The Purification and Characterization of the Drosophila melanogaster Trithorax Protein and its Implications in the Studies of the SET domain Family of Proteins

Methylation at histone H3 lysine 4 (H3K4) is a post-translational modification often associated with transcriptional regulation through altering the structural state of chromatin. The human mixed lineage leukemia protein-1 protein (MLL1) is a known histone methyltransferase that catalyzes the transfer of methyl groups to H3K4. MLL1 works in a core complex with other essential components, proteins WDR5, RbBP5, Ash2L, DPY-30 (WRAD), which is required for H3K4 dimethylation. Trithorax (TRX) protein is the Drosophila melanogaster ortholog to human MLL1, and although structurally similar is unable to perform dimethylation when in complex with the human components. The goal of this study is to understand the structural basis for this difference. We systematically mutated 20 amino acids in TRX the equivalent amino acid in human MLL1 and tested for a gain-of-function H3K4 dimethylation activity. We found 20 amino acid positions in TRX that were highly conserved among intertebrates but were different in vertebrates. Out of the 20 amino acids mutated, 5 showed a gain of dimethylation activity. All of the mutations that showed a gain of dimethylation activity localized to a common SET domain surface. The identified mutations on the common surface identify a location of the dimethyltransferase active site on MLL1

Genome mining-directed discovery of novel 2,5-diketopiperazines from actinobacteria

Natural products derived from cyclodipeptides (CDPs) with a 2,5-diketopiperazine (DKP) skeleton comprise an important class of secondary metabolites, especially indole alkaloids derived from tryptophan-containing CDPs, which are widespread in fungi, bacteria, and plants. They play vital roles in drug discovery and development owing to their significant biological and pharmacological activities. In nature, the DKP cores can be generated by two distinct enzyme groups, that is, the nonribosomal peptide synthetases (NRPSs) and the aminoacyl tRNA-dependent cyclodipeptide synthases (CDPSs). Afterwards, different types of tailoring enzymes, such as cytochrome P450s, FAD-dependent oxidoreductases, cyclodipeptide oxidases (CDOs), prenyltransferases (PTs), and methyltransferases (MTs) are involved in installing a number of functional groups to the DKP scaffolds, thus generating various chemical structures. Although CDPSs belong to a newly defined family of enzymes, a large set of CDPSs have been identified. Among them, only several CDPS-associated biosynthetic pathways have been functionally characterized. In recent years, huge amounts of microbial genome sequences have been released in public databases and revealed numerous silent or cryptic gene clusters hiding in their genomes, including those for 2,5-DKPs, indicating great potential for discovery of novel metabolites. Therefore, full exploration of these untapped gene clusters could be a promising way to expand the chemical range of 2,5-DKPs accessible to the medical industry in the future. In the first project, in cooperation with Dr. Huili Yu, eleven CDPSs from Streptomyces strains were selected for investigation based on phylogenetic analysis. Their functions were characterized via heterologous expression in Escherichia coli. The coding sequences of these CDPSs were individually cloned into pET28a (+) vector and overexpressed in soluBL21 host. The fermentation cultures of generated transformants were then analyzed by LC-MS. Combined with structural elucidation of accumulated products by NMR analysis, nine CDPSs for the assembly of tryptophan-containing cyclodipeptides (cWXs) were identified. Therefore, these nine CDP synthases represented new members of the CDPS family that are responsible for cWX biosynthesis. Among them, there is one cyclo-L-Trp-L-Leu synthase, two cyclo-L-Trp-L-Pro synthases, and three cyclo-L-Trp-L-Trp synthases, as well as three unspecific CDPSs producing up to seven products with cyclo-L-Trp-L-Ala or cyclo-L-Trp-L-Tyr as the major product. Under optimized cultivation conditions, total product yields of generated CDPs in the E. coli supernatants reached 46 to 211 mg/L. In recent years, tryptophan-containing DKPs have received increasing attention due to their promising scaffolds for structural modification. Therefore, our study provides a valid experimental basis for further combination of these CDPSs with other tailoring enzymes to generate more interesting chemical entities in the field of synthetic biology. Afterwards, sequence analysis revealed that eight of nine cWX synthase genes identified in the first project are surrounded by a putative cytochrome P450 gene. Among them, two CDPS genes, gutA24309 from Streptomyces monomycini NRRL B-24309 and gutA3589 from Streptomyces varsoviensis NRRL B-3589, are located in the similar gene loci containing four additional genes coding for three modification enzymes, i.e., CDO, cytochrome P450, and MT. Heterologous expression of these two p450-associated cdps-containing gene clusters in Streptomyces coelicolor led to the identification of eight rare and novel C3-guaninyl indole alkaloids, named guanitrypmycins. Expression of different gene combinations and precursor feeding experiments proved the biosynthetic steps of guanitrypmycins. The CDP skeletons, cyclo-L-Trp-L-Phe and cyclo-L-Trp-L-Tyr assembled by the CDPS GutA, will be dehydrogenated merely at the phenylalanyl/tyrosyl side by the CDO Gut(BC) and subsequently connected with a guanine moiety by the P450 GutD. Furthermore, the MT GutE governs the last modification step to transfer a methyl group to N9′ of the guaninyl residue. Moreover, the non-enzymatic epimerization of the enzymatic pathway products via keto–enol tautomerism increases the structural diversity of guanitrypmycins. In addition, biochemical characterization further confirmed that the P450 enzyme GutD functions as the key biocatalyst and catalyzes the unprecedented regio- and stereospecific 3-guaninylation at the indole ring of the tryptophanyl moiety. Therefore, this study highlights the promise of CDPS-containing pathways as sources of novel biosynthetic transformations and natural products. In analogy, two cdps-p450-containing operons were identified in Saccharopolyspora antimicrobica via genome mining. Heterologous expression, biochemical characterization, together with structural elucidation proved that the two P450 enzymes TtpB1 and TtpB2 catalyze distinct regio- and stereospecific dimerizations of cyclo-L-Trp-L-Trp, which are differing from those previously reported in bacteria. TtpB1 represents the first bacterial P450 that catalyzes the stereospecific C3 (sp3)–C3′ (sp3) bond formation between two monomers, both from the opposite side of H-11/H-11′, while TtpB2 is characterized as the first P450 to mainly catalyze the unusual linkage between C3 (sp3) of a hexahydropyrroloindole unit and N1′ of the tryptophanyl moiety of the second monomer from the H-11 side. Thus, our finding significantly increases the repertoire of DKP-tailoring enzymes. Additionally, in comparison with chemical synthesis, this study provides a simple, direct, and efficient approach for enzymatic one-step preparation of structurally complex DKP dimers

Microbial community composition and mercury cycling in sediments of tagus estuary

Tese de doutoramento, Farmácia (Toxicologia), Universidade de Lisboa, Faculdade de Farmácia, 2016Mercury is a pervasive pollutant well known to cause several disorders in humans and wildlife. The major concern related with mercury pollution is the neurotoxicity associated to methylmercury and its presence in aquatic systems, as it undergoes bioaccumulation and biomagnification in the food chain. In aquatic ecosystems, mercury-resistant microorganisms are the main responsible for methylation of Hg2+ and also for processes of detoxification (reduction of Hg2+ and demethylation of methylmercury). High levels of mercury, including methylmercury, have been shown to exist in the Tagus Estuary. This study aims to give an insight about the involvement of microorganisms in the cycle of mercury in the Tagus Estuary, based on their phenotypic and genetic characterization. To achieve this, mercury-resistant microorganisms were isolated from sediments of four mercury-polluted areas of the Tagus Estuary (Barreiro, Cala do Norte, Rosário and Alcochete) and, after their characterization their potential to transform mercury compounds was evaluated. The isolates encompassed aerobic microorganisms, such as Bacillus sp., Vibrio sp., Aeromonas sp. and Enterobacteriacea sp., and anaerobic microorganisms, such as Clostridium sp., Enterobacteriaceae sp. and the Archaea sulfate-reducing bacteria (e.g. Desulfovibrio desulfuricans). Their resistance to mercury compounds ranged from 0.41-140 μg/mL for Hg2+ and 0.04-50.1 μg/mL for CH3Hg. The genetic system conferring detoxification ability (mer operon genes) was found only in 7% of the isolates, being all aerobes. This set of data indicated the involvement of these microorganisms in the processes of methylation and detoxification of mercury in the Tagus Estuary. To evaluate this hypothesis, isolated microorganisms and microbial communities were incubated with HgCl2. The results showed that these microorganisms are able to reduce Hg2+ into Hg0, resulting in the removal of around 50% of the total added mercury. The highest removal rates were observed among isolates of high contaminated areas (Barreiro and Cala do Norte). It was also observed the formation of organomercurials, including methylmercury. The rate of methylation among the isolates ranged between 1-8%. Moreover, it was found that bacteria isolated from salt marsh are influenced by plants species such as Sacocornia fruticosa and Spartina maritima since the kinetics of Mercury mobility between plant’s roots and the surrounding environment affects mercury-resistant microorganisms’ selection. Thus, these results are the first evidence of the relevance of interaction between bacteria and plants in Hg cycling in the Tagus Estuary. To understand better the conditions promoting methylation and demethylation, three microbial communities (aerobic, anaerobic and sulphate-reducing bacteria communities) were incubated with isotope enriched mercury species (199HgCl and CH3201HgCl). The results showed that microbial communities are actively involved in methylation and demethylation processes, being the methylation directly related with sulphate-reducing bacteria communities with rates up to 0.07% (after 48h), while the demethylation process is strongly promoted (rates up to 100%) by aerobic community. To obtain optimal conditions for mercury reduction, the effects of ambient factors, such as organic matter (glucose), sulphate, iron and chloride, on microbial reduction were evaluated by factorial design methodology. The results revealed that sulphate enhances microbial reduction, while chloride inhibits it. Overall, the results showed that microorganisms of Tagus Estuary are involved in processes that change mercury speciation through reduction and demethylation and formation of methylmercury. The removal is a pathway for detoxification and can be used on the bioremediation strategies. Meanwhile, the formation of methylmercury represents a risk for human health. Thus, this study’s set of data is useful for both risk assessment and bioremediation purposes

Characterization of labrenzin biosynthesis in marine alphaproteobacterium Labrenzia sp. PHM005

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 02-03-202

Analysis of the role of the p47 GTPase IIGP1 in Resistance against Intracellular Pathogens

IIGP1 is a member of the p47 GTPase family of IFNγ-induced proteins, which are among the most potent presently known mediators of cell-autonomous resistance against intracellular bacterial and protozoan pathogens in the mouse. From all studied members of this family IIGP1 is the best characterized with respect to biochemical characteristics and enzymatic activity in vitro, as well as membrane binding properties and dynamic behavior in cells. The role of the protein in intracellular defense was however, unknown and this study was set as an initial attempt to reveal it. This thesis describes the generation of an IIGP1 deficient mouse and analysis of the susceptibility of this animal to pathogens from protozoan and bacterial origin, which employ diverse strategies for host cell invasion and intracellular survival and replication. Despite having intact adaptive immune system, the IIGP1 deficient mice showed higher incidence of development of cerebral malaria after infection with Plasmodium berghei sporozoites. In addition, IIGP1 deficient astrocytes exhibited a partial loss of IFNγ-induced inhibition of Toxoplasma gondii growth. IIGP1 deficient animals were not susceptible to infection with Leishmania major, Listeria monocytogenes, Chlamydia trachomatis and Anaplasma phagocytophilum. From the analysis of the obtained data in the context of the intracellular lifestyle of the pathogens involved in this study, we concluded that IIGP1 seems to be specifically involved in defense against protozoan parasites, which like Pl. berghei and T. gondii reside in non-fusigenic parasitophorous vacuoles after entering cells. The mechanisms of IIGP1-dependent protection of cells against these pathogens remain to be studied