Exploring the mechanism of inhibition of human telomerase by cysteine‐reactive compounds

Abstract

Telomerase is an almost universal cancer target that consists minimally of a core protein human telomerase reverse transcriptase (hTERT) and a RNA component human telomerase RNA (hTR). Some inhibitors of this enzyme are thought to function by the covalent binding to one or several cysteine residues; however, this inhibition mechanism has never been investigated because of the difficulty in producing telomerase. In this study, we use a recent method to produce recombinant hTERT to analyze the effect of cysteine-reactive inhibitors on telomerase. Using mass spectrometry and mutagenesis analysis, we identify several targeted residues in separated domains of the hTERT protein and show that cysteine-reactive reagents abolish the interaction with the CR4/5 region of hTR.

Nucleolin Discriminates Drastically between Long-Loop and Short-Loop Quadruplexes

“…Therefore, an alternative approach was applied, which is based on a well-established workflow for analyzing RNA–protein complexes directly from solution samples. , Then direct trypsinization without DNA digestion should generate bulky DNA–peptide adducts unlikely to be detected by LC-MS/MS. Under these conditions, comparison of the ratio of all peptides in the UV-irradiated solution versus the nonirradiated control solution should show specific peak depletion corresponding to cross-linked peptides. , Following this protocol (shown schematically in Figure S7), we were able to identify a highly significantly reduced peptide in the UV-treated sample by applying a 1% false discovery rate (FDR) for the peptide validation and an intensity threshold in skyline and a p value of <0.05 as the statistical cutoff for the ratio significance (Table S2). The MS analysis indicated that this is a 15-amino acid peptide located in RBD2 of NCL corresponding to the fragment from the Gly at amino acid 429 to the Lys at amino acid 444 (Figure ), which suggests a direct interaction of this domain with the nine-nucleotide loop of the G4 substrate.…”

“…Interestingly, the RBD1 and RBD2 structures of the human NCL have been characterized by NMR56 and are known to be involved in the recognition of the G4 aptamer AS1411. 56 Remarkably, examination of the protein sequence indicates that the depleted peptide is located at the junction between strand β3 and helix α2 of RBD2,56 suggesting these elements might be strong determinants of G4-loop binding. Finally, although RBD1 has not been detected in our analysis, it may act in tandem with RBD2 for G4-loop recognition as has been shown…”

Targeting the Cysteine Redox Proteome in Parkinson’s Disease: The Role of Glutathione Precursors and Beyond

“…hTERT regulation involves transcriptional and post-transcriptional modifications [156,157]. Indeed, ROS overproduction promotes the hTERT export from the nucleus to the cytoplasm, participating in senescence and agerelated neurodegenerative diseases [158,159]. hTERT includes four essential domains (TEN, TRBD, RT, and CTE), and hTR contains two central components needed for its function: the template/pseudoknot (t-PSK) and the CR4/5 domains.…”