Anderson S. Pinheiro

From structure to catalysis: recent developments in the biotechnological applications of lipases.

Microbial lipases are highly appreciated as biocatalysts due to their peculiar characteristics such as the ability to utilize a wide range of substrates, high activity and stability in organic solvents, and regio- and/or enantioselectivity. These enzymes are currently being applied in a variety of biotechnological processes, including detergent preparation, cosmetics and paper production, food processing, biodiesel and biopolymer synthesis, and the biocatalytic resolution of pharmaceutical derivatives, esters, and amino acids. However, in certain segments of industry, the use of lipases is still limited by their high cost. Thus, there is a great interest in obtaining low-cost, highly active, and stable lipases that can be applied in several different industrial branches. Currently, the design of specific enzymes for each type of process has been used as an important tool to address the limitations of natural enzymes. Nowadays, it is possible to “order” a “customized” enzyme that has ideal properties for the development of the desired bioprocess. This review aims to compile recent advances in the biotechnological application of lipases focusing on various methods of enzyme improvement, such as protein engineering (directed evolution and rational design), as well as the use of structural data for rational modification of lipases in order to create higher active and selective biocatalysts.

Oligomerization and Membrane-binding Properties of Covalent Adducts Formed by the Interaction of α-Synuclein with the Toxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde (DOPAL)

Background: The molecular details of the effects of DOPAL on α-synuclein misfolding and oligomerization are unknown. Results: DOPAL forms Schiff-base and Michael-addition adducts with α-synuclein Lys residues. Conclusion: DOPAL modification inhibits aS fibrillization and reduces binding of α-synuclein to synaptic-like vesicles. Significance: DOPAL modification may interfere with the normal functions of α-synuclein and favor the buildup of potentially toxic oligomers. Oxidative deamination of dopamine produces the highly toxic aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), enhanced production of which is found in post-mortem brains of Parkinson disease patients. When injected into the substantia nigra of rat brains, DOPAL causes the loss of dopaminergic neurons accompanied by the accumulation of potentially toxic oligomers of the presynaptic protein α-synuclein (aS), potentially explaining the synergistic toxicity described for dopamine metabolism and aS aggregation. In this work, we demonstrate that DOPAL interacts with aS via formation of Schiff-base and Michael-addition adducts with Lys residues, in addition to causing oxidation of Met residues to Met-sulfoxide. DOPAL modification leads to the formation of small aS oligomers that may be cross-linked by DOPAL. Both monomeric and oligomeric DOPAL adducts potently inhibit the formation of mature amyloid fibrils by unmodified aS. The binding of aS to either lipid vesicles or detergent micelles, which results in a gain of α-helix structure in its N-terminal lipid-binding domain, protects the protein against DOPAL adduct formation and, consequently, inhibits DOPAL-induced aS oligomerization. Functionally, aS-DOPAL monomer exhibits a reduced affinity for small unilamellar vesicles with lipid composition similar to synaptic vesicles, in addition to diminished membrane-induced α-helical content in comparison with the unmodified protein. These results suggest that DOPAL could compromise the functionality of aS, even in the absence of protein oligomerization, by affecting the interaction of aS with lipid membranes and hence its role in the regulation of synaptic vesicle traffic in neurons.

α-Synuclein as an intrinsically disordered monomer – fact or artefact?

Fibrillization of the protein α‐synuclein (α‐syn) is a hallmark of Parkinsons disease and other α‐synucleinopathies. The well‐established idea that α‐syn is a natively disordered monomer prone to forming fibrils was recently challenged by data showing that the protein mostly exists in vitro and in vivo as helically folded tetramers that are resistant to fibrillization. These apparently conflicting findings may be reconciled by the idea that α‐syn exists as a disordered monomer in equilibrium with variable amounts of dynamic oligomeric species. In this context, varying the approaches used for protein purification, such as the method used to lyse cells or the inclusion of denaturing agents, could dramatically perturb this equilibrium and hence alter the relative abundance of the disordered monomer. In the present study, we investigated how the current methods for α‐syn purification affect the structure and oligomeric state of the protein, and we discuss the main pitfalls associated with the production of recombinant α‐syn in Escherichia coli. We demonstrate that α‐syn was expressed in E. coli as a disordered monomer independent of both the cell lysis method and the use of heating/acidification for protein purification. In addition, we provide convincing evidence that the disordered monomer exists in equilibrium with a dynamic dimer, which is not an artefact of the cross‐linking protocol as previously suggested. Unlike the helically folded tetramer, α‐syn dimer is prone to fibrillate and thus it may be an interesting target for anti‐fibrillogenic molecules.

The Metastable State of Nucleocapsids of Enveloped Viruses as Probed by High Hydrostatic Pressure

Enveloped viruses fuse their membranes with cellular membranes to transfer their genomes into cells at the beginning of infection. What is not clear, however, is the role of the envelope (lipid bilayer and glycoproteins) in the stability of the viral particle. To address this question, we compared the stability between enveloped and nucleocapsid particles of thealphavirus Mayaro using hydrostatic pressure and urea. The effects were monitored by intrinsic fluorescence, light scattering, and binding of fluorescent dyes, including bis(8-anilinonaphthalene-1-sulfonate) and ethidium bromide. Pressure caused a drastic dissociation of the nucleocapsids as determined by tryptophan fluorescence, light scattering, and gel filtration chromatography. Pressure-induced dissociation of the nucleocapsids was poorly reversible. In contrast, when the envelope was present, pressure effects were much less marked and were highly reversible. Binding of ethidium bromide occurred when nucleocapsids were dissociated under pressure, indicating exposure of the nucleic acid, whereas enveloped particles underwent no changes. Overall, our results demonstrate that removal of the envelope with the glycoproteins leads the particle to a metastable state and, during infection, may serve as the trigger for disassembly and delivery of the genome. The envelope acts as a “Trojan horse,” gaining entry into the host cell to allow release of a metastable nucleocapsid prone to disassembly.

Pitfalls associated with the use of Thioflavin-T to monitor anti-fibrillogenic activity.

Thioflavin-T (ThT) is a cationic benzothiazole dye that displays enhanced fluorescence upon binding to amyloid fibrils. This property makes ThT the current reagent of choice for the quantification of amyloid fibrils. Herein, we investigate the main pitfalls associated with the use of ThT-based assays to monitor the fibrillation of α-synuclein (α-syn), a protein linked to Parkinsons disease and other α-synucleinopathies. We demonstrated for the first time that ThT interacts with α-syn disordered monomer and accelerates the protein fibrillation in vitro. As a consequence, misleading conclusions may arise from the use of ThT-based real-time assays in the evaluation of anti-fibrillogenic compounds. Interestingly, NMR experiments indicated that C-terminal domain of α-syn is the main region perturbed by ThT interaction, similarly to that found for the pesticide paraquat, a well-documented accelerator of α-syn fibrillation. Moreover, we demonstrated that certain potent inhibitors of α-syn fibrillation, such as oxidized catecholamines and polyphenols, undergo spontaneous oxidation in aqueous solution, generating compounds that strongly quench ThT fluorescence. In light of these findings, we alert for possible artifacts associated to the measure of the anti-fibrillogenic activity based only on ThT fluorescence approach.

NMR solution structure of the reduced form of thioredoxin 1 from Sacharomyces cerevisiae

Keywords NMR Saccharomyces cerevisiae Structure Thioredoxin 2 YeastAbbreviationsTrx thioredoxinBiological contextThioredoxins (Trxs) are ubiquitous heat-stable pro-teins of molecular mass approximately 12,000 Da thatfunction as dithiol oxidoreductases, and are impor-tant for cellular defense against oxidative stress(Monje-Casas et al. 2004). Trxs act as hydrogen do-nors for reductive enzymes such as ribonucleotidereductases (Laurent et al. 1964) and thioredoxinperoxidases (Kang et al. 1998), as a general reductantfor disulfides in proteins (Holmgren 1985), and asregulatory factors for enzymes or receptors inphotosynthetic systems (Buchanan 1991). They alsopromote DNA binding of transcription factors, suchas NF-jB and YAP-1 (Schenk et al. 1994), amongothers.In yeast, there are two cytoplasmic (Trx1 and Trx2)and one mitochondrial (Trx3) isoforms. Some authorsconsider that the preservation of the two cytoplasmicisoforms during evolution is not related to function,meaning that they are redundant. Recently, Monje-Casas et al. (2004) described some differences inexpression levels under normal and oxidative stressconditions. The basal level of both thioredoxins is thesame, but becomes different after treatment withhydrogen peroxide. Trx2 shows an increase of 14 foldin its expression, whereas Trx1 remains unaffected.Gene duplication is widespread, but it is not alwayspreserved in evolution. In Saccharomyces cerevisiaethere might be an explanation for the preservation ofboth Trx1 and Trx2. The parallel evolution of yeastthioredoxins occurred possibly because they exerteddifferent regulatory function. Since regulation occursthrough binding to different cellular targets, Trx1 andTrx2 might be specialized to bind to different targets.Three-dimensional structures of several thioredoxinshave been determined. We have determined the NMRstructure of the two cytoplasmic isoforms. In this note,we report the structure of thioredoxin 2 (PDB ID:2HSY) and compare it to other thioredoxins.Methods and resultsExpression and purificationIndividual colonies of the transformants were grown inLB medium containing 100 lg/ml ampicillin at 37 Cfor

Natural Products: Insights into Leishmaniasis Inflammatory Response

Leishmaniasis is a vector-borne disease that affects several populations worldwide, against which there are no vaccines available and the chemotherapy is highly toxic. Depending on the species causing the infection, the disease is characterized by commitment of tissues, including the skin, mucous membranes, and internal organs. Despite the relevance of host inflammatory mediators on parasite burden control, Leishmania and host immune cells interaction may generate an exacerbated proinflammatory response that plays an important role in the development of leishmaniasis clinical manifestations. Plant-derived natural products have been recognized as bioactive agents with several properties, including anti-protozoal and anti-inflammatory activities. The present review focuses on the antileishmanial activity of plant-derived natural products that are able to modulate the inflammatory response in vitro and in vivo. The capability of crude extracts and some isolated substances in promoting an anti-inflammatory response during Leishmania infection may be used as part of an effective strategy to fight the disease.

Unveiling the role of the pesticides paraquat and rotenone on α-synuclein fibrillation in vitro

Epidemiological data have suggested that exposure to environmental toxins might be associated with the etiology of Parkinsons disease (PD). In this context, certain agrochemicals are able to induce Parkinsonism in different animal models via the inhibition of mitochondrial complex I, which leads to an increase in both oxidative stress and the death of nigrostriatal neurons. Additionally, in vitro experiments have indicated that pesticides are capable of accelerating the fibrillation of the presynaptic protein α-synuclein (aS) by binding directly to the protein. However, the molecular details of these interactions are poorly understood. In the present work we demonstrate that paraquat and rotenone, two agrochemicals that lead to a Parkinsonian phenotype in vivo, bind to aS via solvent effects rather than through specific interactions. In fact, these compounds produced no significant effects on aS fibrillation under physiological concentrations of NaCl. NMR data suggest that paraquat interacts with the C-terminal domain of the disordered aS monomer. This interaction was markedly reduced in the presence of NaCl, presumably due to the disruption of electrostatic interactions between the protein and paraquat. Interestingly, the effects produced by short-term incubation of paraquat with aS on the protein conformation resembled those produced by incubating the protein with NaCl alone. Taken together, our data indicate that the effects of these agrochemicals on PD cannot be explained via direct interactions with aS, reinforcing the idea that the role of these compounds in PD is limited to the inhibition of mitochondrial complex I and/or the up-regulation of aS.

Renin-angiotensin system contributes to naive T-cell migration in vivo.

Angiotensin II (Ang II) plays an important role in the regulation of the T-cell response during inflammation. However, the cellular mechanisms underlying the regulation of lymphocytes under physiologic conditions have not yet been studied. Here, we tested the influence of Ang II on T-cell migration using T cells from BALB/c mice. The results obtained in vivo showed that when Ang II production or the AT1 receptor were blocked, T-cell counts were enhanced in blood but decreased in the spleen. The significance of these effects was confirmed by observing that these cells migrate, through fibronectin to Ang II via the AT1 receptor. We also observed a gradient of Ang II from peripheral blood to the spleen, which explains its chemotactic effect on this organ. The following cellular mechanisms were identified to mediate the Ang II effect: upregulation of the chemokine receptor CCR9; upregulation of the adhesion molecule CD62L; increased production of the chemokines CCL19 and CCL25 in the spleen. These results indicate that the higher levels of Ang II in the spleen and AT1 receptor activation contribute to migration of naive T cells to the spleen, which expands our understanding on how the Ang II/AT1 receptor axis contributes to adaptive immunity.

PWWP domains and their modes of sensing DNA and histone methylated lysines

Chromatin plays an important role in gene transcription control, cell cycle progression, recombination, DNA replication and repair. The fundamental unit of chromatin, the nucleosome, is formed by a DNA duplex wrapped around an octamer of histones. Histones are susceptible to various post-translational modifications, covalent alterations that change the chromatin status. Lysine methylation is one of the major post-translational modifications involved in the regulation of chromatin function. The PWWP domain is a member of the Royal superfamily that functions as a chromatin methylation reader by recognizing both DNA and histone methylated lysines. The PWWP domain three-dimensional structure is based on an N-terminal hydrophobic β-barrel responsible for histone methyl-lysine binding, and a C-terminal α-helical domain. In this review, we set out to discuss the most recent literature on PWWP domains, focusing on their structural features and the mechanisms by which they specifically recognize DNA and histone methylated lysines at the level of the nucleosome.