Unraveling the Na,K-ATPase alpha(4) Subunit Assembling Induced by Large Amounts of C(12)E(8) by Means of Small-Angle X-ray Scattering

In the current work, we studied the effect of the nonionic detergent dodecyloctaethyleneglycol, C(12)E(8), on the structure and oligomeric form of the Na,K-ATPase membrane enzyme (sodium-potassium pump) in aqueous suspension, by means of small-angle X-ray scattering (SAXS). Samples composed of 2 mg/mL of Na,K-ATPase, extracted from rabbit kidney medulla, in the presence of a small amount of C(12)E(8) (0.005 mg/mL) and in larger concentrations ranging from 2.7 to 27 mg/mL did not present catalytic activity. Under this condition, an oligomerization of the alpha subunits is expected. SAXS data were analyzed by means of a global fitting procedure supposing that the scattering is due to two independent contributions: one coming from the enzyme and the other one from C(12)E(8) micelles. In the small detergent content (0.005 mg/mL), the SAXS results evidenced that Na,K-ATPase is associated into aggregates larger than (alpha beta)(2) form. When 2.7 mg/mL of C(12)E(8) is added, the data analysis revealed the presence of alpha(4) aggregates in the solution and some free micelles. Increasing the detergent amount up to 27 mg/mL does not disturb the alpha(4) aggregate: just more micelles of the same size and shape are proportionally formed in solution. We believe that our results shed light on a better understanding of how nonionic detergents induce subunit dissociation and reassembling to minimize the exposure of hydrophobic residues to the aqueous solvent.

Synthesis, characterization and reactivity of new cobalt, palladium and nickel complexes bearing α-diimines and P, O ligands

This work describes the synthesis and characterization of a series of new α-diimine and P,O, β-keto and acetamide phosphines ligands, and their complexation to Ni(II), Co(II),Co(III) and Pd(II) to obtain a series of new compounds aiming to study their structural characteristics and to test their catalytic activity. All the compounds synthesized were characterized by the usual spectroscopic and spectrometric techniques: Elemental Analysis, MALDI-TOF-MS spectrometry, IR, UV-vis, 1H, 13C and 31P NMR spectroscopies. Some of the paramagnetic compounds were also characterized by EPR. For the majority of the compounds it was possible to solve their solid state structure by single crystal X-ray diffraction. Tests for olefin polymerization were performed in order to determine the catalytic activity of the Co(II) complexes. Chapter I presents a brief introduction to homogenous catalysis, highlighting the reactions catalyzed by the type of compounds described in this thesis, namely olefin polymerization and oligomerization and reactions catalyzed by the complexes bearing α-diimines and P,O type ligands. Chapter II is dedicated to the description of the synthesis of new α-diimines cobalt (II) complexes, of general formula [CoX2(α-diimine)], where X = Cl or I and the α-diimines are bis(aryl)acenaphthenequinonediimine) (Ar-BIAN) and 1,4-diaryl-2,3-dimethyl-1,4-diaza-1,3-butadiene (Ar-DAB). Structures solved by single crystal X-ray diffraction were obtained for all the described complexes. For some of the compounds, X-band EPR measurements were performed on polycrystalline samples, showing a high-spin Co(II) (S = 3/2) ion, in a distorted axial environment. EPR single crystal experiments on two of the compounds allowed us to determine the g tensor orientation in the molecular structure. In Chapter III we continue with the synthesis and characterization of more cobalt (II)complexes bearing α-diimines of general formula [CoX2(α-diimine)], with X = Cl or I and α-diimines are bis(aryl)acenaphthenequinonediimine) (Ar-BIAN) and 1,4-diaryl-2,3-dimethyl- 1,4-diaza-1,3-butadiene (Ar-DAB). The structures of three of the new compounds synthesized were determined by single crystal X-ray diffraction. A NMR paramagnetic characterization of all the compounds described is presented. Ethylene polymerization tests were done to determine the catalytic activity of several of the Co(II) complexes described in Chapter II and III and their results are shown. In Chapter IV a new rigid bidentate ligand, bis(1-naphthylimino)acenaphthene, and its complexes with Zn(II) and Pd(II), were synthesized. Both the ligand and its complexes show syn and anti isomers. Structures of the ligand and the anti isomer of the Pd(II) complex were solved by single crystal X-ray diffraction. All the compounds were characterized by elemental analysis, MALDI-TOF-MS spectrometry, and by IR, UV-vis, 1H, 13C, 1H-1H COSY, 1H-13C HSQC, 1H-13C HSQC-TOCSY and 1H-1H NOESY NMR when necessary. DFT studies showed that both conformers of [PdCl2(BIAN)] are isoenergetics and can be obtain experimentally. However, we can predict that the isomerization process is not available in square-planar complex, but is possible for the free ligand. The molecular geometry is very similar in both isomers, and only different orientations for naphthyl groups can be expected. Chapter V describes the synthesis of new P, O type ligands, β-keto phosphine, R2PCH2C(O)Ph, and acetamide phosphine R2PNHC(O)Me, as well as a series of new cobalt(III) complexes namely [(η5-C5H5)CoI2{Ph2PCH2C(O)Ph}], and [(η5- C5H5)CoI2{Ph2PNHC(O)Me}]. Treating these Co(III) compounds with an excess of Et3N, resulted in complexes η2-phosphinoenolate [(η5-C5H5)CoI{Ph2PCH…C(…O)Ph}] and η2- acetamide phosphine [(η5-C5H5)CoI{Ph2PN…C(…O)Me}]. Nickel (II) complexes were also obtained: cis-[Ni(Ph2PN…C(…O)Me)2] and cis-[Ni((i-Pr)2PN…C(…O)Me)2]. Their geometry and isomerism were discussed. Seven structures of the compounds described in this chapter were determined by single crystal X-ray diffraction. The general conclusions of this work can be found in Chapter VI.

Natural and amyloid self-assembly of S100 proteins: structural basis of functional diversity

The S100 proteins are 10-12 kDa EF-hand proteins that act as central regulators in a multitude of cellular processes including cell survival, proliferation, differentiation and motility. Consequently, many S100 proteins are implicated and display marked changes in their expression levels in many types of cancer, neurodegenerative disorders, inflammatory and autoimmune diseases. The structure and function of S100 proteins are modulated by metal ions via Ca2+ binding through EF-hand motifs and binding of Zn2+ and Cu2+ at additional sites, usually at the homodimer interfaces. Ca2+ binding modulates S100 conformational opening and thus promotes and affects the interaction with p53, the receptor for advanced glycation endproducts and Toll-like receptor 4, among many others. Structural plasticity also occurs at the quaternary level, where several S100 proteins self-assemble into multiple oligomeric states, many being functionally relevant. Recently, we have found that the S100A8/A9 proteins are involved in amyloidogenic processes in corpora amylacea of prostate cancer patients, and undergo metal-mediated amyloid oligomerization and fibrillation in vitro. Here we review the unique chemical and structural properties of S100 proteins that underlie the conformational changes resulting in their oligomerization upon metal ion binding and ultimately in functional control. The possibility that S100 proteins have intrinsic amyloid-forming capacity is also addressed, as well as the hypothesis that amyloid self-assemblies may, under particular physiological conditions, affect the S100 functions within the cellular milieu.

Stability of the Transthyretin Molecule as a Key Factor in

Transthyretin (TTR) protects against A-Beta toxicity by binding the peptide thus inhibiting its aggregation. Previous work showed different TTR mutations interact differently with A-Beta, with increasing affinities correlating with decreasing amyloidogenecity of the TTR mutant; this did not impact on the levels of inhibition of A-Beta aggregation, as assessed by transmission electron microscopy. Our work aimed at probing differences in binding to A-Beta by WT, T119M and L55P TTR using quantitative assays, and at identifying factors affecting this interaction. We addressed the impact of such factors in TTR ability to degrade A-Beta. Using a dot blot approach with the anti-oligomeric antibody A11, we showed that A-Beta formed oligomers transiently, indicating aggregation and fibril formation, whereas in the presence of WT and T119M TTR the oligomers persisted longer, indicative that these variants avoided further aggregation into fibrils. In contrast, L55PTTR was not able to inhibit oligomerization or to prevent evolution to aggregates and fibrils. Furthermore, apoptosis assessment showed WT and T119M TTR were able to protect against A-Beta toxicity. Because the amyloidogenic potential of TTR is inversely correlated with its stability, the use of drugs able to stabilize TTR tetrameric fold could result in increased TTR/ABeta binding. Here we showed that iododiflunisal, 3-dinitrophenol, resveratrol, [2-(3,5-dichlorophenyl)amino] (DCPA) and [4- (3,5-difluorophenyl)] (DFPB) were able to increase TTR binding to A-Beta; however only DCPA and DFPB improved TTR proteolytic activity. Thyroxine, a TTR ligand, did not influence TTR/A-Beta interaction and A-Beta degradation by TTR, whereas RBP, another TTR ligand, not only obstructed the interaction but also inhibited TTR proteolytic activity. Our results showed differences between WT and T119M TTR, and L55PTTR mutant regarding their interaction with A-Beta and prompt the stability of TTR as a key factor in this interaction, which may be relevant in AD pathogenesis and for the design of therapeutic TTR-based therapies.

On the track of β-lactam resistance: Studies on the regulation of methicillinresistance in Staphylococcus aureus

Dissertação para obtenção do Grau de Doutor em Biologia

Modulation of α-synuclein aggregation and toxicity

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Contribution to drug discovery and development for tauopathies using yeast as a model

This work aimed to contribute to drug discovery and development (DDD) for tauopathies, while expanding our knowledge on this group of neurodegenerative disorders, including Alzheimer’s disease (AD). Using yeast, a recognized model for neurodegeneration studies, useful models were produced for the study of tau interaction with beta-amyloid (Aβ), both AD hallmark proteins. The characterization of these models suggests that these proteins co-localize and that Aβ1-42, which is toxic to yeast, is involved in tau40 phosphorylation (Ser396/404) via the GSK-3β yeast orthologue, whereas tau seems to facilitate Aβ1-42 oligomerization. The mapping of tau’s interactome in yeast, achieved with a tau toxicity enhancer screen using the yeast deletion collection, provided a novel framework, composed of 31 genes, to identify new mechanisms associated with tau pathology, as well as to identify new drug targets or biomarkers. This genomic screen also allowed to select the yeast strain mir1Δ-tau40 for development of a new GPSD2TM drug discovery screening system. A library of unique 138 marine bacteria extracts, obtained from the Mid-Atlantic Ridge hydrothermal vents, was screened with mir1Δ-tau40. Three extracts were identified as suppressors of tau toxicity and constitute good starting points for DDD programs. mir1Δ strain was sensitive to tau toxicity, relating tau pathology with mitochondrial function. SLC25A3, the human homologue of MIR1, codes for the mitochondrial phosphate carrier protein (PiC). Resorting to iRNA, SLC25A3 expression was silenced in human neuroglioma cells, as a first step towards the engineering of a neural model for replicating the results obtained in yeast. This model is essential to understand the mechanisms of tau toxicity at the mitochondrial level and to validate PiC as a relevant drug target. The set of DDD tools here presented will foster the development of innovative and efficacious therapies, urgently needed to cope with tau-related disorders of high human and social-economic impact.

Untersuchungen zur Dimerisierung von Somatostatin-Rezeptoren mit anderen G-Protein-gekoppelten Rezeptoren

Heterodimerization, G-Protein-Coupled Receptors, somatostatin receptor, internalization, oligomerization, dimerization, homodimerization, GPCR, desensitization, immunoprecipitation

Cracking the BAFF code.

The tumour necrosis factor (TNF) family members B cell activating factor (BAFF) and APRIL (a proliferation-inducing ligand) are crucial survival factors for peripheral B cells. An excess of BAFF leads to the development of autoimmune disorders in animal models, and high levels of BAFF have been detected in the serum of patients with various autoimmune conditions. In this Review, we consider the possibility that in mice autoimmunity induced by BAFF is linked to T cell-independent B cell activation rather than to a severe breakdown of B cell tolerance. We also outline the mechanisms of BAFF signalling, the impact of ligand oligomerization on receptor activation and the progress of BAFF-depleting agents in the clinical setting.

The specificity of TRIM5 alpha-mediated restriction is influenced by its coiled-coil domain.

Retroviruses are both powerful evolutionary forces and dangerous threats to genome integrity. As such, they have imposed strong selective pressure on their hosts, notably triggering the emergence of restriction factors, such as TRIM5 alpha, that act as potent barriers to their cross-species transmission. TRIM5 alpha orthologues from different primates have distinct retroviral restriction patterns, largely dictated by the sequence of their C-terminal PRYSPRY domain, which binds the capsid protein of incoming virions. Here, by combining genetic and functional analyses of human and squirrel monkey TRIM5 alpha, we demonstrate that the coiled-coil domain of this protein, thus far essentially known for mediating oligomerization, also conditions the spectrum of antiretroviral activity. Furthermore, we identify three coiled-coil residues responsible for this effect, one of which has been under positive selection during primate evolution, notably in New World monkeys. These results indicate that the PRYSPRY and coiled-coil domains cooperate to determine the specificity of TRIM5 alpha-mediated capture of retroviral capsids, shedding new light on this complex event.

Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B.

Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic alpha-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic alpha-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B.

Host innate immune responses to microbial pathogens.

Sepsis is among the leading causes of death worldwide and its incidence is increasing. Defined as the host response to infection, sepsis is a clinical syndrome considered to be the expression of a dysregulated immune reaction induced by danger signals that may lead to organ failure and death. Remarkable progresses have been made in our understanding of the molecular basis of host defenses in recent years. The host defense response is initiated by innate immune sensors of danger signals designated under the collective name of pattern-recognition receptors. Members of the family of microbial sensors include the complement system, the Toll-like receptors, the nucleotide-binding oligomerization domainlike receptors, the RIG-I-like helicases and the C-type lectin receptors. Ligand-activated pattern-recognition receptors kick off a cascade of intracellular events resulting in the expression of co-stimulatory molecules and release of effector molecules playing a fundamental role in the initiation of the innate and adaptive immune responses. Fine tuning of proinflammatory and anti-inflammatory reactions is critical for keeping the innate immune response in check. Overwhelming or dysregulated responses induced by infectious stimuli may have dramatic consequences for the host as shown by the profound derangements observed in sepsis. Unfortunately, translational research approaches aimed at the development of therapies targeting newly identified innate immune pathways have not held their promises. Indeed, all recent clinical investigations of adjunctive anti-sepsis treatments had little, if any, impact on morbidity and all-cause mortality of sepsis. Dissecting the mechanisms underlying the transition from infection to sepsis is essential for solving the sepsis enigma. Important components of the puzzle have already been identified, but the hunt must go on in the laboratory and at the bedside.

Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human.

Ligands of the tumor necrosis factor superfamily (TNFSF) (4-1BBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA1, EDA2, FasL, GITRL, LIGHT, lymphotoxin alpha, lymphotoxin alphabeta, OX40L, RANKL, TL1A, TNF, TWEAK, and TRAIL) bind members of the TNF receptor superfamily (TNFRSF). A comprehensive survey of ligand-receptor interactions was performed using a flow cytometry-based assay. All ligands engaged between one and five receptors, whereas most receptors only bound one to three ligands. The receptors DR6, RELT, TROY, NGFR, and mouse TNFRH3 did not interact with any of the known TNFSF ligands, suggesting that they either bind other types of ligands, function in a ligand-independent manner, or bind ligands that remain to be identified. The study revealed that ligand-receptor pairs are either cross-reactive between human and mouse (e.g. Tweak/Fn14, RANK/RANKL), strictly species-specific (GITR/GITRL), or partially species-specific (e.g. OX40/OX40L, CD40/CD40L). Interestingly, the receptor binding patterns of lymphotoxin alpha and alphabeta are redundant in the human but not in the mouse system. Ligand oligomerization allowed detection of weak interactions, such as that of human TNF with mouse TNFR2. In addition, mouse APRIL exists as two different splice variants differing by a single amino acid. Although human APRIL does not interact with BAFF-R, the shorter variant of mouse APRIL exhibits weak but detectable binding to mouse BAFF-R.

Redirecting anti-viral CTL against cancer cells by surface targeting of monomeric MHC class I-viral peptide conjugated to antibody fragments.

To combine the advantage of both the tumor targeting capacity of high affinity monoclonal antibodies (mAbs) and the potent killing properties of cytotoxic T lymphocytes (CTL), we investigated the activity of conjugates made by coupling single Fab' fragments, from mAbs specific for tumor cell surface antigens, to monomeric HLA-A2 complexes containing the immunodominant influenza-matrix peptide 58-66. In solution, the monovalent 95 kDa Fab-HLA-A2/Flu conjugates did not activate influenza-specific CTL. However, when targeted to tumor cells expressing the relevant tumor-associated antigen, the conjugates induced CTL activation and efficient tumor cell lysis, as a result of MHC/peptide surface oligomerization. The highly specific and sensitive in vitro cytotoxicity results presented suggest that injection of Fab-MHC/peptide conjugates could represent a new form of immunotherapy, bridging antibody and T lymphocyte attack on cancer cells.

Transcriptional Activation and Receptor Dimerization is Affected by Mutations in the NR2E3 Ligand-Binding Domain

Purpose: Mutations in the ligand-binding domain (LBD) of NR2E3 cause recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS), Goldmann-Favre syndrome (GFS) and clumped pigmentary retinal degeneration (CPRD). In addition to ligand binding, the LBD contains also essential amino acid sequences for the oligomerization of nuclear receptors. The aim of our studies is to characterize the impact of mutations in the LBD on receptor oligomerization and transcriptional activity of NR2E3. Methods: The different NR2E3 mutants were generated by QuickChange mutagenesis and analyzed in 293T-based transactivation studies and BRET2 (bioluminescence resonance electron transfer) assays. In silico homology modeling of mutant proteins was also performed using available crystallographic data of related nuclear receptors. Results: The mutants p.W234S, p.A256V, p.A256E, p.L263P, p.R309G, p.R311Q, p.R334G, p.L336P, p.L353V, p.R385P and p.M407K, all located in the LBD, showed impaired receptor dimerization at various degrees. Impaired repressor dimerization as assessed by BRET2 assays did not always correlate with impaired repressor function of NR2E3 as assessed by cell-based reporter assays. There were minor differences of transcriptional activity of mutant proteins on mouse S-opsin (opn1sw), mouse cone arrestin (arr3) and human cone arrestin, suggesting that the effect of LBD mutations was independent of the promoter context. Conclusions: Mutational analysis and homology modeling allowed the characterization of potential oligomerization interfaces of the NR2E3 LBD. Additionally, mutations in NR2E3 LBD may cause recessive retinal degenerations by different molecular mechanisms.