Substrate Recognition Properties from an Intermediate Structural State of the UreA Transporter.

Through a combination of comparative modeling, site-directed and classical random mutagenesis approaches, we previously identified critical residues for binding, recognition, and translocation of urea, and its inhibition by 2-thiourea and acetamide in the Aspergillus nidulans urea transporter, UreA. To deepen the structural characterization of UreA, we employed the artificial intelligence (AI) based AlphaFold2 (AF2) program. In this analysis, the resulting AF2 models lacked inward- and outward-facing cavities, suggesting a structural intermediate state of UreA. Moreover, the orientation of the W82, W84, N279, and T282 side chains showed a large variability, which in the case of W82 and W84, may operate as a gating mechanism in the ligand pathway. To test this hypothesis non-conservative and conservative substitutions of these amino acids were introduced, and binding and transport assessed for urea and its toxic analogue 2-thiourea, as well as binding of the structural analogue acetamide. As a result, residues W82, W84, N279, and T282 were implicated in substrate identification, selection, and translocation. Using molecular docking with Autodock Vina with flexible side chains, we corroborated the AF2 theoretical intermediate model, showing a remarkable correlation between docking scores and experimental affinities determined in wild-type and UreA mutants. The combination of AI-based modeling with classical docking, validated by comprehensive mutational analysis at the binding region, would suggest an unforeseen option to determine structural level details on a challenging family of proteins.

Resultados del tratamiento quirúrgico del cáncer de recto y ano en el Hospital Español / Results of the surgical treatment of rectal and anal cancer in the Hospital Español / Resultados do tratamento cirúrgico do câncer de reto e ânus no Hospital Español

La cirugía del cáncer de recto y ano se ha desarrollado considerablemente en las últimas décadas. En función de dichos avances, se ha observado una disminución en la morbimortalidad operatoria, así como también una mejoría en el pronóstico de estos pacientes. El objetivo del presente estudio es exponer y analizar los resultados del tratamiento quirúrgico del cáncer de recto y ano en un servicio universitario. Se realizó un estudio observacional, descriptivo y retrospectivo de todos los pacientes intervenidos por cáncer de recto y ano en el Hospital Español entre 2016 y 2020. Las variables registradas fueron: variables demográficas, clínico-oncológicas, relacionadas a la morbimortalidad operatoria y a la recidiva locorregional, y la sobrevida a 5 años. El procedimiento más realizado fue la resección anterior de recto (RAR) en 11 intervenciones (58%), mientras que las 8 restantes correspondieron a amputaciones abdominoperineales (AAP) (42%). Se diagnosticaron un total de 6 complicaciones intraoperatorias en 5 pacientes, siendo la perforación del tumor la más frecuente, y un total de 18 complicaciones postoperatorias en 11 pacientes, siendo la más frecuente la infección de la herida quirúrgica abdominal. La morbilidad operatoria mayor fue de 31,6% y la mortalidad operatoria a 90 días fue de 0%. La sobrevida global a 5 años fue de 63,2%. Los resultados quirúrgicos en la presente casuística fueron comparables con los de la bibliografía consultada. Destacamos la nula mortalidad a 90 días, con resultados oncológicos similares a los reportados en la literatura.

Rectal and anus surgery have been developed considerably in the last decades. Based on these advancements, it has been observed a decrease in the surgical morbidity and mortality, as well as an improved prognosis of these patients. The aim of the present study is to expose and analyze the results of the anus and rectal surgical treatment in a university service. An observational, descriptive and retrospective study was performed of all the intervened patients for rectum and anus cancer in the Hospital Español between 2016 and 2020. We recorded data about demographic, clinical-oncologic, related to the surgical morbidity and mortality, locoregional relapse and overall 5 year survival. The most performed procedure was the rectum anterior resection in 11 interventions (58%), while the 8 left corresponded to abdominoperineal resection (42%). There was a total of 6 intraoperative complications diagnosed in 5 patients, being the tumor perforation the most frequent one, and a total of 18 postoperative complications diagnosed in 11 patients, being the surgical wound infection the most frequent one. The serious surgical morbidity was 31,6%, while the surgical mortality rate at 90 days was 0%. Overall 5 year survival was 63,2%. The surgical results in the present study about the rectum and anal cancer were comparable with the results reported on the consulted bibliography. We highlight the null mortality within 90 days, with oncologic results similar to the ones reported in the literature.

A cirurgia do câncer retal e anal desenvolveu-se consideravelmente nas últimas décadas. Com base nesses avanços, observou-se diminuição da morbimortalidade operatória, bem como melhora no prognóstico desses pacientes. O objetivo deste estudo é apresentar e analisar os resultados do tratamento cirúrgico do câncer de reto e anal em um serviço universitário. Foi realizado um estudo observacional, descritivo e retrospectivo de todos os pacientes operados por câncer de reto e ânus no Hospital Espanhol entre 2016 e 2020. As variáveis ​​registradas foram: variáveis ​​demográficas, clínico-oncológicas, relacionadas à morbidade e mortalidade operatórias e recorrência locorregional. , e sobrevida em 5 anos. O procedimento mais realizado foi a ressecção anterior do reto (RAR) em 11 intervenções (58%) e as 8 restantes corresponderam a amputações abdominoperineais (AAP) (42%). Foram diagnosticadas 6 complicações intraoperatórias em 5 pacientes, sendo a perfuração tumoral a mais frequente, e um total de 18 complicações pós-operatórias em 11 pacientes, sendo a infecção da ferida operatória abdominal a mais frequente. A morbidade operatória maior foi de 31,6% e a mortalidade operatória em 90 dias foi de 0%. A sobrevida global em 5 anos foi de 63,2%. Os resultados cirúrgicos da presente casuística foram comparáveis ​​aos da bibliografia consultada. Destacamos a mortalidade nula em 90 dias, com resultados oncológicos semelhantes aos relatados na literatura.

A pair of non-optimal codons are necessary for the correct biosynthesis of the Aspergillus nidulans urea transporter, UreA.

In both prokaryotic and eukaryotic genomes, synonymous codons are unevenly used. Such differential usage of optimal or non-optimal codons has been suggested to play a role in the control of translation initiation and elongation, as well as at the level of transcription and mRNA stability. In the case of membrane proteins, codon usage has been proposed to assist in the establishment of a pause necessary for the correct targeting of the nascent chains to the translocon. By using as a model UreA, the Aspergillus nidulans urea transporter, we revealed that a pair of non-optimal codons encoding amino acids situated at the boundary between the N-terminus and the first transmembrane segment are necessary for proper biogenesis of the protein at 37°C. These codons presumably regulate the translation rate in a previously undescribed fashion, possibly contributing to the correct interaction of ureA-translating ribosome-nascent chain complexes with the signal recognition particle and/or other factors, while the polypeptide has not yet emerged from the ribosomal tunnel. Our results suggest that the presence of the pair of non-optimal codons would not be functionally important in all cellular conditions. Whether this mechanism would affect other proteins remains to be determined.

Modelling and mutational analysis of Aspergillus nidulans UreA, a member of the subfamily of urea/H⁺ transporters in fungi and plants.

We present the first account of the structure-function relationships of a protein of the subfamily of urea/H(+) membrane transporters of fungi and plants, using Aspergillus nidulans UreA as a study model. Based on the crystal structures of the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT) and of the Nucleobase-Cation-Symport-1 benzylhydantoin transporter from Microbacterium liquefaciens (Mhp1), we constructed a three-dimensional model of UreA which, combined with site-directed and classical random mutagenesis, led to the identification of amino acids important for UreA function. Our approach allowed us to suggest roles for these residues in the binding, recognition and translocation of urea, and in the sorting of UreA to the membrane. Residues W82, Y106, A110, T133, N275, D286, Y388, Y437 and S446, located in transmembrane helixes 2, 3, 7 and 11, were found to be involved in the binding, recognition and/or translocation of urea and the sorting of UreA to the membrane. Y106, A110, T133 and Y437 seem to play a role in substrate selectivity, while S446 is necessary for proper sorting of UreA to the membrane. Other amino acids identified by random classical mutagenesis (G99, R141, A163, G168 and P639) may be important for the basic transporter's structure, its proper folding or its correct traffic to the membrane.

Translational selection on codon usage in the genus Aspergillus.

Aspergillus is a genus of mold fungi that includes more than 200 described species. Many members of the group are relevant pathogens and other species are economically important. Only one species has been analyzed for codon usage, and this was performed with a small number of genes. In this paper, we report the codon usage patterns of eight completely sequenced genomes which belong to this genus. The results suggest that selection for translational efficiency and accuracy are the major factors shaping codon usage in all of the species studied so far, and therefore they were active in the last common ancestor of the group. Composition and molecular distances analyses show that highly expressed genes evolve slower at synonymous sites. We identified a conserved core of translationally optimal codons and study the tRNA gene pool in each genome. We found that the great majority of preferred triplets match the respective cognate tRNA with more copies in the respective genome. We discuss the possible scenarios that can explain the observed differences among the species analyzed. Finally we highlight the biotechnological application of this research regarding heterologous protein expression.

UreA, the major urea/H+ symporter in Aspergillus nidulans.

We report here the characterization of UreA, a high-affinity urea/H+ symporter of Aspergillus nidulans. The deletion of the encoding gene abolishes urea transport at low substrate concentrations, suggesting that in these conditions UreA is the sole transport system specific for urea in A. nidulans. The ureA gene is not inducible by urea or its precursors, but responds to nitrogen metabolite repression, necessitating for its expression the AreA GATA factor. In contrast to what was observed for other transporters in A. nidulans, repression by ammonium is also operative during the isotropic growth phase. The activity of UreA is down-regulated post-translationally by ammonium-promoted endocytosis. A number of homologues of UreA have been identified in A. nidulans and other Aspergilli, which cluster in four groups, two of which contain the urea transporters characterized so far in fungi and plants. This phylogeny may have arisen by gene duplication events, giving place to putative transport proteins that could have acquired novel, still unidentified functions.