Regulation of interferon and retinoic acid-induced cell death activation through thioredoxin reductase

Interferons (IFNs) and retinoids are potent biological response modifiers. The IFN-beta and all-trans-retinoic acid combination, but not these single agents individually, induces death in several tumor cell lines. To elucidate the molecular basis for these actions, we have employed an antisense knockout approach to identify the gene products that mediate cell death and isolated several genes associated with retinoid-IFN-induced mortality (GRIMs). One of the GRIM cDNAs, GRIM-12, was identical to human thioredoxin reductase (TR). To define the functional relevance of TR to cell death and to define its mechanism of death-modulating functions, we generated mutants of TR and studied their influence on the IFN/RA-induced death regulatory functions of caspases. Wild-type TR activates cell death that was inhibited in the presence of caspase inhibitors or catalytically inactive caspases. A mutant TR, lacking the active site cysteines, inhibits the cell death induced by caspase 8. IFN/all-trans-retinoic acid-induced cytochrome c release from the mitochondrion was promoted in the presence of wild type and was inhibited in the presence of mutant TR. We find that TR modulates the activity of caspase 8 to promote death. This effect is in part caused by the stimulation of death receptor gene expression. These studies identify a new mechanism of cell death regulation by the IFN/all-trans-retinoic acid combination involving redox enzymes.     

Crystal structure of paprika ferredoxin-NADP+ reductase. Implications for the electron transfer pathway

cDNA of Capsicum annuum Yolo Wonder (paprika) has been prepared from total cellular RNA, and the complete gene encoding paprika ferredoxin-NADP(+) reductase (pFNR) precursor was sequenced and cloned from this cDNA. Fusion to a T7 promoter allowed expression in Escherichia coli. Both native and recombinant pFNR were purified to homogeneity and crystallized. The crystal structure of pFNR has been solved by Patterson search techniques using the structure of spinach ferredoxin-NADP(+) reductase as search model. The structure was refined at 2.5-A resolution to a crystallographic R-factor of 19.8% (R(free) = 26.5%). The overall structure of pFNR is similar to other members of the ferredoxin-NADP(+) reductase family, the major differences concern a long loop (residues 167-177) that forms part of the FAD binding site and some of the variable loops in surface regions. The different orientation of the FAD binding loop leads to a tighter interaction between pFNR and the adenine moiety of FAD. The physiological redox partners [2Fe-2S]-ferredoxin I and NADP(+) were modeled into the native structure of pFNR. The complexes reveal a protein-protein interaction site that is consistent with existing biochemical data and imply possible orientations for the side chain of tyrosine 362, which has to be displaced by the nicotinamide moiety of NADP(+) upon binding. A reasonable electron transfer pathway could be deduced from the modeled structures of the complexes.     

Role of the deafness dystonia peptide 1 (DDP1) in import of human Tim23 into the inner membrane of mitochondria

Tim8 and Tim13 of yeast belong to a family of evolutionary conserved zinc finger proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. Mutations in DDP1 (deafness dystonia peptide 1), the human homolog of Tim8, are associated with the Mohr-Tranebjaerg syndrome, a progressive neurodegenerative disorder. We show that DDP1 acts with human Tim13 in a complex in the intermembrane space. The DDP1.hTim13 complex is in direct contact with translocation intermediates of human Tim23 in mammalian mitochondria. The human DDP1.hTim13 complex complements the function of the TIM8.13 complex in yeast and facilitates import of yeast and human Tim23. Thus, the pathomechanism underlying the Mohr-Tranebjaerg syndrome may involve an impaired biogenesis of the human TIM23 complex causing severe pleiotropic mitochondrial dysfunction.     

Microhabitats, thermal heterogeneity, and patterns of physiological stress in the rocky intertidal zone.

Thermal stress has been considered to be among the most important determinants of organismal distribution in the rocky intertidal zone. Yet our understanding of how body temperatures experienced under field conditions vary in space and time, and of how these temperatures translate into physiological performance, is still rudimentary. We continuously monitored temperatures at a site in central California for a period of two years, using loggers designed to mimic the thermal characteristics of mussels, Mytilus californianus. Model mussel temperatures were recorded on both a horizontal and a vertical, north-facing microsite, and in an adjacent tidepool. We periodically measured levels of heat shock proteins (Hsp70), a measure of thermal stress, from mussels at each microsite. Mussel temperatures were consistently higher on the horizontal surface than on the vertical surface, and differences in body temperature between these sites were reflected in the amount of Hsp70. Seasonal peaks in extreme high temperatures ("acute" high temperatures) did not always coincide with peaks in average daily maxima ("chronic" high temperatures), suggesting that the time history of body temperature may be an important factor in determining levels of thermal stress. Temporal patterns in body temperature during low tide were decoupled from patterns in water temperature, suggesting that water temperature is an ineffective metric of thermal stress for intertidal organisms. This study demonstrates that spatial and temporal variability in thermal stress can be highly complex, and "snapshot" sampling of temperature and biochemical indices may not always be a reliable method for defining thermal stress at a site.     

Protein kinase C effectors bind to multidrug ABC transporters and inhibit their activity

P-Glycoprotein and homologous multidrug transporters contain a phosphorylatable linker sequence that was proposed to control drug efflux on the basis that it was indeed phosphorylated in vitro and in vivo, and that inhibitors of protein kinase C (PKC) inhibited both P-glycoprotein phosphorylation and activity. However, site-directed mutagenesis of all phosphorylatable residues did not alter the drug resistance. The present work shows that PKC effectors are able to bind directly to multidrug transporters, from either cancer cells (mouse P-glycoprotein), yeast (Saccharomyces cerevisiae Pdr5p), or protozoan parasite (Leishmania tropica ltmdr1), and to inhibit their energy-dependent drug-efflux activity. The binding of staurosporine and derivatives such as CGP 41251 is prevented by preincubation with ATP, suggesting at least partial interaction at the ATP-binding site. In contrast, more hydrophobic compounds such as calphostin C and CGP 42700 bind outside the ATP-binding site and strongly interfere with drug interaction. A direct correlation is obtained between the efficiencies of PKC effectors to inhibit energy-dependent interaction of rhodamine 6G with yeast Pdr5p, to promote intracellular drug accumulation in various multidrug resistant cells, and to chemosensitize growth of resistant cells. The noncompetitive inhibition by PKC effectors of rhodamine 6G interaction with Pdr5p suggests that the binding might interfere with signal transduction between nucleotide hydrolysis and drug interaction. The overall results indicate that the multidrug transporters from different species display common features for interaction with PKC inhibitors. The hydrophobic derivative of staurosporine, CGP 42700, constitutes a potentially powerful modulator of P-glycoprotein-mediated multidrug resistance.     

The effect of bile salts and calcium on isolated rat liver mitochondria

Intact mitochondria were incubated with and without calcium in solutions of chenodeoxycholate, ursodeoxycholate, or their conjugates. Glutamate dehydrogenase, protein and phospholipid release were measured. Alterations in membrane and organelle structure were investigated by electron paramagnetic resonance spectroscopy. Chenodeoxycholate enhanced enzyme liberation, solubilized protein and phospholipid, and increased protein spin label mobility and the polarity of the hydrophobic membrane interior, whereas ursodeoxycholate and its conjugates did not damage mitochondria. Preincubation with ursodeoxycholate or its conjugate tauroursodeoxycholate for 20 min partially prevented damage by chenodeoxycholate. Extended preincubation even with 1 mM ursodeoxycholate could no longer prevent structural damage. Calcium (from 0.01 mM upward) augmented the damaging effect of chenodeoxycholate (0.15-0.5 mM). The combined action of 0.01 mM calcium and 0.15 mM chenodeoxycholate was reversed by ursodeoxycholate only, not by its conjugates tauroursodeoxycholate and glycoursodeoxycholate. In conclusion, ursodeoxycholate partially prevents chenodeoxycholate-induced glutamate dehydrogenase release from liver cell mitochondria by membrane stabilization. This holds for shorter times and at concentrations below 0.5 mM only, indicating that the different constitution of protein-rich mitochondrial membranes does not allow optimal stabilization such as has been seen in phospholipid- and cholesterol-rich hepatocyte cell membranes, investigated previously.     

Protection of baculovirus-vectors against complement-mediated inactivation by recombinant soluble complement receptor type 1

Baculovirus-based vectors are efficient means for gene transfer into hepatocytes in vitro. However, gene transfer in vivo is hampered by inactivation of baculovirus by the complement system. In this study, we demonstrate protection of baculovirus vectors against complement-mediated inactivation through recombinant soluble complement receptor type 1 (sCR1). Blocking of only the alternative complement pathway by a mutant of sCR1 did not result in baculovirus survival in human serum. The data suggest the use of sCR1 as a potent drug to facilitate baculovirus-mediated gene transfer into hepatocytes in vivo.     

The mitochondrial TIM22 preprotein translocase is highly conserved throughout the eukaryotic kingdom

The Mohr-Tranebjaerg syndrome (MTS), a neurodegenerative syndrome characterized by progressive sensorineural hearing loss, dystonia, mental retardation and blindness, is a mitochondrial disease caused by mutations in the deafness/dystonia peptide 1 (DDP1) gene. DDP1 shows similarity to the yeast proteins Tim9, Tim10 and Tim12, components of the mitochondrial import machinery for carrier proteins. Here, we show that DDP1 belongs to a large family of evolutionarily conserved proteins. We report the identification, chromosomal localization and expressional analysis of six human family members which represent further candidate genes for neurodegenerative diseases.     

The MYC dualism in growth and death

Investigations of intraindividual sequence diversity in mtDNA are a key step in exploring the linkage between somatic mutations in mtDNA and mitochondrial genome evolution. This paper reports a directional cloning procedure enabling the isolation of multiple copies of the D-loop region of the mitochondrial genome from the fish Ameiurus nebulosus. Sequence analysis of 708 D-loop molecules revealed eight mutants, an average intraindividual mutation frequency of 1.12%. Three different types of mutations were detected but each derived from a single mutational event. By contrasting the spectrum of nucleotide variation at multiple biological levels, one can investigate the effects of spontaneous mutations on genome evolution. Such hierarchical analysis suggested shifts in the type and distribution of mtDNA (mitochondrial DNA) mutations at different biological levels, indicating the need to recognize three different rates of mtDNA sequence change from the cellular to population level.