Sérgio A. Alencar

Computational analyses and prediction of guanylin deleterious SNPs

Human guanylin, coded by the GUCA2A gene, is a member of a peptide family that activates intestinal membrane guanylate cyclase, regulating electrolyte and water transport in intestinal and renal epithelia. Deregulation of guanylin peptide activity has been associated with colon adenocarcinoma, adenoma and intestinal polyps. Besides, it is known that mutations on guanylin receptors could be involved in meconium ileus. However, there are no previous works regarding the alterations driven by single nucleotide polymorphisms in guanylin peptides. A comprehensive in silico analysis of missense SNPs present in the GUCA2A gene was performed taking into account 16 prediction tools in order to select the deleterious variations for further evaluation by molecular dynamics simulations (50 ns). Molecular dynamics data suggest that the three out of five variants (Cys104Arg, Cys112Ser and Cys115Tyr) have undergone structural modifications in terms of flexibility, volume and/or solvation. In addition, two nonsense SNPs were identified, both preventing the formation of disulfide bonds and resulting in the synthesis of truncated proteins. In summary the structural analysis of missense SNPs is important to decrease the number of potential mutations to be in vitro evaluated for associating them with some genetic diseases. In addition, data reported here could lead to a better understanding of structural and functional aspects of guanylin peptides.

Shedding some light over the floral metabolism by arum lily (Zantedeschia aethiopica) spathe de novo transcriptome assembly.

Zantedeschia aethiopica is an evergreen perennial plant cultivated worldwide and commonly used for ornamental and medicinal purposes including the treatment of bacterial infections. However, the current understanding of molecular and physiological mechanisms in this plant is limited, in comparison to other non-model plants. In order to improve understanding of the biology of this botanical species, RNA-Seq technology was used for transcriptome assembly and characterization. Following Z. aethiopica spathe tissue RNA extraction, high-throughput RNA sequencing was performed with the aim of obtaining both abundant and rare transcript data. Functional profiling based on KEGG Orthology (KO) analysis highlighted contigs that were involved predominantly in genetic information (37%) and metabolism (34%) processes. Predicted proteins involved in the plant circadian system, hormone signal transduction, secondary metabolism and basal immunity are described here. In silico screening of the transcriptome data set for antimicrobial peptide (AMP) –encoding sequences was also carried out and three lipid transfer proteins (LTP) were identified as potential AMPs involved in plant defense. Spathe predicted protein maps were drawn, and suggested that major plant efforts are expended in guaranteeing the maintenance of cell homeostasis, characterized by high investment in carbohydrate, amino acid and energy metabolism as well as in genetic information.

Venom gland transcriptome analyses of two freshwater stingrays (Myliobatiformes: Potamotrygonidae) from Brazil

Stingrays commonly cause human envenoming related accidents in populations of the sea, near rivers and lakes. Transcriptomic profiles have been used to elucidate components of animal venom, since they are capable of providing molecular information on the biology of the animal and could have biomedical applications. In this study, we elucidated the transcriptomic profile of the venom glands from two different freshwater stingray species that are endemic to the Paraná-Paraguay basin in Brazil, Potamotrygon amandae and Potamotrygon falkneri. Using RNA-Seq, we identified species-specific transcripts and overlapping proteins in the venom gland of both species. Among the transcripts related with envenoming, high abundance of hyaluronidases was observed in both species. In addition, we built three-dimensional homology models based on several venom transcripts identified. Our study represents a significant improvement in the information about the venoms employed by these two species and their molecular characteristics. Moreover, the information generated by our group helps in a better understanding of the biology of freshwater cartilaginous fishes and offers clues for the development of clinical treatments for stingray envenoming in Brazil and around the world. Finally, our results might have biomedical implications in developing treatments for complex diseases.

Prediction of the Impact of Coding Missense and Nonsense Single Nucleotide Polymorphisms on HD5 and HBD1 Antibacterial Activity against Escherichia coli

Defensins confer host defense against microorganisms and are important for human health. Single nucleotide polymorphisms (SNPs) in defensin gene‐coding regions could lead to less active variants. Using SNP data available at the dbSNP database and frequency information from the 1000 Genomes Project, two DEFA5 (L26I and R13H) and eight DEFB1 (C35S, K31T, K33R, R29G, V06I, C12Y, Y28* and C05*) missense and nonsense SNPs that are located within mature regions of the coded defensins were retrieved. Such SNPs are rare and population restricted. In order to assess their antibacterial activity against Escherichia coli, two linear regression models were used from a previous work, which models the antibacterial activity as a function of solvation potential energy, using molecular dynamics data. Regarding only the antibacterial predictions, for HD5, no biological differences between wild‐type and its variants were observed; while for HBD1, the results suggest that the R29G, K31T, Y28* and C05* variants could be less active than the wild‐type one. The data here reported could lead to a substantial improvement in knowledge about the impact of missense SNPs in human defensins and their world distribution.

Structural impact analysis of missense SNPs present in the uroguanylin gene by long-term molecular dynamics simulations.

The guanylate cyclase activator 2B, also known as uroguanylin, is part of the guanylin peptide family, which includes peptides such as guanylin and lymphoguanylin. The guanylin peptides could be related to sodium absorption inhibition and water secretion induction and their dysfunction may be related to various pathologies such as chronic renal failure, congestive heart failure and nephrotic syndrome. Besides, uroguanylin point mutations have been associated with essential hypertension. However, currently there are no studies on the impact of missense SNPs on uroguanylin structure. This study applied in silico SNP impact prediction tools to evaluate the impact of uroguanylin missense SNPs and to filter those considered as convergent deleterious, which were then further analyzed through long-term molecular dynamics simulations of 1μs of duration. The simulations suggested that all missense SNPs considered as convergent deleterious caused some kind of structural change to the uroguanylin peptide. Additionally, four of these SNPs were also shown to cause modifications in peptide flexibility, possibly resulting in functional changes.

HD5 and HBD1 variants’ solvation potential energy correlates with their antibacterial activity against Escherichia coli

The structure‐activity relationship of defensins is not clear. It is known that point mutations in HD5 and HBD1 could modify their activities; however, these mutations do not seem to alter their three‐dimensional structures. Here, applying molecular dynamics simulations, this relationship was studied in depth. There are modifications in flexibility, solvent accessible surface area and radius of gyration, but these properties are not reflected in the activity. Only alterations in the solvation potential energy were correlated to antibacterial activity against Escherichia coli. Data here reported could lead to a better understanding of structural and functional aspects of α‐ and β‐defensins.

Theoretical structural characterization of lymphoguanylin: A potential candidate for the development of drugs to treat gastrointestinal disorders

Guanylin peptides (GPs) are small cysteine-rich peptide hormones involved in salt absorption, regulation of fluids and electrolyte homeostasis. This family presents four members: guanylin (GN), uroguanylin (UGN), lymphoguanylin (LGN) and renoguanylin (RGN). GPs have been used as templates for the development of drugs for the treatment of gastrointestinal disorders. Currently, LGN is the only GP with only one disulfide bridge, making it a remarkable member of this family and a potential drug template; however, there is no structural information about this peptide. In fact, LGN is predicted to be highly disordered and flexible, making it difficult to obtain structural information using in vitro methods. Therefore, this study applied a series of 1μs molecular dynamics simulations in order to understand the structural behavior of LGN, comparing it to the C115Y variant of GN, which shows the same Cys to Tyr modification. LGN showed to be more flexible than GN C115Y. While the negatively charged N-terminal, despite its repellent behavior, seems to be involved mainly in pH-dependent activity, the hydrophobic core showed to be the determinant factor in LGNs flexibility, which could be essential in its activity. These findings may be determinant in the development of new medicines to help in the treatment of gastrointestinal disorders. Moreover, our investigation of LGN structure clarified some issues in the structure-activity relationship of this peptide, providing new knowledge of guanylin peptides and clarifying the differences between GN C115Y and LGN.

Computational Investigation of Growth Hormone Receptor Trp169Arg Heterozygous Mutation in a Child With Short Stature

Mutations in the growth hormone receptor (GHR) gene can cause disruption of the growth hormone signaling pathway, resulting in growth deficiency due to growth hormone (GH) resistance. Both recessive and apparently dominant mutations have been described in the literature. In order to shed some light on the molecular mechanism of partial growth hormone resistance caused by heterozygous mutations, we performed an in‐depth in silico analysis of a mutation found in a girl with a previous diagnosis of idiopathic short stature. An array of algorithms was used to predict pathogenicity and potential impact on the protein, and molecular modeling, docking and dynamics were used to determine structural consequences. The results suggest that both of the possible single mutation‐containing heteromeric GH–GHR complexes, as well as the double GHR mutant complex result in perturbation of complex structures, with altered ability of the GHR dimers to interact with the GH peptide. J. Cell. Biochem. 118: 4762–4771, 2017.

Comparative transcriptome analyses of magainin I-susceptible and -resistant Escherichia coli strains

Antimicrobial peptides (AMPs) have attracted considerable attention because of their multiple and complex mechanisms of action toward resistant bacteria. However, reports have increasingly highlighted how bacteria can escape AMP administration. Here, the molecular mechanisms involved in Escherichia coli resistance to magainin I were investigated through comparative transcriptomics. Sub-inhibitory concentrations of magainin I were used to generate four experimental groups, including magainin I-susceptible E. coli, in the absence (C) and presence of magainin I (CM); and magainin I-resistant E. coli in the absence (R) and presence of magainin I (RM). The total RNA from each sample was extracted; cDNA libraries were constructed and further submitted for Illumina MiSeq sequencing. After RNA-seq data pre-processing and functional annotation, a total of 103 differentially expressed genes (DEGs) were identified, mainly related to bacterial metabolism. Moreover, down-regulation of cell motility and chaperone-related genes was observed in CM and RM, whereas cell communication, acid tolerance and multidrug efflux pump genes (ABC transporter, major facilitator and resistance-nodulation cell division superfamilies) were up-regulated in these same groups. DEGs from the C and R groups are related to basal levels of expression of homeostasis-related genes compared to CM and RM, suggesting that the presence of magainin I is required to change the transcriptomics panel in both C and R E. coli strains. These findings show the complexity of E. coli resistance to magainin I through the rearrangement of several metabolic pathways involved in bacterial physiology and drug response, also providing information on the development of novel antimicrobial strategies targeting resistance-related transcripts and proteins herein described.

In silico analyses of deleterious missense SNPs of human apolipoprotein E3

ApoE3 is the major chylomicron apolipoprotein, binding in a specific liver peripheral cell receptor, allowing transport and normal catabolism of triglyceride-rich lipoprotein constituents. Point mutations in ApoE3 have been associated with Alzheimer’s disease, type III hyperlipoproteinemia, atherosclerosis, telomere shortening and impaired cognitive function. Here, we evaluate the impact of missense SNPs in APOE retrieved from dbSNP through 16 computational prediction tools, and further evaluate the structural impact of convergent deleterious changes using 100 ns molecular dynamics simulations. We have found structural changes in four analyzed variants (Pro102Arg, Arg132Ser, Arg176Cys and Trp294Cys), two of them (Pro102Arg and Arg176Cys) being previously associated with human diseases. In all cases, except for Trp294Cys, there was a loss in the number of hydrogen bonds between CT and NT domains that could result in their detachment. In conclusion, data presented here could increase the knowledge of ApoE3 activity and be a starting point for the study of the impact of variations on APOE gene.