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

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

Lipovitellin inhibition of Artemia trypsin-like proteinase: A role for a storage protein in regulating proteinase activity during development

Artemia trypsin-like proteinase has been reported previously to be highly inhibited in the embryo (B. Ezquieta and C. G. Vallejo (1985) Comp. Biochem. Physiol.82B, 731–736). We report now that Artemia lipovitellin, the major storage protein complex, inhibits the proteinase. We have carried out an in vitro study of the characteristics of the inhibition. Lipovitellin, a glycolipoprotein of high molecular mass (650 kDa), behaves initially as a substrate but after a limited proteolysis becomes an inhibitor of the proteinase. The enzyme although inhibited in the hydrolysis of the protein substrate retains activity toward low molecular weight substrates. The residual activity on the protein substrate is inhibited by small inhibitors of the proteinase. These features of lipovitellin inhibition are reminiscent of the trap mechanism of α2-macroglobulin inhibition, previously proposed as suitable for regulating proteolytic processes involved in development. Inhibition by lipovitellin is greater at low temperatures and has been determined at 17 and 37 °C, in the lower and higher part of the viable temperature range of Artemia development. At high temperature the proteinase hydrolyzes the inhibitor quite efficiently and the inhibition is lower. The inhibition by lipovitellin appears specific for Artemia trypsin-like proteinase when compared with other control pairs protein/proteinase. The results may provide support for an additional role of storage proteins as developmental inhibitors of proteinases.This research was supported by a grant (0834/81) from Comisión Asesora de Investigación Científica y Técnica and a fellowship to B.E. from Caja de Ahorros de Madrid

Diguanosine 5′,5‴ -P1,P4-tetraphosphate causes specific inhibition and desensitization of Artemia trypsin-like proteinase in the hydrolysis of a high-affinity, arginine-rich substrate

Diguanosine tetraphosphate (Gp4G) inhibits A rtemia trypsin-like proteinase. The inhibition is quite specific and presents a K i about 1 pM. Gp4G modifies both maximum velocity and affinity of the proteinase. The proteinase, without losing activity, can be desensitized to the inhibition by Gp4G at low concentrations (1-10 p M). Gp4G inhibits the hydrolysis of the high-affinity, arginine-rich substrate, protamine, to a lesser extent that of polylysine and does not affect the catalysis of other basic proteins. The results may suggest the involvement of Gp4G in the regulation of the hydrolysis of polypeptides containing clusters of arginine.This work has been supported by Comisión Asesora de Investigación Científica y Técnica, grant No. 0834/81. B.E. held a fellowship from Caja de Ahorros de Madrid. We are indebted to Drs. A. Sols and C. Gancedo for helpful criticism

Artemia trypsin-like proteinase: Developmental activation is inhibited by a lysosomotropic agent

The exponential increase in activity of Artemia trypsin-like proteinase observed during larval development has been suggested to depend on the activation of an inactive precursor found in embryos associated with lipovitellin (Ezquieta and Vallejo (1985) Comp. Biochem. Physiol. 82B, 731-736). We here show that the inhibition of the lysosomal function by chloroquine prevents the activation of Artemia trypsin-like proteinase in vivo

The trypsin-like proteinase of Artemia: Yolk localization and developmental activation

1. A trypsin-like proteinase has been found in Artemia dormant embryos localized in yolk granules associated to lipovitellin. 2. During development, and in parallel with an exponential increase in the activity of the proteinase, we have observed (a) gradual changes in its subcellular localization from the yolk granules to the cytosol, (b) a gradual decrease in the molecular weight of the cytosolic enzyme, and (c) a gradual improvement in the accessibility of substrates and inhibitors to the proteinase active centre. 3. The partially purified cytosolic proteinase reacted with anti-lipovitellin antiserum. The results are discussed in relation to the yolk origin of the proteinase and its activation during development.This work was supported by a grant from Comisión Asesora de Investigación Científica y Técnica Grant No. 0834/81. B.E. has a fellowship from Caja de Ahorros de Madrid

Use of nonradioactive labeling to detect large gene rearrangements in 21-hydroxylase deficiency Uso de marcação não radiativa para identificação de grandes rearranjos gênicos na deficiência da 21-hidroxilase

PURPOSE: To establish the Southern blotting technique using hybridization with a nonradioactive probe to detect large rearrangements of CYP21A2 in a Brazilian cohort with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH-21OH). METHOD: We studied 42 patients, 2 of them related, comprising 80 non-related alleles. DNA samples were obtained from peripheral blood, digested by restriction enzyme Taq I, submitted to Southern blotting and hybridized with biotin-labeled probes. RESULTS: This method was shown to be reliable with results similar to the radioactive-labeling method. We found CYP21A2 deletion (2.5%), large gene conversion (8.8%), CYP21AP deletion (3.8%), and CYP21A1P duplication (6.3%). These frequencies were similar to those found in our previous study in which a large number of cases were studied. Good hybridization patterns were achieved with a smaller amount of DNA (5 mug), and fragment signs were observed after 5 minutes to 1 hour of exposure. CONCLUSIONS: We established a non-radioactive (biotin) Southern blot/hybridization methodology for CYP21A2 large rearrangements with good results. Despite being more arduous, this technique is faster, requires a smaller amount of DNA, and most importantly, avoids problems with the use of radioactivity.<br>OBJETIVO: Padronizar a técnica de Southern blotting usando hibridização com material não radioativo para detectar grandes rearranjos no gene CYP21A2 em uma amostra da população brasileira com hiperplasia adrenal congênita. MÉTODO: Foram estudados 42 pacientes, 2 dos quais aparentados, totalizando 80 alelos não relacionados. As amostras de DNA foram obtidas de sangue periférico, digeridas com enzima de restrição Taq I, realizado Southern blotting e hibridizadas com sonda marcada com biotina. RESULTADOS: O método se mostrou eficaz, com resultados similares aos encontrados ao utilizar a metodologia com material radioativo. Foram encontradas 2,5% de deleção do CYP21A2, 8,8% de grandes conversões, 3,8% de deleção do CYP21A1P e 6,3% de duplicação do CYP21A1P. Estas freqüências foram similares às encontradas em nosso estudo prévio, onde um número significante de casos foi estudado. Um bom padrão de hibridização foi alcançado utilizando menor quantidade de DNA (5mg) e a emissão de sinais foi observada entre 5 minutos e 1 hora de exposição. CONCLUSÕES: Padronizamos uma técnica de Southern blotting/ hibridização com material não radioativo (biotina) para a pesquisa de grandes rearranjos no gene CYP21A2 com bons resultados. Apesar de ser mais trabalhoso, este método é mais rápido, utiliza menores quantidades de DNA e, principalmente, evita problemas com o uso de radioatividade