Investigation on solubilization protocols in the refolding of the thioredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach

The lack of efficient refolding methodologies must be overcome to take full advantage of the fact that bacteria express high levels of aggregated recombinant proteins. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, dissociating aggregates, which makes HHP a useful tool to solubilize proteins for subsequent refolding. A process of refolding was set up by using as a model TsnC, a thioredoxin that catalyzes the disulfide reduction to a dithiol, a useful indication of biological activity. The inclusion bodies (IB) were dissociated at 2.4 kbar. The effect of incubation of IB suspensions at 1–800 bar, the guanidine hydrochloride concentration, the oxidized/reduced glutathione (GSH/GSSG) ratios, and the additives in the refolding buffer were analyzed. To assess the yields of fully biologically active protein obtained for each tested condition, it was crucial to analyze both the TsnC solubilization yield and its enzymatic activity. Application of 2.4 kbar to the IB suspension in the presence of 9 mM GSH, 1 mM GSSG, 0.75 M guanidine hydrochloride, and 0.5 M arginine with subsequent incubation at 1 bar furnished high refolding yield (81%). The experience gained in this study shall help to establish efficient HHP-based protein refolding processes for other proteins.This work was supported by grants from the State of São Paulo Research Foundation – FAPESP (Process 10/13353-0) and National Council for Scientific and Technological Development – CNPq (Process 479816/2007-7) and fellowship 134597/2010-9 from National Council for Scientific and Technological Development – CNPq

Kinetic characterization and search for inhibitors of Ohr (Organic Hydroperoxide Resistance protein) from Xylella fastidiosa

A Xylella fastidiosa é uma bactéria gram-negativa, colonizadora do xilema e é o agente responsável por doenças em plantas cultivadas. No Brasil, a principal doença causada por esta bactéria é a CVC (Clorose Variegada dos Citros), a qual traz grandes prejuízos à produção de laranja dos estados de São Paulo e Minas Gerais. Apesar do atual controle da doença, ainda não se desenvolveu um método específico para o controle da bactéria. Durante a interação planta-patógeno ocorre uma geração exacerbada de oxidantes por parte do hospedeiro, na tentativa de eliminar o patógeno de seu organismo. Dessa forma, os patógenos são expostos a hidroperóxidos derivados de ácidos graxos, formados a partir da ação de lipoxigenases ou ainda pela reação direta de lipídeos com espécies oxidantes. Durante o processo evolutivo, foram selecionados mecanismos de defesa contra estas espécies oxidantes por parte dos patógenos. Dentre estes mecanismos, encontra-se a enzima Ohr (Organic Hydroperoxide Resistance protein), uma peroxidase baseada em resíduos de cisteínas, dependente de grupos lipoil e que possui alta atividade frente à hidroperóxidos orgânicos. Esta proteína provavelmente atua na proteção da célula bacteriana e possui algumas particularidades que fazem dela um alvo em potencial para o desenvolvimento de drogas. Os objetivos deste projeto foram caracterizar possíveis substratos fisiológicos de Ohr de X. fastidiosa, e ainda, buscar moléculas capazes de inibir a atividade peroxidásica desta enzima. Inicialmente demonstramos que Ohr é capaz de reduzir hidroperóxidos de ácido graxo com alta eficiência (kcat/KM ~ 106 M-1.s-1)e, além disso, estes hidroperóxidos são capazes de inativar Ohr em um processo dose dependente, provavelmente devido à alta afinidade entre estes e a enzima. Porém, a enzima não apresentou atividade frente à hidroperóxido de fosfolipídeo (fosfatidilcolina) e hidroperóxido de colesterol. Ademais, elucidamos a estrutura de Ohr na conformação oxidada (ponte dissulfeto), auxiliando no entendimento da dinâmica do ciclo catalítico da enzima. Por fim, selecionamos um composto capaz de inibir a atividade peroxidásica de Ohr in vitro, e temos indícios de que este é capaz de afetar o crescimento bacteriano em situação de estresse oxidativo.Xylella fastidiosa is a gram-negative bacterium that colonizes the xylem and is the causative agent for several plant diseases. In Brazil, the main disease caused by this bacterium is the CVC (Citrus Variegated Chlorosis), which provokes large losses to the orange production in São Paulo and Minas Gerais states. Despite the current disease control, it has not been yet developed a specific method to eliminate the bacterium. During the plant-pathogen interactions, hosts produce an exacerbated amount of oxidants, in an attempt to eliminate the pathogen. Among them, fatty acids hydroperoxides are formed by the lipoxygenase action or even by the direct reaction between lipids and oxidant species. During the evolutionary process, pathogen defense mechanisms against oxidative species have evolved. Among them, Ohr (Organic Hydroperoxide Resistance protein) that is a Cys-based, lipoyl dependent peroxidase, displaying high activity towards organic hydroperoxides. This protein probably plays a central role in oxidative stress response and presnts some particularities, which make it a potential target for drug design. The objectives of this project were to characterize possible physiological substrates of Ohr from X. fastidiosa and search for molecules capable of inhibiting its peroxidase activity. Initially, we demonstrated that Ohr reduced fatty acid hydroperoxides with high efficiency (kcat/KM ~ 106 M-1.s-1). Moreover, these hydroperoxides inactivated Ohr in a dose-dependent manner, probably due to the high affinity between them and the enzyme. However, the enzyme did not display any activity towards phospholipids (posphatidilcholine) hydroperoxides and cholesterol hydroperoxide. Besides, we elucidated the structure of Ohr in the oxidized form (disulfide bond), which gave us insights on the dynamics of structural elements in the catalytic site. Ultimately, we identified a compound that was able to inhibit the peroxidase activity of Ohr in vitro, and we gained evidences that this compound can affect the bacterial growth under oxidative stress

A 14.7 kDa Protein from <i>Francisella tularensis subsp. novicida</i> (Named FTN_1133), Involved in the Response to Oxidative Stress Induced by Organic Peroxides, Is Not Endowed with Thiol-Dependent Peroxidase Activity

<div><p><i>Francisella</i> genus comprises Gram-negative facultative intracellular bacteria that are among the most infectious human pathogens. A protein of 14.7 KDa named as FTN_1133 was previously described as a novel hydroperoxide resistance protein in <i>F. tularensis subsp. novicida</i>, implicated in organic peroxide detoxification and virulence. Here, we describe a structural and biochemical characterization of FTN_1133. Contrary to previous assumptions, multiple amino acid sequence alignment analyses revealed that FTN_1133 does not share significant similarity with proteins of the Ohr/OsmC family or any other Cys-based, thiol dependent peroxidase, including conserved motifs around reactive cysteine residues. Circular dichroism analyses were consistent with the <i>in silico</i> prediction of an all-α-helix secondary structure. The pK_a of its single cysteine residue, determined by a monobromobimane alkylation method, was shown to be 8.0±0.1, value that is elevated when compared with other Cys-based peroxidases, such as peroxiredoxins and Ohr/OsmC proteins. Attempts to determine a thiol peroxidase activity for FTN_1133 failed, using both dithiols (DTT, thioredoxin and lipoamide) and monothiols (glutathione or 2-mercaptoethanol) as reducing agents. Heterologous expression of <i>FTN_1133</i> gene in <i>ahpC</i> and <i>oxyR</i> mutants of <i>E. coli</i> showed no complementation. Furthermore, analysis of <i>FTN_1133</i> protein by non-reducing SDS-PAGE showed that an inter-molecular disulfide bond (not detected in Ohr proteins) can be generated under hydroperoxide treatment, but the observed rates were not comparable to those observed for other thiol-dependent peroxidases. All the biochemical and structural data taken together indicated that FTN_1133 displayed distinct characteristics from other thiol dependent peroxidases and, therefore, suggested that FTN_1133 is not directly involved in hydroperoxide detoxification.</p></div

Squematic electron flow of systems that were used to test a possible thiol-dependent peroxidase activity of recombinant FTN_1133.

<p><b>A.</b> Consumption of hydroperoxides determined by FOX assay, using DTT, β-mercaptoethanol or glutathione as electron donors. <b>B.</b> Oxidized DTT assay was used to monitor the ability of thioredoxin to support a putative FTN_1133 activity. In this case, thioredoxin would provide electrons to reduce FTN_1133 instead of DTT, which would be engaged to recycle the thioredoxin protein; <b>C.</b> For lipoamide-coupled assay, electrons originated from NADH are transferred to a Dihydrolipoamide dehydrogenase (Lpd), that through lipoamide would reduce FTN_1133; <b>D.</b> In the GR/Grx-coupled assay, electrons originated from NADPH would flow to FTN_1133, through GR/Grx system. β-ME, β-mercaptoethanol; Trx_E.c., Thioredoxin from <i>E. coli</i>; LpD_X.f., Dihydrolipoamide Dehydrogenase from <i>X. fastidiosa</i>; GR_S.c., Glutathione Reductase from baker's yeast <i>S. cerevisae</i>; GrxC_X.f., Glutaredoxin C, from <i>X. fastidiosa</i>.</p

Sequence alignment of FTN_1133.

<p>FTN_1133 sequence was run against non-redundant database of NCBI using PSI-Blast algorithm operating in a default mode. The alignment of sequences from PSI-Blast output was generated by using the Kalign algorithm and processed by Jalview. The location of the cysteine residue on <i>Francisella</i> sequences is indicated by red boxes. Dark and light blue shadings backgrounds illustrate regions with greater than 80% of similarity presented among all sequences. Protein sequence ID FTN_1133 from <i>F. tularensis novicida</i> U112 has 100% amino acid identity with FTE_0530 (<i>F. tularensis novicida</i> FTE), FTG_0533 (<i>F. tularensis novicida</i> FTG), FN3523_1168 (<i>F. tularensis novicida</i> 3523) and FNFX1_1179 (<i>F. tularensis novicida</i> Fx1). Protein sequence IDFTT_1152 from <i>F. tularensis tularensis SCHU4</i> has 100% identity with FTL_0803 (<i>F. tularensis holarctica</i> LVS), FTF_1152, (<i>F. tularensis tularensis</i> FSC198), FTH_0797 (<i>F. tularensis holarctica</i> OSU18), FTW_1191 (<i>F. tularensis tularensis</i> WY96_3418), FTA_0849 (<i>F. tularensis holarctica</i> FTNF002_00), FTM_0836 (<i>F. tularensis mediasiatica</i> FSC147), FTU_1185 (<i>F. tularensis tularensis</i> TIGB03), FTV_1101 (<i>F. tularensis tularensis</i> TI0902), FTS_0796 (<i>F. tularensis holarctica</i> FSC200) and FTHG_00746 (<i>F. tularensis holarctica</i> 257). Protein sequences IDOOM_1726, Fphi_1909 and F7308_1240 are from <i>F. noatunensis orientalis</i> str. Toba 04, <i>F. philomiragia philomiragia</i> ATCC 25017 and <i>Francisella sp.</i> TX077308, respectively. Protein sequence ID, gi|517110088 is from <i>Fangia hongkongensis</i> and protein sequences ID, gi|515947316 and gi|510837960, are from <i>Piscirickettsia salmonis</i>.</p

FTN_1133 secondary structure.

<p><b>A.</b> Cartoon of predicted FTN_1133 secondary structure. Blue traces represent predicted loop regions and red traces represent predicted α-helix region of FTN_1133 sequence. <b>B.</b> Circular dichroism spectra of FTN_1133 were collected with 3 and 6 µM in the presence of NaCl (100 mM), sodium phosphate pH 7.4 (20 mM) and 10% glycerol using a 0.01 cm cell in a JASCO J-710 spectropolarimeter. <b>C.</b> Estimates of secondary structure elements of FTN_1133 from data obtained from item B., using SELCON3, CONTINLL, CDSSTR algorithms. NRSMD, denotes the <u>N</u>ormalized <u>R</u>oot-<u>M</u>ean-<u>S</u>quare <u>D</u>eviation.</p

Assay for FTN_1133 DTT-dependent peroxidase activity.

<p>Decomposition of different peroxides was monitored during 40<b>A.</b>, <b>B.</b> and <b>C.</b>, CuOOH, tBOOH and H₂O₂ decomposition, respectively, in the presence of FTN_1133 (10 µM), HEPES-HCl pH 7.4 (50 mM), DTT (0.5 mM), sodium azide (0.1 mM) and DTPA (0.1 mM). <b>D.</b>, <b>E.</b> and <b>F.</b>, CuOOH, tBOOH and H₂O₂ decomposition, respectively, in the presence of OsmC (10 µM), HEPES-HCl pH 7.4 (50 mM), DTT (0.5 mM), sodium azide (0.1 mM) and DTPA (0.1 mM). All reactions were started by addition of 200 µM of peroxide. Blue line, (blue triangle) enzyme+DTT+peroxide (catalyzed reaction); Green line, (green triangle) enzyme+peroxide without DTT; Black line, (black square) only peroxide and red line, (red circle) peroxide+DTT (uncatalyzed reaction). The figure is representative of at least two independent set of experiments, each one done in technical triplicates.</p

Inter-molecular disulfide bond formation in FTN_1133 upon hydroperoxide treatment. A.

<p>Non-reducing SDS-PAGE of freshly purified recombinant FTN_1133 (5 µM) that was incubated in the absence or presence of 0.06, 0.2, 0.5, 2, 10 and 100 mM of DTT during 10 minutes at 37°C. <b>B.</b> Representative non-reducing SDS-PAGE showing that the treatment of FTN_1133 with increasing amount of hydroperoxides. FTN_1133 inter-molecular disulfide bond formation was assessed by the appearance of a band corresponding to the dimer (∼34 kDa). Treatments, using pre-reduced FTN_1133 (5 µM), were performed for 10 minutes at 37°C with 0, 1, 5, 10, 20, 50 and 100 µM of CuOOH (a), tBOOH (b) or H₂O₂ (c). <b>C.</b> Time course of FTN_1133 oxidation towards hydroperoxide treatment. The assay was carried out with pre-reduced FTN_1133 (5 µM) treated with 100 µM CuOOH (b), tBOOH (c) or H₂O₂ (d), during 0, 0.5, 1, 2, 3, 4 and 22 hours at 30°C in a buffer containing 0.5 M of NaCl, 20 mM of sodium phosphate pH 7.4 and 1 mM of DTPA. (a) Represent the control reaction (no addition of hydroperoxide). Immediately after hydroperoxides treatments, all samples were alkylated with NEM (100 mM) for 30 minutes at room temperature to avoid oxidation artifacts due to protein denaturation by SDS. The figure is representative of three independent set of experiments.</p

Structural and Biochemical Comparative Analysis among Cys-Based Ohr/OsmC Protein Family: Insights on Their High Reactivity Towards Hydroperoxides

Fapesp, INCT Redoxoma, CNPq, CAPE