Local anesthetics block transient expression of inducible functions for transformation in Streptococcus sanguis.

Procaine and tetracaine reversibly inhibit transformation by preventing the transient expression of competence-specific, inducible functions, which are usually triggered in response to cellular stimulation with competence protein. Affinity studies with 14C-labeled procaine showed that the anesthetic bound to cell surface macromolecules specifically in the initiation phases of competence-specific events and blocked transfer of information imparted by cellular membrane receptor(s) upon interaction with competence protein.     

Distribution of Steroid 1-Dehydrogenation and Side-Chain Degradation Enzymes in the Spores of Fusarium solani: Causes of Metabolic Lag and Carbohydrate Independence

The spores of a strain of Fusarium solani 1-dehydrogenate ring A and cleave the 17beta-acetyl side chain of 17alpha-hydroxypregn-4-ene-3,20-dione (17alpha-hydroxyprogesterone) to give 17alpha-hydroxypregna-1,4-diene-3,20-dione (the 1-dehydro analogue) and little androsta-1,4-diene-3,4-diene-3,17-dione (androstadienedione). A 4-h lag period is observed in the course of metabolism, and there are no requirements for external additives. Exoenzymes or surface enzymes bound to the cell outside the plasma membrane, either in the periplasmic space or bound to the cell wall, cannot be detected. The spore activity is not destroyed by treatment with aqueous HCl (pH 1.50), indicating that the 1-dehydrogenation and side-chain degradation enzymes are located away from the surface of the spores. Phenethyl alcohol destroys the spore permeability barriers, and it is also likely that it exposes its enzymes to acid inactivation. The action of phenethyl alcohol is reversible at low concentrations and irreversible at high concentrations. This investigation shows that: (i) the spore 1-dehydrogenating and side-chain-degrading enzymes appear to be bound to, or imbedded in, the plasma membrane; (ii) the lag period observed in the course of metabolism of the steroid by the spores might be required for enzyme activation or diffusion of the substrate through the cell wall; and (iii) the internal metabolities of the spores, that might be required for the conversion process, appear to be present in a nondiffusible form or bound to intrasporal macromolecules.     

In vivo binding of 2,3,6,2′,3′,6′-hexachlorobiphenyl and 2,4,5,2′,4′,5′-hexachlorobiphenyl to mouse liver macromolecules

The role of metabolic activation in the binding of polychlorinated biphenyls (PCBs) to cellular macromolecules was investigated in vivo by comparing the relative binding of 2,4,5,2′,4′,5′-[U-¹⁴C]hexachlorobiphenyl (2,4,5), a slowly metabolized PCB, with that of 2,3,6,2′,3′,6′-[U-¹⁴C]hexachlorobiphenyl (2,3,6), a rapidly metabolized PCB, and the appropriate controls. Each hexachlorobiphenyl was administered to mice, orally for 5 days (7.28 mg/kg/day). Following the dosing schedule, animals were killed at 1, 5 and 8 days. The concentration of each PCB was determined in liver, muscle and kidney and in purified macromolecules isolated from those tissues. The concentration of 2,4,5 was consistently higher than the concentration of 2,3,6 in all tissues studied. However, the amount of 2,3,6 bound to the purified macromolecules was consistently at least one order of magnitude greater than that of 2,4,5. The greatest binding was observed in RNA followed by protein and DNA, respectively. The purity of the macromolecules and the presence of PCB-derived radioactivity at the monomer level were confirmed. This is the first report of ¹⁴C-labeled PCB being bound to purified RNA, DNA, and proteins isolated from the tissues of animals treated in vivo. The binding is thought to be covalent and to be the result of metabolic activation.     

Role of Membrane Association and Atg14-Dependent Phosphorylation in Beclin-1-Mediated Autophagy

During autophagy, a double membrane envelops cellular material for trafficking to the lysosome. Human beclin-1 and its yeast homologue, Atg6/Vps30, are scaffold proteins bound in a lipid kinase complex with multiple cellular functions, including autophagy. Several different Atg6 complexes exist, with an autophagy-specific form containing Atg14. However, the roles of Atg14 and beclin-1 in the activation of this complex remain unclear. We here addressed the mechanism of beclin-1 complex activation and reveal two critical steps in this pathway. First, we identified a unique domain in beclin-1, conserved in the yeast homologue Atg6, which is involved in membrane association and, unexpectedly, controls autophagosome size and number in yeast. Second, we demonstrated that human Atg14 is critical in controlling an autophagy-dependent phosphorylation of beclin-1. We map these novel phosphorylation sites to serines 90 and 93 and demonstrate that phosphorylation at these sites is necessary for maximal autophagy. These results help clarify the mechanism of beclin-1 and Atg14 during autophagy.     

The release of radioactive nucleic acids and mucoproteins by trypsin and ethylenediaminetetra-acetate treatment of baby-hamster cells in tissue culture

Monolayers of baby-hamster kidney cells were grown on glass in tissue culture and harvested with trypsin or EDTA in order to investigate the cell surface macromolecules removed by these cell-disaggregating agents. The release of nucleic acids from the cells during the harvesting procedure was monitored by labelling the cellular RNA with [5-(3)H]uridine and the cellular DNA with [2-(14)C]thymidine. Treatment of the cells with EDTA was found to cause an increase in the permeability of the plasma membrane with 7.6% of the cellular RNA, but less than 1% of the cellular DNA, being released. Moreover, 61% of the cells harvested with EDTA were permeable to Trypan Blue. With crude trypsin, lysis of the cell occurred with the release of similar amounts of RNA and DNA amounting to about 11% of the total cellular nucleic acid. In contrast, crystalline trypsin released only 1% of the cellular nucleic acids. Since virtually all the cells (99%) after harvesting in crystalline trypsin were impermeable to Trypan Blue, this method was suitable for obtaining cell surface macromolecules without contamination by intracellular damage. [1-(14)C]Glucosamine was incorporated by the cells only into bound hexosamines and sialic acids. [By monitoring the release of radioactivity in high-molecular-weight material in such experiments a measure of the release of macromolecules containing amino sugars was obtained.] Of the total macromolecules containing amino sugars in the cells 33%, 24% and 13% were released when the cells were harvested with crude trypsin, crystalline trypsin or EDTA respectively. Crystalline trypsin also released 39% of the total sialic acid of the cell, whereas less than 1% of the cellular sialic acid was present in the EDTA-treated fraction. It is concluded that the macromolecules containing amino sugars released with crude trypsin and EDTA are likely to be heavily contaminated with intracellular material. However, the macromolecules released by crystalline trypsin appear to come from the cell surface.     

The 14-3-3 proteins in regulation of cellular metabolism

Thirty years ago, it was discovered that 14-3-3 proteins could activate enzymes involved in amino acid metabolism. In the following decades, 14-3-3s have been shown to be involved in many different signaling pathways that modulate cellular and whole body energy and nutrient homeostasis. Large scale screening for cellular binding partners of 14-3-3 has identified numerous proteins that participate in regulation of metabolic pathways, although only a minority of these targets have yet been subject to detailed studies. Because of the wide distribution of potential 14-3-3 targets and the resurging interest in metabolic pathway control in diseases like cancer, diabetes, obesity and cardiovascular disease, we review the role of 14-3-3 proteins in the regulation of core and specialized cellular metabolic functions. We cite illustrative examples of 14-3-3 action through their direct modulation of individual enzymes and through regulation of master switches in cellular pathways, such as insulin signaling, mTOR- and AMP dependent kinase signaling pathways, as well as regulation of autophagy. We further illustrate the quantitative impact of 14-3-3 association on signal response at the target protein level and we discuss implications of recent findings showing 14-3-3 protein membrane binding of target proteins.     

Metabolic activation of the pneumotoxin, 3-methylindole, by vaccinia-expressed cytochrome P450s

Twelve human cytochrome P450s and one mouse P450 were produced in HepG2 cells using vaccinia virus cDNA expression and analyzed for their ability to bioactivate the pneumotoxin, 3-methylindole (3MI), to an electrophilic metabolite(s) which alkylated cellular macromolecules. Cell lysates containing CYP2C8, CYP3A4, CYP2A6 and CYP2F1 metabolized 3MI to an intermediate(s) that became covalently bound to lysate material. A control lysate produced from cells which had been infected with a wild-type vaccinia virus was not able to bioactivate 3MI. The mouse 1A2 enzyme metabolized 3MI at a rate of 75.4 pmol/mg protein/minute, while the rate of metabolism in the lysate containing the human 1A2 P450 enzyme was not different from that in the control lysate. Therefore, the catalytic capabilities of orthologous P450 enzymes to activate 3MI cannot be extrapolated among different species. These results indicate that human P450s are capable of bioactivating 3MI to a metabolite which binds to cellular macromolecules suggesting that this compound may be toxic to humans.     

Role of deoxyribonucleic acid ligase in a doxyribonucleic acid membrane fraction extracted from pneumococci.

Deoxyribonucleic acid (DNA) ligase has been detected in a DNA membrane fraction extracted from Pneumococcus. The specific activity of the enzyme in this fraction is 10-fold greater than in the remaining cell extract. It remains firmly bound (with other enzymes) to the complex after a purification procedure in which a considerable percentage of the macromolecules are dissociated. The ligase acts in two ways in the DNA membrane fraction in vitro. One, it catalyzes the linkage of small-molecular-weight pieces of newly synthesized DNA into heavier-molecular-weight DNA strands as shown by others (M Gellert, 1976; R. Okazaki, A. Sugino, S. Hirose, T. Okazaki, Y. Imae, R. Kainuma-Kuroda, T. Ogawa, M. Arisawa, and Y. Kurosowa, 1973; B. Olivera and I. Lehman, 14; and A. Sugino, S. Hirose, and R. Okazaki, 1972) and, two, it protects DNA from degradation by deoxyribonucleases. This latter effect is due to a competition between the ability of the nucleases to degrade DNA and the ability of DNA ligase to seal the nicks produced by these degradative enzymes. The ligase acts cooperatively with other enzymes in the DNA membrane fraction to synthesize DNA.     

Processing of somatostatin-28 to somatostatin-14 by rat hypothalamic synaptosomal membranes

The conversion of synthetic somatostatin-28 (S-28) to somatostatin- 14 (S-14, SRIF) by subcellular fractions of rat hypothalamus has been investigated. The conversion products were identified by two techniques: (1) two separate RIAs using antibodies directed toward the central (RIA R149) or the N- terminal (RIA S39) region of the S-14 molecule, (2) gel chromatography of the reaction mixture followed by analysis of the column fractions by RIA R149. Maximal S-28 to S-14 converting activity was observed with the particulate fraction of the lysed synaptosomal pellet sedimenting at the density interface 8–16% Ficoll in 0.32 M sucrose in a discontinuous sucrose/ Ficoll gradient. Concomitant with conversion, degradation of total somatostatin-like immunoreactivity (SLI) was also observed with this fraction ($\text{t}\text{1}\text{2}\text{～ 24}\text{min}$). Relatively little converting activity was found in the remaining subcellular fractions. These data suggest that hypothalamic synaptosomes contain membrane bound enzymes which are able to catalyze the conversion of S-28 to S-14. Tissue specific differences in this converting activity may account for the reported variability in the S-28:S-14 ratios in different tissues.     

The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated Rho kinase signalling

A yeast two-hybrid screen performed to identify binding partners of the CaR (calcium-sensing receptor) intracellular tail identified the adaptor protein 14-3-3θ as a novel binding partner that bound to the proximal membrane region important for CaR expression and signalling. The 14-3-3θ protein directly interacted with the CaR tail in pull-down studies and FLAG-tagged CaR co-immunoprecipitated with EGFP (enhanced green fluorescent protein)-tagged 14-3-3θ when co-expressed in HEK (human embryonic kidney)-293 or COS-1 cells. The interaction between the CaR and 14-3-3θ did not require a putative binding site in the membrane-proximal region of the CaR tail and was independent of PKC (protein kinase C) phosphorylation. Confocal microscopy demonstrated co-localization of the CaR and EGFP-14-3-3θ in the ER (endoplasmic reticulum) of HEK-293 cells that stably expressed the CaR (HEK-293/CaR cells), but 14-3-3θ overexpression had no effect on membrane expression of the CaR. Overexpression of 14-3-3θ in HEK-293/CaR cells attenuated CaR-mediated Rho signalling, but had no effect on ERK (extracellular-signal-regulated kinase) 1/2 signalling. Another isoform identified from the library, 14-3-3ζ, exhibited similar behaviour to that of 14-3-3θ with respect to CaR tail binding, cellular co-localization and impact on receptor-mediated signalling. However, unlike 14-3-3θ, this isoform, when overexpressed, significantly reduced CaR plasma membrane expression. Results indicate that 14-3-3 proteins mediate CaR-dependent Rho signalling and may modulate the plasma membrane expression of the CaR.     

Structure-function analysis of CD14 as a soluble receptor for lipopolysaccharide

CD14 is a glycophosphatidylinositol-linked protein expressed by myeloid cells and also circulates as a plasma protein lacking the glycophosphatidylinositol anchor. Both membrane and soluble CD14 function to enhance activation of cells by lipopolysaccharide (LPS), which we refer to as receptor function. We have previously reported the LPS binding and cell activation functions of a group of five deletion mutants of CD14 (Viriyakosol, S., and Kirkland, T.N. (1995) J. Biol. Chem. 270, 361-368). We have now studied the functional impact of these mutations on soluble CD14. We found that some deletions that abrogated LPS binding in membrane CD14 have no effect on LPS binding in soluble CD14. In fact, some of the soluble CD14 deletion mutants bound LPS with an apparent higher affinity than wild-type CD14. Furthermore, we found that all five deletions essentially ablated soluble CD14 LPS receptor function, whereas only two of the deletions completely destroyed membrane CD14 LPS receptor function. Some of the mutants were able to compete with wild-type CD14 in soluble CD14-dependent assays of cellular activation. We concluded that the soluble and membrane forms of CD14 have different structural determinants for LPS receptor function.     

Release of anchored membrane enzymes by lipoamidase

Lipoamidase is found to be able to release various membrane-anchored enzymes from the membrane compartment of pig brain. Released enzymes revealed their intact enzyme activities in the soluble fraction. Lipoamidase could release at least two types of anchored enzymes, i.e. glycosyl-phosphatidylinositol-bonded and myristoylated enzymes, but not integral membrane bound enzymes. The reaction was competitively inhibited by lipoyllysine. This releasing mechanism found in the membranes may play important roles in the secretory mechanism of extracellular enzymes and also in the cellular signal-transduction system through topological changes in cellular enzymes.     

Functional and biochemical disposition of 7,12-dimethylbenz[a]anthracene in murine lymphoid cells

The metabolism of the polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA) was studied in murine lymphocytes. This carcinogen has previously been shown to be immunosuppressive to lymphocytes regardless of their ability to be induced via the Ah locus and receptor. Experiments were designed to quantify the generation of metabolites of DMBA by lymphocytes incubated with [14C]DMBA and to ascertain whether radioactivity was covalently bound to cellular macromolecules in DMBA-exposed lymphocytes. No significant metabolism of DMBA was detected in culture supernatants, except when cultures were incubated in the presence of Arochlor-induced rat liver 9000 x g supernatants (S9). Covalent binding of 14C to cellular macromolecules was enhanced approximately eightfold in the presence of S9. Inhibition of monooxygenase activity by alpha-naphthoflavone did not modulate the immunosuppressive character of DMBA. Furthermore, addition of S9 did not amplify or ablate DMBA-mediated suppression of lymphocyte proliferation to the mitogen concanavalin A (Con A). Selected metabolites of DMBA were evaluated for immunosuppressive effects in cultures stimulated with mitogens and cellular alloantigens. 7-Hydroxymethyl-12-methylbenz[a]anthracene (OHMe) and 5,6-dihydro-5,6-dihydroxybenz[a]anthracene (Diol) were found to cause only slightly greater suppression of lymphocyte responses than DMBA. Thus, it appears that metabolites of DMBA were not responsible for the immunosuppression observed in lymphocyte cultures and that lymphocytes were not equipped to metabolize any significant amount of DMBA. These data lend support to the hypothesis that parent compound alone is responsible for the immunosuppressive effects observed in murine lymphocyte culture.     

Evidence for an in vivo modification of mitochondrial proteins by coenzyme A.

Following denaturation of mitochondrial proteins by sodium dodecyl sulfate, a [1-14C]pantothenic acid-derived radioactivity proved to be acid precipitable in the outer membrane, the intermembrane space, the inner membrane and in the matrix of rat liver mitochondria, where it had the highest specific radioactivity of 541 +/- 29 cpm/100 micrograms protein. This tightly and/or covalently bound protein radioactivity could be released by incubation in the presence of dithioerythreitol; it was identified as [14C]coenzyme A by its HPLC retention time, its absorption spectrum and its radioactivity. This acid-stable and thiol-labile coenzyme A-binding apparently refers to specific protein binding sites. With the purified, homogeneous mitochondrial matrix enzymes acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase) (EC 2.3.1.9, acetyl-CoA:acetyl-CoA C-acetyltransferase) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16) coenzyme A was found exclusively, e.g., in the modified, partially-active forms A1 und A2 of acetyl-CoA acetyltransferase and not in the unmodified fully-active enzyme. Thus it is evident that this coenzyme A modification is transient. We suggest that coenzyme A-modification is a signal involved in the assembly or the degradation process of distinct mitochondrial matrix proteins.     

Nucleotide sequence of nuclear 5.7S RNA of mouse cells

Viable tumor cells were examined by ¹⁹F-NMR spectroscopy after treatment with 4-trifluoromethyl 2,6-dinitrophenyl sulphonate (CF₃-DNBS), which is an analog of 2,4,6-trinitrophenyl sulphonate (TNBS). The presence of a strong ¹⁹F-NMR signal from treated cells suggested the binding of the “probe”. Treatment of labelled cells with proteolytic enzymes significantly decreased the signals, suggesting that the label was essentially bound to the cell surface macromolecules. A proportion of the material bound to the cell was removable by dialysis of cell extracts against a structural analog, suggesting that some CF₃-DNBS-cell membrane bonds were not covalent. The existence of such non-covalent bonds has also been confirmed with soluble proteins. By the same approach, it was also found that ¹⁴C-TNBS formed covalent and non-covalent bonds with tumor cells.     

14-3-3 regulates actin dynamics by stabilizing phosphorylated cofilin

The functionality of the actin cytoskeleton depends on a dynamic equilibrium between filamentous and monomeric actin. Proteins of the ADF/cofilin family are essential for the high rates of actin filament turnover observed in motile cells through regulation of actin polymerization/depolymerization cycles. Rho GTPases act through p21-activated kinase-1 (Pak-1) and Rho kinase to inhibit cofilin activity via the LIM kinase (LIMK)-mediated phosphorylation of cofilin on Ser3. We report the identification of 14-3-3zeta as a novel phosphocofilin binding protein involved in the maintenance of the cellular phosphocofilin pool. A Ser3 phosphocofilin binding protein was purified from bovine brain and was identified as 14-3-3zeta by mass spectrometry. The phosphorylation-dependent interaction between cofilin and 14-3-3zeta was confirmed in pulldown and coimmunoprecipitation experiments. Both Ser3 phosphorylation and a 14-3-3 recognition motif in cofilin are necessary for 14-3-3 binding. The expression of 14-3-3zeta increases phosphocofilin levels, and the coexpression of 14-3-3zeta with LIMK further elevates phosphocofilin levels and potentiates LIMK-dependent effects on the actin cytoskeleton. This potentiation of cofilin action appears to be a result of the protection of phosphocofilin from phosphatase-mediated dephosphorylation at Ser3 by bound 14-3-3zeta. Taken together, these results suggest that 14-3-3zeta proteins may play a dynamic role in the regulation of cellular actin structures through the maintenance of phosphocofilin levels.     

Preferential in vitro binding of high mobility group proteins 14 and 17 to nucleosomes containing active and DNase I sensitive single-copy genes

High mobility group (HMG) proteins 14 and 17 bind to mononucleosomes in vitro, but the exact nature of this binding has not been clearly established. A new method was developed to allow direct membrane transfer of DNA from HMG 14/17 bound and unbound nucleosomes, which have been separated by acrylamide gel electrophoresis. Hybridization analysis of membranes obtained by this method revealed that the HMG 14/17 bound nucleosomes of avian erythrocytes and rat hepatic tumor (HTC) cells were enriched, about 2-fold, in actively transcribed genes and also inactive but DNase I sensitive genes. Nucleosomes containing inactive, DNase I resistant genes were bound by HMG 14/17, but not preferentially. Several factors that have been reported to greatly influence the binding of HMG 14/17 to nucleosomes in vitro were tested and shown to not account for the preferential binding to DNase I sensitive chromatin. These factors include nucleosomal linker DNA length, single-stranded DNA nicks, and DNA bulk hypomethylation. An additional factor, histone acetylation, was preferentially associated with the HMG 14/17 bound chromatin fraction of avian erythrocytes, but it was not associated with the HMG 14/17 bound chromatin fraction of metabolically active HTC cells. The latter finding was true for all kinetic forms of histone acetylation.     

Key residues of loop 3 in the interaction with the interface residue at position 14 in triosephosphate isomerase from Trypanosoma brucei

Cysteine 14 is an interface residue that is fundamental for the catalysis and stability of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM). Its side chain is surrounded by a deep pocket of 11 residues that are part of loop 3 of the adjacent monomer. Mutation of this residue to serine (producing single mutant C14S) yields a wild-type-like enzyme that is resistant to the action of sulfhydryl reagents methylmethane thiosulfonate (MMTS) and 5,5-dithiobis(2-nitrobenzoate) (DTNB). This mutant enzyme was a starting point for probing by cysteine scanning the role of four residues of loop 3 in the catalysis and stability of the enzyme. Considering that the conservative substitution of either serine or alanine with cysteine would minimally alter the structure and properties of the environment of the residue in position 14, we made double mutants C14S/A69C, C14S/S71C, C14S/A73C, and C14S/S79C. Three of these double mutants were similar in their kinetic parameters to wild-type TbTIM and the single mutant C14S, but double mutant C14S/A73C showed a greatly reduced k cat. All enzymes had similar CD spectra, but all mutants had thermal stabilities lower than that of wild-type TbTIM. Intrinsic fluorescence was also similar for all enzymes, but the double mutants bound up to 50 times more 1-anilino-8-naphthalene sulfonate (ANS) and were susceptible to digestion with subtilisin. The double mutants were also susceptible to inactivation by sulfhydryl reagents. Double mutant C14S/S79C exhibited the highest sensitivity to MMTS and DTNB, bound a significant amount of ANS, and had the highest sensitivity to subtilisin. Thus, the residues at positions 73 and 79 are critical for the catalysis and stability of TbTIM, respectively.     

The chemistry of phospholipid binding by the Saccharomyces cerevisiae phosphatidylinositol transfer protein Sec14p as determined by EPR spectroscopy

The major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p is the founding member of a large eukaryotic protein superfamily. Functional analyses indicate Sec14p integrates phospholipid metabolism with the membrane trafficking activity of yeast Golgi membranes. In this regard, the ability of Sec14p to rapidly exchange bound phospholipid with phospholipid monomers that reside in stable membrane bilayers is considered to be important for Sec14p function in cells. How Sec14p-like proteins bind phospholipids remains unclear. Herein, we describe the application of EPR spectroscopy to probe the local dynamics and the electrostatic microenvironment of phosphatidylcholine (PtdCho) bound by Sec14p in a soluble protein-PtdCho complex. We demonstrate that PtdCho movement within the Sec14p binding pocket is both anisotropic and highly restricted and that the C5 region of the sn-2 acyl chain of bound PtdCho is highly shielded from solvent, whereas the distal region of that same acyl chain is more accessible. Finally, high field EPR reports on a heterogeneous polarity profile experienced by a phospholipid bound to Sec14p. Taken together, the data suggest a headgroup-out orientation of Sec14p-bound PtdCho. The data further suggest that the Sec14p phospholipid binding pocket provides a polarity gradient that we propose is a primary thermodynamic factor that powers the ability of Sec14p to abstract a phospholipid from a membrane bilayer.