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

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

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