Bairong Shen

Characterization of CA XIII, a novel member of the carbonic anhydrase isozyme family.

The carbonic anhydrase (CA) gene family has been reported to consist of at least 11 enzymatically active members and a few inactive homologous proteins. Recent analyses of human and mouse databases provided evidence that human and mouse genomes contain genes for still another novel CA isozyme hereby named CA XIII. In the present study, we modeled the structure of human CA XIII. This model revealed a globular molecule with high structural similarity to cytosolic isozymes, CA I, II, and III. Recombinant mouse CA XIII showed catalytic activity similar to those of mitochondrial CA V and cytosolic CA I, with kcat/Km of 4.3 × 107 m–1 s–1, and kcat of 8.3 × 104 s–1. It is very susceptible to inhibition by sulfonamide and anionic inhibitors, with inhibition constants of 17 nm for acetazolamide, a clinically used sulfonamide, and of 0.25 μm, for cyanate, respectively. Using panels of cDNAs we evaluated human and mouse CA13 gene expression in a number of different tissues. In human tissues, positive signals were identified in the thymus, small intestine, spleen, prostate, ovary, colon, and testis. In mouse, positive tissues included the spleen, lung, kidney, heart, brain, skeletal muscle, and testis. We also investigated the cellular and subcellular localization of CA XIII in human and mouse tissues using an antibody raised against a polypeptide of 14 amino acids common for both human and mouse orthologues. Immunohistochemical staining showed a unique and widespread distribution pattern for CA XIII compared with the other cytosolic CA isozymes. In conclusion, the predicted amino acid sequence, structural model, distribution, and activity data suggest that CA XIII represents a novel enzyme, which may play important physiological roles in several organs.

Characterization of CA XV, a new GPI-anchored form of carbonic anhydrase

The main function of CAs (carbonic anhydrases) is to participate in the regulation of acid-base balance. Although 12 active isoenzymes of this family had already been described, analyses of genomic databases suggested that there still exists another isoenzyme, CA XV. Sequence analyses were performed to identify those species that are likely to have an active form of this enzyme. Eight species had genomic sequences encoding CA XV, in which all the amino acid residues critical for CA activity are present. However, based on the sequence data, it was apparent that CA XV has become a non-processed pseudogene in humans and chimpanzees. RT-PCR (reverse transcriptase PCR) confirmed that humans do not express CA XV. In contrast, RT-PCR and in situ hybridization performed in mice showed positive expression in the kidney, brain and testis. A prediction of the mouse CA XV structure was performed. Phylogenetic analysis showed that mouse CA XV is related to CA IV. Therefore both of these enzymes were expressed in COS-7 cells and studied in parallel experiments. The results showed that CA XV shares several properties with CA IV, i.e. it is a glycosylated glycosylphosphatidylinositol-anchored membrane protein, and it binds CA inhibitor. The catalytic activity of CA XV is low, and the correct formation of disulphide bridges is important for the activity. Both specific and non-specific chaperones increase the production of active enzyme. The results suggest that CA XV is the first member of the alpha-CA gene family that is expressed in several species, but not in humans and chimpanzees.

Genome wide analysis of pathogenic SH2 domain mutations.

The authors have made a genome‐wide analysis of mutations in Src homology 2 (SH2) domains associated with human disease. Disease‐causing mutations have been detected in the SH2 domains of cytoplasmic signaling proteins Bruton tyrosine kinase (BTK), SH2D1A, Ras GTPase activating protein (RasGAP), ZAP‐70, SHP‐2, STAT1, STAT5B, and the p85α subunit of the PIP3. Mutations in the BTK, SH2D1A, ZAP70, STAT1, and STAT5B genes have been shown to cause diverse immunodeficiencies, whereas the mutations in RASA1 and PIK3R1 genes lead to basal carcinoma and diabetes, respectively. PTPN11 mutations cause Noonan sydrome and different types of cancer, depending mainly on whether the mutation is inherited or sporadic. We collected and analyzed all known pathogenic mutations affecting human SH2 domains by bioinformatics methods. Among the investigated protein properties are sequence conservation and covariance, structural stability, side chain rotamers, packing effects, surface electrostatics, hydrogen bond formation, accessible surface area, salt bridges, and residue contacts. The majority of the mutations affect positions essential for phosphotyrosine ligand binding and specificity. The structural basis of the SH2 domain diseases was elucidated based on the bioinformatic analysis. Proteins 2008.

RankViaContact: ranking and visualization of amino acid contacts.

SUMMARY RankViaContact is a web service for calculation of residue-residue contact energies in proteins based on a coarse-grained model, and for visualization of interactions. The service provides information about ranked contact energies of residues, coordination numbers and the relative solvent accessibility of selected residues, as well as sequence and structure information. The program can be used to design stabilizing mutations, to analyze residue-residue contacts and to study the consequences of mutations. AVAILABILITY http://bioinf.uta.fi/Rank.htm.

PON-Sol: prediction of effects of amino acid substitutions on protein solubility

MOTIVATION Solubility is one of the fundamental protein properties. It is of great interest because of its relevance to protein expression. Reduced solubility and protein aggregation are also associated with many diseases. RESULTS We collected from literature the largest experimentally verified solubility affecting amino acid substitution (AAS) dataset and used it to train a predictor called PON-Sol. The predictor can distinguish both solubility decreasing and increasing variants from those not affecting solubility. PON-Sol has normalized correct prediction ratio of 0.491 on cross-validation and 0.432 for independent test set. The performance of the method was compared both to solubility and aggregation predictors and found to be superior. PON-Sol can be used for the prediction of effects of disease-related substitutions, effects on heterologous recombinant protein expression and enhanced crystallizability. One application is to investigate effects of all possible AASs in a protein to aid protein engineering. AVAILABILITY AND IMPLEMENTATION PON-Sol is freely available at http://structure.bmc.lu.se/PON-Sol The training and test data are available at http://structure.bmc.lu.se/VariBench/ponsol.php CONTACT mauno.vihinen@med.lu.se SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

A novel mutation in CD40 and its functional characterization.

CD40 is a costimulatory protein expressed on the surface of many different cells. It delivers signals regulating diverse cellular responses, including proliferation, differentiation, growth suppression, and cell death. In this study, we report a novel CD40 mutant (c.234C>A or p.H78Q) that is expressed in the U266 cell line and in freshly isolated tumor cells. Three‐dimensional structural model and Scatchard analysis revealed that the mutated residue located in a region is important for binding to CD40L (CD154). Functional analysis indicated that the mutated CD40 was translocated to the CD40 signalosome and involved in CD40 signal transduction. In conclusion, the mutation in CD40 can lead to an alteration of function, including the change of antigen epitope and the binding affinity with CD40L. Hum Mutat 30, 985–994, 2009.

PON-tstab: Protein Variant Stability Predictor. Importance of Training Data Quality

Several methods have been developed to predict effects of amino acid substitutions on protein stability. Benchmark datasets are essential for method training and testing and have numerous requirements including that the data is representative for the investigated phenomenon. Available machine learning algorithms for variant stability have all been trained with ProTherm data. We noticed a number of issues with the contents, quality and relevance of the database. There were errors, but also features that had not been clearly communicated. Consequently, all machine learning variant stability predictors have been trained on biased and incorrect data. We obtained a corrected dataset and trained a random forests-based tool, PON-tstab, applicable to variants in any organism. Our results highlight the importance of the benchmark quality, suitability and appropriateness. Predictions are provided for three categories: stability decreasing, increasing and those not affecting stability.

NDDVD: an integrated and manually curated Neurodegenerative Diseases Variation Database

Abstract Neurodegenerative diseases (NDDs) are associated with genetic variations including point substitutions, copy number alterations, insertions and deletions. At present, a few genetic variation repositories for some individual NDDs have been created, however, these databases are needed to be integrated and expanded to all the NDDs for systems biological investigation. We here build a relational database termed as NDDVD to integrate all the variations of NDDs using Leiden Open Variation Database (LOVD) platform. The items in the NDDVD are collected manually from PubMed or extracted from the existed variation databases. The cross-disease database includes over 6374 genetic variations of 289 genes associated with 37 different NDDs. The patterns, conservations and biological functions for variations in different NDDs are statistically compared and a user-friendly interface is provided for NDDVD at: http://bioinf.suda.edu.cn/NDDvarbase/LOVDv.3.0. URL: http://bioinf.suda.edu.cn/NDDvarbase/LOVDv.3.0