Keshava N. Kumar

Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA).

The anti-apoptotic effect of Bcl-2 is well established, but the detailed mechanisms are unknown. In the present study, we show in vitro a direct interaction of Bcl-2 with the rat skeletal muscle SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), leading to destabilization and inactivation of the protein. Recombinant human Bcl-2D21, a truncated form of Bcl-2 with a deletion of 21 residues at the C-terminal membrane-anchoring region, was expressed and affinity-purified as a glutathione S-transferase fusion protein. Bcl-2D21 co-immunoprecipitated and specifically interacted with SERCA in an in vitro-binding assay. The original level of Bcl-2 in sarcoplasmic reticulum vesicles was very low, i.e. hardly detectable by immunoblotting with specific antibodies. The addition of Bcl-2D21 to the sarcoplasmic reticulum resulted in the inhibition of the Ca2+-ATPase activity dependent on the Bcl-2D21/SERCA molar ratio and incubation time. A complete inactivation of SERCA was observed after 2.5 h of incubation at approx. 2:1 molar ratio of Bcl-2D21 to SERCA. In contrast, Bcl-2D21 did not significantly change the activity of the plasma-membrane Ca2+-ATPase. The redox state of the single Cys158 residue in Bcl-2D21 and the presence of GSH did not affect SERCA inhibition. The interaction of Bcl-2D21 with SERCA resulted in a conformational transition of SERCA, assessed through a Bcl-2-dependent increase in SERCA thiols available for the labelling with a fluorescent reagent. This partial unfolding of SERCA did not lead to a higher sensitivity of SERCA towards oxidative inactivation. Our results suggest that the direct interaction of Bcl-2 with SERCA may be involved in the regulation of apoptotic processes in vivo through modulation of cytoplasmic and/or endoplasmic reticulum calcium levels required for the execution of apoptosis.

Nitric oxide: Its identity and role in blood pressure control

In response to biochemical factors like catecholamines, bradykinins, histamine and physical factors like shear stress, endothelial cells release a non prostanoid factor, called endothelium derived relaxing factor (EDRF), which relaxes vascular smooth muscle. Since this discovery in 1980, many additional agents have been shown to stimulate release of EDRF from endothelium. Biological and chemical evidence has supported the proposal that EDRF is actually nitric oxide (NO). Research on the synthesis, inhibition and physiological roles of nitric oxide (NO) has led to studies of its involvement in blood pressure homeostasis and immune functions.

The 71 kDa glutamate-binding protein is increased in cerebellar granule cells after chronic ethanol treatment

Besides the N-methyl-D-aspartate (NMDA) receptor proteins NR1 and NR2, another complex of proteins which has been shown to contain ligand-binding sites characteristic of NMDA receptors is expressed in cerebellar granule cells. One of the proteins in the latter complex is the 71 kDa glutamate-binding protein (GBP). To determine the role of the GBP in the response to NMDA, primary cultures of cerebellar granule cells were treated with an antisense oligonucleotide complementary to mRNA for this protein. This treatment substantially reduced both mRNA and protein levels of the GBP, as well as the response of the cells to NMDA, measured as an increase in intracellular Ca2+ with fura-2 fluorescence. The antisense oligonucleotide treatment did not alter the Ca2+ responses to KC1 or kainate. Chronic ethanol exposure has previously been shown to increase NMDA receptor function and the density of binding sites for the NMDA receptor channel blocker, dizocilpine, in cerebellar granule cells. Chronic exposure of the cells to 100mM ethanol is now shown to result in significant increases in mRNA and protein levels for the GBP (45% and 100%, respectively). Ethanol treatment did not affect mRNA levels for NR1 or NR2A, caused only a small increase (20%) in protein levels for NR1, and resulted in a decrease (30%) in NR2A protein. Although a role of the NMDA receptor NR1/NR2 subunits cannot be ruled out, these results are compatible with the hypothesis of involvement of the GBP in the chronic ethanol-induced increase in NMDA receptor function in cerebellar granule cells.

Isolation of glutamate-binding proteins from rat and bovine brain synaptic membranes and immunochemical and immunocytochemical characterization

We previously isolated two glutamate-binding proteins from rat brain synaptic membranes of 71,000 and 63,000 mol. wt [Chen et al. (1988) J. biol. Chem. 263, 417-426]. In the present study, the 71,000 and 63,000 mol. wt glutamate-binding proteins were purified from rat and bovine brain synaptic membranes by affinity chromatographic separation on an L-glutamate-derived Trisacryl matrix. The major protein component in the purified fractions was a 71,000 mol. wt protein as judged by sodium dodecyl sulfate gel electrophoresis. This fraction represented enrichment of bovine brain glutamate-binding proteins by a factor of 7000-8000 when compared with brain homogenates. The ligand binding characteristics of the proteins purified by this new procedure are very similar to those of the proteins purified by the procedures we described in previous studies. Polyclonal antibodies were raised in mice against the purified, native rat and bovine brain glutamate-binding proteins. These two sets of antibodies interacted specifically with the glutamate-binding proteins but not with any glutamate-metabolizing enzymes. Both sets of antibodies labeled a 71,000 mol. wt protein in Western blots of synaptic membranes obtained from either rat or bovine brain, an indication of homology between these proteins. Both sets of antibodies produced immunoprecipitation of approximately 70-75% of all glutamate-binding proteins from solubilized synaptic membrane proteins. These observations are an indication that the glutamate-binding proteins described above are the most common glutamate-binding entities in both bovine and rat brain synaptic membranes and that they can be easily purified in a one-step chromatographic procedure.

Ion channel properties of a protein complex with characteristics of a glutamate/N-methyl-D-aspartate receptor

The functional reconstitution of glutamate receptor proteins purified from mammalian brain has been difficult to accomplish. However, channels activated by l‐glutamate (L‐Glu) and N‐methyl‐d‐aspartate (NMDA) were detected in planar lipid bilayer membranes (PLMs) following the reconstitution of a complex of proteins with binding sites for NMDA receptor (NMDAR) ligands. The presence of glycine was necessary for optimal activation. A linear current‐voltage relationship was observed with the reversal potential being zero. Channels activated by L‐Glu had conductances of 23, 47 and 65 pS, and were suppressed partially by competitive and fully by non‐competitive inhibitors of NMDARs. Magnesium had little effect on the reconstituted channels.

Ethanol-Induced Inhibition of [3H]Thienylcyclohexylpiperidine (TCP) Binding to NMDA Receptors in Brain Synaptic Membranes and to a Purified Protein Complex

Abstract: N‐Methyl‐d‐asparate receptors (NMDARs) are a major target of ethanol effects in the nervous system. Haloperidol‐insensitive, but dizocilpine (MK‐801)‐sensitive, binding of N‐[1‐(2‐[3H]thienyl)cyclohexyl]piperidine ([3H]TCP) to synaptic membranes has the characteristics of ligand interaction with the ion channel of NMDARs. In the present studies, ethanol produced a concentration‐dependent decrease in the maximal activation of [3H]TCP binding to synaptic membranes by NMDA and Gly, but a moderate change in the activation by l‐Glu when l‐Glu was present at concentrations < 100 µM. However, ethanol (100 mM) inhibited completely the activation of [3H]TCP binding produced by high concentrations of l‐Glu (200–400 µM). It also inhibited strongly the activation of [3H]TCP binding by spermidine or spermidine plus Gly. In a purified complex of proteins that has l‐Glu‐, Gly‐, and [3H]TCP‐binding sites, ethanol (100 mM) decreased significantly the maximal activation of [3H]TCP binding produced by either l‐Glu or Gly. Activation constants (Kact) for l‐Glu and Gly acting on the purified complex were 12 and 28 µM, respectively. Ethanol had no significant effect on the Kact of l‐Glu but caused an increase in the Kact of Gly. These studies have identified at least one protein complex in neuronal membranes whose response to both l‐Glu and Gly is inhibited by ethanol. These findings may explain some of the effects of acute and chronic ethanol treatment on the function and expression of the subunits of this complex in brain neurons.

Immunocytochemical and in situ hybridization studies of the expression and distribution of three subunits of a complex with N-methyl-d-aspartate receptor-like properties

A group of four proteins with recognition sites for L-glutamate, N-methyl-D-aspartate, glycine, and competitive and non-competitive inhibitors of N-methyl-D-aspartate receptors was previously purified from rat brain synaptic membranes. The biochemical and immunochemical characteristics of this complex, as well as the sequences of the complementary DNAs of three subunits, are distinct from those of other glutamate receptors, transporters, or enzymes. The function of this complex has not yet been defined, but it appears to be involved in glutamate-induced neuronal excitation and toxicity. It is not known whether all protein components of the complex are expressed in the same populations of brain cells. In the present study, immunohistochemical and in situ hybridization were used to map the distribution of the glutamate-binding, glycine/thienylcyclohexylpiperidine-binding, and carboxypiperazinyl-propylphosphonate-binding protein subunits of the complex. These proteins were abundantly expressed in pyramidal neurons of the hippocampus and cerebral cortex, and in granule cells of the dentate gyrus, cerebellum, and olfactory tubercle. Based on these results, it was concluded that the three subunits of the complex have similar patterns of expression in rat brain. The distribution of one subunit of the complex, glutamate-binding protein, was traced throughout the rat brain, thus providing a potential map of the expression of the complex in rodent brain. In addition, probes were developed in the present study that should be useful in future explorations of the role of these proteins in brain function and of the possible co-localization of the protein subunits in single cells or cell processes.

A synaptic membrane glycine-, glutamate- and thienylcyclohexylpiperidine-binding protein: isolation and immunochemical characterization

Antibodies raised against a 43 kDa component of a complex of synaptic membrane proteins with ligand binding sites characteristic of glutamate/N-methyl-D-aspartate (NMDA) receptors, were used previously to clone a cDNA for a glycine-, glutamate-, and thienylcyclohexylpirperidine (TCP)-binding protein, pGlyBP (Kumar et al., Biochem. Biophys. Res. Commun. 216, 390-398, 1995). In the present studies, the antibodies were shown to label a 60 kDa protein, in synaptic membranes, that was relatively hydrophilic as demonstrated by its predominant separation in the detergent-depleted phase of proteins solubilized with Triton X-114. A 55-60 kDa protein was purified from rat brain synaptic membranes by chromatographic separation through matrices derivatized with 5,7-di-chlorokynurenic acid (5,7-DCK) followed by chromatography on a matrix derivatized with 8-hydroxyquinoline (8-OHQ). The isolated fractions were highly enriched in strychnine-insensitive [3H]glycine, NMDA- and glutamate-sensitive L-[3H]glutamate, and MK-801-sensitive [3H]TCP binding sites. The purified protein bound [3H]glycine with a stoichiometry of 1.1-1.2 mol glycine per mol protein and exhibited both high (KD = 280 nM) and low affinity (KD = 30 microM) glycine binding sites. Glycine binding was inhibited by D-serine and R-(+)-3-amino-1-hydroxypyrrolidin-2-one(R-(+)-HA-966). The KD values for high and low affinity sites of glycine binding as well as those for the inhibition by R-(+)-HA-966 were very similar to the KDs for glycine binding to the expressed pGlyBP. Both L-glutamate and glycine activated [3H]TCP binding to the isolated proteins, but with relatively low affinity. The anti-43 kDa antibodies reacted strongly with the 55-60 kDa protein. Based on these results, it appears that the 60 kDa glycoprotein in brain synaptic membranes described in the present study is the same protein as the cloned pGlyBP.

Immunochemical and immunohistochemical characterization of a synaptic membrane protein that binds the competitive antagonists of NMDA receptors

An approximately 54-kDa protein that has binding sites for the competitive N-methyl-D-aspartate (NMDA) receptor antagonists 3-((+-)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) and (+-)-(E)-2-amino-4-propyl-5-phosphonopentenoic acid (CGP 39653) was purified from rat brain synaptic membranes. Polyclonal antibodies to this protein reacted specifically with an approximately 54-kDa protein in synaptic membranes and immunoextracted approximately 60% of [3H]CGP 39653 binding sites associated with solubilized membrane proteins. The antibodies also labeled antigenic sites in the perikaryon and apical and basilar dendrites of pyramidal neurons of the hippocampus and cerebral cortex.

A rat brain bicistronic gene with an internal ribosome entry site codes for a phencyclidine-binding protein with cytotoxic activity

The cloning and characterization of the gene for the fourth subunit of a glutamate-binding protein complex in rat brain synaptic membranes are described. The cloned rat brain cDNA contained two open reading frames (ORFs) encoding 8.9- (PRO1) and 9.5-kDa (PRO2) proteins. The cDNA sequence matched contiguous genomic DNA sequences in rat chromosome 17. Both ORFs were expressed within the structure of a single brain mRNA and antibodies against unique sequences in PRO1- and PRO2-labeled brain neurons in situ, indicative of bicistronic gene expression. Dicistronic vectors in which ORF1 and ORF2 were substituted by either two different fluorescent proteins or two luciferases indicated concurrent, yet independent translation of the two ORFs. Transfection with noncapped mRNA led to cap-independent translation of only ORF2 through an internal ribosome entry sequence preceding ORF2. In vitro or cell expression of the cloned cDNA led to the formation of multimeric protein complexes containing both PRO1 and PRO2. These complexes had low affinity (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801)-sensitive phencyclidine-binding sites. Overexpression of PRO1 and PRO2 in CHO cells, but not neuroblastoma cells, caused cell death within 24–48 h. The cytotoxicity was blocked by concurrent treatment with MK-801 or by two tetrahydroisoquinolines that bind to phencyclidine sites in neuronal membranes. Co-expression of two of the other subunits of the protein complex together with PRO1/PRO2 abrogated the cytotoxic effect without altering PRO1/PRO2 protein levels. Thus, this rare mammalian bicistronic gene coded for two tightly interacting brain proteins forming a low affinity phencyclidine-binding entity in a synaptic membrane complex.