BackgroundGenomic mutations caused by cytotoxic agents used in cancer chemotherapy may cause secondary malignancies as well as contribute to the evolution of treatment-resistant tumour cells. The stable diploid genome of the chicken DT40 lymphoblast cell line, an established DNA repair model system, is well suited to accurately assay genomic mutations.ResultsWe use whole genome sequencing of multiple DT40 clones to determine the mutagenic effect of eight common cytotoxics used for the treatment of millions of patients worldwide. We determine the spontaneous mutagenesis rate at 2.3 × 10–10 per base per cell division and find that cisplatin, cyclophosphamide and etoposide induce extra base substitutions with distinct spectra. After four cycles of exposure, cisplatin induces 0.8 mutations per Mb, equivalent to the median mutational burden in common leukaemias. Cisplatin-induced mutations, including short insertions and deletions, are mainly located at sites of putative intrastrand crosslinks. We find two of the newly defined cisplatin-specific mutation types as causes of the reversion of BRCA2 mutations in emerging cisplatin-resistant tumours or cell clones. Gemcitabine, 5-fluorouracil, hydroxyurea, doxorubicin and paclitaxel have no measurable mutagenic effect. The cisplatin-induced mutation spectrum shows good correlation with cancer mutation signatures attributed to smoking and other sources of guanine-directed base damage.ConclusionThis study provides support for the use of cell line mutagenesis assays to validate or predict the mutagenic effect of environmental and iatrogenic exposures. Our results suggest genetic reversion due to cisplatin-induced mutations as a distinct mechanism for developing resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0963-7) contains supplementary material, which is available to authorized users.
The most preferred residue in the substrates of human immunodeficiency virus (HIV-1) protease is glutamic acid in the P2' position. The catalytic importance of this charged residue has been studied to obtain a detailed insight into the mechanism of action, which will promote drug design to combat the virus. To this end, we have synthesized Lys-Ala-Arg-Val-Leu*Phe(NO2)-Glu-Ala-Nle (substrate E) and its counterpart containing the neutral Gln (substrate Q) in place of Glu. Kinetic analyses have shown that the specificity rate constants (kcat/Km) display bell-shaped pH dependencies for both substrates, but the pH-independent limiting value is 35-40-fold higher with substrate E than with substrate Q. In contrast to the pH-rate profiles of kcat/Km, there is a striking difference between the pH dependencies of Km and kcat for the two substrates. This indicates different ground state and transition state stabilizations in the two reactions. Solvent kinetic deuterium isotope effects show that the rate-limiting step for the hydrolysis of substrate E is a chemical step coupled with proton transfer whereas with substrate Q it is a physical step, presumably a conformational change. Accordingly, the charged residue in P2' alters the rate-limiting step and the nature of the enzyme-substrate complex, resulting in different mechanisms for the two substrates.
The flexibility of prolyl oligopeptidase has been investigated using molecular dynamics (MD) and molecular framework approaches to delineate the route of the substrate to the active site. The selectivity of the enzyme is mediated by a seven-bladed beta-propeller that in the crystal structure does not indicate the possible passage for the substrate to the catalytic center. Its open topology however, could allow the blades to move apart and let the substrate into the large central cavity. Flexibility analysis of prolyl oligopeptidase structure using the FIRST (Floppy Inclusion and Rigid Substructure Topology) approach and the atomic fluctuations derived from MD simulations demonstrated the rigidity of the propeller domain, which does not permit the substrate to approach the active site through this domain. Instead, a smaller tunnel at the inter-domain region comprising the highly flexible N-terminal segment of the peptidase domain and a facing hydrophilic loop from the propeller (residues 192-205) was identified by cross-correlation analysis and essential dynamics as the only potential pathway for the substrate. The functional importance of the flexible loop has been also verified by kinetic analysis of the enzyme with a split loop. Catalytic effect of engineered disulfide bridges was rationalized by characterizing the concerted motions of the two domains.
Proteases have a variety of strategies for selecting substrates in order to prevent uncontrolled protein degradation. A recent crystal structure determination of prolyl oligopeptidase has suggested a way for substrate selection involving an unclosed seven-bladed β-propeller domain. We have engineered a disulfide bond between the first and seventh blades of the propeller, which resulted in the loss of enzymatic activity. These results provided direct evidence for a novel strategy of regulation in which oscillating propeller blades act as a gating filter during catalysis, letting small peptide substrates into the active site while excluding large proteins to prevent accidental proteolysis.
The activity of human immunodeficiency virus protease is markedly increased at elevated salt concentration. The structural basis of this effect has been explored by several independent methods by using both the wild-type enzyme and its triple mutant (Q7K/L33I/ L63I) (Mildner, and the transition midpoint (D1 ⁄2 ) between the folded and unfolded states increase, indicating that the salt stabilizes the enzyme structure. These equilibrium data are supported by kinetic studies on the urea-mediated unfolding by measuring fluorescence change, red shifting in the maximum of the emission spectrum, and farand near-UV CD. The salt effects observed in urea-mediated unfolding reactions prevail upon heat denaturation. All these findings support the existence of a twostate equilibrium between the folded and unfolded proteins. The pH dependence of fluorescence intensity indicated that the conformation of human immunodeficiency virus type 1 protease should change in the catalytically competent pH region. It is concluded that preferential hydration stabilizes the protease structure in the presence of salt, providing entropic contribution to enhance the catalytic activity.AThe protease encoded by HIV-1 1 is involved in the specific processing of large viral polyproteins into individual structural proteins and enzymes. This prompted extensive investigations on HIV-1 protease as a potential therapeutic target of AIDS. The enzyme is a member of the aspartic protease family and exists as homodimer of identical polypeptide chains, each consisting of 99 amino acid residues (cf. Refs. 1 and 2). High salt concentration was found to enhance the catalytic activity, primarily by lowering the Michaelis constant (K m ), and it was suggested that the salting-out effect of NaCl decreased K m and increased k cat /K m , the specificity rate constant (3, 4). We have pointed out that K m values may be markedly dependent on pH, while the rate enhancement by salt is practically independent of K m and pH (5). Therefore, other factors, for example the conformational stability of the protein, may also be important. Indeed, raising the NaCl concentration frequently stabilizes the protein structure by preferential hydration (6). However, the effects of ionic strength on the conformational stability of HIV-1 protease have not yet been studied. To reveal whether or not the enhanced catalytic activity is associated with a more stable structure, we have examined the stability of the enzyme against urea, pH, and heat denaturation at different salt concentrations. A mutant enzyme designed to resist autolysis (7) has been used in most experiments. EXPERIMENTAL PROCEDURESHIV-1 Protease-The enzyme was expressed in Escherichia coli and purified as described (5). The construction containing three mutations (Q7K/L33I/L63I) in the HIV-1 protease was kindly supplied by Dr. Tomasselli (7), and the enzyme was purified from inclusion bodies with the same method as used for the wild-type protease. The protein concentration was determined at 280 nm (5). The activity of enzym...
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