Flow cytometric analysis of mitochondria from CA1 and CA3 regions of rat hippocampus reveals differences in permeability transition pore activation

Mitochondria are important in the pathophysiology of several neurodegenerative diseases, and mitochondrial production of reactive oxygen species (ROS), membrane depolarization, permeability changes and release of apoptogenic proteins are involved in these processes. Following brain insults, cell death often occurs in discrete regions of the brain, such as the subregions of the hippocampus. To analyse mitochondrial structure and function in such subregions, only small amounts of mitochondria are available. We developed a protocol for flow cytometric analysis of very small samples of isolated brain mitochondria, and analysed mitochondrial swelling and formation of ROS in mitochondria from the CA1 and CA3 regions of the hippocampus. Calcium-induced mitochondrial swelling was measured, and fluorescent probes were used to selectively stain mitochondria (nonyl acridine orange), to measure membrane potential (tetramethylrhodamine-methyl-ester, 1,1',3,3,3',3'-hexamethylindodicarbocyanine-iodide) and to measure production of ROS (2',7'-dichlorodihydrofluorescein-diacetate). We found that formation of ROS and mitochondrial permeability transition pore activation were higher in mitochondria from the CA1 than from the CA3 region, and propose that differences in mitochondrial properties partly underlie the selective vulnerability of the CA1 region to brain insults. We also conclude that flow cytometry is a useful tool to analyse the role of mitochondria in cell death processes.     

Topology of the membrane-associated hepatitis C virus protein NS4B

Hepatitis C virus (HCV) belongs to the Hepacivirus genus in the Flaviviridae family. Among the least known viral proteins in this family is the nonstructural protein NS4B, which has been suggested to be a part of the replication complex. Hydrophobicity plots indicate a common profile among the NS4B proteins from different members of the Flaviviridae family, suggesting a common function. In order to gain a deeper understanding of the nature of HCV NS4B, we have determined localization and topology of this protein by using recombinant HCV NS4B constructs. The protein localized to the endoplasmic reticulum (ER), but also induced a pattern of cytoplasmic foci positive for markers of the ER. Computer predictions of the membrane topology of NS4B suggested that it has four transmembrane segments. The N and C termini were anticipated to be localized in the cytoplasm, because they are processed by the cytoplasmic NS3 protein. By introducing glycosylation sites at various positions in HCV NS4B, we show that the C terminus is cytoplasmic and the loop around residue 161 is lumenal as predicted. Surprisingly, the N-terminal tail was translocated into the lumen in a considerable fraction of the NS4B molecules, most likely by a posttranslational process. Interestingly, NS4B proteins of the yellow fever and dengue viruses also have their N termini located in the ER lumen due to an N-terminal signal peptide not found in NS4B of HCV. A shared topology achieved in two different ways supports the notion of a common function for NS4B in FLAVIVIRIDAE:     

The crystal structure of Leishmania major 3-mercaptopyruvate sulfurtransferase. A three-domain architecture with a serine protease-like triad at the active site

Leishmania major 3-mercaptopyruvate sulfurtransferase is a crescent-shaped molecule comprising three domains. The N-terminal and central domains are similar to the thiosulfate sulfurtransferase rhodanese and create the active site containing a persulfurated catalytic cysteine (Cys-253) and an inhibitory sulfite coordinated by Arg-74 and Arg-185. A serine protease-like triad, comprising Asp-61, His-75, and Ser-255, is near Cys-253 and represents a conserved feature that distinguishes 3-mercaptopyruvate sulfurtransferases from thiosulfate sulfurtransferases. During catalysis, Ser-255 may polarize the carbonyl group of 3-mercaptopyruvate to assist thiophilic attack, whereas Arg-74 and Arg-185 bind the carboxylate group. The enzyme hydrolyzes benzoyl-Arg-p-nitroanilide, an activity that is sensitive to the presence of the serine protease inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone, which also lowers 3-mercaptopyruvate sulfurtransferase activity, presumably by interference with the contribution of Ser-255. The L. major 3-mercaptopyruvate sulfurtransferase is unusual with an 80-amino acid C-terminal domain, bearing remarkable structural similarity to the FK506-binding protein class of peptidylprolyl cis/trans-isomerase. This domain may be involved in mediating protein folding and sulfurtransferase-protein interactions.     

Properties of the apo-form of the NADP(H)-binding domain III of proton-pumping Escherichia coli transhydrogenase: implications for the reaction mechanism of the intact enzyme

Proton-translocating nicotinamide nucleotide transhydrogenases contain an NAD(H)-binding domain (dI), an NADP(H)-binding domain (dIII) and a membrane domain (dII) with the proton channel. Separately expressed and isolated dIII contains tightly bound NADP(H), predominantly in the oxidized form, possibly representing a so-called "occluded" intermediary state of the reaction cycle of the intact enzyme. Despite a K(d) in the micromolar to nanomolar range, this NADP(H) exchanges significantly with the bulk medium. Dissociated NADP(+) is thus accessible to added enzymes, such as NADP-isocitrate dehydrogenase, and can be reduced to NADPH. In the present investigation, dissociated NADP(H) was digested with alkaline phosphatase, removing the 2'-phosphate and generating NAD(H). Surprisingly, in the presence of dI, the resulting NADP(H)-free dIII catalyzed a rapid reduction of 3-acetylpyridine-NAD(+) by NADH, indicating that 3-acetylpyridine-NAD(+) and/or NADH interacts unspecifically with the NADP(H)-binding site. The corresponding reaction in the intact enzyme is not associated with proton pumping. It is concluded that there is a 2'-phosphate-binding region in dIII that controls tight binding of NADP(H) to dIII, which is not a required for fast hydride transfer. It is likely that this region is the Lys424-Arg425-Ser426 sequence and loops D and E. Further, in the intact enzyme, it is proposed that the same region/loops may be involved in the regulation of NADP(H) binding by an electrochemical proton gradent.     

Interaction of scavenger receptor class B type I with peroxisomal targeting receptor Pex5p

Scavenger receptor class B type I (SR-BI) is an HDL receptor that mediates selective HDL lipid uptake. Peroxisomes play an important role in lipid metabolism and peroxisomal targeting signal type 1 (PTS1)-containing proteins are translocated to peroxisomes by the peroxisomal targeting import receptor, Pex5p. We have previously identified a PTS1 motif in the intracellular domain of rat SR-BI. Here, we examine the possible interaction between Pex5p and SR-BI. Expression of a Flag-tagged intracellular domain of SR-BI resulted in translocation to the peroxisome as demonstrated by double labeling with anti-Flag IgG and anti-catalase IgG analyzed by confocal microscopy. Immunoprecipitation experiments with anti-SR-BI antibody showed that Pex5p co-precipitated with SR-BI. However, when an antibody against Pex5p was used for immunoprecipitation, only the 57kDa, non-glycosylated form, of SR-BI co-precipitated. We conclude that the PTS1 domain of SR-BI is functional and can mediate peroxisomal interaction via Pex5p, in vitro.     

Roles of individual amino acids in helix 14 of the membrane domain of proton-translocating transhydrogenase from Escherichia coli as deduced from cysteine mutagenesis

Proton-translocating nicotinamide nucleotide transhydrogenase is a membrane-bound protein composed of three domains: the hydrophilic NAD(H)-binding domain, the hydrophilic NADP(H)-binding domain, and the hydrophobic membrane domain. The latter harbors the proton channel. In Escherichia coli transhydrogenase, the membrane domain is composed of 13 transmembrane alpha helices, of which especially helices 13 and 14 contain conserved residues. To characterize the roles of the individual residues betaLeu240 to betaSer260 in helix 14, these were mutated as single mutants to cysteines in the cysteine-free background, and in the case of betaGly245, betaGly249, and betaGly252, also to leucines. In addition to the residues forming the helix, residues betaAsn238 and betaAsp239 were also mutated. Except for betaI242C, all mutants were normally expressed, purified, and characterized with respect to, e.g., catalytic activities and proton pumping. The results show that mutation of the conserved glycines betaGly245, betaGly249, and betaGly252, located on the same face of the helix, led to a general inhibition of all activities, especially in the case of betaGly252, suggesting a role of these glycines in helix-helix interactions. In contrast, mutation of the conserved serines betaSer250, betaSer251, and betaSer256 led to enhanced activities of all reactions, including the cyclic reaction which was mediated by bound NADP(H). Mutation of the remaining residues resulted in intermediate inhibitory effects. The results strongly support an important regulatory role of the membrane domain on the NADP(H)-binding site.     

Identification of a novel calreticulin isoform (Crt2) in human and mouse

The human REIC gene is a recently found mortalization-related gene and a candidate tumor suppressor gene expression of which is largely attenuated in many immortalized and tumor-derived cell lines (Biochem. Biophys. Res. Commun. 268 (2000) 20-24). To gain insight into the mechanisms of the down-regulation, we investigated the genomic structure and promoter activity of the human REIC gene. The gene, identical with the DKK-3 gene, resides on chromosome 11p15.1, consists of nine exons, and has two promoters. Methylation in the main promoter region was detected in 11 out of 21 cell lines tested (52%) derived from a variety of human tumors, in which the expression of the REIC gene was decreased. In ten of these 11 cell lines the minor promoter was also methylated. Similarly, the REIC gene expression was decreased in 14 of 24 fresh non-small cell lung cancer specimens (58%) compared to that in corresponding non-cancerous tissue, though allelic loss and tumor-specific mutation were rare. Of these 14 tumors, at least five tumors exhibited heavy methylation of the REIC promoter region. These results indicate that the down-regulation of the REIC gene expression is ascribed to the aberrant promoter hyper-methylation at least in a subset of human tumors. The expression was restored upon treatment of SQ5 cells with 5-aza-deoxycytidine, confirming DNA methylation as the mode of downregulation. A notable single nucleotide polymorphism in the coding region (cSNP) with an amino acid substitution of glycine (GGG) to arginine (AGG) was found at codon 335 of the REIC gene. However, the distribution of the cSNP showed no significant difference between lung cancer patients and healthy population.     

The interdomain region of dengue NS5 protein that binds to the viral helicase NS3 contains independently functional importin beta 1 and importin alpha/beta-recognized nuclear localization signals

Dengue virus NS5 protein is a multifunctional RNA-dependent RNA polymerase that is essential for virus replication. We have shown previously that the 37- amino acid interdomain spacer sequence (residues (369)X(2)KKX(14)KKKX(11)RKX(3)405) of Dengue2 NS5 contains a functional nuclear localization signal (NLS). In this study, beta-galactosidase fusion proteins carrying point mutations of the positively charged residues or truncations of the interdomain linker region (residues 369-389 or residues 386-405) were analyzed for nuclear import and importin binding activities to show that the N-terminal part of the linker region (residues 369-389, a/bNLS) is critical for nuclear localization and is recognized with high affinity by the conventional NLS-binding importin alpha/beta heterodimeric nuclear import receptor. We also show that the importin beta-binding site (residues 320-368, bNLS) adjacent to the a/bNLS, previously identified by yeast two-hybrid analysis, is functional as an NLS, recognized with high affinity by importin beta, and able to target beta-galactosidase to the nucleus. Intriguingly, the bNLS is highly conserved among Dengue and related flaviviruses, implying a general role for the region and importin beta in the infectious cycle.