Using protein design algorithms to understand the molecular basis of disease caused by protein–DNA interactions: the Pax6 example

Quite often a single or a combination of protein mutations is linked to specific diseases. However, distinguishing from sequence information which mutations have real effects in the protein’s function is not trivial. Protein design tools are commonly used to explain mutations that affect protein stability, or protein–protein interaction, but not for mutations that could affect protein–DNA binding. Here, we used the protein design algorithm FoldX to model all known missense mutations in the paired box domain of Pax6, a highly conserved transcription factor involved in eye development and in several diseases such as aniridia. The validity of FoldX to deal with protein–DNA interactions was demonstrated by showing that high levels of accuracy can be achieved for mutations affecting these interactions. Also we showed that protein-design algorithms can accurately reproduce experimental DNA-binding logos. We conclude that 88% of the Pax6 mutations can be linked to changes in intrinsic stability (77%) and/or to its capabilities to bind DNA (30%). Our study emphasizes the importance of structure-based analysis to understand the molecular basis of diseases and shows that protein–DNA interactions can be analyzed to the same level of accuracy as protein stability, or protein–protein interactions

Structure-Based Analysis of Five Novel Disease-Causing Mutations in 21-Hydroxylase-Deficient Patients

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism, and accounts for 90–95% of CAH cases. The affected enzyme, P450C21, is encoded by the CYP21A2 gene, located together with a 98% nucleotide sequence identity CYP21A1P pseudogene, on chromosome 6p21.3. Even though most patients carry CYP21A1P-derived mutations, an increasing number of novel and rare mutations in disease causing alleles were found in the last years. In the present work, we describe five CYP21A2 novel mutations, p.R132C, p.149C, p.M283V, p.E431K and a frameshift g.2511_2512delGG, in four non-classical and one salt wasting patients from Argentina. All novel point mutations are located in CYP21 protein residues that are conserved throughout mammalian species, and none of them were found in control individuals. The putative pathogenic mechanisms of the novel variants were analyzed in silico. A three-dimensional CYP21 structure was generated by homology modeling and the protein design algorithm FoldX was used to calculate changes in stability of CYP21A2 protein. Our analysis revealed changes in protein stability or in the surface charge of the mutant enzymes, which could be related to the clinical manifestation found in patients

Tecno X : Estudiantes latinoamericanos aplicando tecnologías

TECNOx busca desde 2015 aglutinar una comunidad de innovación basada en la interdisciplina, equidad & justicia tecnológica, libre acceso y enfocada a problemas relevantes para las sociedades latinoamericanas. A través de un formato de competencia tecnológica semestral para equipos de estudiantes, nos proponemos nuclear una comunidad de tecnólogos latinoamericanos, expertos en innovación de los ámbitos público, privado y ciudadano. Se trata de una experiencia formativa no tradicional, inmersiva e intensiva, donde los participantes disponen de un contexto en el cual aprender haciendo y hacer en comunidad. Al finalizar cada edición, nos juntamos para celebrar en un festejo en el que compartimos lo aprendido, procuramos mejorar los proyectos entre todos y premiamos las nuevas habilidades desarrolladas.Facultad de Informátic

Molecular basis of thermal stability in truncated (2/2) hemoglobins

International audienceBackground: Understanding the molecular mechanism through which proteins are functional at extreme high and low temperatures is one of the key issues in structural biology. To investigate this phenomenon, we have focused on two instructive truncated hemoglobins from Thermobifida fusca (Tf-trHbO) and Mycobacterium tuberculosis (Mt-trHbO); although the two proteins are structurally nearly identical, only the former is stable at high temperatures. Methods: We used molecular dynamics simulations at different temperatures as well as thermal melting profile measurements of both wild type proteins and two mutants designed to interchange the amino acid residue, either Pro or Gly, at E3 position. Results: The results show that the presence of a Pro at the E3 position is able to increase (by 8°) or decrease (by 4°) the melting temperature of Mt-trHbO and Tf-trHbO, respectively. We observed that the ProE3 alters the structure of the CD loop, making it more flexible. Conclusions: This gain in flexibility allows the protein to concentrate its fluctuations in this single loop and avoid unfolding. The alternate conformations of the CD loop also favor the formation of more salt-bridge interactions, together augmenting the protein's thermostability. General significance: These results indicate a clear structural and dynamical role of a key residue for thermal stability in truncated hemoglobins

Structure-based analysis of five novel disease-causing mutations in 21-hydroxylase-deficient patients

Fil: Minutolo, Carolina. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Nadra, Alejandro D. Universidad de Buenos Aires. Departamento de Fisiología Biología Molecular y Celular; Argentina.Fil: Fernández, Cecilia. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Taboas, Melisa. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Buzzalino, Noemí. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Casali, Bárbara. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Belli, Susana. Hospital Durand. División Endocrinología; Argentina.Fil: Charreau, Eduardo H. Instituto de Biología y Medicina Experimental; Argentina.Fil: Alba, Liliana. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Dain, Liliana. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism, and accounts for 90–95% of CAH cases. The affected enzyme, P450C21, is encoded by the CYP21A2 gene, located together with a 98% nucleotide sequence identity CYP21A1P pseudogene, on chromosome 6p21.3. Even though most patients carry CYP21A1P-derived mutations, an increasing number of novel and rare mutations in disease causing alleles were found in the last years. In the present work, we describe five CYP21A2 novel mutations, p.R132C, p.149C, p.M283V, p.E431K and a frameshift g.2511_2512delGG, in four non-classical and one salt wasting patients from Argentina. All novel point mutations are located in CYP21 protein residues that are conserved throughout mammalian species, and none of them were found in control individuals. The putative pathogenic mechanisms of the novel variants were analyzed in silico. A three-dimensional CYP21 structure was generated by homology modeling and the protein design algorithm FoldX was used to calculate changes in stability of CYP21A2 protein. Our analysis revealed changes in protein stability or in the surface charge of the mutant enzymes, which could be related to the clinical manifestation found in patients

Transcriptomic analysis and molecular docking reveal genes involved in the response of Aedes aegypti larvae to an essential oil extracted from Eucalyptus

Background: Aedes aegypti (L.) is an urban mosquito, vector of several arboviruses that cause severe diseases in hundreds of million people each year. The resistance to synthetic insecticides developed by Ae. aegypti populations worldwide has contributed to failures in vector control campaigns, increasing the impact of arbovirus diseases. In this context, plant-derived essential oils with larvicidal activity could be an attractive alternative for vector control. However, the mode of action and the detoxificant response of mosquitoes to plant derived compounds have not been established, impairing the optimization of their use. Methods and findings: Here we compare gene expression in Ae. aegypti larvae after 14 hrs of exposure to Eucalyptus camaldulensis essential oil with a control group exposed to vehicle (acetone) for the same lapse, by using RNA-Seq. We found differentially expressed genes encoding for cuticle proteins, fatty-acid synthesis, membrane transporters and detoxificant related gene families (i.e. heat shock proteins, cytochromes P450, glutathione transferases, UDP-glycosyltransferases and ABC transporters). Finally, our RNA-Seq and molecular docking results provide evidence pointing to a central involvement of chemosensory proteins in the detoxificant response in mosquitoes. Conclusions and significance: Our work contributes to the understanding of the physiological response of Ae. aegypti larvae to an intoxication with a natural toxic distilled from Eucalyptus leafs. The results suggest an involvement of most of the gene families associated to detoxification of xenobiotics in insects. Noteworthy, this work provides important information regarding the implication of chemosensory proteins in the detoxification of a natural larvicide. Understanding the mode of detoxification of Eucalyptus distilled compounds could contribute to their implementation as a tool in mosquito control.Los datos de la investigación están disponibles en la base de datos NCBI SRA (número de acceso Bioproject PRJNA671513) (link en "Documentos relacionados")Centro Regional de Estudios Genómico