Biomedical subjects

Luca D D'Andrea

Publications and source records attributed to Luca D D'Andrea.

3 recordsLinked to original sources

PubMed2006

A novel type of zinc finger DNA binding domain in the Agrobacterium tumefaciens transcriptional regulator Ros.

Transcriptional factors bearing a Cys(2)His(2) zinc finger were thought to be confined to eukaryotes, but recent studies have suggested their presence also in prokaryotes. In this paper, we report the first complete functional characterization of the DNA binding domain present in the putative Cys(2)His(2) zinc finger-containing prokaryotic transcriptional regulator Ros from Agrobacterium tumefaciens. We demonstrate that in the single zinc binding motif present in the Ros protein the metal ion is coordinated by two cysteines (Cys79 and Cys82) and two histidines (His92 and His97), separated by a shorter spacer with respect to the eukaryotic classical Cys(2)His(2) domains. The Cys(2)His(2) zinc finger motif is essential for Ros DNA binding and is part of a larger DNA binding domain which includes four basic regions located on either side of the finger, one at the N-terminus and three at the C-terminus. The one described here is a novel type of DNA binding domain containing a noncanonical Cys(2)His(2) zinc finger motif which, by sequence alignment, seems to be conserved in all the bacterial putative zinc finger proteins identified so far. Interestingly, basic amino acids have been shown to be important in stabilizing the DNA binding of eukaryotic single Cys(2)His(2) zinc finger domains, confirming that the modality of DNA binding using a single zinc finger motif flanked by basic residues is widespread throughout the living kingdom from eukaryotic, both animal and plant, to prokaryotic, even if in each kingdom it presents its peculiarity.

Agrobacterium tumefaciens↗

PubMed2004

Assignment of the binding site for haptoglobin on apolipoprotein A-I.

Haptoglobin (Hpt) was previously found to bind the high density lipoprotein (HDL) apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme lecithin:cholesterol acyltransferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I structure was analyzed to detect the site bound by Hpt. ApoA-I was treated by cyanogen bromide or hydroxylamine; the resulting fragments, separated by electrophoresis or gel filtration, were tested by Western blotting or enzyme-linked immunosorbent assay for their ability to bind Hpt. The ApoA-I sequence from Glu113 to Asn184 harbored the binding site for Hpt. Biotinylated peptides were synthesized overlapping such a sequence, and their Hpt binding activity was determined by avidin-linked peroxidase. The highest activity was exhibited by the peptide P2a, containing the ApoA-I sequence from Leu141 to Ala164. Such a sequence contains an ApoA-I domain required for binding cells, promoting cholesterol efflux, and stimulating LCAT. The peptide P2a effectively prevented both binding of Hpt to HDL-coated plastic wells and Hpt-dependent inhibition of LCAT, measured by anti-Hpt antibodies and cholesterol esterification activity, respectively. The enzyme activity was not influenced, in the absence of Hpt, by P2a. Differently from ApoA-I or HDL, the peptide did not compete with hemoglobin for Hpt binding in enzyme-linked immunosorbent assay experiments. The results suggest that Hpt might mask the ApoA-I domain required for LCAT stimulation, thus impairing the HDL function. Synthetic peptides, able to displace Hpt from ApoA-I without altering its property of binding hemoglobin, might be used for treatment of diseases associated with defective LCAT function.

Amino Acid Sequence↗

PubMed2003

TPR proteins: the versatile helix.

Tetratrico peptide repeat (TPR) proteins have several interesting properties, including their folding characteristics, modular architecture and range of binding specificities. In the past five years, many 3D structures of TPR domains have been solved, revealing at a molecular level the versatility of this basic fold. Here, we discuss the structure of TPRs and highlight the diversity of arrangements and functions that are associated with these ubiquitous domains. Genomic analyses of the distribution of TPR domains are presented along with implications for protein engineering.

Amino Acid Sequence↗