Clostridium difficile is an anaerobic, gram‐positive bacterium and a common gastrointestinal pathogen. Two virulence factors of C. difficile known to mediate disease are Toxin A (TcdA) and Toxin B (TcdB). These toxins are glucosyltransferase‐containing multi‐domain proteins that enter the host's epithelial cells by binding to cell–surface carbohydrate antigens. Once inside the cell, the toxins undergo a pH‐induced conformational change and release their glucosyltransferase domains (GTD), leading to disruption of cell cytoskeleton integrity. Existing treatments for C. difficile such as fecal transplants and antibiotics have not proven highly effective. Passive immunotherapy with inhibitory antibodies recognizing TcdA and TcdB is a promising alternative. Recombinant Camelidae antibodies (V H Hs) that neutralize the toxins' function have been identified. TcdA's receptor‐binding domain has seven repetitive elements known to interact with cell‐surface carbohydrates. The V H H antibodies A20.1 and A26.8, which are known to neutralize TcdA, do not block the carbohydrate‐binding site on TcdA directly, suggesting a novel mode of TcdA inhibition. The Ashbury College MSOE Center for BioMolecular Modeling SMART Team used 3‐D modelling and printing technology to examine the structure‐function relationships of a TcdA fragment (TcdA‐A2) simultaneously bound to A20.1 and A26.8. The structure shows that the V H Hs target distinct epitopes on the toxin and neutralize TcdA by a novel mechanism independent of the carbohydrate binding sites, providing a rationale for designing highly potent biparatopic TcdA‐neutralizing antibodies. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
The lamin A/C (LMNA) gene codes for A type lamins which are key components of the nuclear lamina and are involved in maintaining the structure of the nucleus and its processes. Mutations in LMNA cause a group of diseases known as laminopathies. For this project, the lamin A/C variant affecting amino acid 192 is studied. The causal mutation results in an amino acid change from glycine [G] (smaller, non‐polar, uncharged) to aspartic acid [D] (larger, polar, charged). This D192G variant is linked to a severe form of a cardiac disease called Dilated Cardiomyopathy (DCM), where cardiomyocytes are presented with multiple nuclear abnormalities. In vitro experiments with the D192G variant showed abnormal nuclear localization of Protein Kinase C alpha (PKC‐α). PKC‐α is the major protein kinase C isoform found in the heart and skeletal muscles. This enzyme phosphorylates a host of proteins and uses A‐type lamins to reach its nuclear targets. In order to help elucidate the potential role of PKC‐α as the link between the disease phenotype and lamin mutation, a structural comparison between the wild type lamin A and the D192G variant was performed in silico. Moreover, the PKC‐α binding site on the wild type and mutant lamin A were compared in order to determine if a conformation change has occurred. Since there is currently no full‐length crystal structure for either the wild type or D192G lamin A/C variant, the I‐TASSER software suite was used to generate simulated protein structures. The top five structures for the WT and variant simulations were examined. The final models selected to most likely represent reality were chosen based on various factors such as computed C‐score, what is known in literature and human analysis of the simulations. The results show that the D192G variant predominantly takes a globular form which is very comparable to the structure of the wild type lamin A found in the nucleoplasm. This globular form also potentially explains the lamin aggregates found in cells expressing the D192G variant. However, this globular variant conformation significantly differs when compared to the known and prevalent wild type lamin A conformation which is the globular head‐alpha helical rod‐globular tail structure. In particular, the conformation of PKCα binding site on lamin A is also significantly altered. The Ashbury College MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine the structure‐function relationships of the wild type lamin A/C protein and the D192G variant.
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