Drug-dependent inhibition of nucleotide hydrolysis in the heterodimeric ABC multidrug transporter PatAB from Streptococcus pneumoniae.

The bacterial heterodimeric ATP-binding cassette (ABC) multidrug exporter PatAB has a critical role in conferring antibiotic resistance in multidrug-resistant infections by Streptococcus pneumoniae. As with other heterodimeric ABC exporters, PatAB contains two transmembrane domains that form a drug translocation pathway for efflux and two nucleotide-binding domains that bind ATP, one of which is hydrolysed during transport. The structural and functional elements in heterodimeric ABC multidrug exporters that determine interactions with drugs and couple drug binding to nucleotide hydrolysis are not fully understood. Here, we used mass spectrometry techniques to determine the subunit stoichiometry in PatAB in our lactococcal expression system and investigate locations of drug binding using the fluorescent drug-mimetic azido-ethidium. Surprisingly, our analyses of azido-ethidium-labelled PatAB peptides point to ethidium binding in the PatA nucleotide-binding domain, with the azido moiety crosslinked to residue Q521 in the H-like loop of the degenerate nucleotide-binding site. Investigation into this compound and residue's role in nucleotide hydrolysis pointed to a reduction in the activity for a Q521A mutant and ethidium-dependent inhibition in both mutant and wild type. Most transported drugs did not stimulate or inhibit nucleotide hydrolysis of PatAB in detergent solution or lipidic nanodiscs. However, further examples for ethidium-like inhibition were found with propidium, novobiocin and coumermycin A1, which all inhibit nucleotide hydrolysis by a non-competitive mechanism. These data cast light on potential mechanisms by which drugs can regulate nucleotide hydrolysis by PatAB, which might involve a novel drug binding site near the nucleotide-binding domains.

Study of the intracellular delivery mechanism of a pH-sensitive peptide modified with enhanced green fluorescent protein.

The targeted delivery of therapeutic agents is a promising approach to enhance the efficacy and reduce the toxicity of cancer treatments. Understanding the intracellular endocytic mechanisms of a cell penetrating peptide (CPP) in an acidic environment is important for targeted delivery of macromolecules to tumours. In this study, we constructed a pH-sensitive CPP-based delivery system for the intracellular delivery of macromolecules. A pH-sensitive CPP, HBHAc, was fused with a model protein, enhanced green fluorescent protein (EGFP), through recombinant DNA technology. We found that is essential that negatively charged proteoglycans on the cell surface interact with HBHAc-EGFP prior to the cellular uptake of HBHAc-EGFP. The uptake was significantly restricted at 4 °C under pH conditions of both 6.5 and 7.5. The increased positive charge of HBHAc-EGFP under the acidic condition leads to a pH-dependent cellular uptake, and we observed that the internalisation of HBHAc-EGFP was significantly higher at pH 6.5 than at pH 7.5 (p < .05). Thus, with pH-sensitive activity, HBHAc is expected to improve tumour-targeted intracellular protein delivery. Moreover, our findings provide a new insight that the endocytic pathway may change under different pH conditions and suggest that this unique phenomenon benefits pH-sensitive drug delivery for tumour therapy.

Expression of tumor suppressor p53 facilitates DNA repair but not UV-induced G2/M arrest or apoptosis in Chinese hamster ovary CHO-K1 cells.

Tumor suppressor p53 is an essential regulator in mammalian cellular responses to DNA damage including cell cycle arrest and apoptosis. Our study with Chinese hamster ovary CHO-K1 cells indicates that when p53 expression and its transactivation capacity was inhibited by siRNA, UVC-induced G2/M arrest or apoptosis were unaffected as revealed by flow cyotmetric analyses and other measurements. However, inhibition of p53 rendered the cells slower to repair UV-induced damages upon a plasmid as shown in host cell reactivation assay. Furthermore, the nuclear extract (NE) of p53 siRNA-treated cells was inactive to excise the UV-induced DNA adducts as analyzed by comet assay. Consistently, the immunodepletion of p53 also deprived the excision activity of the NE in the similar experiment. Thus, tumor suppressor p53 of CHO-K1 cells may facilitate removal of UV-induced DNA damages partly via its involvement in the repair mechanism.