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C de Chiara

Publications and source records attributed to C de Chiara.

5 recordsLinked to original sources
PubMed2003

The AXH module: an independently folded domain common to ataxin-1 and HBP1.

Ataxin-1 (ATX1), a human protein responsible for spinocerebellar ataxia type 1 in humans, shares a region of homology, named AXH module, with the apparently unrelated transcription factor HBP1. Here, we describe the first characterisation of the AXH module in terms of its structural properties and stability. By producing protein constructs spanning the AXH modules of ATX1 and HBP1 and by comparing their properties, we have identified the minimal region sufficient for forming independently folded units (domains). Knowledge of the AXH domain boundaries allows us to map many of the interactions of ATX1 with other molecules onto the AXH module. We further show that the AXH of ATX1 is a dimerisation domain and is able to recognise RNA with the same nucleotide preference previously described for the full-length protein. AXH is therefore a novel protein-protein and RNA binding motif.

Amino Acid Motifs↗
PubMed2003

PcF protein from Phytophthora cactorum and its recombinant homologue elicit phenylalanine ammonia lyase activation in tomato.

The phytotoxic protein PcF (Phytophthora cactorum-Fragaria) is a 5.6-kDa cysteine-rich, hydroxyproline-containing protein that is secreted in limited amounts by P. cactorum, an oomycete pathogen of tomato, strawberry and other relevant crop plants. Although we have shown that pure PcF triggers plant reactivity, its mechanism of action is not yet understood. Here we show that PcF, like other known fungal protein elicitors involved in pathogen-plant interaction, stimulates the activity of the defense enzyme phenylalanine ammonia lyase (EC 4.3.1.5) in tomato seedlings. Recognizing that a key step in understanding the mechanism of action of PcF at a molecular level is knowledge of its three-dimensional structure, we overexpressed this protein extracellularly in Pichia pastoris. The preliminary structural and functional characterization of a recombinant PcF homologue, N4-rPcF, is reported. Interestingly, although N4-rPcF is devoid of proline hydroxylation and has four additional amino acid residues attached to its N terminus, its secondary structure and biological activity are indistinguishable from wild-type PcF.

Amino Acid Sequence↗
PubMed1997

Thioredoxin from Bacillus acidocaldarius: characterization, high-level expression in Escherichia coli and molecular modelling.

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx) was purified to homogeneity by anion-exchange chromatography and gel-filtration chromatography, based on its ability to catalyse the dithiothreitol-dependent reduction of bovine insulin disulphides. The protein has a molecular mass of 11577 Da, determined by electrospray mass spectrometry, a pI of 4.2, and its primary structure was obtained by automated Edman degradation after cleavage with trypsin and cyanogen bromide. The sequences of known bacterial Trxs were aligned at the active site: BacTrx has an identity ranging from 45 to 53% with all sequences except that of the unusual Anabaena strain 7120 Trx (37% identity). The gene coding for BacTrx was isolated by a strategy based on PCR gene amplification and cloned in a plasmid downstream of a lac-derived promoter sequence; the recombinant clone was used as the expression vector for Escherichia coli. The expression was optimized by varying both the time of cell growth and the time of exposure to the inducer isopropyl beta-d-thiogalactoside; expressed BacTrx represents approx. 5% of the total cytosolic protein. CD spectra and differential scanning calorimetry measurements demonstrated that BacTrx is endowed with a higher conformational heat stability than the Trx from E. coli. Nanogravimetry experiments showed a lower content of bound water in BacTrx than in E. coli Trx, and a transition temperature approx. 10 degrees C higher for BacTrx. The three-dimensional model of the oxidized form of BacTrx was constructed by a comparative molecular modelling technique, using E. coli Trx and Anabaena strain 7120 Trx as reference proteins. Increased networks of ion-pairs and shorter loops emerged as major features of the BacTrx structure compared with those of the template proteins. The findings are discussed in the light of the current knowledge about molecular determinants of protein stability.

Amino Acid Sequence↗
PubMedDate not supplied

[Ebstein's disease].

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Adolescent↗