Structure and lipid interactions of membrane-associated glycosyltransferases : Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes

This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway. From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine.

Processos Oxidativos Avançados para Remediação de Águas Contaminadas por Pesticidas

Este trabalho teve como objectivo inicial o estudo de processos oxidativos avançados de forma a remediar e tratar águas contaminadas por pesticidas. No entanto, ao longo do trabalho experimental, constatou-se que os produtos resultantes da degradação de pesticidas são muitas vezes mais tóxicos do que os compostos que lhes deram origem e que, por isso, degradar um composto nem sempre é o melhor para o ambiente. Assim, neste trabalho, procurou-se estudar o processo de degradação com o objectivo de minimizar o impacto ambiental dos pesticidas na água e no ambiente em geral. A parte experimental deste trabalho foi dividida em duas etapas, sendo que, em ambas, a voltametria de onda quadrada e a espectrofotometria de UV/Vis foram os métodos de análise utilizados, para acompanhar o processo de fotodegradação. Na primeira etapa estudou-se a relação entre a estrutura química dos pesticidas MCPA, MCPP, 2.4-D e Dicloroprop e a sua fotodegradação. Soluções aquosas dos pesticidas enunciados foram submetidas a irradiação UV/vis, com incrementos variáveis de tempo de irradiação. Os resultados obtidos, nesta etapa, permitiram constatar diferenças na percentagem de degradação dos diferentes pesticidas. Dos pesticidas estudados verificou-se uma maior fotodegradação para o MCPA e MCPP seguido do Dicloroprop e finalmente o 2.4-D que se degradou menos. Os dados obtidos sugerem que a fotodegradação destes pesticidas está intimamente ligada com a estrutura das moléculas. A presença de um maior número de grupos cloro ligados ao anel aromático nos pesticidas 2,4-D e Dicloroprop faz com que estes sejam mais estáveis e por isso se degradam menos que o MCPA e o MCPP. Por outro lado, o facto de o 2,4-D apresentar um potencial de oxidação mais elevado do que o Dicloroprop, faz com que este seja mais difícil de degradar, o que justifica a diferença entre os dois. Desta forma, foi possível concluir que a estrutura dos pesticidas condiciona o processo de degradação, como esperado. Na segunda etapa, estudou-se a estabilização dos pesticidas MCPA e MCPP após encapsulação, com 2-hidroxipropil-β-ciclodextrina (HP-β-CD), em água desionizada e em água do rio. Para tal, submeteram-se as soluções aquosas dos pesticidas com e sem ciclodextrina, a irradiação UV/vis, também com incrementos variáveis de tempo. No caso do MCPA verificou-se que, tanto para água desionizada como para água do rio, que este herbicida encapsulado se degrada bastante menos do que o MCPA livre. O encapsulamento permitiu reduzir quase para metade a taxa de fotodegradação. Assim, confirmou-se que a HP-β-CD permite estabilizar este pesticida, tornando-o mais resistente à fotodegradação. Desta forma, originam-se menos produtos de degradação, os quais podem ser mais tóxicos, e reduz-se de o impacto ambiental deste herbicida. Verificou-se também que o MCPA livre se degrada mais em água do rio do que em água desionizada, provavelmente devido à matéria orgânica presente nesta água, que promove o processo de degradação. No que respeita ao MCPP também se constatou que este herbicida se degrada menos encapsulado do que livre, em água desionizada e em água do rio. Neste caso, conseguiu-se reduzir pouco a taxa de fotodegradação, mas, ainda assim se verifica uma estabilização deste pesticida através do encapsulamento. No entanto, tornou-se mais evidente a estabilização do MCPP após encapsulação em água do rio, já que apresenta uma taxa de fotodegradação menor. Este facto demonstra que a HP-β-CD permite estabilizar também este pesticida, tornando-o mais resistente à fotodegradação, e reduzindo seu impacto ambiental.

Redução do Impacto Ambiental de Poluentes Orgânicos Persistentes

Nos dias de hoje a contaminação dos solos e águas subterrâneas com pesticidas através da agricultura tornou-se um problema. Problema este, considerado ainda maior nas áreas onde o abastecimento de água potável é quase totalmente à base de água subterrânea, causando deste modo risco para a saúde humana devido à exposição directa de possíveis resíduos de pesticidas na água potável. É certo que a persistência dos pesticidas no solo é importante para obter um bom controlo sob as ervas daninhas durante a sua época de crescimento, contudo o uso desses pesticidas contamina não só o solo como as águas superficiais. As questões acerca do uso de pesticidas na actualidade continuarão a persistir, uma vez que existem muitos factores e características inerentes a este processo que necessitam de ser abordadas e mais importante que isso estudadas, como por exemplo a sua degradação e toxicidade. Neste trabalho efectuou-se o encapsulamento de pesticidas em moléculas de β – ciclodextrina (β-CD). O que se pretende com este encapsulamento, é aumentar a hidrofilicidade do pesticida de forma a garantir que este persista o tempo suficiente permitindo um bom controlo das ervas daninhas, tendo sempre em conta as preocupações inerentes ao uso dos pesticidas, como por exemplo a dificuldade de biodegradação. O estudo centrou-se em torno de dois dos pesticidas mais utilizados em Portugal: MCPA e Bentazona. Estes herbicidas foram encapsulados individualmente na β-CD formando assim complexos, mais solúveis e eventualmente mais estáveis quimicamente garantindo uma redução dos efeitos dos pesticidas no meio ambiente. Este estudo foi dividido essencialmente em duas partes: a síntese e caracterização dos complexos pesticida-β-CD e posteriormente a avaliação da estabilidade química em solução aquosa e da solubilidade dos complexos formados. A utilização de diversas técnicas analíticas nomeadamente DSC, FTIR, Espectrofotometria de UV, HPLC e Electroquímica permitiram concluir que o pesticida MCPA encapsula pela acção da β-CD aquando do complexo formado em solução etanólica e numa proporção estequiométrica MCPA:β-CD de 1:2 respectivamente. Obteve-se para as várias soluções estudadas, todas elas com concentrações diferentes de β-CD, uma constante de estabilidade de 102,4. No caso da Bentazona, os resultados preliminares obtidos indiciam claramente a formação de um complexo com a β-CD para o complexo formado em solução etanólica.

Neurofilaments and NFL-TBS.40-63 peptide penetrate oligodendrocytes through clathrin-dependent endocytosis to promote their growth and survival in vitro.

International audience

Biosynthesis and mode of action of ribosomally synthesized and post-translationally modified antimicrobial peptides

One of the greatest sources of biologically active compounds is natural products. Often these compounds serve as platforms for the design and development of novel drugs and therapeutics. The overwhelming amount of genomic information acquired in recent years has revealed that ribosomally synthesized and post-translationally modified natural products are much more widespread than originally anticipated. Identified in nearly all forms of life, these natural products display incredible structural diversity and possess a wide range of biological functions that include antimicrobial, antiviral, anti-inflammatory, antitumor, and antiallodynic activities. The unique pathways taken to biosynthesize these compounds offer exciting opportunities for the bioengineering of these complex molecules. The studies described herein focus on both the mode of action and biosynthesis of antimicrobial peptides. In Chapter 2, it is demonstrated that haloduracin, a recently discovered two-peptide lantibiotic, possesses nanomolar antimicrobial activity against a panel of bacteria strains. The potency of haloduracin rivals that of nisin, an economically and therapeutically relevant lantibiotic, which can be attributed to a similar dual mode of action. Moreover, it was demonstrated that this lantibiotic of alkaliphile origin has better stability at physiological pH than nisin. The molecular target of haloduracin was identified as the cell wall peptidoglycan precursor lipid II. Through the in vitro biosynthesis of haloduracin, several analogues of Halα were prepared and evaluated for their ability to inhibit peptidoglycan biosynthesis as well as bacterial cell growth. In an effort to overcome the limitations of in vitro biosynthesis strategies, a novel strategy was developed resulting in a constitutively active lantibiotic synthetase enzyme. This methodology, described in Chapter 3, enabled the production of fully-modified lacticin 481 products with proteinogenic and non-proteinogenic amino acid substitutions. A number of lacticin 481 analogues were prepared and their antimicrobial activity and ability to bind lipid II was assessed. Moreover, site-directed mutagenesis of the constitutively active synthetase resulted in a kinase-like enzyme with the ability to phosphorylate a number of peptide substrates. The hunt for a lantibiotic synthetase enzyme responsible for installing the presumed dehydro amino acids and a thioether ring in the natural product sublancin, led to the identification and characterization of a unique post-translational modification. The studies described in Chapter 4, demonstrate that sublancin is not a lantibiotic, but rather an unusual S-linked glycopeptide. Its structure was revised based on extensive chemical, biochemical, and spectroscopic characterization. In addition to structural investigation, bioinformatic analysis of the sublancin gene cluster led to the identification of an S-glycosyltransferase predicted to be responsible for the post-translational modification of the sublancin precursor peptide. The unprecedented glycosyltransferase was reconstituted in vitro and demonstrated remarkable substrate promiscuity for both the NDP-sugar co-substrate as well as the precursor peptide itself. An in vitro method was developed for the production of sublancin and analogues which were subsequently evaluated in bioactivity assays. Finally, a number of putative biosynthetic gene clusters were identified that appear to harbor the necessary genes for production of an S-glycopeptide. An additional S-glycosyltransferase with more favorable intrinsic properties including better expression, stability, and solubility was reconstituted in vitro and demonstrated robust catalytic abilities.

Caracterização físico-química de complexo de inclusão entre hidroximetilnitrofurazona e hidroxipropil-beta-ciclodextrina

Hydroxymethylnitrofurazone (NFOH) is a prodrug that is active against Trypanosoma cruzi. It however presents low solubility and high toxicity. Hydroxypropyl-beta-cyclodextrin (HP-beta-CD) can be used as a drug-delivery system for NFOH modifying its physico-chemical properties. The aim of this work is to characterize the inclusion complex between NFOH and HP-beta-CD. The rate of NFOH release decreases after complexation and thermodynamic parameters from the solubility isotherm studies revealed that a stable complex is formed (deltaGº= 1.7 kJ/mol). This study focuses on the physico-chemical characterization of a drug-delivery formulation that comes out as a potentially new therapeutic option for Chagas disease treatment.

Non-inclusion complexes between riboflavin and cyclodextrins

Objectives To investigate the molecular interaction between beta-cyclodextrin (beta CD) or hydroxypropyl-beta-cyclodextrin (HP beta CD) and riboflavin (RF), and to test the anticancer potential of these formulations. Methods The physicochemical characterization of the association between RF and CDs was performed by UV-vis absorption, fluorescence, differential scanning calorimetry and NMR techniques. Molecular dynamics simulation was used to shed light on the mechanism of interaction of RF and CDs. Additionally, in-vitro cell culture tests were performed to evaluate the cytotoxicity of the RFCD complexes against prostate cancer cells. Key findings Neither beta CD nor HP beta CD led to substantial changes in the physicochemical properties of RF (with the exception of solubility). Additionally, rotating frame Overhauser effect spectroscopy experiments detected no spatial correlations between hydrogens from the internal cavity of CDs and RF, while molecular dynamics simulations revealed out-of-ring RFCD interactions. Notwithstanding, both RF beta CD and RFHP beta CD complexes were cytotoxic to PC3 prostate cancer cells. Conclusions The interaction between RF and either beta CD or HP beta CD, at low concentrations, seems to be made through hydrogen bonding between the flavonoid and the external rim of both CDs. Regardless of the mechanism of complexation, our findings indicate that RFCD complexes significantly increase RF solubility and potentiate its antitumour effect.

Desenvolvimento e caraterização de hidrogéis físicos e químicos a partir de polissacarídeos naturais

Dissertação de Mestrado, Engenharia Biológica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

Surface photochemistry of pesticides: an approach using diffuse reflectance and chromatography techniques

The photochemistry of pesticides triadimenol and triadimefon was studied on cellulose and beta-cyclodextrin (beta-CD) in controlled and natural conditions, using diffuse reflectance techniques and chromatographic analysis. The photochemistry of triadimenol occurs from the chlorophenoxyl moiety, while the photodegradation of triadimefon also involves the carbonyl group. The formation of 4-chlorophenoxyl radical is one of the major reaction pathways for both pesticides and leads to 4-chlorophenol. Triadimenol also undergoes photooxidation and dechlorination, leading to triadimefon and dechlorinated triadimenol, respectively. The other main reaction process of triadimefon involves alpha-cleavage from the carbonyl group, leading to decarbonylated compounds. Triadimenol undergoes photodegradation at 254 nm but was found to be stable at 313 nm, while triadimefon degradates in both conditions. Both pesticides undergo photochemical decomposition under solar radiation, being the initial degradation of rate per unit area of triadimefon 1 order of magnitude higher than the observed for triadimenol in both supports. The degradation rates of the pesticides were somewhat lower in beta-CD than on cellulose. Photoproduct distribution of triadimenol and triadimefon is similar for the different irradiation conditions, indicating an intramolecular energy transfer from the chlorophenoxyl moiety to the carbonyl group in the latter pesticide.

Functional relationship of colorectal cancer-associated glycans with malignant phenotype and transcriptome changes

Glycosyltransferases ST6GAL1 and B4GALNT2 (and their cognate antigens Sia6LacNAc and Sda, respectively) are associated with colorectal cancer (CRC) but it is not fully clear their biological and clinical significance. We explored the clinical relevance of both glycosyltransferases by interrogating The Cancer Genome Atlas (TCGA) database while the phenotypic/transcriptomic effects of ST6GAL1/B4GALNT2 overexpression were studied in genetically modified CRC cell lines. Transcriptomic data from CRC patients in TCGA database suggested a moderate impact of ST6GAL1 on CRC progression, although it was not possible to define a clear role for this glycosyltransferase. Transcriptomic analysis of ST6GAL1-transduced cell lines revealed a much deeper effect of ST6GAL1 on gene expression in SW948 than in SW48. The overexpression of ST6GAL1 induced opposite effects on soft agar growth and wound healing in both cell lines. These results indicate that the impact of a cancer-associated glycosyltransferase change on phenotype/transcriptome can be extremely variable, depending on the molecular context of the tumor cell. On the contrary, transcriptomic analysis of B4GALNT2-modified cell lines together with TCGA database survey demonstrated a strong impact of B4GALNT2 on the transcriptional activity of CRC cells, in particular its association with a better prognosis. We suggest an anti-tumoral role of B4GALNT2 in CRC. We also investigated the glycan changes related to ST6GAL1/B4GALNT2 expression in a small cohort of tissues/plasma as well as the N-glycomic profile of CRC, normal and polyp tissues. We found an increase of ST6GAL1 activity in CRC and inflammatory bowel disease plasma samples comparing with plasma from healthy donors. A different Sda protein carrier pattern was observed between healthy donors and CRC plasma samples. β-arrestin 1 is a possible candidate as Sda carrier protein in plasma samples although future validation studies are needed. The alterations found in the N-glycan pattern highlight the importance of N-glycome as a molecular signature in cancer.

Impact of Glycosyltransferases on the Phenotype, Signaling and Transcriptome of Colorectal Cancer Cell Lines. Focus on the role of glycosyltransferases B4GALNT2 and FUT6 and their cognate Sda and sLex antigens

In colorectal cancer (CRC), two carbohydrate structures are modulated: the Sda antigen, synthesized by B4GALNT2, and sLex antigen, mainly synthesized by FUT6. sLex antigen is often overexpressed and associated with worse prognosis; B4GALNT2/Sda antigen are dramatically downregulated but their role in tumor progression and development is not fully clear. TCGA interrogation revealed a dramatic down-regulation of B4GALNT2 mRNA in CRC, compared with normal samples. Patients with higher B4GALNT2 mRNA in CRC samples displayed longer survival. Yet, methylation and miRNA expression play a relevant role in B4GALNT2 downregulation in CRC. To clarify the mechanisms linking the B4GALNT2/Sda expression level to CRC phenotype, three different CRC cell lines were modified to express B4GALNT2: LS174T cell line, in which the constitutively expressed sLex antigen was partially replaced by Sda; SW480/SW620 pair, both lacking Sda and sLex antigens. In LS174T cells, the expression of B4GALNT2 reduced the ability to grow in poor adherence conditions and the expression of ALDH, a stemness marker. In SW620 cells, B4GALNT2 expression impacted on the main aspects of malignancy. In SW480 cells the expression of B4GALNT2 left unchanged the proliferation rate and the wound healing ability. To clarify the impact of sLex on CRC phenotype, the SW480/SW620 pair were permanently transfected to express FUT6 cDNA. In both cell lines, overexpression of FUT6/sLex boosted the clonogenic ability in standard growth conditions. Conversely, the growth in soft agar and the capacity to close a wound were enhanced only in SW620 cells. Transcriptome analysis of CRC cell lines transfected either with B4GALNT2 or FUT6 showed a relevant impact of both enzymes on gene expression modulation. Overall, current data may help to personalize therapies for CRC patients according to the B4GALNT2 levels and support a causal effect of this glycosyltransferase on reducing malignancy independently of sLex inhibition.