Intracellular GSH levels rather than ROS levels are tightly related to AMA-induced HeLa cell death

Antimycin A (AMA) inhibits succinate oxidase and NADH oxidase, and also inhibits mitochondrial electron transport between cytochromes b and c. We investigated the involvement of ROS and GSH in AMA-induced HeLa cell death. AMA increased the intracellular H(2)O(2) and O(2)(*-) levels and reduced the intracellular GSH content. ROS scavengers (Tempol, Tiron, Trimetazidine and NAC) did not down-regulate the production of ROS and inhibit apoptosis in AMA-treated cells. Treatment with NAC and N-propylgallate showing the enhancement of GSH depletion in AMA-treated cells significantly intensified the levels of apoptosis. Calpain inhibitors I and II (calpain inhibitor III) and Ca(2+)-chelating agent (EGTA/AM) significantly reduced H(2)O(2) levels in AMA-treated HeLa cells. However, treatment with calpain inhibitor III intensified the levels of O(2)(*-) in AMA-treated cells. In addition, calpain inhibitor III strongly depleted GSH content with an enhancement of apoptosis in AMA-treated cells. Conclusively, the changes of ROS by AMA were not tightly correlated with apoptosis in HeLa cells. However, intracellular GSH levels are tightly related to AMA-induced cell death.     

Autophagy modulates keratin-containing inclusion formation and apoptosis in cell culture in a context-dependent fashion

The major pathways for protein degradation are the proteasomal and lysosomal systems. Derangement of protein degradation causes the formation of intracellular inclusions, and apoptosis and is associated with several diseases. We utilized hepatocyte-derived cell lines to examine the consequences of the cytoplasmic hepatocyte Mallory-Denk body-like inclusions on organelle organization, autophagy and apoptosis, and tested the hypothesis that autophagy affects inclusion turnover. Proteasome inhibitors (PIs) generate keratin-containing Mallory-Denk body-like inclusions in cultured cells and cause reorganization of mitochondria and other organelles, autophagy and apoptosis. In cultured hepatoma cells, caspase inhibition blocks PI-induced apoptosis but not inclusion formation or autophagy activation. Autophagy induction by rapamycin decreases the extent of PI-induced inclusions and apoptosis in Huh7 and OUMS29 cells. Surprisingly, blocking of autophagy sequestration by 3 methyl adenine or beclin 1 siRNA, but not bafilomycin A1 inhibition of autophagic degradation, also inhibits inclusion formation in the tested cells. Therefore, autophagy can be upstream of apoptosis and may promote or alleviate inclusion formation in cell culture in a context-dependent manner via putative autophagy-associated molecular triggers. Manipulation of autophagy may offer a strategy to address the importance of inclusion formation and its significance in inclusion-associated diseases.     

The importance of conserved amino acid residues in p94 protease sub-domain IIb and the IS2 region for constitutive autolysis

p94/calpain 3, a skeletal muscle-specific member of calpain protease family, is characterized by apparent Ca(2+)-independence during exhaustive autolysis and concomitant proteolysis of non-self substrates. The purpose of our study was to comprehensively profile the structural basis of p94 enabling activation in the cytosol without an extra Ca(2+). Ca(2+)-dependent p94 mutants were screened using "p94-trapping", which is an application of yeast genetic reporter system called "proteinase-trapping". Several amino acids were revealed as critical for apparent Ca(2+)-independent p94 activity. These results highlight the importance of conserved amino acids in domain IIb as well as in the p94-specific IS2 region.     

The [4Fe-4S] cluster of quinolinate synthase from Escherichia coli: investigation of cluster ligands

Nicotinamide adenine dinucleotide (NAD) derives from quinolinic acid which is synthesized in Escherichia coli from l-aspartate and dihydroxyacetone phosphate through the concerted action of l-aspartate oxidase and the [4Fe-4S] quinolinate synthase (NadA). Here, we addressed the question of the identity of the cluster ligands. We performed in vivo complementation experiments as well as enzymatic, spectroscopic and structural in vitro studies using wild-type vs. Cys-to-Ala mutated NadA proteins. These studies reveal that only three cysteine residues, the conserved Cys113, Cys200 and Cys297, are ligands of the cluster. This result is in contrast to the previous proposal that pointed the three cysteines of the C(291)XXC(294)XXC(297) motif. Interestingly, we demonstrated that Cys291 and Cys294 form a disulfide bridge and are important for activity.     

Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane during mitochondrial reactive oxygen stress in macrophages

Uncoupling protein-2 (UCP2) is a member of the inner mitochondrial membrane anion-carrier superfamily. Although mRNA for UCP2 is widely expressed, protein expression is detected in only a few cell types, including macrophages. UCP2 functions by an incompletely defined mechanism, to reduce reactive oxygen species production during mitochondrial electron transport. We observed that the abundance of UCP2 in macrophages increased rapidly in response to treatments (rotenone, antimycin A and diethyldithiocarbamate) that increased mitochondrial superoxide production, but not in response to superoxide produced outside the mitochondria or in response to H₂O₂. Increased UCP2 protein was not accompanied by increases in ucp2 gene expression or mRNA abundance, but was due to enhanced translational efficiency and possibly stabilization of UCP2 protein in the inner mitochondrial membrane. This was not dependent on mitochondrial membrane potential. These findings extend our understanding of the homeostatic function of UCP2 in regulating mitochondrial reactive oxygen production by identifying a feedback loop that senses mitochondrial reactive oxygen production and increases inner mitochondrial membrane UCP2 abundance and activity. Reactive oxygen species-induction of UCP2 may facilitate survival of macrophages and retention of function in widely variable tissue environments.     

Dynamic regulation of uncoupling protein 2 content in INS-1E insulinoma cells

Uncoupling protein 2 (UCP2) regulates glucose-stimulated insulin secretion in pancreatic beta-cells. UCP2 content, measured by calibrated immunoblot in INS-1E insulinoma cells (a pancreatic beta-cell model) grown in RPMI medium, and INS-1E mitochondria, was 2.0 ng/million cells (7.9 ng/mg mitochondrial protein). UCP2 content was lower in cells incubated without glutamine and higher in cells incubated with 20 mM glucose, and varied from 1.0-4.4 ng/million cells (2.7-14.5 ng/mg mitochondrial protein). This dynamic response to nutrients was achieved by varied expression rates against a background of a very short UCP2 protein half-life of about 1 h.     

Glycyrrhetinic acid as inhibitor or amplifier of permeability transition in rat heart mitochondria

Glycyrrhetinic acid (GE), a hydrolysis product of glycyrrhizic acid, one of the main constituents of licorice root, is able, depending on its concentration, to prevent or to induce the mitochondrial permeability transition (MPT) (a phenomenon related to oxidative stress) in rat heart mitochondria (RHM). In RHM, below a threshold concentration of 7.5 μM, GE prevents oxidative stress and MPT induced by supraphysiological Ca²⁺ concentrations. Above this concentration, GE induces oxidative stress by interacting with a Fe-S centre of Complex I, thus producing ROS, and amplifies the opening of the transition pore, once again induced by Ca²⁺. GE also inhibits Ca²⁺ transport in RHM, thereby preventing the oxidative stress induced by the cation. However, the reduced amount of Ca²⁺ transported in the matrix is sufficient to predispose adenine nucleotide translocase for pore opening. Comparisons between observed results and the effects of GE in rat liver mitochondria (RLM), in which the drug induces only MPT without exhibiting any protective effect, confirm that it interacts in a different way with RHM, suggesting tissue specificity for its action. The concentration dependence of the opposite effects of GE, in RHM but not RLM, is most probably due to the existence of a different, more complex, pathway by means of which GE reaches its target. It follows that high GE concentrations are necessary to stimulate the oxidative stress capable of inducing MPT, because of the above effect, which prevents the interaction of low concentrations of GE with the Fe-S centre. The reported results also explain the mechanism of apoptosis induction by GE in cardiomyocytes.     

Discovery of isoquinolinone and naphthyridinone-based inhibitors of poly(ADP-ribose) polymerase-1 (PARP1) as anticancer agents: Structure activity relationship and preclinical characterization

The exploitation of GLU988 and LYS903 residues in PARP1 as targets to design isoquinolinone (I & II) and naphthyridinone (III) analogues is described. Compounds of structure I have good biochemical and cellular potency but suffered from inferior PK. Constraining the linear propylene linker of structure I into a cyclopentene ring (II) offered improved PK parameters, while maintaining potency for PARP1. Finally, to avoid potential issues that may arise from the presence of an anilinic moiety, the nitrogen substituent on the isoquinolinone ring was incorporated as part of the bicyclic ring. This afforded a naphthyridinone scaffold, as shown in structure III. Further optimization of naphthyridinone series led to identification of a novel and highly potent PARP1 inhibitor 34, which was further characterized as preclinical candidate molecule. Compound 34 is orally bioavailable and displayed favorable pharmacokinetic (PK) properties. Compound 34 demonstrated remarkable antitumor efficacy both as a single-agent as well as in combination with chemotherapeutic agents in the BRCA1 mutant MDA-MB-436 breast cancer xenograft model. Additionally, compound 34 also potentiated the effect of agents such as temozolomide in breast cancer, pancreatic cancer and Ewing’s sarcoma models.     

The isolation and characterization of 3-(2-carboxyethyl)cytosine following in vitro reaction of β-propiolactone with calf thymus DNA

The new adduct 3-(2-carboxyethyl)cytosine (3-CEC) was isolated following in vitro reaction of the carcinogen β-propiolactone (BPL) with calf thymus DNA. The structure of 3-CEC was confirmed by synthesis from BPL and dCyd. Reaction of BPL with cCyd (pH 7.0–7.5, 37°C) gave 3-(2-carboxyethyl)deoxycytidine (3-CEdCyd) (9% yield) and 3,N⁴-bis(2-carboxyethyl)deoxycytidine (3,N⁴-BCEdCyd) (0.6% yield). 3-CEdCyd and 3,N⁴-BCEdCyd were hydrolyzed (1.5 N HC1, 100°C, 2 h) to 3-CEC and 3,N⁴-bis(2-carboxyethyl)cytosine (3,N⁴-BCEC), respectively. The structure of 3-CEC was assigned on the basis of UV and NMR spectra and the electron impact (EI) mass spectra of 3-CEC and a tri-trimethylsilyl (TMS) derivative of 3 CEC as well as deuterated (d₂₇) tri-TMS derivative of 3-CEC. The structure of 3,N⁴-BCEC was assigned on the basis of UV spectra and the EI mass spectra of a tri-TMS derivative. EI and isobutane chemical ionization mass spectra of 3-methylcytosine (3-MeCyt) and a di-TMS derivative of 3-MeCyt were obtained and were helpful in deducing the structures of 3-CEC and 3,N⁴-BCEC. This is the first report of the alkylation by BPL of an exocyclic atom on a base in DNA. Compound 3,N⁴-BCEC was not detected in BPL-reacted calf thymus DNA. The relative amounts of 1-(2-carboxyethyl)adenine (1-CEA), 7-(2-carboxyethyl)guanine (7-CEG), 3-(2-carboxyethyl)thymine (3-CET) and 3-CEC isolated from BPL-reacted DNA following perchloric acid hydrolysis were 0.23, 1.00, 0.39 and 0.41 respectively, when the alkylation reaction was conducted in phosphate buffer at 0–5°C and pH 7.5 and 0.10, 1.00, 0.29 and 0.28 respectively when the reaction was conducted in H₂O at 37°C and pH 7.0–7.5.