Mitochondrial carrier homolog 2 is a target of tBID in cells signaled to die by tumor necrosis factor alpha

BID, a proapoptotic BCL-2 family member, plays an essential role in the tumor necrosis factor alpha (TNF-alpha)/Fas death receptor pathway in vivo. Activation of the TNF-R1 receptor results in the cleavage of BID into truncated BID (tBID), which translocates to the mitochondria and induces the activation of BAX or BAK. In TNF-alpha-activated FL5.12 cells, tBID becomes part of a 45-kDa cross-linkable mitochondrial complex. Here we describe the biochemical purification of this complex and the identification of mitochondrial carrier homolog 2 (Mtch2) as part of this complex. Mtch2 is a conserved protein that is similar to members of the mitochondrial carrier protein family. Our studies with mouse liver mitochondria indicate that Mtch2 is an integral membrane protein exposed on the surface of mitochondria. Using blue-native gel electrophoresis we revealed that in viable FL5.12 cells Mtch2 resides in a protein complex of ca. 185 kDa and that the addition of TNF-alpha to these cells leads to the recruitment of tBID and BAX to this complex. Importantly, this recruitment was partially inhibited in FL5.12 cells stably expressing BCL-X(L). These results implicate Mtch2 as a mitochondrial target of tBID and raise the possibility that the Mtch2-resident complex participates in the mitochondrial apoptotic program.     

Utilization of amylose-lipid complexes as molecular nanocapsules for conjugated linoleic Acid

Amylose-conjugated linoleic acid (CLA) complexes were produced by water/dimethyl sulfoxide (DMSO) and KOH/HCl complexation methods. The formation of amylose V form was confirmed by X-ray diffraction (XRD), and complexes formed at 30, 60, and 90 degrees C exhibit melting temperatures exceeding 88 degrees C. Atomic force microscopy (AFM) images showed distinct difference in complex organization, with complexes formed in water/DMSO showing spherical shape with typical diameter of 150 nm. Complexes formed by KOH/HCl showed elongated structure with typical width of 43-160 nm. Water/DMSO complexes exhibit superior protection to CLA against oxidation. All complexes showed high retention of CLA in simulated stomach conditions, and the digestion of complexes by amylases results in high hydrolysis and CLA release by pancreatin and alpha-amylase. Only moderate release was detected following hydrolysis by amyloglucosidase and beta-amylase. It is therefore suggested that amylose-CLA complexes can serve as molecular nanocapsules for protection and delivery of CLA.     

Proapoptotic BID is an ATM effector in the DNA-damage response

The "BH3-only" proapoptotic BCL-2 family members are sentinels of intracellular damage. Here, we demonstrated that the BH3-only BID protein partially localizes to the nucleus in healthy cells, is important for apoptosis induced by DNA damage, and is phosphorylated following induction of double-strand breaks in DNA. We also found that BID phosphorylation is mediated by the ATM kinase and occurs in mouse BID on two ATM consensus sites. Interestingly, BID-/- cells failed to accumulate in the S phase of the cell cycle following treatment with the topoisomerase II poison etoposide; reintroducing wild-type BID restored accumulation. In contrast, introducing a nonphosphorylatable BID mutant did not restore accumulation in the S phase and resulted in an increase in cellular sensitivity to etoposide-induced apoptosis. These results implicate BID as an ATM effector and raise the possibility that proapoptotic BID may also play a prosurvival role important for S phase arrest.     

Inhibition of glycogen synthase kinase-3beta by bivalent zinc ions: insight into the insulin-mimetic action of zinc

Zinc is an important trace element found in most body tissues as bivalent cations and has essential roles in human health. The insulin-like effect of zinc cations raises the possibility that they inhibit glycogen synthase kinase-3beta (GSK-3beta), a serine/threonine protein kinase linked with insulin resistance and type 2 diabetes. Here we show that physiological concentrations of zinc ions directly inhibit GSK-3beta in vitro in an uncompetitive manner. Treatment of HEK-293 cells with zinc enhanced glycogen synthase activity and increased the intracellular levels of beta-catenin, providing evidence for inhibition of endogenous GSK-3beta by zinc. Moreover, zinc ions enhanced glucose uptake 3-fold in isolated mouse adipocytes, an increase similar to activation with saturated concentrations of insulin. We propose that the in vivo insulin-mimetic actions of zinc are mediated via direct inhibition of endogenous GSK-3beta.     

Enhanced Oxidative Stress and Altered Antioxidants in Brains of Bcl-2-Deficient Mice

Abstract: Bcl-2 is an antiapoptotic protein located in the outer mitochondrial membrane. Cellular perturbations associated with programmed cell death may be the consequence of disrupted mitochondrial function as well as excessive production of reactive oxygen species (ROS). Numerous studies indicate that Bcl-2 is involved in opposing cell death induced by oxidative stimuli, but its mode of action is uncertain. We reexamined the role of Bcl-2 by using a loss-of-function model, Bcl-2 knockout mice. Brains from Bcl-2 -deficient mice had a 43% higher content of oxidized proteins and 27% lower number of cells in the cerebellum relative to wild-type mice. Incubation of cerebellar neurons from Bcl-2 +/+ brains with 0.5 m M dopamine caused 25% cell death, whereas in Bcl-2 -deficient cells, it resulted in 52% death; glial cells provided protection in both cultures. Splenocytes from Bcl-2 -deficient mice were also killed more effectively by dopamine as well as paraquat. Bcl-2 -deficient mice did not survive intraperitoneal injection of MPTP, which caused a decrease in dopamine level in the striatum of Bcl-2 +/− brains, which was more significant than in wild-type mice. When compared with Bcl-2 +/+ brains, brains of 8-day-old Bcl-2 -deficient mice had higher activities of the antioxidant enzymes GSH reductase (192%) and GSH transferase (142%), whereas at the age of 30 days, GSH peroxidase was significantly lower (66%). Activities of GSH transferase and GSH reductase increased significantly (158 and 262%, respectively) from day 8 to day 30 in Bcl-2 +/+ mice, whereas GSH peroxidase decreased (31%) significantly in Bcl-2 −/− animals. In summary, our results demonstrated enhanced oxidative stress and susceptibility to oxidants as well as altered levels of antioxidant enzymes in brains of Bcl-2 -deficient mice. It is concluded that Bcl-2 affects cellular levels of ROS, which may be due to an effect either on their production or on antioxidant pathways.     

Regulation of Photosystem II core protein phosphorylation at the substrate level: Light induces exposure of the CP43 chlorophyll a protein complex to thylakoid protein kinase(s)

Light induces conformational changes in the CP43 chl-a-protein antenna complex in isolated PS II core-complexes exposing phosphorylation site(s) to PS II core-associated protein kinase(s), to added solubilized thylakoid protein kinase(s), as well as to tryptic cleavage. The substrate-activation effect is demonstrated by exposure of the PS II cores to light during the kinase assay as well as by preillumination of the PS II cores in which the endogenous kinase(s) has been inactivated by treatment with N-ethylmaleimid. In the latter case, phosphorylation was performed in darkness following addition of the solubilized protein kinase(s). The solubilized protein kinase(s) does not require light activation. The apparent molecular masses of the main protein kinase(s) associated with the PS II cores (about 31–35 kDa and 45 kDa) differ from that of the major protein kinase present in solubilized preparations obtained from spinach thylakoids (64 kDa). The light-induced exposure of CP43 increases with the light intensity in the range of 20–100 μmol photons m⁻² s⁻¹ as demonstrated by preillumination of N-ethylmaleimid treated cores followed by addition of the solubilized protein kinase(s) and performing the phosphorylation assay in darkness. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fragile-X Carrier Screening and the Prevalence of Premutation and Full-Mutation Carriers in Israel

Fragile-X syndrome is caused by an unstable CGG trinucleotide repeat in the FMR1 gene at Xq27. Intermediate alleles (51-200 repeats) can undergo expansion to the full mutation on transmission from mother to offspring. To evaluate the effectiveness of a fragile-X carrier-screening program, we tested 14,334 Israeli women of child-bearing age for fragile-X carrier status between 1992 and 2000. These women were either preconceptional or pregnant and had no family history of mental retardation. All those found to be carriers of premutation or full-mutation alleles were offered genetic counseling and also prenatal diagnosis, if applicable. We identified 207 carriers of an allele with >50 repeats, representing a prevalence of 1:69. There were 127 carriers with >54 repeats, representing a prevalence of 1:113. Three asymptomatic women carried the fully mutated allele. Among the premutation and full-mutation carriers, 177 prenatal diagnoses were performed. Expansion occurred in 30 fetuses, 5 of which had an expansion to the full mutation. On the basis of these results, the expected number of avoided patients born to women identified as carriers, the cost of the test in this study (U.S. $100), and the cost of lifetime care for a mentally retarded person (>$350,000), screening was calculated to be cost-effective. Because of the high prevalence of fragile-X premutation or full-mutation alleles, even in the general population, and because of the cost-effectiveness of the program, we recommend that screening to identify female carriers should be carried out on a wide scale.     

Hemispheric asymmetry of rapid chloride responses to inositol trisphosphate and calcium in Xenopus oocytes

Shallow injection of inositol 1,4,5-trisphosphate (IP₃) near the animal pole of the Xenopus oocyte resulted in a large depolarizing current that decayed rapidly. A similar injection near the vegetal pole produced a much smaller response characterized by a significantly slower rate of decay. Injection of CaCl₂ near the animal pole of the oocyte resulted in a large depolarizing current characterized by rapid rise and decay times. Injection near the vegetal pole of the cell produced responses that exhibited similar amplitudes but much longer rise and decay times. The protein kinase C (PK-C) activator, β-phorbol 12-myristate 13-acetate (PMA), significantly enhanced the rapid responses to IP₃ injections at either hemisphere but did not affect the amplitudes of the responses to CaCl₂. The PK-C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) had no effect on the responses to CaCl₂. These results imply an asymmetric distribution of calcium stores and chloride channels between the two hemispheres of the oocyte.