Probing the telomere-binding activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

摘要

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-studied, ubiquitous glycolytic enzyme that also exhibits several non-glycolytic activities, including DNA repair, membrane fusion, apoptotic signaling, tRNA transport and microtubule bundling. GAPDH binds telomere repeats, and in human A549 non small-cell lung adenocarcinoma cells, overexpression of GAPDH protects telomeres from chemotherapeutic agents. The maintenance of telomeric DNA in A549 cells appears to play a role in chemotherapeutic resistance. This study addresses the binding of GAPDH to human telomeric DNA and determines the extent to which GAPDH maintains the viability of A549 cells treated with the chemotherapeutic agents gemcitabine (GMZ) and doxorubicin (DOX). Molecular analysis of the GAPDH-telomere interaction shows GAPDH recognizes G5, G6 and T1 nucleotides of the telomeric-DNA repeat (5'-TTAGGG-3'), and binding to the single strand (ss) - telomeric DNA oligonucleotide (5'-(AGGGTT) 3-3' with high affinity (Kd = 45 nM). Furthermore, the DNA-binding site on GAPDH comprises both the enzyme catalytic site and NAD+-binding site. The stoichiometry of binding is 2:1 (DNA:GAPDH) and GAPDH forms a high-molecular weight complex when bound to a synthetic ss-telomeric DNA oligonucleotide. Protection of telomeres by GAPDH in A549 cells increases cell viability following treatment with the chemotherapeutic agents GMZ and DOX. The data from these experiments combined with high-resolution crystal data allowed for construction of a putative model of GAPDH binding to ss-telomeric DNA.