No abstract

Diacylglycerol kinase (DAGK) is a 13-kDa integral membrane protein that spans the lipid bilayer three times and which is active in some micellar systems. In this work DAGK was purified using metal ion chelate chromatography, and its structural properties in micelles and organic solvent mixtures studies were examined, primarily to address the question of whether the structure of DAGK can be determined using solution NMR methods. Cross-linking studies established that DAGK is homotrimeric in decyl maltoside (DM) micelles and mixed micelles. The aggregate detergent-protein molecular mass of DAGK in both octyl glucoside and DM micelles was determined to be in the range of 100-110 kDa-much larger than the sum of the molecular weights of the DAGK trimers and the protein-free micelles. In acidic organic solvent mixtures, DAGK-DM complexes were highly soluble and yielded relatively well-resolved NMR spectra. NMR and circular dichroism studies indicated that in these mixtures the enzyme adopts a kinetically trapped monomeric structure in which it irreversibly binds several detergent molecules and is primarily alpha-helical, but in which its tertiary structure is largely disordered. Although these results provide new information regarding the native oligomeric state of DAGK and the structural properties of complex membrane proteins in micelles and organic solvent mixtures, the results discourage the notion that the structure of DAGK can be readily determined at high resolution with solution NMR methods.

Escherichia coli diacylglycerol kinase (DAGK) is a 13.2 kDa enzyme which spans the cytoplasmic membrane three times. Functional DAGK was purified to homogeneity using a polyhistidine tag and Ni(II)-chelate chromatography. Transmission Fourier transform infrared spectroscopy (FT-IR) of DAGK in phosphatidylcholine multilayers led to the conclusion that > or = 90 of DAGK's native 121 residues are alpha-helical, consistent with a model in which DAGK consists of two amphipathic alpha-helices and three transmembrane helices. Polarized attenuated total reflection FT-IR studies of DAGK in oriented multilamellae yielded data consistent with a topological arrangement in which the three transmembrane helices are well-aligned with the bilayer normal while the two amphipathic helices are approximately parallel with the membrane plane. The ability of DAGK to spontaneously insert into preformed lipid vesicles was examined using a novel assay system involving DAGK-catalyzed phosphorylation of a fluorescently tagged diacylglycerol. When micellar DAGK is diluted into L alpha-phase vesicles spontaneous insertion of the enzyme is fairly efficient (ca. 30%). DAGK refolding and insertion from delipidated urea-solubilized DAGK into lipid vesicles is also modestly efficient (3.8 +/- 2.1%) above the gel to liquid crystalline phase transition temperature. The insertion studies indicate that the difference in energy barriers (delta delta G++) between pathways leading to catalytically productive folding and insertion of DAGK relative to unproductive pathways is < 4 kcal/mol. However, additional studies carried out with mutant forms of DAGK indicated that the differences between refolding/insertion pathways for DAGK in vivo and in vitro can be significant.

Type 2 diabetes involves insulin resistance and β-cell failure leading to inadequate insulin secretion. An important component of β-cell failure is cell loss by apoptosis. Apoptosis repressor with caspase recruitment domain (ARC) is an inhibitor of apoptosis that is expressed in cardiac and skeletal myocytes and neurons. ARC possesses the unusual property of antagonizing both the extrinsic (death receptor) and intrinsic (mitochondria/endoplasmic reticulum [ER]) cell death pathways. Here we report that ARC protein is abundant in cells of the endocrine pancreas, including >99.5% of mouse and 73% of human β-cells. Using genetic gain- and loss-of-function approaches, our data demonstrate that ARC inhibits β-cell apoptosis elicited by multiple inducers of cell death, including ER stressors tunicamycin, thapsigargin, and physiological concentrations of palmitate. Unexpectedly, ARC diminishes the ER stress response, acting distal to protein kinase RNA-like ER kinase (PERK) and inositol-requiring protein 1α, to suppress C/EBP homologous protein (CHOP) induction. Depletion of ARC in isolated islets augments palmitate-induced apoptosis, which is dramatically rescued by deletion of CHOP. These data demonstrate that ARC is a previously unrecognized inhibitor of apoptosis in β-cells and that its protective effects are mediated through suppression of the ER stress response pathway.

No abstract

No abstract

Mitochondria isolated from ischemic cardiac tissue exhibit diminished rates of respiration and ATP synthesis. The present study was undertaken to determine whether cytochrome c release was responsible for ischemia-induced loss in mitochondrial function. Rat hearts were perfused in Langendorff fashion for 60 min (control) or for 30 min followed by 30 min of no flow ischemia. Mitochondria isolated from ischemic hearts in a buffer containing KCl exhibited depressed rates of maximum respiration and a lower cytochrome c content relative to control mitochondria. The addition of cytochrome c restored maximum rates of respiration, indicating that the release of cytochrome c is responsible for observed declines in function. However, mitochondria isolated in a mannitol/sucrose buffer exhibited no ischemia-induced loss in cytochrome c content, indicating that ischemia does not on its own cause the release of cytochrome c. Nevertheless, state 3 respiratory rates remained depressed, and cytochrome c release was enhanced when mitochondria from ischemic relative to perfused tissue were subsequently placed in a high ionic strength buffer, hypotonic solution, or detergent. Thus, events that occur during ischemia favor detachment of cytochrome c from the inner membrane increasing the pool of cytochrome c available for release. These results provide insight into the sequence of events that leads to release of cytochrome c and loss of mitochondrial respiratory activity during cardiac ischemia/reperfusion.

Reaction rates were determined between disulfide reagents of varying hydrophobicity and single-cysteine mutants of diacylglycerol kinase, an integral membrane protein. Polar reagents reacted most rapidly with surface-exposed sites. However, a very non-polar reagent also reacted more rapidly with exposed cysteines than with membrane sites. Moreover, this non-polar reagent usually reacted more slowly with membrane sites than did more polar reagents. These results are consistent with the notion that disulfide exchange reactions involving buried cysteines of diacylglycerol kinase are very slow in the membrane interior, such that the competing rates of reactions which occur when normally buried cysteine sites make motional excursions to hydrated regions of the interface can be significant.z 2000 Federation of European Biochemical Societies.