when compared to aristolochic acid and p-bromophenacyl bromide (p-BPB). In an attempt to elucidate how HP-2 interacts with PrTX-III, mass spectrometry, circular dichroism and intrinsic fluorescence analysis were performed. Docking scores of the ligands (HP-2, aristolochic acid and p-BPB) using PrTX-III as target were also calculated.Results: HP-2 inhibited the enzymatic activity of PrTX-III (IC50 11.34 +/- 0.28 mu g/mL) although it did not form a stable chemical complex in the active site, since mass spectrometry measurements showed no difference between native (13,837.34 Da) and HP-2 treated PrTX-III (13,856.12 Da). A structural analysis of PrTX-III after treatment with HP-2 showed a decrease in dimerization and a slight protein unfolding. In the platelet aggregation assay, HP-2 previously incubated with PrTX-III inhibited the aggregation when compared with untreated protein. PrTX-III chemical treated with aristolochic acid and p-BPB, two standard PLA(2) inhibitors, showed low inhibitory effects when compared with the HP-2 treatment. Docking scores corroborated these results, showing higher affinity of HP-2 for the PrTX-III target (PDB code: 1GMZ) than aristolochic acid and p-BPB. HP-2 previous incubated with the platelets inhibits the aggregation induced by untreated PrTX-III as well as arachidonic acid.Conclusion: HP-2 changes the structure of PrTX-III, inhibiting the enzymatic activity of this enzyme. In addition, PrTX-III platelet aggregant activity was inhibited by treatment with HP-2, p-BPB and aristolochic acid, and these results were corroborated by docking scores.