F.J. Navarro

A novel family of Ig-like receptors for HLA class I molecules that modulate function of lymphoid and myeloid cells.

We review what is presently known about structure, cellular distribution, biochemical characteristics, and function of a new family of human cell‐surface receptors referred to as immunoglobulin‐like transcripts (ILTs), leukocyte Ig‐like receptors (LIRs), or monocyte/macrophage Ig‐like receptors (MIRs). These receptors are genetically, structurally, and functionally related to a group of natural killer (NK) cell receptors for HLA class I molecules known as killer cell Ig‐like receptors (KIRs). Distinct ILT/LIR/MIR isotypes are differentially expressed on lymphocytes, monocytes, macrophages, dendritic cells, and granulocytes; at least some of them recognize HLA class I molecules. Whereas some isotypes either inhibit or induce cell activation, others may be secreted as soluble receptors. ILT/LIR/MIR receptors may allow all immune cells to monitor class I expression on other cells and to respond in its absence, just as NK cells do. In addition, they may contribute to homeostasis by establishing activation thresholds that can be overcome only by relevant triggering stimuli and not by bystander cells. J. Leukoc. Biol. 66: 375–381; 1999.

Vitamin E and selenium deficiency induces expression of the ubiquinone-dependent antioxidant system at the plasma membrane

We have used a model of dietary deficiency that leads to a chronic oxidative stress to evaluate responses that are adaptations invoked to boost cellular defense systems. Long‐Evans hooded rats were fed with a diet lacking vitamin E (E) and selenium (Se) for 7 wk from weaning leading to animals deficient in both nutrients (−E −Se). In the absence of an electron donor, liver plasma membranes from these rats were more sensitive to lipid peroxidation, although they contained 40% greater amounts of ubiquinone than the plasma membranes from rats consuming diets with sufficient vitamin E and Se (+E +Se). The incubation of plasma membranes with NAD(P)H resulted in protection against peroxidation, and this effect was more pronounced in −E −Se membranes. Deficiency was accompanied by a twofold increase in redox activities associated with trans plasma membrane electron transport such as ubiquinone reductase and ascorbate free radical reductase. Staining with a polyclonal antibody against pig liver cytochrome b5 reductase, which acts as one ubiquinone reductase in the plasma membrane, showed an increased expression of the enzyme in membranes from −E −Se rats. Little DT‐diaphorase activity was measured in +E +Se plasma membranes, but this activity was dramatically increased in −E −Se plasma membranes. No such increase was found in liver cytosols, which contained elevated activity of calcium‐independent phospholipase A2. Thus, ubiquinone‐dependent antioxidant protection in + E + Se plasma membranes is based primarily on NADH‐cytochrome b5 reductase, whereas additional protection needed in −E −Se plasma membranes is supported by the increase of ubiquinone levels, increased expression of the cytochrome b5 reductase, and translocation of soluble DT‐diaphorase to the plasma membrane. Our results indicate that, in the absence of vitamin E and Se, enhancement of ubiquinone‐dependent reductase systems can fulfill the membrane antioxidant protection.—Navarro, F., Navas, P., Burgess, J. R., Bello, R. I., de Cabo, R., Arroyo, A., Villalba, J. M. Vitamin E and selenium deficiency induces expression of the ubiquinone‐dependent antioxidant system at the plasma membrane. FASEB J. 12, 1665–1673 (1998)

Paired inhibitory and triggering NK cell receptors for HLA class I molecules

Human natural killer (NK) cells specifically interact with major histocompatibility complex (MHC) class I molecules employing different receptor systems, shared with subsets of alphabeta and gammadelta T lymphocytes. Killer cell immunoglobulin-like receptors (KIRs) recognize groups of human leukocyte antigen (HLA) class Ia proteins displaying common structural features at the alpha-1 domain; among them, KIR2DL4 has been proposed to specifically interact with the class Ib molecule HLA-G1. Members of a related family of immunoglobulin (Ig)-like receptors (ILT2 or LIR-1 and ILT4 or LIR-2), expressed by other leukocyte lineages, interact with a broad spectrum of class Ia molecules and HLA-G1. On the other hand, CD94/NKG2-A(-C) and NKG2D lectin-like receptors, respectively, recognize the class Ib molecules HLA-E and MICA. A recurrent finding within the different receptor families is the existence of pairs of homologous molecules that often share the same ligands but display divergent functions. Inhibitory receptors tend to exhibit an affinity for HLA molecules higher than their activating counterparts. Recruitment of SH2 domain-bearing tyrosine phosphatases (SHP) by cytoplasmic phosphorylated immunoreceptor tyrosine-based inhibition motifs (ITIMs) is a crucial event for the inhibitory signalling pathway. By contrast, triggering receptors assemble with homodimers of immune tyrosine-based activation motif (ITAM)-bearing adaptor molecules (i.e., DAP12, CD3 xi) that engage tyrosine kinases (ZAP70 and syk).

Role of cytochrome b5 reductase on the antioxidant function of coenzyme Q in the plasma membrane

Cytochrome b5 reductase purified from liver plasma membrane reduces coenzyme Q (CoQ) in reconstituted liposomes in the absence of cytochrome b5. Both CoQ and its reductase are responsible for the reduction of the ascorbate free radical at the cell surface. Thus, NADH-CoQ reductase represents a partial reaction of NADH-AFR reductase in the plasma membrane. Cytochrome b5 reductase maintains CoQ and ascorbate in their reduced state to support antioxidations. Reduced CoQ prevents lipid peroxidation in liposomes and plasma membranes. Also, oxidized CoQ can prevent lipid peroxidations in the presence of cytochrome b5 reductase and NADH. Addition of CoQ to intact cells prevents serum withdrawal-induced lipid peroxidation and apoptosis. The prevention of apoptosis by CoQ is independent of the bcl-2 protein content in the cell. Antioxidants that act at the plasma membrane as CoQ and ascorbate would represent a first barrier to protect lipids from oxidative stress and subsequent apoptosis. Cytochrome b5 reductase is then an enzyme leading this function at the plasma membrane. These data support the idea that when the plasma membrane barrier fails, bcl-2 protein would be required to prevent cell death.

ANTIOXIDANT ASCORBATE IS STABILIZED BY NADH-COENZYME Q10 REDUCTASE IN THE PLASMA MEMBRANE

Plasma membranes isolated from K562 cells contain an NADH-ascorbate free radical reductase activity and intact cells show the capacity to reduce the rate of chemical oxidation of ascorbate leading to its stabilization at the extracellular space. Both activities are stimulated by CoQ10 and inhibited by capsaicin and dicumarol. A 34-kDa protein (p34) isolated from pig liver plasma membrane, displaying NADH-CoQ10 reductase activity and its internal sequence being identical to cytochrome b5 reductase, increases the NADH-ascorbate free radical reductase activity of K562 cells plasma membranes. Also, the incorporation of this protein into K562 cells by p34-reconstituted liposomes also increased the stabilization of ascorbate by these cells. TPA-induced differentiation of K562 cells increases ascorbate stabilization by whole cells and both NADH-ascorbate free radical reductase and CoQ10 content in isolated plasma membranes. We show here the role of CoQ10 and its NADH-dependent reductase in both plasma membrane NADH-ascorbate free radical reductase and ascorbate stabilization by K562 cells. These data support the idea that besides intracellular cytochrome b5-dependent ascorbate regeneration, the extracellular stabilization of ascorbate is mediated by CoQ10 and its NADH-dependent reductase.

Neutral magnesium-dependent sphingomyelinase from liver plasma membrane: purification and inhibition by ubiquinol.

Plasma membranes isolated from pig liver contained almost no acid sphingomyelinase but significant neutral magnesium-dependent sphingomyelinase that was activated by phosphatidylserine. We report here the purification to apparent homogeneity of neutral sphingomyelinase of about 87 kDa from liver plasma membranes. The purified enzyme strictly required magnesium and had a neutral optimal pH. In contrast with neutral sphingomyelinase purified from other sources (such as brain), the enzyme purified from from liver plasma membrane was not inhibited by GSH and, strikingly, it was not activated by phosphatidylserine. Liver sphingomyelinase was inhibited by several lipophilic antioxidants in a dose-dependent way. Ubiquinol-10 was more effective than α-tocopherol, α-tocopherylquinone, α-tocopherylquinone, and ubiquinone-10, and inhibition was noncompetitive. Differential inhibition of neutral sphingomyelinase by antioxidants did not correlate with different levels of protection against lipid peroxidation. The purified sphingomyelinase was not inhibited significantly by ubiquinone-10 and ubiquinol-10, but ubiquinol-0 and ubiquinone-0 inhibited by 30 and 60% respectively. Our results demonstrate a direct inhibitory effect of ubiquinol on the plasma membrane n-SMase and support the participation of this molecule in the regulation of ceramide-mediated signaling.

Interactions between ascorbyl free radical and coenzyme Q at the plasma membrane.

A role for coenzyme Q in the stabilization of extracellular ascorbate by intact cells has beenrecently recognized. The aim of this work was to study the interactions between reducedubiquinone in the plasma membrane and the ascorbyl free radical, as an approach to understandubiquinone-mediated ascorbate stabilization at the cell surface. K-562 cells stabilized ascorbateand decreased the steady-state levels of the semiascorbyl radical. The ability of cells to reduceascorbyl free radical was inhibited by the quinone analogs capsaicin and chloroquine andstimulated by supplementing cells with coenzyme Q10. Purified plasma membranes also reducedascorbyl free radical in the presence of NADH. Free-radical reduction was notobserved inquinone-depleted plasma membranes, but restored after its reconstitution with coenzyme Q10.Addition of reduced coenzyme Q10 to depleted membranes allowed them toreduce the signalof the ascorbyl free radical without NADH incubation and the addition of an extra amount ofpurified plasma membrane quinone reductase further stimulated this activity. Reduction wasabolished by treatment with the reductase inhibitor p-hydroximercuribenzoate and by blockingsurface glycoconjugates with the lectin wheat germ agglutinin, which supports the participationof transmembrane electron flow. The activity showed saturation kinetics by NADH andcoenzyme Q, but not by the ascorbyl free radical in the range of concentrations used. Our resultssupport that reduction of ascorbyl free radicals at the cell surface involves coenzyme Qreduction by NADH and the membrane-mediated reduction of ascorbyl free radical.

Down-regulation of Eukaryotic Nitrate Transporter by Nitrogen-dependent Ubiquitinylation

In the yeast Hansenula polymorpha, the YNT1 gene encodes the high affinity nitrate transporter, which is repressed by reduced nitrogen sources such as ammonium or glutamine. Ynt1 protein is degraded in response to glutamine in the growth medium. Ynt1 disappears independently of YNT1 glutamine repression as shown in strains where YNT1 repression is abolished. Ynt1-green fluorescent protein chimera and a mutant defective in vacuolar proteinase A (Δpep4) showed that Ynt1 is degraded in the vacuole in response to glutamine. The central hydrophilic domain of Ynt1 contains PEST-like sequences whose deletion blocked Ynt1 down-regulation. Site-directed mutagenesis showed that Lys-253 and Lys-270, located in this sequence, were involved in internalization and subsequent vacuolar degradation of Ynt1. Ynt1-ubiquitin conjugates were induced by glutamine and not nitrate. We conclude that glutamine triggers Ynt1 down-regulation via ubiquitinylation of lysines in the central hydrophilic domain, and proteolysis in the vacuole.

Ubiquinol regeneration by plasma membrane ubiquinone reductase

SummarySeveral enzyme systems have been proposed to play a role in the maintenance of ubiquinol in membranes other than the inner mitochondrial membrane. The aim of this study was to investigate the mechanisms involved in NADH-driven regeneration of antioxidant ubiquinol at the plasma membrane. Regeneration was measured by quantifying the oxidized and reduced forms of ubiquinone by electrochemical detection after separation by high-performance liquid chromatography. Plasma membrane incubation with NADH resulted in the consumption of endogenous ubiquinone, and a parallel increase in ubiquinol levels. The activity showed saturation kinetics with respect to the pyridine nucleotides and was moderately inhibited byp-hydroxymercuribenzoate. Only a slight inhibition was achieved with dicumarol at concentrations reported to fully inhibit DT-diaphorase. Salt-extracted membranes displayed full activity of endogenous ubiquinol regeneration, supporting the participation of an integral membrane protein. In liposomes-reconstituted systems, the purified cytochromeb5 reductase catalyzed the reduction of the natural ubiquinone homologue coenzyme Q10 at rates accounting for the activities observed in whole plasma membranes, and decreased the levels of lipid peroxidation. Our data demonstrate the role of the cytochromeb5 reductase in the regeneration of endogenous ubiquinol.

Phosphorylation of the Yeast Nitrate Transporter Ynt1 Is Essential for Delivery to the Plasma Membrane during Nitrogen Limitation

Ynt1 is the sole high affinity nitrate transporter of the yeast Hansenula polymorpha. It is highly regulated by the nitrogen source, by being down-regulated in response to glutamine by repression of the YNT1 gene and Ynt1 ubiquitinylation, endocytosis, and vacuolar degradation. On the contrary, we show that nitrogen limitation stabilizes Ynt1 levels at the plasma membrane, requiring phosphorylation of the transporter. We determined that Ser-246 in the central intracellular loop plays a key role in the phosphorylation of Ynt1 and that the nitrogen permease reactivator 1 kinase (Npr1) is necessary for Ynt1 phosphorylation. Abolition of phosphorylation led Ynt1 to the vacuole by a pep12-dependent end4-independent pathway, which is also dependent on ubiquitinylation, whereas Ynt1 protein lacking ubiquitinylation sites does not follow this pathway. We found that, under nitrogen limitation, Ynt1 phosphorylation is essential for rapid induction of nitrate assimilation genes. Our results suggest that, under nitrogen limitation, phosphorylation prevents Ynt1 delivery from the secretion route to the vacuole, which, aided by reduced ubiquitinylation, accumulates Ynt1 at the plasma membrane. This mechanism could be part of the response that allows nitrate-assimilatory organisms to cope with nitrogen depletion.