André Pina

## Background
Non-B DNA conformations are molecular structures that do not follow the canonical DNA double helix. Mutagenetic instability in nuclear and mitochondrial DNA (mtDNA) genomes has been associated with simple non-B DNA conformations, as hairpins or more complex structures, as G-quadruplexes. One of these structures is Structure A, a cloverleaf-like non-B conformation predicted for a 93-nt (nucleotide) stretch of the mtDNA control region 5′-peripheral domain. Structure A is embedded… Exibir mais

## Background
Non-B DNA conformations are molecular structures that do not follow the canonical DNA double helix. Mutagenetic instability in nuclear and mitochondrial DNA (mtDNA) genomes has been associated with simple non-B DNA conformations, as hairpins or more complex structures, as G-quadruplexes. One of these structures is Structure A, a cloverleaf-like non-B conformation predicted for a 93-nt (nucleotide) stretch of the mtDNA control region 5′-peripheral domain. Structure A is embedded in a hot spot for the 3′ end of human mtDNA deletions revealing its importance in influencing the mutational instability of the mtDNA genome.

## Methods
To better characterize Structure A, we predicted its 3D conformation using state-of-art methods and algorithms. The methodologic workflow consisted in the prediction of non-B conformations using molecular dynamics simulations. The conservation scores of alignments of the Structure A region in humans, primates, and mammals, was also calculated.

## Results
Our results show that these computational methods are able to measure the stability of non-B conformations by using the level of base pairing during molecular dynamics. Structure A showed high stability and low flexibility correlated with high conservation scores in mammalian, more specifically in primate lineages.

##Conclusions
We showed that 3D non-B conformations can be predicted and characterized by our methodology. This allowed the in-depth analysis of the structure A, and the main results showed the structure remains stable during the simulations.

## General significance
The fine-scale atomic molecular determination of this type of non-B conformation opens the way to perform computational molecular studies that can show their involvement in mtDNA cellular mechanisms. Exibir menos

Increased levels of cathepsin B (CatB), a cysteine protease, have been associated with different types of tumors, including prostate cancer. Hence, the identification of novel targeting ligands homing CatB may be a promising approach for the development of CatB-targeted therapies. In this work, a methodology called systematic evolution of ligands by EXponential enrichment (SELEX) was used to generate and isolate new aptamers targeting CatB. After 8 rounds of selection, the pool was sequenced… Exibir mais

Increased levels of cathepsin B (CatB), a cysteine protease, have been associated with different types of tumors, including prostate cancer. Hence, the identification of novel targeting ligands homing CatB may be a promising approach for the development of CatB-targeted therapies. In this work, a methodology called systematic evolution of ligands by EXponential enrichment (SELEX) was used to generate and isolate new aptamers targeting CatB. After 8 rounds of selection, the pool was sequenced, and the 10 most prevalent sequences, along with their corresponding reverse complementary sequences, were further analyzed. In order to assess the binding affinity of the aptamers towards the CatB, bioinformatics tools were used. After generating the 3D structures for each aptamer, the HADDOCK software was used for the docking studies between them and CatB. Molecular dynamics simulations and free binding energy calculations by molecular mechanics-generalized born surface area (MM-GBSA) allowed selection of a new aptamer with potential to target CatB. Cell binding assays against the PC-3 prostate cancer cell line confirmed the in silico predictions, further validating the newly discovered aptamer as a promising targeting ligand for CatB-overexpressing cancer cells. Exibir menos

The catalytic mechanism of Pdx2 was herein studied with atomic detail employing the computational ONIOM hybrid QM/MM methodology. Pdx2 employs a Cys-His-Glu catalytic triad to deaminate glutamine to glutamate and ammonia–the source of the nitrogen of pyridoxal 5’-phosphate(PLP). This enzyme is, therefore, a rate-limiting step in the PLP biosynthetic pathway of Malaria and Tuberculosis pathogens. For this reason,Pdx2 is considered a novel and promising drug target to treat these diseases. The… Exibir mais

The catalytic mechanism of Pdx2 was herein studied with atomic detail employing the computational ONIOM hybrid QM/MM methodology. Pdx2 employs a Cys-His-Glu catalytic triad to deaminate glutamine to glutamate and ammonia–the source of the nitrogen of pyridoxal 5’-phosphate(PLP). This enzyme is, therefore, a rate-limiting step in the PLP biosynthetic pathway of Malaria and Tuberculosis pathogens. For this reason,Pdx2 is considered a novel and promising drug target to treat these diseases. The results herein obtained show that the catalytic mechanism of Pdx2 occurs in six steps that can be divided into four stages: (i) activation of Cys87, (ii) deamination of glutamine with the formation of the glutamyl-thioester intermediate, (iii) hydrolysis of the formed intermediate, and (iv) enzymatic turnover. The kinetic data available in the literature(19.1–19.5 kcal mol-1) agrees very well with the calculated free energy barrier of the hydrolytic step(18.2 kcal.mol-1), which is the rate-limiting step of the catalytic process when substrate is readily available in the active site. This catalytic mechanism differs from other known amidases in three main points: i)it requires the activation of the nucleophile Cys87 to a thiolate; ii)the hydrolysis occurs in a single step and therefore does not require the formation of a second tetrahedral reaction intermediate, as it is proposed, and iii)Glu198 does not have a direct role in the catalytic process. Exibir menos

FCUL, Lisbon, Portugal

André F. Pina, Sérgio F. Sousa, Nuno M. F. S. A. Cerqueira

October 5-12
Università della Svizzera italiana, Lugano, Switzerland

André F. Pina, Sérgio F. Sousa, Nuno M. F. S. A. Cerqueira

Enzymes catalyze a plethora of chemical reactions in nature. These biocatalysts offer a variety of benefits that no other synthetic catalyst can offer: high catalytic power, regio- and stereo-selectivity, ability to work at mild conditions, and to react in environmentally friendly manufacturing processes. However, enzymes are not normally optimally suited for industrial applications. It is important to improve their properties to favor a more efficient application of enzymes in an industrial… Exibir mais

Enzymes catalyze a plethora of chemical reactions in nature. These biocatalysts offer a variety of benefits that no other synthetic catalyst can offer: high catalytic power, regio- and stereo-selectivity, ability to work at mild conditions, and to react in environmentally friendly manufacturing processes. However, enzymes are not normally optimally suited for industrial applications. It is important to improve their properties to favor a more efficient application of enzymes in an industrial setting while also expanding the range of potential chemical reactions that they can catalyze. Problems like the scarce nature of oil reserves, together with increasing concerns on man-made environmental damage, have led to a boost in the need for the development and optimization of new and improved tailor-made enzymes for industrial applications. In this review, we focus our analysis on different enzymes, containing a pyridoxal-5’-phosphate (PLP) cofactor, that have great potential as biocatalysts in a variety of industries and applications. The selected PLP enzymes are the ω-transaminases, lysine decarboxylase, threonine aldolase, L-tyrosine phenol-lyase, α-amino-ϵ-caprolactam racemases, and cystathionine β-lyases. For each of these enzymes, the catalytic mechanism, industrial application, as well as, the advantages and disadvantages of their application is reviewed in detail. In general, this review highlights the immense biocatalytic potential, rich chemistry and diverse set of applications that different enzymes sharing a common element (PLP) can have. Exibir menos

CIM-FMUP, Porto, Portugal

André Pina, Sérgio F. Sousa, Nuno M. F. S. A. Cerqueira

FCT/UNL, Lisbon, Portugal

André F. Pina, Sérgio F. Sousa, Nuno M. F. S. A. Cerqueira

Long, S., Resende, D. I., Kijjoa, A., Silva, A. M., Pina, A., Fernández-Marcelo, T., Vasconcelos, M. H., Sousa, E., Pinto, M. M. (2018)

Faculty of Medicine of the University of Porto (FMUP), Porto, Portugal

Lopes, N., Long, S., Resende, D., Kijjoa, A., Silva, A., Pina, A., Fernández-Marcelo, T., Vasconce-
los, M. H., Pinto, M., Sousa, E.

Faculty of Pharmacy of the University of Lisbon (FFUL), Lisbon, Portugal

Long, S., Resende, D., Kijjoa, A., Silva, A., Pina, A., Fernández-Marcelo, T., Vasconcelos, M. H., Sousa, E., Pinto, M.

Faculty of Medicine of the University of Porto (FMUP), Porto, Portugal

Castro, I., Pina, A., Sousa, D., Lopes-Rodrigues, V., Caires, H., Xavier, C. P. R., Vasconcelos, M. H.