Biomedical subjects

Gary Laco

Publications and source records attributed to Gary Laco.

3 recordsLinked to original sources

PubMed2006

Molecular replacement with pseudosymmetry and model dissimilarity: a case study.

Crystals of human T-cell leukemia virus protease (HTLV-1 PR) have been very difficult to prepare and only native data extending to 2.6 angstroms resolution could be collected. Initial attempts to solve the structure with a variety of low-sequence-identity models utilizing proteases from other retroviruses and using a number of molecular-replacement programs were unsuccessful. The structure was finally solved using Phaser, revealing extensive pseudosymmetry and significant deviations from the starting models, features that were likely to be responsible for the initial failures. The steps taken to solve this structure and some of its intriguing crystallographic aspects are discussed.

Aspartic Acid Endopeptidases↗

PubMed2004

Phosphorylation of DNA topoisomerase I by the c-Abl tyrosine kinase confers camptothecin sensitivity.

DNA topoisomerase I (topo I) is involved in the regulation of DNA supercoiling, gene transcription, recombination, and DNA repair. The anticancer agent camptothecin specifically targets topo I. The mechanisms responsible for the regulation of topo I in cells, however, are not known. This study demonstrates that c-Abl-dependent phosphorylation up-regulates topo I activity. The c-Abl SH3 domain bound directly to the N-terminal region of topo I. The results demonstrate that c-Abl phosphorylated topo I at Tyr268 in core subdomain II. c-Abl-mediated phosphorylation of topo I Tyr268 in vitro and in cells conferred activation of the topo I isomerase function. Moreover, activation of c-Abl by treatment of cells with ionizing radiation was associated with c-Abl-dependent phosphorylation of topo I and induction of topo I activity. The functional significance of the c-Abl/topo I interaction is supported by the findings that (i) mutant topo I(Y268F) exhibited loss of c-Abl-induced topo I activity, and (ii) c-Abl-/- cells were deficient in the accumulation of protein-linked DNA breaks. In addition, loss of topo I phosphorylation in c-Abl-deficient cells conferred resistance to camptothecin-induced apoptosis. These findings collectively support a model in which c-Abl-mediated phosphorylation of topo I is functionally important to topo I activity and sensitivity to topo I poisons.

Amino Acid Sequence↗

PubMed2003

Differential induction of topoisomerase I-DNA cleavage complexes by the indenoisoquinoline MJ-III-65 (NSC 706744) and camptothecin: base sequence analysis and activity against camptothecin-resistant topoisomerases I.

Camptothecin (CPT) and its derivatives target mammalian DNA topoisomerase I (top1) and are among the most effective novel anticancer drugs. However, the activity of CPTs is limited by several factors, including drug inactivation by lactone ring opening, tumor drug resistance, and toxicity in patients. Novel top1 inhibitors are being searched to overcome such limitations and expand the anticancer spectrum of camptothecins. MJ-III-65 (NSC 706744) is among the most promising indenoisoquinolines to date. In this study, we show that MJ-III-65 enhances top1 cleavage complexes by both inhibiting their reversal (religation) more efficiently than CPT and by enhancing their formation. The top1 DNA cleavage complexes induced by MJ-III-65 exhibit a different distribution pattern compared with CPT and exhibit different base sequence preferences immediately around the top1 cleavage sites. Although CPTs have a preference for thymine at the (-1) position and guanine at the (+1) position of the top1-mediated DNA cleavage sites, MJ-III-65 can accommodate different base pairs at the (-1), (+1), or (+2) position, with a preference for a cytosine at the (-1) position on the scissile strand. Another difference with CPTs is the activity of MJ-III-65 against CPT-resistant top1 enzymes, implying that the amino acid residue interactions with top1 are different for MJ-III-65 and CPTs. As with CPT, MJ-III-65 is inactive against vaccinia top1. This study shows the specific molecular interactions of MJ-III-65 with top1 and demonstrates that MJ-III-65 is a potentially useful top1 inhibitor that enhances and traps top1 cleavage sites not sensitive to CPTs.

Animals↗