The Global Sequence Signature algorithm unveils a structural network surrounding heavy chain CDR3 Loop in Camelidae variable domains. Backgroundof the article: A large fraction of camelid (camels and llamas) antibodies is composed of heavy chain-only homodimers, able to recognise antigens with their variable domain. Events in somatic assembly and maturation of antibodies such as hypermutations and rearrangement of variable loops (CDRs - complementary determining regions) and selection among a wide range of framework variants are generally considered to be random processes. Methods: An original algorithmic approach (Global Sequence Signature-GSS) was developed, able to take into account multiple functional and/or local sequence properties to detect scattered evolutionary constraints into sequences. Results: Using the GSS approach, we show that the length of the main hypervariable loop (CDR3) is linked to the nature of 19 surrounding residues on the scaffold. Surprisingly, the relation between CDR3 size and scaffold residues strongly depends on the considered species, illustrating either significant differences in selection mechanisms or functional constraints during antibody maturation. Conclusions: Combined with the statistical coupling analysis (SCA) approach at the level of scaffold residues, this study has unravelled a robust interaction network on antibody structure surrounding the CDR3 loop. General significance: In addition to the general applicability of the GSS algorithm, which can bring together functional and sequence data to locate hot spots of constrained evolution, the relationship between CDR3 and scaffold discussed here should be taken into account in protein engineering when designing antibody libraries. In addition, an open web site was issued, with tutorial and software for exploiting the algorythm in biochemistry, molecular biology and biotechnology: KASTELIC Damjana, SOLER Nicolas, KOMEL Radovan, POMPON Denis: Global sequences signature analysis of camelidae VH/VHH sequences. [S. l.: s. n., 2013]. http://gss.mrc-lmb.cam.ac.uk/. [COBISS.SI-ID 30498265].
Mouse knockout of the cholesterogenic cytochrome P450 lanosterol 14[alpha]-Demethylase (CYP51) resembles Antley-Bixler syndrome: Antley-Bixler syndrome (ABS) represents a group of heterogeneous disorders characterized by skeletal, cardiac and urogenital abnormalities that have frequently been associated with mutations in fibroblast growth factor receptor2 or cytochrome P450 reductase genes. In some ABS patients, reduced activity of the cholesterogenic cytochrome P450 CYP51A1, an ortholog of the mouse CYP51, and accumulation of lanosterol and 24,25-dihydrolanosterol has been reported, but the role of CYP51A1 in the ABS etiology has remained obscure. To test whether Cyp51 could be involved in generating an ABS-like phenotype, a mouse knockout model was developed that exhibited several prenatal ABS-like features leading to lethality at embryonic day 15. Cyp51(-/-) mice had no functional Cyp51 mRNA and no immunodetectable CYP51 protein. The two CYP51 enzyme substrates (lanosterol and 24,25-dihydrolanosterol) were markedly accumulated. Cholesterol precursors downstream of the CYP51 enzymatic step were not detected, indicating that the targeting in this study blocked de novo cholesterol synthesis. This was reflected in the up-regulation of 10 cholesterol synthesis genes, with the exception of 7-dehydrocholesterol reductase. Lethality was ascribed to heart failure due to hypoplasia, ventricle septum, epicardial and vasculogenesis defects, suggesting that Cyp51 deficiency was involved in heart development and coronary vessel formation. As the most likely downstream molecular mechanisms, alterations were identified in the sonic hedgehog and retinoic acid signaling pathways. Cyp51 knockout mice provide evidence that Cyp51 is essential for embryogenesis and present a potential animal model for studying ABS syndrome in humans. THIS IS THE FIRST TRANSGENIC MOUSE ENTIRELY CONSTRUCTED IN SLOVENIA.
Brain-derived neurotrophic factor (BDNF) plays a pivotal role in the pathophysiology of suicidal behavior and BDNF levels are decreased in the brain and plasma of suicide subjects. So far, the mechanisms leading to downregulation of BDNF expression are poorly understood. We tested the hypothesis that alterations of DNA methylation could be involved in the dysregulation of BDNF gene expression in the brain of suicide subjects. In the study forty-four suicide completers and 33 nonsuicide control subjects of white ethnicity were involved. Three independent quantitative methylation techniques were performed on postmortem samples of brain tissue. BDNF messenger RNA levels were determined by quantitative real-time polymerase chain reaction. We measured the DNA methylation degree at BDNF promoter IV and the genome-wide DNA methylation levels in the brain's Wernicke area. Postmortem brain samples from suicide subjects showed a statistically significant increase of DNA methylation at specific CpG sites in BDNF promoter/exon IV compared with nonsuicide control subjects (P ( .001). Most of the CpG sites lying in the -300/+500 region, on both strands, had low or no methylation, with the exception of a few sites located near the transcriptional start site that had differential methylation, while genome-wide methylation levels were comparable among the subjects. The mean methylation degree at the 4 CpG sites analyzed by pyrosequencing was always less than 12.9% in the 33 nonsuicide control subjects, while in 13 of 44 suicide victims (30%), the mean methylation degree ranged between 13.1% and 34.2%. Higher methylation degree corresponded to lower BDNF messenger RNA levels. CONCLUSIONS: BDNF promoter/exon IV is frequently hypermethylated in the Wernicke area of the postmortem brain of suicide subjects irrespective of genome-wide methylation levels, indicating that a gene-specific increase in DNA methylation could cause or contribute to the downregulation of BDNF expression in suicide subjects. THE REPORTED DATA REVEAL A NOVEL LINK BETWEEN EPIGENETIC ALTERATION IN THE BRAIN AND SUICIDAL BEHAVIOR.
Results of the previous research were published in J. Med. Chem. (2008) and were also exposed by SciBX (joint publication of Nature in BioCentury) as promising for the design of new antifungal compounds (The Distilery – Infectious Disease, SciBX, 19 June 2008, vol. 1, no. 21, p. 13). Here, in this paper, we discuss the biological role of two alternative redox partners of CYP53A15 enzyme, gene cloned in our previous research and mathematical model for the protein constructed. The content of the paper is as follows: Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.
We determined the crystal structure of the full-length cAMP phosphodiesterase Rv0805 from Mycobacterium tuberculosis. Furthermore we showed, that localization of Rv0805 to the bacterial cell wall is dependent on its C terminus, and expression of either wild type or mutationally inactivated Rv0805 in M. smegmatis alters cell permeability to hydrophobic cytotoxic compounds. Rv0805 may therefore play a key role in the pathogenicity of Mycobacteria, not only by hydrolyzing bacterial cAMP, but also by moonlighting as a protein that can alter cell wall functioning. Enzyme Rv0805 is being exposed as potential target for the design of new anti-tuberculosis agents.