Neurofibromatosis in the gluteus and posterior region of the thigh: case report / Neurofibromatose em glúteo e região posterior da coxa: relato de caso

Introduction: Neurofibromatosis is an autosomal dominant disorder, and type 1 is associated with an increased risk of tumor formation with neurocutaneous involvement. The variable evolution, often with limiting tumors, in addition to the significant incidence of cases requiring treatment, makes it fundamental to discuss procedures already performed in medical practice for early, careful, and individualized recognition of the diagnosis and treatment of the patient. The report aims to present a surgical case of neurofibromatosis, calling attention to the surgical technique, the characteristics of the disease, and the importance of the procedure in the quality of life of patients limited by the condition. Case Report: A 23-year-old male patient with a large mass neurofibroma in the gluteus and posterior surface of the right leg, in addition to café au lait stains in the distal third of the legs. He was treated with surgery to remove the tumor and a flap and graft in the affected region. The procedures were performed by a multidisciplinary team, allowing the total removal of the tumor mass, with subsequent skin grafting in the hip and thigh lesion on the right side and the fasciocutaneous flap in VY in the area. There were no significant complications in the immediate postoperative period. Conclusion: Neurofibromas can become limiting and impair patients' quality of life with neurofibromatosis type 1; therefore, early management and diagnosis are essential. Although the condition does not present a cure, there is a need for research into less invasive and preventive treatments for injuries.

Introdução: A neurofibromatose é um distúrbio autossômico dominante e o tipo 1 está associado a um aumento do risco de formação de tumores com acometimento neurocutâneo. A evolução variável, muitas vezes com tumorações limitantes, além da incidência significativa de casos que necessitam de tratamento, torna fundamental a discussão de condutas já realizadas na prática médica para um reconhecimento precoce, cuidadoso e individualizado do diagnóstico e do tratamento do enfermo. O relato objetiva apresentar um caso cirúrgico de neurofibromatose, chamando atenção para a técnica cirúrgica, as características da doença e a importância do procedimento na qualidade de vida de pacientes limitados pela afecção. Relato de Caso: Paciente de 23 anos, sexo masculino, com neurofibroma de grande massa em glúteo e face posterior da perna direita, além de manchas café com leite em terço distal de pernas. Foi tratado com uma cirurgia de retirada do tumor, além de retalho e enxerto na região acometida. Os procedimentos foram realizados por equipe multidisciplinar, possibilitando a retirada total da massa tumoral, com posterior realização de enxerto de pele na lesão do quadril e coxa em lado direito, e o retalho fasciocutâneo em V-Y na área. Não houve complicações significativas nos pós-operatórios imediatos.

Digital technology and its impacts on the sleep quality and academic performance during the pandemic.

BACKGROUND: Sleep deficits caused by the overuse of digital technology is observed among medical students. Due to the coronavirus disease 2019 (COVID-19) pandemic, an emergency remote teaching method was put into practice, which may have resulted in changes in the sleep-wake cycle. The balance between the influences of external and internal synchronizers can be affected by sudden alterations in daily life, including changes in nightly habits and sleep quality, which can lead to increased levels of anxiety and reduced functional performance, for example. OBJECTIVE: To understand the relationship between the use of digital technology, changes in the circadian cycle, and academic performance during the pandemic. METHODS: The present is an analytical, cross-sectional, observational study in which a sample of 123 medical students filled out an online questionnaire on self-perception regarding sleep quality and academic performance before and during the pandemic. RESULTS: Assessing changes in sleep quality and productivity, the study revealed that 100% of the students made continuous use of screens before bedtime. Thus, during the period of social distancing and remote classes, 77.2% of the students reported "poor" or "very poor academic performance, which was probably related to the fact that 65.9% of these students were unable to maintain their productivity due to daytime sleepiness. CONCLUSIONS: The prolonged use of screens was associated with poor sleep quality and changes in academic performance, with significant psychological impact. Thus, it is worth emphasizing the importance of sleep hygiene in light of the new forms of teaching implemented during the COVID-19 pandemic.

INTRODUçãO: Observa-se um déficit de sono ocasionado pelo uso excessivo de tecnologias digitais entre estudantes de medicina. Em face da pandemia da doença do coronavírus 2019 (coronavirus disease 2019, COVID-19, em inglês), um método de ensino à distância foi adotado, e pode ter acarretado mudanças no ciclo de sono e vigília. O equilíbrio entre as influências dos sincronizadores externos e internos pode ser afetado por mudanças bruscas na vida diária, isto inclui alterações nos hábitos noturnos e na qualidade do sono, que podem causar aumento dos níveis de ansiedade e redução do desempenho funcional. OBJETIVO: Compreender a relação entre o uso de tecnologias digitais, alterações no ciclo circadiano, e desempenho acadêmico durante a pandemia. MéTODOS: Trata-se de um estudo observacional, analítico e transversal, no qual uma amostra de 123 estudantes de medicina responderam a um questionário online sobre a autopercepção referente à qualidade do sono e ao desempenho acadêmico antes e durante a pandemia. RESULTADOS: Com a avaliação das alterações na qualidade do sono e na produtividade, o estudo revelou que 100% dos alunos faziam uso contínuo de telas antes do horário de dormir. Assim, durante o período de distanciamento social e aulas remotas, 77,2% dos alunos relataram que o desempenho acadêmico era "ruim" ou "péssimo", o que provavelmente estava relacionado ao fato de que 65,9% desses alunos que não conseguiram manter sua produtividade por conta de sonolência diurna. CONCLUSõES: O uso de telas por tempo prolongado foi associado a mudanças relacionadas à baixa qualidade do sono e a mudanças no desempenho acadêmico, com impacto psicológico significativo. Assim, vale ressaltar a importância da higiene do sono diante das novas formas de ensino implantadas durante a pandemia da COVID-19.

Digital technology and its impacts on the sleep quality and academic performance during the pandemic / Tecnologias digitais e seus impactos na qualidade do sono e desempenho acadêmico em tempos de pandemia

Abstract Background Sleep deficits caused by the overuse of digital technology is observed among medical students. Due to the coronavirus disease 2019 (COVID-19) pandemic, an emergency remote teaching method was put into practice, which may have resulted in changes in the sleep-wake cycle. The balance between the influences of external and internal synchronizers can be affected by sudden alterations in daily life, including changes in nightly habits and sleep quality, which can lead to increased levels of anxiety and reduced functional performance, for example. Objective To understand the relationship between the use of digital technology, changes in the circadian cycle, and academic performance during the pandemic. Methods The present is an analytical, cross-sectional, observational study in which a sample of 123 medical students filled out an online questionnaire on self-perception regarding sleep quality and academic performance before and during the pandemic. Results Assessing changes in sleep quality and productivity, the study revealed that 100% of the students made continuous use of screens before bedtime. Thus, during the period of social distancing and remote classes, 77.2% of the students reported "poor" or "very poor academic performance, which was probably related to the fact that 65.9% of these students were unable to maintain their productivity due to daytime sleepiness. Conclusions The prolonged use of screens was associated with poor sleep quality and changes in academic performance, with significant psychological impact. Thus, it is worth emphasizing the importance of sleep hygiene in light of the new forms of teaching implemented during the COVID-19 pandemic.

Resumo Introdução Observa-se um déficit de sono ocasionado pelo uso excessivo de tecnologias digitais entre estudantes de medicina. Em face da pandemia da doença do coronavírus 2019 (coronavirus disease 2019, COVID-19, em inglês), um método de ensino à distância foi adotado, e pode ter acarretado mudanças no ciclo de sono e vigília. O equilíbrio entre as influências dos sincronizadores externos e internos pode ser afetado por mudanças bruscas na vida diária, isto inclui alterações nos hábitos noturnos e na qualidade do sono, que podem causar aumento dos níveis de ansiedade e redução do desempenho funcional. Objetivo Compreender a relação entre o uso de tecnologias digitais, alterações no ciclo circadiano, e desempenho acadêmico durante a pandemia. Métodos Trata-se de um estudo observacional, analítico e transversal, no qual uma amostra de 123 estudantes de medicina responderam a um questionário online sobre a autopercepção referente à qualidade do sono e ao desempenho acadêmico antes e durante a pandemia. Resultados Com a avaliação das alterações na qualidade do sono e na produtividade, o estudo revelou que 100% dos alunos faziam uso contínuo de telas antes do horário de dormir. Assim, durante o período de distanciamento social e aulas remotas, 77,2% dos alunos relataram que o desempenho acadêmico era "ruim" ou "péssimo", o que provavelmente estava relacionado ao fato de que 65,9% desses alunos que não conseguiram manter sua produtividade por conta de sonolência diurna. Conclusões O uso de telas por tempo prolongado foi associado a mudanças relacionadas à baixa qualidade do sono e a mudanças no desempenho acadêmico, com impacto psicológico significativo. Assim, vale ressaltar a importância da higiene do sono diante das novas formas de ensino implantadas durante a pandemia da COVID-19.

A cell surface arabinogalactan-peptide influences root hair cell fate.

Root hairs (RHs) develop from specialized epidermal trichoblast cells, whereas epidermal cells that lack RHs are known as atrichoblasts. The mechanism controlling RH cell fate is only partially understood. RH cell fate is regulated by a transcription factor complex that promotes the expression of the homeodomain protein GLABRA 2 (GL2), which blocks RH development by inhibiting ROOT HAIR DEFECTIVE 6 (RHD6). Suppression of GL2 expression activates RHD6, a series of downstream TFs including ROOT HAIR DEFECTIVE 6 LIKE-4 (RSL4) and their target genes, and causes epidermal cells to develop into RHs. Brassinosteroids (BRs) influence RH cell fate. In the absence of BRs, phosphorylated BIN2 (a Type-II GSK3-like kinase) inhibits a protein complex that regulates GL2 expression. Perturbation of the arabinogalactan peptide (AGP21) in Arabidopsis thaliana triggers aberrant RH development, similar to that observed in plants with defective BR signaling. We reveal that an O-glycosylated AGP21 peptide, which is positively regulated by BZR1, a transcription factor activated by BR signaling, affects RH cell fate by altering GL2 expression in a BIN2-dependent manner. Changes in cell surface AGP disrupts BR responses and inhibits the downstream effect of BIN2 on the RH repressor GL2 in root epidermis.

The Lazy Life of Lipid-Linked Oligosaccharides in All Life Domains.

Lipid-linked oligosaccharides (LLOs) play an important role in the N-glycosylation pathway as the donor substrate of oligosaccharyltransferases (OSTs), which are responsible for the en bloc transfer of glycan chains onto a nascent polypeptide. The lipid component of LLO in both eukarya and archaea consists of a dolichol, and an undecaprenol in prokarya, whereas the number of isoprene units may change between species. Given the potential relevance of LLOs and their related enzymes to diverse biotechnological applications, obtaining reliable LLO models from distinct domains of life could support further studies on complex formation and their processing by OSTs, as well as protein engineering on such systems. In this work, molecular modeling techniques, such as quantum mechanics calculations, molecular dynamics simulations, and metadynamics were employed to study eukaryotic (Glc3-Man9-GlcNAc2-PP-Dolichol), bacterial (Glc1-GalNAc5-Bac1-PP-Undecaprenol), and archaeal (Glc1-Man1-Gal1-Man1-Glc1-Gal1-Glc1-P-Dolichol) LLOs in membrane bilayers. Microsecond molecular dynamics simulations and metadynamics calculations of LLOs revealed that glycan chains are more prone to interact with the membrane lipid head groups, while the PP linkages are positioned at the lipid phosphate head groups level. The dynamics of isoprenoid chains embedded within the bilayer are described, and membrane dynamics and related properties are also investigated. Overall, there are similarities regarding the structure and dynamics of the eukaryotic, the bacterial, and the archaeal LLOs in bilayers, which can support the comprehension of their association with OSTs. These data may support future studies on the transferring mechanism of the oligosaccharide chain to an acceptor protein.

Duffy binding-like 1α adhesin from Plasmodium falciparum recognizes ABH histo-blood group saccharide in a type specific manner.

The ability of erythrocytes, infected by Plasmodium falciparum, to adhere to endothelial cells (cytoadherence) and to capture uninfected erythrocyte (rosetting) is the leading cause of death by severe malaria. Evidences link the binding of the adhesin Duffy Binding Like1-α (DBL1α) domain to the ABH histo-blood antigens with formation of rosettes. Inspired by this very close relationship between the disease susceptibility and individual blood type, here we investigate the structural requirements involved in the interaction of DBL1α with A, B and H histo-blood determinants and their subtypes. Our results evidence the high preference of DBL1α to A epitopes, in comparison to B and H epitopes. DBL1α interacts with ABH epitopes in subtype specific manner, presenting a remarkable affinity for type 2 structures, Fucα1-2Galß1-4GlcNAcß1, particularly the A2 epitope. The contacts made by DBL1α binding pocket and the ABH histo-blood groups were mapped by theoretical methods and supported by NMR experiments.

Alpha-hemolysin nanopore allows discrimination of the microcystins variants.

Microcystins (MCs) are a class of cyclic heptapeptides with more than 100 variants produced by cyanobacteria present in surface waters. MCs are potent hepatotoxic agents responsible for fatal poisoning in animals and humans. Several techniques are employed in the detection of MCs, however, there is a shortage of methods capable of discriminating variants of MCs. In this work we demonstrate that the α-hemolysin (αHL) nanopore can detect and discriminate the variants (LR, YR and RR) of MCs in aqueous solution. The discrimination process is based on the analysis of the residence times of each variant of MCs within the unitary nanopore, as well as, on the amplitudes of the blockages in the ionic current flowing through it. Simulations of molecular dynamics and calculation of the electrostatic potential revealed that the variants of MCs present different charge distribution and correlated with the three patterns on the amplitudes of the blockages in the ionic current. Additionally, molecular docking analysis indicates different patterns of interaction of the variants of MCs with two specific regions of the nanopore. We conclude that αHL nanopore can discriminate variants of microcystins by a mechanism based mainly on electrostatic interaction. Finally, we propose the use of nanopore-based technology as a promising method for analyzing microcystins in aqueous solutions.

Polymyxin Binding to the Bacterial Outer Membrane Reveals Cation Displacement and Increasing Membrane Curvature in Susceptible but Not in Resistant Lipopolysaccharide Chemotypes.

Lipid-A is the causative agent of Gram-negative sepsis and is responsible for an increasingly high mortality rate among hospitalized patients. Compounds that bind Lipid-A can limit this inflammatory process. The cationic antimicrobial peptide polymyxin B (Pmx-B) is one of the simplest molecules capable of selectively binding to Lipid-A and may serve as a model for further development of Lipid-A binding agents. Gram-negative bacteria resistance to Pmx-B relies on the upregulation of a number of regulatory systems, which promote chemical modifications of the lipopolysaccharide (LPS) structure and leads to major changes in the physical-chemical properties of the outer membrane. A detailed understanding of how the chemical structure of the LPS modulates macroscopic properties of the outer membrane is paramount for the design and optimization of novel drugs targeting clinically relevant strains. We have performed a systematic investigation of Pmx-B binding to outer membrane models composed of distinct LPS chemotypes experimentally shown to be either resistant or susceptible to the peptide. Molecular dynamics simulations were carried out for Pmx-B bound to the penta- and hexa-acylated forms of Lipid-A (more susceptible) and Lipid-A modified with 4-amino-4-deoxy-l-arabinose (resistant) as well as the penta-acylated form of LPS Re (less susceptible). The present simulations show that upon binding to the bacterial outer membrane surface, Pmx-B promotes cation displacement and structural changes in membrane curvature and integrity as a function of the LPS chemotype susceptibility or resistance to the antimicrobial peptide.

Staphylococcus aureus δ-toxin in aqueous solution: Behavior in monomeric and multimeric states.

δ-Toxin is a 26 amino acid peptide capable of lysing several mammalian cell types and subcellular structures. Structurally, δ-toxin predominantly exhibits a α-helical secondary structure in membranes but, in aqueous solution, it adopts varying helical content. As no atomic-level data is available for this peptide in aqueous solutions and for the water-to-membrane transition, this work aims to characterize δ-toxin behavior in these conditions through molecular dynamics simulations in triplicates employing four different parameter sets. Our results, validated on previous experimental data, suggest that the peptide has from 4 to 16 residues folded as α-helix in aqueous solution, and a water-to-membrane foldamer comprising residues 14-18. Considering a previously proposed stable tetramer form in aqueous solutions, protein-protein docking and molecular dynamics studies were performed, suggesting that δ-toxin increases its α-helical content in such organization. The obtained results are expected to contribute in future studies on δ-toxin aggregation and interaction to biomembranes.

Insights into the effects of glycosylation and the monosaccharide-binding activity of the plant lectin CrataBL.

CrataBL is a glycoprotein isolated from Crataeva tapia bark, containing two N-glycosylation sites. It has been identified to present lectin activity with some specificity for binding glucose over galactose. However, to date, no information on the effects of glycosylation or CrataBL monosaccharide-binding sites and monosaccharide specificity has been obtained. Thus, molecular docking and molecular dynamics simulations were employed to characterize the glycosylated CrataBL conformation and dynamics in aqueous solutions, as well as the molecular basis for its binding specificity. The obtained results indicate both local and distant conformational stabilization effects of N-linked glycans over CrataBL protein moiety. Regarding its lectin activity, molecular docking calculations were performed in two possible binding sites, identified through sequence-based, structure-based and evolutionary information, using α- and ß-anomeric states of the monosaccharides. The obtained poses were further refined through molecular dynamics simulations, suggesting that positively-charged amino acids dictate the binding preference for glucose over galactose in both sites. In addition, a possible preference for ß-monosaccharides was proposed. Such data are expected to contribute to a better comprehension of the lectins monosaccharide-binding activities and carbohydrate-binding site structures.

Mapping Putative B-Cell Zika Virus NS1 Epitopes Provides Molecular Basis for Anti-NS1 Antibody Discrimination between Zika and Dengue Viruses.

B-cell epitope sequences from Zika virus (ZIKV) NS1 protein have been identified using epitope prediction tools. Mapping these sequences onto the NS1 surface reveals two major conformational epitopes and a single linear one. Despite an overall average sequence identity of ca. 55% between the NS1 from ZIKV and the four dengue virus (DENV) serotypes, epitope sequences were found to be highly conserved. Nevertheless, nonconserved epitope-flanking residues are responsible for a dramatically divergent electrostatic surface potential on the epitope regions of ZIKV and DENV2 serotypes. These findings suggest that strategies for differential diagnostics on the basis of short linear NS1 sequences are likely to fail due to immunological cross-reactions. Overall, results provide the molecular basis of differential discrimination between Zika and DENVs by NS1 monoclonal antibodies.

Conformational stability of the epidermal growth factor (EGF) receptor as influenced by glycosylation, dimerization and EGF hormone binding.

The epidermal growth factor receptor (EGFR) is an important transmembrane glycoprotein kinase involved the initiation or perpetuation of signal transduction cascades within cells. These processes occur after EGFR binds to a ligand [epidermal growth factor (EGF)], thus inducing its dimerization and tyrosine autophosphorylation. Previous publications have highlighted the importance of glycosylation and dimerization for promoting proper function of the receptor and conformation in membranes; however, the effects of these associations on the protein conformational stability have not yet been described. Molecular dynamics simulations were performed to characterize the conformational preferences of the monomeric and dimeric forms of the EGFR extracellular domain upon binding to EGF in the presence and absence of N-glycan moieties. Structural stability analyses revealed that EGF provides the most conformational stability to EGFR, followed by glycosylation and dimerization, respectively. The findings also support that EGF-EGFR binding takes place through a large-scale induced-fitting mechanism. Proteins 2017; 85:561-570. © 2016 Wiley Periodicals, Inc.

Glycosylation is crucial for a proper catalytic site organization in human glucocerebrosidase.

Gaucher disease, an autosomal recessive disorder, is caused by a deficiency of glucocerebrosidase (GCase) enzyme, a peripheral membrane-associated glycoprotein that hydrolyses glucosylceramide in lysosomes. Glycosylation is essential for the development of a catalytically active enzyme, specifically in the first site, located at Asn19. However, both the molecular basis of the relevance of N-glycosylation over GCase activity and the effects of glycosylation over its structure and dynamics are still not fully understood. Thus, the present work evaluated GCase enzyme in increasing glycosylation content using triplicate unbiased molecular dynamics simulations. Accordingly, the N-linked glycan chains caused local conformational stabilization effects over the protein, as well as in regions flanking the enzyme catalytic dyad. In the case of the Asn19-linked glycan, it also occurred around region 438-444, where one of the most prevalent GCase mutations is found. Markedly, an increasing catalytic dyad organization was related to increasing glycosylation contents, offering the first atomic-level explanation for the experimental observation that GCase activity is controlled by glycosylation, especially at Asn19.

Inhibition of the hemolytic activity caused by Staphylococcus aureus alpha-hemolysin through isatin-Schiff copper(II) complexes.

A great number of pathogens secrete pore-forming proteins during infection. Such molecules, from either bacterial or viral origin, are considered important virulence factors, which makes them attractive targets in the study of new therapeutic agents. Thus, the inhibitory activity of isatin-Schiff base copper(II) complexes was evaluated against membrane damage activity of Staphylococcus aureus α-hemolysin (α-HL). For this purpose, a standard hemolysis assay with rabbit erythrocytes and micromolar concentrations of the compounds was employed. Additionally, planar artificial lipid membranes with a single α-HL ion channel and molecular docking studies were used to elucidate the molecular mechanism of the complexes. Accordingly, the compounds were observed to possess a significant anti-hemolytic activity, capable of interacting with the constriction region of α-HL channel and blocking it in a potential dependent manner. Based on these results, it is expected that such isatin-Schiff base Copper(II) complexes may be employed as cotherapeutic agents for the treatment of staphylococcal infections.

The Calcium Goes Meow: Effects of Ions and Glycosylation on Fel d 1, the Major Cat Allergen.

The major cat allergen, Fel d 1, is a structurally complex protein with two N-glycosylation sites that may be filled by different glycoforms. In addition, the protein contains three putative Ca2+ binding sites. Since the impact of these Fel d 1 structure modifications on the protein dynamics, physiology and pathology are not well established, the present work employed computational biology techniques to tackle these issues. While conformational effects brought upon by glycosylation were identified, potentially involved in cavity volume regulation, our results indicate that only the central Ca2+ ion remains coordinated to Fel d 1 in biological solutions, impairing its proposed role in modulating phospholipase A2 activity. As these results increase our understanding of Fel d 1 structural biology, they may offer new support for understanding its physiological role and impact into cat-promoted allergy.

Extension and validation of the GROMOS 53A6(GLYC) parameter set for glycoproteins.

An extension of the GROMOS 53A6GLYC force field for carbohydrates to encompass glycoprotein linkages is presented. The set includes new atomic charges and incorporates adequate torsional potential parameters for N-, S-, C-, P-, and O-glycosydic linkages, offering compatibility with the GROMOS force field family for proteins. Validation included the description of glycosydic linkage geometries between amino acid and monosaccharide residues, comparison of NMR-derived protein-carbohydrate and carbohydrate-carbohydrate nuclear overhauser effect (NOE) signals for glycoproteins and the effects of glycosylation on protein flexibility and dynamics.

Atomic model and micelle dynamics of QS-21 saponin.

QS-21 is a saponin extracted from Quillaja saponaria, widely investigated as a vaccine immunoadjuvant. However, QS-21 use is mainly limited by its chemical instability, significant variety in molecular composition and low tolerance dose in mammals. Also, this compound tends to form micelles in a concentration-dependent manner. Here, we aimed to characterize its conformation and the process of micelle formation, both experimentally and computationally. Therefore, molecular dynamics (MD) simulations were performed in systems containing different numbers of QS-21 molecules in aqueous solution, in order to evaluate the spontaneous micelle formation. The applied methodology allowed the generation of micelles whose sizes were shown to be in high agreement with small-angle X-ray scattering (SAXS). Furthermore, the ester linkage between fucose and acyl chain was less solvated in the micellar form, suggesting a reduction in hydrolysis. This is the first atomistic interpretation of previous experimental data, the first micellar characterization of saponin micelles by SAXS and first tridimensional model of a micelle constituted of saponins, contributing to the understanding of the molecular basis of these compounds.

Structural glycobiology of heparin dynamics on the exosite 2 of coagulation cascade proteases: Implications for glycosaminoglycans antithrombotic activity.

fIIa and fXa are two of the main targets of antithrombin, a serine proteases inhibitor that plays a major role in the regulation of blood clotting. The formation of ternary complexes between such molecules and glycosaminoglycans, as heparin, is the main path for inhibiting those enzymes, which may occur through two distinct mechanisms of action. While these serine proteases present distinct susceptibilities to these paths, in which fIIa demands an interaction with heparin, neither the molecular basis of this differential inhibition nor the role of fIIa glycosylation on this process is fully understood. Thus, the present work evaluated through molecular dynamics simulations the effects of glycosylation on fIIa and the consequences of heparin binding to both proteases function and dynamics. Based on the obtained data, fIIa N-linked glycan promoted an increase in the active site pocket size by stabilizing regions that encircle it, while heparin binding was observed to reverse such an effect. Additionally, heparin orientation observed on the surface of fIIa, but not fXa, allows a linear long-chain heparin binding to antithrombin in ternary complexes. Finally, the enzymes catalytic triad organization was disrupted due to a strong glycosaminoglycan binding to the proteases exosite 2. Such data support an atomic-level explanation for the higher inhibition constant of the antithrombin-heparin complex over fIIa than fXa, as well as for the different susceptibilities of those enzymes for antithrombin mechanisms of action.

Effects of glycosylation and pH conditions in the dynamics of human arylsulfatase A.

Arylsulfatase A (ARSA) is a lysosomal sulfatase that catalyzes the hydrolysis of cerebroside sulfate. Its deficiency results in Metachromatic Leukodystrophy, whereas a minor condition called ARSA pseudodeficiency occurs in healthy individuals, which has been associated with the substitution of the glycosylated Asn350 by a Ser and with the loss of the polyadenylation signal. In this work, we have investigated ARSA dynamics employing molecular dynamics simulations in response to (1) different pH's, as, beyond its natural lysossomal environment, it has been recently identified in cytoplasmatic medium and (2) glycan occupancies, including its normal glycosylation state, presenting three high mannose-type oligosaccharides. Accordingly, four systems were studied considering ARSA under different conditions: (1) nonglycosylated at pH ∼ 7 (ARSApH7); (2) non-glycosylated at pH ∼ 5 (ARSApH5); (3) triple glycosylated at pH ∼ 5 (ARSAglyc,pH5); and (4) ARSA-N350S mutant at pH ∼ 5 (ARSAN350S,pH5). Lowering pH and increasing glycosylation was found to reduce the flexibility of the enzyme. In addition, at acidic pH, the glycosylated enzyme presented a higher secondary conformational stability when compared to its nonglycosylated counterpart, supporting experimental findings on triple glycosylation as the essential state of ARSA. The N350S mutant exhibited a consistent degree of unfolding, which may be related to its in vitro reduced stability. Finally, the obtained data are discussed in the search for structural evidences able to contribute to the understanding of biological activity of ARSA and molecular etiology of ARSA pseudodeficiency, as determined by ARSA-N350S in the absence of polyadenylation defect.