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Klebsiella pneumoniae is an opportunistic pathogen, which concerns public health systems worldwide, as multiple antibiotic-resistant strains are frequent. One of its pathogenicity factors is the Type VI Secretion System (T6SS), a macromolecular complex assembled through the bacterial membranes. T6SS injects effector proteins inside target cells. Such effectors confer competitive advantages or modulate the target cell signaling and metabolism to favor bacterial infection. The VgrG protein is a T6SS core component. It may present a variable C-terminal domain carrying an additional effector function. Kp52.145 genome encodes three VgrG proteins, one of them with a C-terminal extension (VgrG4-CTD). VgrG4-CTD is 138 amino acids long, does not contain domains of known function, but is conserved in some Klebsiella, and non-Klebsiella species. To get insights into its function, recombinant VgrG4-CTD was used in pulldown experiments to capture ligands from macrophages and lung epithelial cells. A total of 254 proteins were identified: most of them are ribosomal proteins. Cytoskeleton-associated and proteins involved in the phagosome maturation pathway were also identified. We further showed that VgrG4-CTD binds actin and induces actin remodeling in macrophages. This study presents novel clues on the role of K. pneumoniae T6SS in pathogenesis.

Solid forms can influence the dissolution, stability, and processability of active pharmaceutical ingredients. However, besides the internal arrangement, crystal habit studies are important, since ...

International audience; There is increasing support for water molecules playing a role in signal propagation through G protein-coupled receptors (GPCRs). However, exploration of the hydration features of GPCRs is still in its infancy. Here, we combined site-specific labeling with unnatural amino acids to molecular dynamics to delineate how local hydration of the ghrelin receptor growth hormone secretagogue receptor (GHSR) is rearranged upon activation. We found that GHSR is characterized by a specific hydration pattern that is selectively remodeled by pharmacologically distinct ligands and by the lipid environment. This process is directly related to the concerted movements of the transmembrane domains of the receptor. These results demonstrate that the conformational dynamics of GHSR are tightly coupled to the movements of internal water molecules, further enhancing our understanding of the molecular bases of GPCR-mediated signaling.

Dorsal-ventral patterning of the Drosophila embryo depends on the NFκB superfamily transcription factor Dorsal (Dl). Toll receptor activation signals for degradation of the IκB inhibitor Cactus (Cact), leading to a ventral-to-dorsal nuclear Dl gradient. Cact is critical for Dl nuclear import, as it binds to and prevents Dl from entering the nuclei. Quantitative analysis of cact mutants revealed an additional Cact function to promote Dl nuclear translocation in ventral regions of the embryo. To investigate this dual Cact role, we developed a predictive model based on a reaction-diffusion regulatory network. This network distinguishes non-uniform Toll-dependent Dl nuclear import and Cact degradation, from the Toll-independent processes of Cact degradation and reversible nuclear-cytoplasmic Dl flow. In addition, it incorporates translational control of Cact levels by Dl. Our model successfully reproduces wild-type data and emulates the Dl nuclear gradient in mutant dl and cact allelic combinations. Our results indicate that the dual role of Cact depends on the dynamics of Dl-Cact trimers along the dorsal-ventral axis: In the absence of Toll activation, free Dl-Cact trimers retain Dl in the cytoplasm, limiting the flow of Dl into the nucleus; in ventral-lateral regions, Dl-Cact trimers are recruited by Toll activation into predominant signaling complexes and promote Dl nuclear translocation. Simulations suggest that the balance between Toll-dependent and Toll-independent processes are key to this dynamics and reproduce the full assortment of Cact effects. Considering the high evolutionary conservation of these pathways, our analysis should contribute to understanding NFκB/c-Rel activation in other contexts such as in the vertebrate immune system and disease., Author summary In Drosophila, Toll pathway establishes spatially distinct gene expression territories that define the embryonic dorsal-ventral axis. Toll activation leads to degradation of the IκB inhibitor Cactus, releasing the NFκB superfamily transcription factor Dorsal for nuclear entry. Recently, quantitative analysis of cact mutants revealed that Cact displays an additional function to promote Dl nuclear translocation in ventral regions of the embryo. To understand this novel activity, we developed a predictive theoretical model that shows that the kinetics of Dorsal-Cactus complex formation prior to their recruitment to Toll-signaling complexes is an essential regulatory hub. Cactus controls the balance between the recruitment of these complexes by active Toll receptor and association-dissociation events that generate free Dorsal for direct nuclear import.

Vibrio parahaemolyticus strains recovered from human diarrheal stools (one in 1975 and two in 2001) and environmental sources (four, between 2008 and 2010) were investigated for the presence of virulence genes (trh, tdh, and vpadF), pandemic markers (orf8, toxRSnew), and with respect to their pathogenic potential in two systemic infection models. Based only on the presence or absence of these genetic markers, they were classified as follows: the environmental strains were non-pathogenic, whereas among the clinical strains, the one isolated in 1975 was pathogenic (non-pandemic), and the other two were pathogenic (pandemic). The pathogenic potential of the strains was evaluated in mice and Galleria mellonella larvae infection models, and except for the clinical (pathogenic, non-pandemic) isolate, the others produced lethal infection in both organisms, regardless of their source, serotype, and genotype (tdh, orf8, toxRSnew, and vpadF). Based on mice and larval mortality rates, the strains were then grouped according to virulence (high, intermediate, and avirulent), and remarkably similar results were obtained by using these models: The clinical strain (pathogenic and non-pandemic) was classified as avirulent, and other strains (four non-pathogenic and two pandemic) were considered of high or intermediate virulence. In summary, these findings demonstrate that G. mellonella larvae can indeed be used as an alternative model to study the pathogenicity of V. parahaemolyticus. Moreover, they raise doubts about the use of traditional virulence markers to predict pathogenesis of the species and show that reliable models are indispensable to determine the pathogenic potential of environmental isolates considered non-pathogenic, based on the absence of the long-standing virulence indicators.

One of the most abundant proteins in V. cholerae O1 cells grown under inorganic phosphate (Pi) limitation is PstS, the periplasmic Pi-binding component of the high-affinity Pi transport system Pst2 (PstSCAB), encoded in pst2 operon (pstS-pstC2-pstA2-pstB2). Besides its role in Pi uptake, Pst2 has been also associated with V. cholerae virulence. However, the mechanisms regulating pst2 expression and the non-stoichiometric production of the Pst2 components under Pi-limitation are unknown. A computational-experimental approach was used to elucidate the regulatory mechanisms behind pst2 expression in V. cholerae O1. Bioinformatics analysis of pst2 operon nucleotide sequence revealed start codons for pstS and pstC genes distinct from those originally annotated, a regulatory region upstream pstS containing potential PhoB-binding sites and a pstS-pstC intergenic region longer than predicted. Analysis of nucleotide sequence between pstS-pstC revealed inverted repeats able to form stem-loop structures followed by a potential RNAse E-cleavage site. Another putative RNase E recognition site was identified within the pstA-pstB intergenic sequence. In silico predictions of pst2 operon expression regulation were subsequently tested using cells grown under Pi limitation by promoter-lacZ fusion, gel electrophoresis mobility shift assay and quantitative RT-PCR. The experimental and in silico results matched very well and led us to propose a pst2 promoter sequence upstream of pstS gene distinct from the previously annotated. Furthermore, V. cholerae O1 pst2 operon transcription is PhoB-dependent and generates a polycistronic mRNA molecule that is rapidly processed into minor transcripts of distinct stabilities. The most stable was the pstS-encoding mRNA, which correlates with PstS higher levels relative to other Pst2 components in Pi-starved cells. The relatively higher stability of pstS and pstB transcripts seems to rely on the secondary structures at their 3' untranslated regions that are known to block 3'-5' exonucleolytic attacks.

Modeling expression patterns of Drosophila, in space and time, plays a critical role to understand the development of multicellular organisms. In confocal microscopy, to produce precise quantitative data it is frequently necessary to process and analyze large amounts of digital images. Automatic preprocessing is a crucial step in this scenario, essential to standardize significant features such as orientation, size, position, direction, lighting condition and texture of embryo images. Even though a lot of efforts have been made, a robust embryo standardization strategy is still needed. In this paper, we propose the method Embrystandar. It is designed to remove background artifacts and standardize the direction and orientation of a Drosophila embryo through a sequence of automatic operations. To test its potential for large-scale image processing, Embrystandar was applied in different databases. It showed to be robust and precise, reaching more than 90% success rate.

Dengue is an important mosquito-borne disease and a global public health problem. The disease is caused by dengue virus (DENV), which is a member of the Flaviviridae family and contains a positive single-stranded RNA genome that encodes a single precursor polyprotein that is further cleaved into structural and non-structural proteins. Among these proteins, the non-structural 3 (NS3) protein is very important because it forms a non-covalent complex with the NS2B cofactor, thereby forming the functional viral protease. NS3 also contains a C-terminal ATPase/helicase domain that is essential for RNA replication. Here, we identified 47 NS3-interacting partners using the yeast two-hybrid system. Among those partners, we highlight several proteins involved in host energy metabolism, such as apolipoprotein H, aldolase B, cytochrome C oxidase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH directly binds full-length NS3 and its isolated helicase and protease domains. Moreover, we observed an intense colocalization between the GAPDH and NS3 proteins in DENV2-infected Huh7.5.1 cells, in NS3-transfected BHK-21 cells and in hepatic tissue from a fatal dengue case. Taken together, these results suggest that the human GAPDH-DENV NS3 interaction is involved in hepatic metabolic alterations, which may contribute to the appearance of steatosis in dengue-infected patients. The interaction between GAPDH and full-length NS3 or its helicase domain in vitro as well as in NS3-transfected cells resulted in decreased GAPDH glycolytic activity. Reduced GAPDH glycolytic activity may lead to the accumulation of metabolic intermediates, shifting metabolism to alternative, non-glycolytic pathways. This report is the first to identify the interaction of the DENV2 NS3 protein with the GAPDH protein and to demonstrate that this interaction may play an important role in the molecular mechanism that triggers hepatic alterations.

High-mobility group B (HMGB) proteins have highly conserved, unique DNA-binding domains, HMG boxes, that can bind non-B-type DNA structures, such as bent, kinked and unwound structures, with high affinity. HMGB proteins also promote DNA bending, looping and unwinding. In this study, we determined the role of the Aedes aegypti single HMG-box domain protein AaHMGB; characterized its structure, spatiotemporal expression levels, subcellular localization, and nucleic acid binding activities; and compared these properties with those of its double-HMG-box counterpart protein, AaHMGB1. Via qRT-PCR, we showed that AaHMGB is expressed at much higher levels than AaHMGB1 throughout mosquito development. In situ hybridization results suggested a role for AaHMGB and AaHMGB1 during embryogenesis. Immunolocalization in the midgut revealed that AaHMGB is exclusively nuclear. Circular dichroism and fluorescence spectroscopy analyses showed that AaHMGB exhibits common features of α-helical structures and is more stably folded than AaHMGB1, likely due to the presence of one or two HMG boxes. Using several DNA substrates or single-stranded RNAs as probes, we observed significant differences between AaHMGB and AaHMGB1 in terms of their binding patterns, activity and/or specificity. Importantly, we showed that the phosphorylation of AaHMGB plays a critical role in its DNA-binding activity. Our study provides additional insight into the roles of single- versus double-HMG-box-containing proteins in nucleic acid interactions for better understanding of mosquito development, physiology and homeostasis.

Cyanobacteria tend to become the dominant phytoplankton component in eutrophic freshwater environments during warmer seasons. However, general observations of cyanobacterial adaptive advantages in these circumstances are insufficient to explain the prevalence of one species over another in a bloom period, which may be related to particular strategies and interactions with other components of the plankton community. In this study, we present an integrative view of a mixed cyanobacterial bloom occurring during a warm, rainy period in a tropical hydropower reservoir. We used high-throughput sequencing to follow temporal shifts in the dominance of cyanobacterial genera and shifts in the associated heterotrophic bacteria community. The bloom occurred during late spring-summer and included two distinct periods. The first period corresponded to Microcystis aeruginosa complex (MAC) dominance with a contribution from Dolichospermum circinale; this pattern coincided with high water retention time and low transparency. The second period corresponded to Cylindrospermopsis raciborskii and Synechococcus spp. dominance, and the reservoir presented lower water retention time and higher water transparency. The major bacterial phyla were primarily Cyanobacteria and Proteobacteria, followed by Actinobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes. Temporal shifts in the dominance of cyanobacterial genera were associated with physical features of the water (retention time and transparency) and with shifts in the associated heterotrophic bacteria. The MAC bloom was associated with a high abundance of Bacteroidetes, particularly Cytophagales. In the second bloom period, Planctomycetes increased in relative abundance, five Planctomycetes OTUs were positively correlated with Synechococcus or C. raciborskii OTUs. Our results suggest specific interactions of the main cyanobacterial genera with certain groups of the heterotrophic bacterial community; the results suggest that considering biotic interactions may lead to a better understanding of the shifts in cyanobacterial dominance.

Ornithine lipids (OLs) are phosphorus-free lipids found in many bacteria grown under phosphate deprivation, a condition that activates the PhoBR system and leads to phosphate uptake and metabolism. Two OL synthesis pathways have already been described. One depends on OlsB and OlsA acyltransferases to add, respectively, the first and second acyl chains to an ornithine molecule. The other pathway is carried out by OlsF, a bifunctional enzyme responsible for both acylation steps. Although Vibrio cholerae lacks olsBA genes, an olsF homologue (vc0489) was identified in its genome. In this work we demonstrated that V. cholerae produces OLs and expresses vc0489 in response to phosphate depletion, in a PhoBR-dependent manner. In Escherichia coli, under similar condition, vc0489 expression leads to OL accumulation. These results indicate a strong connection between OL synthesis and VC0489 from V. cholerae and, for the first time, a direct regulation of an olsF homologue by the PhoBR system.

Proteins are found in solution as ensembles of conformations in dynamic equilibrium. Exploration of functional motions occurring on micro- to millisecond time scales by molecular dynamics (MD) simulations still remains computationally challenging. Alternatively, normal mode (NM) analysis is a well-suited method to characterize intrinsic slow collective motions, often associated with protein function, but the absence of anharmonic effects preclude a proper characterization of conformational distributions in a multidimensional NM space. Using both methods jointly appears to be an attractive approach that allows an extended sampling of the conformational space. In line with this view, the MDeNM (molecular dynamics with excited normal modes) method presented here consists of multiple-replica short MD simulations in which motions described by a given subset of low-frequency NMs are kinetically excited. This is achieved by adding additional atomic velocities along several randomly determined linear combinations of NM vectors, thus allowing an efficient coupling between slow and fast motions. The relatively high-energy conformations generated with MDeNM are further relaxed with standard MD simulations, enabling free energy landscapes to be determined. Two widely studied proteins were selected as examples: hen egg lysozyme and HIV-1 protease. In both cases, MDeNM provides a larger extent of sampling in a few nanoseconds, outperforming long standard MD simulations. A high degree of correlation with motions inferred from experimental sources (X-ray, EPR, and NMR) and with free energy estimations obtained by metadynamics was observed. Finally, the large sets of conformations obtained with MDeNM can be used to better characterize relevant dynamical populations, allowing for a better interpretation of experimental data such as SAXS curves and NMR spectra.

A putative porin function has been assigned to VCA1008 of Vibrio cholerae. Its coding gene, vca1008, is expressed upon colonization of the small intestine in infant mice and human volunteers, and is essential for infection. In vitro, vca1008 is expressed under inorganic phosphate limitation and, in this condition, VCA1008 is the major outer membrane protein of the bacterium. Here, we provide the first functional characterization of VCA1008 reconstituted into planar lipid bilayers. Our main findings were: 1) VCA1008 forms an ion channel that, at high voltage (~ ± 100mV), presents a voltage-dependent activity and displays closures typical of trimeric porins, with a conductance of 4.28 ± 0.04 nS (n = 164) in 1M KCl; 2) It has a preferred selectivity for anions over cations; 3) Its conductance saturates with increasing inorganic phosphate concentration, suggesting VCA1008 contains binding site(s) for this anion; 4) Its ion selectivity is controlled by both fixed charged residues within the channel and diffusion along the pore; 5) Partitioning of poly (ethylene glycol)s (PEGs) of different molecular mass suggests that VCA1008 channel has a pore exclusion limit of 0.9 nm.

Ischaemic preconditioning (IPC) provides myocardial resistance to ischaemia/reperfusion (I/R) injuries. The protection afforded by IPC is not limited to the target tissue but extends to remote tissues, suggesting a mechanism mediated by humoral factors. The aim of the present study was to identify the humoral factors that are responsible for the cardioprotection induced by the coronary effluent transferred from IPC to naive hearts. Isolated rat hearts were submitted to IPC (three cycles of 5 min I/R) before 30-min global ischaemia and 60-min reperfusion. The coronary effluent (Efl_IPC) collected during IPC was fractionated by ultrafiltration in different molecular weight ranges ( 50 kDa) and evaluated for cardioprotective effects by perfusion before I/R in naive hearts. Only the

The evolutionarily conserved Toll signaling pathway controls innate immunity across phyla and embryonic patterning in insects. In the Drosophila embryo Toll is required to establish gene expression domains along the dorsal-ventral axis. Pathway activation induces degradation of the IκB inhibitor Cactus, resulting in a ventral-to-dorsal nuclear gradient of the NFκB effector Dorsal. Here we investigate how cactus modulates Toll signals through its effects on the Dorsal gradient and Dorsal target genes. Quantitative analysis using a series of loss and gain-of-function conditions shows that the ventral and lateral aspects of the Dorsal gradient can behave differently respective to Cactus fluctuations. In lateral and dorsal embryo domains loss of Cactus allows more Dorsal to translocate to the nucleus. Unexpectedly, cactus loss-of-function alleles decrease Dorsal nuclear localization ventrally, where Toll signals are high. Overexpression analysis suggests that this ability of Cactus to enhance Toll stems from mobilization of a free Cactus pool induced by the Calpain A protease. These results indicate that Cactus acts to bolster Dorsal activation, in addition to its role as an NFκB inhibitor, ensuring a correct response to Toll signals.

The evolutionarily conserved Toll signaling pathway controls innate immunity across phyla and embryonic patterning in insects. In the Drosophila embryo Toll is required to establish gene expression domains along the dorsal-ventral axis. Pathway activation induces degradation of the IκB inhibitor Cactus resulting in a nuclear gradient of the NFκB effector Dorsal. Here we investigate howcactusmodulates Toll signals through its effects on the Dorsal gradient and Dorsal target genes. Quantitative analysis using a series of loss and gain-of-function conditions shows that the ventral and lateral aspects of the Dorsal gradient behave differently respective to Cactus fluctuations. Unexpectedly, Cactus favors Dorsal nuclear localization required as response to high Toll signals at the ventral side of the embryo. Furthermore, N-terminal deleted Cactus mimics these effects, indicating that the ability of Cactus to favor Toll stems from mobilization of a free Cactus pool induced by the Calpain A protease. These results indicate that unexplored mechanisms are at play to ensure a correct response to high Toll signals.Summary:The IκB protein Cactus favors high Toll signals, revealing that the ventral and lateral aspects of the Dorsal/NFκB nuclear gradient behave differently respective to Cactus concentrations in the Drosophila embryo.

The evolutionarily conserved Toll signaling pathway controls innate immunity across phyla and embryonic patterning in insects. In the Drosophila embryo, Toll is required to establish gene expression domains along the dorsal-ventral axis. Pathway activation induces degradation of the IκB inhibitor Cactus, resulting in a ventral-to-dorsal nuclear gradient of the NFκB effector Dorsal. Here, we investigate how cactus modulates Toll signals through its effects on the Dorsal gradient and on Dorsal target genes. Quantitative analysis using a series of loss- and gain-of-function conditions shows that the ventral and lateral aspects of the Dorsal gradient can behave differently with respect to Cactus fluctuations. In lateral and dorsal embryo domains, loss of Cactus allows more Dorsal to translocate to the nucleus. Unexpectedly, cactus loss-of-function alleles decrease Dorsal nuclear localization ventrally, where Toll signals are high. Overexpression analysis suggests that this ability of Cactus to enhance Toll stems from the mobilization of a free Cactus pool induced by the Calpain A protease. These results indicate that Cactus acts to bolster Dorsal activation, in addition to its role as a NFκB inhibitor, ensuring a correct response to Toll signals.

RND (resistance-nodulation-division) transporters are found in several Gram-negative bacteria species. These proteins form a complex along with membrane fusion proteins and outer membrane factors. This complex acts as an efflux pump, transporting several different molecules directly from the cytoplasm to the extracellular medium. Although the crystallographic structure of each protein of the complex is known, the complete complex assembly is not yet fully characterized. In 2014, a model for the complete system, based upon a cryoelectron-microscopy map, was proposed. In the present work, we propose an alternative model that also satisfies the volume obtained from the cryoelectron-microscopy assay. In this model, the AcrA helical domains are interleaved to the helical domains of the TolC protein, increasing the diameter of the formed pore. We believe that this model represents a better model for RND-type efflux pump and might contribute to the characterization of this system.

International audience; Describing biological macromolecular energetics from computer simulations can pose major challenges, and often necessitates enhanced conformational sampling. We describe the calculation of conformational free-energy profiles along carefully chosen collective coordinates: “consensus” normal modes, developed recently as robust alternatives to conventional normal modes. In an application to the HIV-1 protease, we obtain efficient sampling of significant flap opening movements governing inhibitor binding from relatively short simulations, in close correspondence with experimental results.

Cyclin-dependent kinases (CDKs) and their associated regulatory cyclins are central for timely regulation of cell-cycle progression. They constitute attractive pharmacological targets for development of anticancer therapeutics, since they are frequently deregulated in human cancers and contribute to sustained, uncontrolled tumor proliferation. Characterization of their structural/dynamic features is essential to gain in-depth insight into structure-activity relationships. In addition, the identification of druggable pockets or key intermediate conformations yields potential targets for the development of novel classes of inhibitors. Structural studies of CDK2/cyclin A have provided a wealth of information concerning monomeric/heterodimeric forms of this kinase. There is, however, much less structural information for other CDK/cyclin complexes, including CDK4/cyclin D1, which displays an alternative (open) position of the cyclin partner relative to CDK, contrasting with the closed CDK2/cyclin A conformation. In this study, we carried out normal-mode analysis and enhanced sampling simulations with our recently developed method, molecular dynamics with excited normal modes, to understand the conformational equilibrium on these complexes. Interestingly, the lowest-frequency normal mode computed for each complex described the transition between the open and closed conformations. Exploration of these motions with an explicit-solvent representation using molecular dynamics with excited normal modes confirmed that the closed conformation is the most stable for the CDK2/cyclin A complex, in agreement with their experimentally available structures. On the other hand, we clearly show that an open↔closed equilibrium may exist in CDK4/cyclin D1, with closed conformations resembling that captured for CDK2/cyclin A. Such conformational preferences may result from the distinct distributions of frustrated contacts in each complex. Using the same approach, the putative roles of the Thr160 phosphoryl group and the T-loop conformation were investigated. These results provide a dynamic view of CDKs revealing intermediate conformations not yet characterized for CDK members other than CDK2, which will be useful for the design of inhibitors targeting critical conformational transitions.

Estudo da influência do campo de torção e da matriz nematica no passo de um cristal liquido colesterico Study of the influence of the torsion field and the nematic matrix on the pitch of a cholesteric liquid crystal

GRASPs (from Golgi ReAssembly and Stacking Proteins) are proteins involved in the organization and maintenance of the Golgi complex. While the extension of this role has been questioned in the past few years, many other functions have been assigned to them. In particular, we highlight the participation in tethering vesicles that need to move along the Golgi and processes of unconventional protein secretion. Structurally, GRASPs can be divided in an N-terminal domain called GRASP and a non-conserved, highly disordered C-terminal domain, termed SPR. Structural information, hitherto, has been scarce, which motivated the beginning of this project. To study GRASPs, we used the only GRASP (called Grh1) of a model organism: the yeast Saccharomyces cereviseae. We showed that Grh1 contains regions of intrinsic disorder also in its GRASP domain, being classified as a molten globule. Besides, Grh1 is capable of forming amyloid-like fibrils when in specific conditions in vitro, such as low pH and moderately elevated temperature. With the aim of investigating a possible relationship between fibril formation and the role played by Grh1 in unconventional protein secretion, part of the present thesis was done in vivo, and it was possible to demonstrate that the yeast GRASP fibrillates in starvation and heat-shock conditions. Here, we discuss the use of Fluorescence Lifetime Imaging Microscopy as a valid technique to help detect fibril formation in cell and also the possible implications of fibrillation for the formation of the Compartments for Unconventional Protein Secretion (CUPS). This work also contains initial experiments that point to a liquid-liquid phase separation of Grh1, an observation in consonance with the findings of intrinsic disorder and the recent proposition that the Golgi is actually an organelle phase separated from the cytosol. Finally, we present initial experiments of the characterization of Bug1, the golgin partner of Grh1. There are no structural data available on golgins in solution yet and their purification always presented an obstacle on obtaining them. We describe here a protocol capable of purifying Bug1 in high quantities, therefore paving the way for many other discoveries in the fields of protein secretion and liquid-liquid phase separation. GRASPs (de Golgi ReAssembly and Stacking Proteins) são proteínas inicialmente envolvidas na organização e manutenção do complexo de Golgi. Enquanto este papel vem sendo questionado nos últimos anos, muitas outras funções vem sendo a elas atribuídas. Em especial, destacamos a participação no ancoramento de vesículas de secreção que precisam atravessar o Golgi e processos de secreção não convencional. Estruturalmente, GRASPs podem ser divididas em um domínio N-terminal chamado GRASP e um domínio C-terminal que é altamente desordenado e não conservado, chamado SPR. Informação estrutural sobre GRASPs, até agora, tem sido muito escassa, o que motivou o início deste trabalho. Para estudar GRASPs, usamos a única GRASP da levedura Saccharomyces cereviseae (Grh1), um organismo modelo. Demonstramos que Grh1 possui regiões de desordem intrínseca também no domínio GRASP, sendo considerada uma proteína do tipo molten globule. Além disso, Grh1 é capaz de formar fibras do tipo amiloide quando em condições específicas in vitro, como baixo pH e temperatura levemente elevada. No objetivo de investigar uma possível correlação entre a formação de fibras e a função desempenhada por Grh1 em processos de secreção não convencional, parte desse trabalho foi realizada in vivo, e foi possível mostrar que a GRASP de levedura fibrila em condições específicas de privação de nutrientes e choque térmico. Aqui, discutimos o uso da microscopia do tempo de vida de fluorescência como uma técnica válida para auxiliar na detecção de formação de fibras in cell, e também as possíveis implicações da formação de fibras por GRASPs para formação de Compartimentos para Secreção Não Convencional (do inglês CUPS). Esse trabalho também contém experimentos iniciais que apontam para uma separação de fase líquido-líquido sofrida poe Grh1, o que estaria em consonância com os achados de desordem intrínseca e a recente proposta de que o Golgi seria, na verdade, uma organela em fase separada do citosol. No final deste trabalho, apresentamos ainda experimentos iniciais de caracterização de Bug1, a golgina parceira de Grh1. Não existem dados estruturais disponíveis para nenhuma golgina em solução, e sua purificação sempre se mostrou um obstáculo. Descrevemos aqui um protocolo através do qual foi possível se purificar Bug1 em grandes quantidades, abrindo caminho assim para que muitas outras descobertas sejam feitas no que diz respeito à secreção de proteínas e separação de fase líquido-líquido.

A galectina-4 humana (HGal-4), pertencente à família das galectinas, possui dois domínios de reconhecimento de carboidratos (CRDs) com alta afinidade para β-galactosídeos e se encontra amplamente distribuída em células normais e neoplásicas de diferentes organismos. Suas funções snglobam uma grande variedade de eventos celulares, tais como processos inflamatórios, neoplásicos, progressão tumoral e metástase. Entretanto, muitas perguntas sobre suas interações com diferentes carboidratos, a especificidade destas interações e o papel específico das galectinas permanecem ainda sem resposta. No presente trabalho, propomos a investigação das interações galectina-glicano da galectina-4 humana e de seus domínios CRDs independentes (CRD-I e CRD-II) através de um conjunto de métodos biofísicos. Através do método de dicroísmo circular (CD), usando várias regiões espectrais, e fluorescência fomos capazes de entender mudanças ocorrentes na estrutura secundária e terciária das protéinas quando da interação com lactose/sacarose. Estes dados, juntamente com testes de hemaglutinação, mostraram que a glectina-4 e os CRDs respondem de forma distinta à ligação com açúcar. Por diferentes técnicas (fluorescência, ITC e MST) determinamos as constantes de dissociação para os domínios CRDs (Kd ~0,5 mM) e para HGal-4 e, de forma qualitativa, os valores obtidos indicaram possíveis estados oligoméricos dessas proteínas. A investigação da interação proteína-membrana da HGal-4 foi feita, primeiramente, com miméticos de membranas e monitorada pela técnica de RPE em crescente complexidade de composição de tais miméticos, indo desde composições mais simples, passando por lipid rafts na presença de diferentes glicolipídeos (GM1, LPS) e chegando-se à interação com células tumorais (U87MG, T98G e HT-29). Tais experimentos mostraram que galectina-4 reconhece e se liga naqueles modelos onde existem glicanos complexos na superfície. Investigamos também a participação de HGal-4 endógena e exógena no tratamento quimioterápico de células tumorais e verificamos um papel importante de HGal-4 para células HT-29. Finalizando esta tese, apresentamos o trabalho realizado em um ano de estágio na University of Oxford, durante o qual, investigamos a estrutura da região C-terminal de um receptor da família GPCR, qual seja o receptor de neurotensina NTS1. Aqui, mais uma vez, foi empregada a técnica de RPE que aliada à produção/marcação de mutantes do receptor, permitiu determinar que a hélice H8 se estabiliza quando em proteolipossomos. Human galectin-4 (HGal-4), a member of the galectin family, contains two carbohydrate recognition domains (CRDs) with high affinity for β-galactosides and is widely distributed in normal and neoplastic cells of different organisms. Its functions include a wide variety of cellular events such as inflammation, cancer, cell adhesion, tumor progression and metastasis. However, many questions about their interactions with different carbohydrates, the specificity of these interactions and the specific role of galectins remain unanswered. In this study, we propose the investigation of galectin-glycan interactions of human galectin-4 and its independent CRDs (CRD-I and-II) through a combination of biophysical methods. From circular dichroism (CD), measured in different spectral ranges, and fluorescence experiments we were able to understand changes in secondary and terciary structure of the protein while interacting with lactose/sucrose. These results along with hemagglutination assays showed that galectin-4 and its CRDs respond differently to sugar binding. From fluorescence, ITC and MST measurements we determined the dissociation constants for the CRDs (Kd ~0.5 mM) and for HGal-4. These values qualitatively indicated the formation of potential oligomers of CRDs and of HGal-4. The investigation of the HGal-4 interaction with membranes was firstly performed using mimetic membranes and monitored by EPR spectroscopy. The composition of the mimetic membranes was gradually increased so that to span simple compositions (such as DMPC), passing by lipid rafts in the presence of different glycolipds (GM1, LPS) up to interactions with tumor cells (U87MG, T98G e HT-29). These experiments showed that galectin-4 recognizes and binds to membrane models constituted by complex glycans on their surface. We also investigated the involvement of endogenous and exogenous HGal-4 in chemotherapies of tumor cells and found an important role of HGal-4 in the case of HT-29 cells. At last, we presented the work done in an one-year internship at the University of Oxford, during which we investigated the C-terminal region of the GPCR family receptor, the neurotensin receptor NTS1. Here, we used once again the EPR technique combined with the production/spin-labelling of mutants of the receptors, and determined that helix H8 was stabilized upon receptor reconstitution in proteolipossomes.

A Análise de Acoplamentos Estatísticos é uma técnica computacional capaz de identificar resíduos importantes para a estrutura e função de proteínas em uma família por meio da quantificação de conservação posicional, correlação entre posições e identificação de grupos de resíduos correlacionados entre si. Neste trabalho, a análise de acoplamentos estatísticos foi implementada e aplicada ao estudo das proteínas tirosina fosfatases. Em conjunto com as proteínas tirosina quinases (PTKs), que adicionam um grupo fosforil a um resíduo de tirosina em uma proteína, as proteínas tirosina fosfatases (PTPs), que o removem, são responsáveis por diversos processos de sinalização celular. Elas são um caso de evolução convergente, onde um subgrupo (as proteínas tirosina fosfatases de baixo peso molecular) não apresenta homologia às chamadas PTPs \"clássicas\", capazes de defosforilar apenas resíduos de tirosina, e às fosfatases de especifidicade dupla, capazes de defosforilar também resíduos de serina e treonina, além de substratos não-protéicos. Em comum, as três subfamílias apresentam apenas o motivo CX5R, característico para todas as PTPs. Através do estudo das três subfamílias utilizando a análise de acoplamentos estatísticos, foi possível obter uma descrição detalhada de suas características conservadas e correlacionadas, relacionando-as ao conhecimento acumulado sobre proteínas tirosina fosfatases e a questões em aberto como a regulação por dimerização, a especificidade e mutações relacionadas a patologias. Foi possível também apresentar um método capaz de distinguir proteínas tirosina fosfatases de baixo peso molecular das arsenato redutases, derivadas das primeiras por evolução divergente. Adicionalmente, a técnica foi aplicada ao estudo das hexoquinases, às superóxido dismutases e às peroxidases. A tese descreve também estudos desenvolvidos pelo autor na área de cristalografia de proteínas a determinação das estruturas da Transtirretina humana em complexo com genisteína, da holo-Hexoquinase PI de S. cerevisae, do complexo IL-22/IL-22R1 e da Laminarinase de R. marinus. The statistical coupling analysis is a computational technique which can identify important residues for the structure and function of proteins in a family by quantifying positional conservation, correlation between positions and identifying groups of self-correlating residues. Its implementation in this research group was applied to the study of the protein tyrosine phosphatases. Together with the protein tyrosine kinases (PTKs), which add a phosphoryl group to a tyrosine residue in proteins, the protein tyrosine phosphatases (PTPs), which remove it, are responsible for a variety of cell signaling processes. They are a case of convergent evolution, since one subgroup (the low molecular weight protein tyrosine phosphatases) are not homologous to the classical phosphatases, which can only dephosphorilate tyrosine residues, and the dual-specificity phosphatases, which can also dephosphorilate serine and threonine residues, and also non-proteinaceous substrates. All three sub-families have, in common, the CX5R motif, a characteristic of all PTPs. By applying the statistical coupling analysis to the study of the three sub-families, it was possible to obtain a detailed depiction of their conserved and correlated characteristics, relating them to the accumulated knowledge on protein tyrosine phosphatases and open questions such as protein regulation by dimerization, specificity and disease-related mutations. It was also possible to present a method to distinguish between low molecular weight phosphatases and arsenate reductases, which are derived by the former by divergent evolution. In addition, the technique was applied to the study of hexokinases, superoxide dismutases and peroxidases. The thesis also describe studies developed by the author in the field of protein crystallography the structure determination of human transthyretin in complex with genistein, holo-hexokinase PI from S. cerevisae, the IL-22/IL-22R1 complex and the laminarinase from R. marinus.

Bioinformática estrutural de proteínas modificadas por eventos de splicing alternativo Structural Bioinformatics of Proteins modified by Alternative Splicing

O programa THOR de dinâmica e mecânica molecular foi aprimorado com a introdução de técnicas que permitem a modelagem de sistemas biomoleculares na presença do solvente representado explicitamente. Essas técnicas incluem o emprego de condições de contorno periódicas, raio de corte, potencial eletrostático modificado e método shake para vínculos de distância. Além disso, a técnica da lista de vizinhos foi implementada devido ao grande aumento no número de átomos do sistema, pela inclusão explícita do solvente. Alguns sistemas foram modelados e a influência do solvente sobre suas propriedades conformacionais foi analisada, respondendo às expectativas baseadas em previsões teóricas e evidências experimentais. The computational package THOR for molecular mechanic and dynamics simulation was improved by the introduction of techniques for modeling of biomolecular systems with explicit representation of solvent molecules. Those techniques include the application of periodic boundary conditions, the use of a cut-off sphere for the potential, with modifications in the electrostatic potential, and the utilization of the method shake to impose distance constraints. Furthermore, a neighbours list was implemented, due to the high number of atoms to be dealt with when the solvent is explicity represented. Simulations were performed on some systems of biological interest, and the influence of the solvent on their conformational properties was analysed. The results are in good agreement with theoretical previsions and experimental data.