Viability and mitochondrial bioenergetic functions in human colon cancer cells are not affected by treatment with peptides mtCPP1, UPF25, mtgCPP, and mtCPP1gHO

The popularity of the specially synthesized cell-penetrating peptides (CPPs) in cancer treatment has grown recently. The main aim of this study was to investigate the effects of four mitochondrially targeted antioxidant CPPs on the viability and bioenergetic function of mitochondria in human adenocarcinoma Caco-2 cells. The number of viable cells was measured by MTT and trypan blue assays. Respirometry and the permeabilized cell technique were applied to measure the mitochondrial function in this cell line. We did not observe any significant effect of CPPs on the mitochondrial reserve respiratory capacity, the function of respiratory chain complexes, and the inclination of these cancer cells to aerobic glycolysis. mtgCPP peptide with the highest antioxidant activity demonstrated improved mitochondrial coupling efficiency. CPPs do not affect mitochondrial function directly but can be considered in therapeutics as a drug-delivery molecule

Disruption of the Ran System by Cysteine Oxidation of the Nucleotide Exchange Factor RCC1

Transport regulation by the Ran GTPase requires its nuclear localization and GTP loading by the chromatin-associated exchange factor RCC1. These reactions generate Ran protein and Ran nucleotide gradients between the nucleus and the cytoplasm. Cellular stress disrupts the Ran gradients, but the specific mechanisms underlying this disruption have not been elucidated. We used biochemical approaches to determine how oxidative stress disrupts the Ran system. RCC1 exchange activity was reduced by diamide-induced oxidative stress and restored with dithiothreitol. Using mass spectrometry, we found that multiple solvent-exposed cysteines in RCC1 are oxidized in cells treated with diamide. The cysteines oxidized in RCC1 included Cys93, which is solvent exposed and unique because it becomes buried upon contact with Ran. A Cys93Ser substitution dramatically reduced exchange activity through an effect on RCC1 binding to RanGDP. Diamide treatment reduced the size of the mobile fraction of RCC1-green fluorescent protein in cells and inhibited nuclear import in digitonin-permeabilized cell assays. The Ran protein gradient was also disrupted by UV-induced stress but without affecting RCC1 exchange activity. Our data suggest that stress can disrupt the Ran gradients through RCC1-dependent and RCC1-independent mechanisms, possibly dependent on the particular stress condition.

Glucose Metabolism in Legionella pneumophila: Dependence on the Entner-Doudoroff Pathway and Connection with Intracellular Bacterial Growth

ABSTRACT Glucose metabolism in Legionella pneumophila was studied by focusing on the Entner-Doudoroff (ED) pathway with a combined genetic and biochemical approach. The bacterium utilized exogenous glucose for synthesis of acid-insoluble cell components but manifested no discernible increase in the growth rate. Assays with permeabilized cell preparations revealed the activities of three enzymes involved in the pathway, i.e., glucokinase, phosphogluconate dehydratase, and 2-dehydro-3-deoxy-phosphogluconate aldolase, presumed to be encoded by the glk, edd, and eda genes, respectively. Gene-disrupted mutants for the three genes and the ywtG gene encoding a putative sugar transporter were devoid of the ability to metabolize exogenous glucose, indicating that the pathway is almost exclusively responsible for glucose metabolism and that the ywtG gene product is the glucose transporter. It was also established that these four genes formed part of an operon in which the gene order was edd-glk-eda-ywtG, as predicted by genomic information. Intriguingly, while the mutants exhibited no appreciable change in growth characteristics in vitro, they were defective in multiplication within eukaryotic cells, strongly indicating that the ED pathway must be functional for the intracellular growth of the bacterium to occur. Curiously, while the deficient glucose metabolism of the ywtG mutant was successfully complemented by the ywtG + gene supplied in trans via plasmid, its defect in intracellular growth was not. However, the latter defect was also manifested in wild-type cells when a plasmid carrying the mutant ywtG gene was introduced. This phenomenon, resembling so-called dominant negativity, awaits further investigation.