Some characteristics of fusogenic activity of cytoplasmic latrotoxin-like brain protein

We studied fusion of negatively charged artificial phospholipid vesicles (liposomes) in the presence of two electrophoretic fractions (molecular mass of about 90 and 50 kdalton) of latrotoxin-like (L) protein. It was shown that both fractions are capable of causing liposome fusion in acidic media. Treatment of native preparations of L protein with NEM depressed their fusogenic activity. Some common characteristics of L protein and well-known fusogenic proteins allow us to account for the possibility of participation of L protein in fusion of the membranes in the cell.     

Membranotropic properties of latrotoxin-like protein: Studies on liposomes

Fusion and changes in permeability of artificial phospholipid vesicles (liposomes) caused by the influence of α-latrotoxin-like protein (L protein) from the gray matter of the bovine brain were studied using a hydrophobic fluorescent probe, R18. It was shown that L protein stimulates fusion of negatively charged liposomes. This effect becomes stronger in acidified media. The influence of L protein on the permeability of phosphatidylcholine liposome membrane is also a pH-dependent process.     

Effect of vanadate on brain protein phosphorylation

The effect of vanadate on the phosphorylation of synaptosomal membrane proteins prepared from rat cerebral cortex was studied. Vanadate concentrations of 10^–6, 10^–5, and 10^–4 M increased the endogenous phosphorylation activity by 25%, 37%, and 75%, respectively. Increasing the ATP concentration in the assay medium from 50 to 500 M did not influence the above effect. A commercial preparation of the purified protein kinase was stimulated 40% by 10^–3 M vanadate. Calcium-calmodulin dependent activity was stimulated only 20% by 10^–5 M vanadate. The effect was not enhanced by further increasing vanadate concentration. Addition of calcium ions (above 50 M) suppressed the vanadate effect, while an inhibition was observed at high Ca²⁺ concentration (2.5 mM). Below 50 M calcium ions stimulated phosphorylation activity in the absence of vanadate and did not affect the stimulatory action of vanadate. Cyclic AMP-dependent endogenous phosphorylation was also stimulated by vanadate. Activation by cAMP could not be observed at vanadate concentrations above 10^–6 M. Possible mechanisms of the vanadate effect are discussed.     

The ability of latrotoxin-like brain protein to induce fusion of negatively charged liposomes

A latrotoxin-like protein isolated from the bovine brain promoted fusion of negatively charged liposomes consisting of phosphatidylcholine, phosphatidylethanolamine, and cardiolipin in a molar ratio of 2:3:5. The fusogenic effect significantly increased at mild acidic pH 6.0 and under denaturation (4 M urea, 0.1% SDS). Using ANS as the fluorescent probe, it was found that hydrophobicity of the latrotoxin-like protein increases along with the fusogenic activity. We hypothesize the existence in the protein molecule of conformational changes promoting the fusion, and the possible participation of the protein in neurosecretion processes.Neirofiziologiya/Neurophysiology, Vol. 25, No. 5, pp. 329–334, September–October, 1993.     

Endorphin-regulated protein phosphorylation in brain membranes

This study was initiated to determine whether opioid peptides exert direct effects on the phosphorylation of specific proteins in membranes from rat neostriatum. It was found that low concentrations of β-endorphin (0.1–10nM) inhibit the phosphorylation of specific proteins designated F and H (M.W. 47,000 and 10–20,000 respectively). In addition, β-endorphin produced an overall stimulation of phosphate incorporation into other membrane proteins, the phosphorylation of which is dependent on calcium ions. The stimulatory effects were blocked by naloxone, but the inhibitory effects were not. The regulation of membrane protein-phosphorylation by endorphins may constitute a biochemical mechanism mediating for some of the physiological affects of these peptides on neuronal function.     

Lysosomal enzyme binding receptor protein from monkey brain and its phosphorylation.

The lysosomal enzyme binding receptor protein isolated from monkey brain by phosphomannan-Sepharose affinity chromatography was phosphorylated by [gamma-32P] ATP by protein kinases tightly associated with the receptor protein. A greater than 200 kDa protein was phosphorylated on both serine and tyrosine residues and a approximately 45 kDa protein was phosphorylated on only serine residues as evidenced by SDS-gel electrophoresis, autoradiography and phosphoamino acid analysis [(Panneerselvam, Ramamoorthy & Balasubramanian (1987) Biochem Biophys Res Commun, 147, 927-935)]. 125I-labelled lysosomal enzymes could be cross-linked to the receptor protein in the presence of disuccinimidyl suberate. Phosphorylation of the receptor on both serine and tyrosine residues was inhibited by quercetin, polylysine and polymyxin B. Catalytic subunit of cyclic AMP-dependent protein kinase preferentially phosphorylated the approximately 45 kDa protein. In the presence of Triton X-100, phosphorylation of a few additional protein bands on non-tyrosine residues was observed. There was a marked reduction in the efficiency of binding lysosomal enzymes by the phosphorylated receptor protein in comparison to the unphosphorylated receptor protein.     

Effect of feeding protein deficient diet on phospholipid turnover and protein kinase C mediated protein phosphorylation in rat brain

Feeding of protein deficient diet is known to alter the transmembrane signalling in brain of rat by reducing total protein kinase C (PKC) activity. Phospholipid metabolism regulates the activation of PKC through generation of second messengers and the extent of PKC activation accordingly influences the magnitude of phosphorylation of its endogenous substrate proteins. Thus it was speculated that ingestion of protein deficient diet may modify the turnover rate of membrane phospholipids and magnitude of phosphorylation of endogenous substrate proteins of PKC. The experiments were conducted on rats fed on three different types of laboratory prepared diets viz. casein (20% casein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threonine) for 28 days. The metabolism of phosphoinositides (PIs) and phosphatidyl choline (PC) was studied by equilibrium labeling with [3H] myo inositol and [14C methyl] choline chloride respectively. The phosphorylation of endogenous substrate proteins of PKC was studied by using 32P-gamma-ATP followed by SDS-PAGE and autoradiography. The results suggest that in deficient group, there is an increased incorporation of [3H] myo inositol in PIs and inositol phosphate pool in comparison to the casein group. The phosphatidyl inositol (PI) turnover reduced, although there was a marginal increase in the phosphatidyl inositol monophosphate (PIP) and phosphatidyl inositol bis phosphate (PIP2). Supplementation of diet showed a reversal of the pattern towards control to a considerable extent. In the deficient group, PC metabolism showed an increased incorporation of [14C methyl] choline in choline phospholipids but decreased incorporation in phosphoryl choline in comparison with the casein group. The increase in total PC contents was significant but marginal in residue contents. The turnover rate of PC increased only marginally and that of residue declined. Supplementation of diet reduced the total contents of PC and residue, but the turnover rate of PC and residue remained still higher. Phosphorylation of endogenous proteins showed four different proteins of 78, 46, 33 and 16 kDa to be the substrates of PKC in casein group. In deficient group, phosphorylation of these proteins increased markedly while supplementation of diet had a reversing effect rendering the values to be intermediate between casein and the supplemented group. The changes in phospholipid metabolism and in phosphorylation of endogenous substrate proteins of PKC suggest that dietary protein deficiency causes alterations in transmembrane signalling mechanism in rat brain. These effects are partially reversed by improving the quality of proteins in the diet.     

Biochemical investigation of Tau protein phosphorylation status and its solubility properties in Drosophila

Tau hyperphosphorylation and insoluble aggregate formation are two cellular features of tauopathies. However, the contribution of Tau protein hyperphosphorylation and its aggregation to Tau pathology still remain controversial. Overexpression of human tau transgenes in the Drosophila eye is toxic and causes neuronal degeneration. We showed that human Tau protein was phosphorylated by endogenous protein kinases in flies, and overexpression of either GSK3beta or Cdk5 enhanced tau-induced toxicity. Using a dominant-negative approach, we showed that kinase activity is important for the enhancement of tau-induced toxicity. Interestingly, such enhancement was accompanied with hyperphosphorylation and alteration of protein solubility properties of Tau. This situation was reminiscent of that observed in pre-tangle neurons in tauopathies patients. We also observed age-dependent Tau aggregate formation in aged transgenic flies. In summary, tau-induced toxicity is enhanced when the human Tau protein undergoes hyperphosphorylation, and we further demonstrated that aging contributes to Tau aggregate formation. Our data also underscore the utilization of transgenic Drosophila Tau models for the studies of pre-tangle events in tauopathies.     

Modulation of brain protein phosphorylation by the S-100 protein

The effects of the nervous system specific protein, S-100, on protein phosphorylation in rat brain is examined. The S-100 protein inhibits the phosphorylation of several soluble brain proteins in a calcium dependent fashion. The most potent effect exhibited by S-100 was on the phosphorylation of a protein having a molecular weight of 73,000. The data suggest that the calcium binding S-100 protein, for which a function has not yet been assigned, may modulate calcium dependent phosphorylation of selected brain proteins.     

Effect of radioprotectors on cAMP-dependent protein phosphorylation]

The capacity of radioprotectors (serotonin, aminoethylisothiuronium) to stimulate in radioprotective doses cAMP-dependent protein phosphorylation of the cytozol and nuclei of the mouse liver and of the spleen cytozol was shown in vivo. Radioprotectors were incapable of modulating in vitro the activity of protein kinases and the stimulating effect of cAMP. The data presented and previous investigations suggest that the activation of cAMP-dependent phosphorylation by radioprotectors was due to the increase of intracellular cAMP concentration under the effect of radioprotectors.     

Effect of aldosterone on renal ribosomal protein phosphorylation

The phosphorylation of ribosomes in kidney cortex slices was measured during 60 min of incubation 2 h after intravenous injection of aldosterone into adrenalectomized rats. No significant differences from controls were observed in the incorporation of [³²P] into total ribosomes, ribosomal RNA or ribosomal protein. The phosphorylation of ribosomal proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis was also compared in renal cortical slices from aldosterone-treated and control animals by a double-labeling technique using [³²P] and [³³P]. After aldosterone administration the phosphorylation of at least two proteins with low mobilities (high molecular weights) increased, while that of at least two others with higher mobilities (lower molecular weights) decreased.     

Effect of protein S-100 on phosphorylation of nuclear proteins of cells of rat brain and liver

The effect of acidic neurospecific protein S-100 on the phosphorylation of brain and liver nuclear proteins with 1 and 10 microM ATP was investigated. It was shown that protein S-100 increases the phosphorylation of brain nuclear proteins, while antigen D, another acidic neurospecific protein half-identical to 14-3-2 protein, inhibits this process. Ca2+ and cAMP at concentration of 10(-6) M do not affect the phosphorylation of brain nuclear proteins. In control assays the tracer 32P is presumably incorporated into high molecular weight nuclear protein fractions (Mr greater than 40000). After addition of protein S-100 the tracer is mainly incorporated into these proteins as well independently of ATP concentration (1 or 10 microM). The phosphorylation of nuclear proteins with molecular weights above 100000 is mostly increased in this case. At ATP concentration of 1 microM protein S-100 decreases histone phosphorylation 2.3 times but does not affect that of non-histone proteins. However, at 10 microM ATP the inhibitory action of this protein on histone phosphorylation is absent. The possible mechanisms of protein S-100 action on nuclear proteins phosphorylation are discussed.     

Effect of sulfhydryl-disulfide state on protein phosphorylation: phosphorylation of bovine serum albumin

Bovine serum albumin (BSA) was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase under general protein phosphorylation conditions. The optimal pH for this phosphorylation was 9.0. The K0.5 (the concentration required for 50% of maximal phosphorylation) for BSA at pH 7.5 was 15 microM. One mole of phosphate was incorporated per mole of BSA, and only one phosphopeptide fragment was obtained after extensive proteolysis with trypsin. BSA phosphorylation required dithiothreitol or GSH, but GSH was only one-fiftieth as effective as dithiothreitol. GSSG counteracted the effect of dithiothreitol and GSH. Phosphorylation increased in a time-dependent and dithiothreitol concentration-dependent manner when BSA was preincubated with dithiothreitol. The increase in the incorporation of 32P correlated with the appearance of up to six free sulfhydryl groups. The effect of dithiothreitol on BSA appeared reversible, since reoxidation of reduced BSA decreased its susceptibility to phosphorylation. These experiments showed that this in vitro phosphorylation is dependent on the sulfhydryl-disulfide state of BSA. The possible implications of the sulfhydryl-disulfide state of proteins in the regulation of phosphorylation are discussed.     

Phosphatidylserine translocation into brain mitochondria: Involvement of a fusogenic protein associated with mitochondrial membranes

Data reported in the literature indicate that lipid movement between intracellular organelles can occur through contacts and close physical association of membranes (Vance, J.E. 1990. J Biol Chem 265: 7248-7256). The advantage of this mechanism is that the direct interaction of membranes provides the translocation event without the involvement of lipid-transport systems. However, pre-requisite for the functioning of this machinery is the presence of protein factors controlling membrane association and fusion. In the present work we have found that liposomes fuse to mitochondria at acidic pH and that the pre-treatment of mitochondria with pronase inhibits the fusogenic activity. Mixing of ¹⁴C-phosphatilyserine (PS) labeled liposomes with mitochondria at pH 6.0 results in the translocation of ¹⁴C-PS into mitochondria and in its decarboxylation to¹⁴ C-phosphatidylethanolamine through the PS decarboxylase activity localized on the outer surface of the inner mitochondrial membrane. Incorporation of ¹⁴C-PS is inhibited by the pre-treatment of mitochondria with pronase or with EEDQ, a reagent for the derivatization of the protonated form of carboxylic groups. These results indicate the presence of a protein associated with mitochondria which is able to trigger the fusion of liposomes to the mitochondrial membrane. A partial purification of a mitochondrial fusogenic glycoprotein is described in this work. The activity of the fusogenic protein appears to be dependent on the extent of protonation of the residual carboxylic groups and is influenced by the glucidic moiety, as demonstrated by its interaction with Concanavalin A. The purifed protein is able to promote the recover of the¹⁴ C-PS import from liposomes to pronase-treated mitochondria. Therefore, the protein is candidate to be an essential component in the machinery for the mitochondrial import of PS. (Mol Cell Biochem 175: 71–80, 1997)     

Effect of protein kinase A-mediated phosphorylation on the structure and association properties of the enteropathogenic Escherichia coli Tir virulence protein

Enteropathogenic Escherichia coli virulence is dependent on delivery of the translocated intimin receptor protein (Tir) into host cells. Tir phosphorylation on a single tyrosine (Tyr-474) is essential in mediating cytoskeletal rearrangement correlated with disease. Tir is also phosphorylated on other residues, with cAMP-dependent kinase (PKA) modification shown to play a role in Tir function. However, the mechanism by which nontyrosine phosphorylation affects Tir function remains unclear. In this study, analytical ultracentrifugation, SDS and native gel electrophoresis revealed that both Tir and its C-terminal domain (residues 385-550 of Tir that include the PKA substrate sites) exist in an equilibrium of monomers, dimers, and in the case of Tir, higher oligomers. PKA phosphorylation (1:300, PKA/C-Tir, mol/mol) shifted the equilibrium of C-Tir, but not Tir, predominantly to the dimeric state, whereas, at 100-fold higher concentrations of PKA the phosphorylated C-Tir was largely monomeric. This monomeric state was also produced at the lower PKA concentration and physiological ionic strength. Phosphorylation-mediated effects were achieved without significant changes in secondary structure as determined by circular dichroism spectroscopy. The data presented indicate that PKA-mediated phosphorylation induces changes in the association properties of the C-terminal domain of Tir that may facilitate Tir-Tir interactions and subsequently C-Tir-host protein interactions in vivo.     

In vitro phosphorylation of S-100 protein by brain nuclear protein kinases

A three-fold increased ³²P incorporation was observed when S-100 protein was added to a nuclear protein kinase preparation (NPKP) from brain. The specificity of the reaction was indicated by two observations: an increase in ³²P incorporation was not found either with 14–302 protein or when S-100 was added to liver NPKP. SDS-gel analysis shows prominent incorporation of ³²P by brain NPKP into an endogenous brain protein having a molecular weight near 45000 daltons, and, in the presence of S-100, predominantly into S-100 protein itself. Liver NPKP in the presence of S-100, showed an increased incorporation of ³²P into endogenous proteins without any phosphorylation of S-100.     

Reconstitution and fusogenic properties of Sendai virus envelopes

Sendai virus membranes were reconstituted by detergent dialysis, using the non-ionic detergents Triton X-100 and octyl glucoside. Membrane reassembly was determined by measuring the surface-density-dependent efficiency of resonance energy transfer between two fluorescent phospholipid analogues, which were co-reconstituted with the viral envelopes. The functional incorporation of the viral proteins was established by monitoring the ability of the reconstitution products to fuse with erythrocyte membranes, utilizing assays based on either resonance energy transfer or on relief of fluorescence selfquenching. The persistent adherence of residual Triton X-100 with the reconstituted membrane was revealed by an artificial detergent-effect on the resonance energy transfer efficiency and the occurrence of hemolysis of human erythrocytes under conditions where fusion does not occur. Properly reconstituted Sendai virus envelopes were obtained with octyl glucoside. The fusion activity of the viral envelopes was dependent on the initial concentration of octyl glucoside used to disrupt the virus and the rate of detergent removal. Rapid removal of detergent by dialysis against large volumes of dialysis buffer (ratio 1:850) or by gel filtration produced reconstituted membranes capable of inducing hemagglutination but significant fusion activity was not detected. By decreasing the volume ratio of dialysate versus dialysis buffer to 1:250 or 1:25, fusogenic viral envelopes were obtained. The initial fusion kinetics of the reconstituted viral membrane and the parent virus were different in that both the onset and the initial rate of fusion of the reconstituted membranes were faster, whereas the extents to which both particles eventually fused with the target membrane were similar. The differences in the initial fusion kinetics lead us to suggest that the details of the fusion mechanism between Sendai virus and the target membrane involve factors other than the mere presence of glycoproteins F and HN in the viral bilayer. Finally, the results also indicate that determination of the viral fusion activity in a direct manner, rather than by an indirect assay, such as hemolysis, is imperative for a proper evaluation of the functional properties retained upon viral reconstitution.