Chemical specificity and physical properties of the lipid bilayer in the regulation of protein kinase C by anionic phospholipids: evidence for the lack of a specific binding site for phosphatidylserine.

The association of protein kinase C (PKC) with membranes was found not to be specific for phosphatidyl-L-serine (PS). In particular, a synthetic phospholipid, dansyl-phosphatidylethanolamine, proved to be fully functional in the association of PKC with lipid bilayers and in mediating the interaction of this enzyme with diacylglycerol. Dansyl-phosphatidylethanolamine was also able to activate the enzyme in a Ca2+-dependent fashion. Differences in the ability to bind and activate PKC observed for an array of anionic lipids were not larger than alterations caused by changes in acyl chain composition. Thus, although different lipids interact to different extents with PKC, there are no specific binding sites for the PS headgroup on the enzyme. We found that lipids with a greater tendency to form inverted phases increased the binding of PKC to bilayers. However, these changes in lipid structure cannot be considered separately from the miscibility of lipid components in the membrane. For pairs of lipids with similar acyl chains, the dependence on PS concentration is sigmoidal, while for dissimilar acyl chains there is much less dependence of binding on PS concentration. The results can be explained in terms of differences in the lateral distribution of components in the membrane.

Core binding factor beta-smooth muscle myosin heavy chain chimeric protein involved in acute myeloid leukemia forms unusual nuclear rod-like structures in transformed NIH 3T3 cells.

Patients with the M4Eo subtype of acute myeloid leukemia almost invariably are found to have an inversion of chromosome 16 in their leukemic cells, which results in a gene fusion between the transcription factor called core binding factor beta (CBFbeta) on 16q and a smooth muscle myosin heavy chain (SMMHC) gene on 16p. Subcellular localizations of the wild-type CBFbeta and the CBFbeta-SMMHC fusion protein were determined by immunofluorescence of NIH 3T3 cells that overexpress wild-type or fusion protein. Normal CBFbeta showed an unexpected perinuclear pattern consistent with primary localization in the Golgi complex. The CBFbeta-SMMHC fusion protein had a very different pattern. Nuclear staining included rod-like crystalline structures as long as 11 microm. The heterodimeric partner of CBFbeta, CBFalpha, formed part of this complex. Cytoplasmic staining included stress fibers that colocalized with actin, probably as a consequence of the myosin heavy chain component of the fusion protein. Deletion of different regions of the CBFbeta portion of the fusion protein showed that binding to CBFalpha was not required for nuclear translocation. However, deletion of parts of the SMMHC domain of the fusion protein involved in myosin-mediated filament formation resulted in proteins that did not form rod-like structures. These observations confirm previous indirect evidence that the CBFbeta-SMMHC fusion protein is capable of forming macromolecular nuclear aggregates and suggests possible models for the mechanism of leukemic transformation.

Binding of the sigma 70 protein to the core subunits of Escherichia coli RNA polymerase, studied by iron-EDTA protein footprinting.

We have used a nonspecific protein cleaving reagent to map the interactions between subunits of the multisubunit enzyme RNA polymerase (Escherichia coli). We developed suitable conditions for using an untethered Fe-EDTA reagent, which does not bind significantly to proteins. Comparison of the cleaved fragments of the subunits from the core enzyme (alpha 2 beta beta') and the holoenzyme (core+sigma 70) shows that absence of the sigma 70 subunit is associated with the appearance of several cleavage sites on the subunits beta (within 10 residues of sequence positions 745, 764, 795, and 812) and beta' (within 10 residues of sequence positions 581, 613, and 728). A cleavage site near beta residue 604 is present in the holoenzyme but absent in the core, demonstrating that a conformational change occurs when sigma 70 binds. No differences are observed for the alpha subunit.

X-ray structure of a vanadium-containing enzyme: chloroperoxidase from the fungus Curvularia inaequalis.

The chloroperoxidase (EC 1.11.1.-) from the fungus Curvularia inaequalis belongs to a class of vanadium enzymes that oxidize halides in the presence of hydrogen peroxide to the corresponding hypohalous acids. The 2.1 A crystal structure (R = 20%) of an azide chloroperoxidase complex reveals the geometry of the catalytic vanadium center. Azide coordinates directly to the metal center, resulting in a structure with azide, three nonprotein oxygens, and a histidine as ligands. In the native state vanadium will be bound as hydrogen vanadate(V) in a trigonal bipyramidal coordination with the metal coordinated to three oxygens in the equatorial plane, to the OH group at one apical position, and to the epsilon 2 nitrogen of a histidine at the other apical position. The protein fold is mainly alpha-helical with two four-helix bundles as main structural motifs and an overall structure different from other structures. The helices pack together to a compact molecule, which explains the high stability of the protein. An amino acid sequence comparison with vanadium-containing bromoperoxidase from the seaweed Ascophyllum nodosum shows high similarities in the regions of the metal binding site, with all hydrogen vanadate(V) interacting residues conserved except for lysine-353, which is an asparagine.

The peptide binding site of the substance P (NK-1) receptor localized by a photoreactive analogue of substance P: presence of a disulfide bond.

Substance P (SP) is a neuropeptide that mediates multiple physiological responses including transmission of painful stimuli and inflammation via an interaction with a receptor of known primary sequence. To identify the regions of the SP receptor, also termed the NK-1 receptor, involved in peptide recognition, we are using analogues of SP containing the photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa). In the present study, we used radioiodinated Bpa8-SP to covalently label with high efficiency the rat SP receptor expressed in a transfected mammalian cell line. To identify the amino acid residue that serves as the site of covalent attachment, a membrane preparation of labeled receptor was subjected to partial enzymatic cleavage by trypsin. A major digestion product of 22 kDa was identified. Upon reduction with 2-mercaptoethanol the mass of this product decreased to 14 kDa. The 22-kDa tryptic fragment was purified in excellent yield by preparative SDS/PAGE under nonreducing conditions. Subcleavage with Staphylococcus aureus V8 protease and endoproteinase ArgC yielded fragments of 8.2 and 9.0 kDa, respectively. Upon reductive cleavage, the V8 protease fragment decreased to 3.0 kDa while the endoproteinase ArgC fragment decreased to 3.2 kDa. Taking into consideration enzyme specificity, molecular size, determination of the presence or absence of N-glycosylation sites, and recognition by antibodies to specific sequences of the SP receptor, the V8 protease fragment is Thr-173 to Glu-183, while the endoproteinase ArgC fragment is Val-178 to Arg-190. These two fragments share the common sequence Val-Val-Cys-Met-Ile-Glu (residues 178-183). The site of covalent attachment of radioiodinated Bpa8-SP is thus restricted to a residue within this overlap sequence. The data presented here also establish that the cysteine residue in this sequence Cys-180, which is positioned in the middle of the second extracellular loop, participates in a disulfide bond that links the first and second extracellular loops of the receptor.

Phosphinate analogs of D-, D-dipeptides: slow-binding inhibition and proteolysis protection of VanX, a D-, D-dipeptidase required for vancomycin resistance in Enterococcus faecium.

VanX is a D-Ala-D-Ala dipeptidase that is essential for vancomycin resistance in Enterococcus faecium. Contrary to most proteases and peptidases, it prefers to hydrolyze the amino substrate but not the related kinetically and thermodynamically more favorable ester substrate D-Ala-D-lactate. The enzymatic activity of VanX was previously found to be inhibited by the phosphinate analogs of the proposed tetrahedral intermediate for hydrolysis of D-Ala-D-Ala. Here we report that such phosphinates are slow-binding inhibitors. D-3-[(1-Aminoethyl)phosphinyl]-D-2-methylpropionic acid I showed a time-dependent onset of inhibition of VanX and a time-dependent return to uninhibited steady-state rates upon dilution of the enzyme/inhibitor mixture. The initial inhibition constant Ki after immediate addition of VanX to phosphinate I to form the E-I complex is 1.5 microM but is then lowered by a relatively slow isomerization step to a second complex, E-I*, with a final K*i of 0.47 microM. This slow-binding inhibition reflects a Km/K*i ratio of 2900:1. The rate constant for the slow dissociation of complex E-I* is 0.24 min-1. A phosphinate analog with an ethyl group replacing what would be the side chain of the second D-alanyl residue in the normal tetrahedral adduct gives a K*i value of 90 nM. Partial proteolysis of VanX reveals two protease-sensitive loop regions that are protected by the intermediate analog phosphinate, indicating that they may be part of the VanX active site.

Mutagenesis of palmitoylation sites in endothelial nitric oxide synthase identifies a novel motif for dual acylation and subcellular targeting.

The endothelial nitric oxide synthase (ec-NOS) plays a key role in the transduction of signals from the bloodstream to the underlying smooth muscle. ecNOS undergoes a complex series of covalent modifications, including myristoylation and palmitoylation, which appear to play a role in ecNOS membrane association. Mutagenesis of the myristoylation site, which prevents both myristoylation and palmitoylation, blocks ecNOS targeting to cell membranes. Further, as described for some G-protein alpha subunits, both membrane association and palmitoylation of ecNOS are dynamically regulated: in response to agonists, the enzyme undergoes partial redistribution to the cell cytosol concomitant with depalmitoylation. To clarify the role of palmitoylation in determining ecNOS subcellular localization, we have constructed palmitoylation-deficient mutants of ecNOS. Serine was substituted for cysteine at two potential palmitoylation sites (Cys-15 and Cys-26) by site-directed mutagenesis. Immunoprecipitation of ecNOS mutants following cDNA transfection and biosynthetic labeling with [3H]palmitate revealed that mutagenesis of either cysteine residue attenuated palmitoylation, whereas replacement of both residues completely eliminated palmitoylation. Analysis of N-terminal deletion mutations of ecNOS demonstrated that the region containing these two cysteine residues is both necessary and sufficient for enzyme palmitoylation. The cysteines thus identified as the palmitoylation sites for ecNOS are separated by an unusual (Gly-Leu)5 sequence and appear to define a sequence motif for dual acylation. We analyzed the subcellular distribution of ecNOS mutants by differential ultracentrifugation and found that mutagenesis of the ecNOS palmitoylation sites markedly reduced membrane association of the enzyme. These results document that ecNOS palmitoylation is an important determinant for the subcellular distribution of ecNOS and identify a new motif for the reversible palmitoylation of signaling proteins.

Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) is expressed in the adrenal cortex and gonads and regulates the expression of several P450 steroid hydroxylases in vitro. We examined the role of SF-1 in the adrenal glands and gonads in vivo by a targeted disruption of the mouse SF-1 gene. All SF-1-deficient mice died shortly after delivery. Their adrenal glands and gonads were absent, and persistent Mullerian structures were found in all genotypic males. While serum levels of corticosterone in SF-1-deficient mice were diminished, levels of adrenocorticotropic hormone (ACTH) were elevated, consistent with intact pituitary corticotrophs. Intrauterine survival of SF-1-deficient mice appeared normal, and they had normal serum level of corticosterone and ACTH, probably reflecting transplacental passage of maternal steroids. We tested whether SF-1 is required for P450 side-chain-cleavage enzyme (P450scc) expression in the placenta, which expresses both SF-1 and P450scc, and found that in contrast to its strong activation of the P450scc gene promoter in vitro, the absence of SF-1 had no effect on P450scc mRNA levels in vivo. Although the region targeted by our disruption is shared by SF-1 and by embryonal long terminal repeat-binding protein (ELP), a hypothesized alternatively spliced product, we believe that the observed phenotype reflects absent SF-1 alone, as PCR analysis failed to detect ELP transcripts in any mouse tissue, and sequences corresponding to ELP are not conserved across species. These results confirm that SF-1 is an important regulator of adrenal and gonadal development, but its regulation of steroid hydroxylase expression in vivo remains to be established.

Peptide conjugation to an in vitro-selected DNA ligand improves enzyme inhibition.

An in vitro selection technique was used to identify a specific high-affinity DNA ligand targeted to human neutrophil elastase (HNE). 1H NMR data and a comparative analysis of the selected sequences suggest that the DNA folds into a G-quartet structure with duplexed ends. The high-affinity binding DNA alone did not inhibit the enzymatic activity of HNE. The DNA was covalently attached to a tetrapeptide, N-methoxysuccinyl-Ala-Ala-Pro-Val, that is a weak competitive inhibitor of HNE. HNE was inhibited by this DNA-peptide conjugate nearly five orders of magnitude more effectively than by the peptide alone. These results demonstrate that in vitro-selected nucleic acids can be used as a vehicle for molecular delivery.

Forced evolution of glutathione S-transferase to create a more efficient drug detoxication enzyme.

Glutathione S-transferases (EC 2.5.1.18) in mammalian cells catalyze the conjugation, and thus, the detoxication of a structurally diverse group of electrophilic environmental carcinogens and alkylating drugs, including the antineoplastic nitrogen mustards. We proposed that structural alteration of the nonspecific electrophile-binding site would produce mutant enzymes with increased efficiency for detoxication of a single drug and that these mutants could serve as useful somatic transgenes to protect healthy human cells against single alkylating agents used in cancer chemotherapy protocols. Random mutagenesis of three regions (residues 9-14, 102-112, and 210-220), which together compose the glutathione S-transferase electrophile-binding site, followed by selection of Escherichia coli expressing the enzyme library with the nitrogen mustard mechlorethamine (20-500 microM), yielded mutant enzymes that showed significant improvement in catalytic efficiency for mechlorethamine conjugation (up to 15-fold increase in kcat and up to 6-fold increase in kcat/Km) and that confer up to 31-fold resistance, which is 9-fold greater drug resistance than that conferred by the wild-type enzyme. The results suggest a general strategy for modification of drug- and carcinogen-metabolizing enzymes to achieve desired resistance in both prokaryotic and eukaryotic plant and animal cells.

The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.

Phosphorylation of G-protein-coupled receptors plays an important role in regulating their function. In this study the G-protein-coupled receptor phosphatase (GRP) capable of dephosphorylating G-protein-coupled receptor kinase-phosphorylated receptors is described. The GRP activity of bovine brain is a latent oligomeric form of protein phosphatase type 2A (PP-2A) exclusively associated with the particulate fraction. GRP activity is observed only when assayed in the presence of protamine or when phosphatase-containing fractions are subjected to freeze/thaw treatment under reducing conditions. Consistent with its identification as a member of the PP-2A family, the GRP is potently inhibited by okadaic acid but not by I-2, the specific inhibitor of protein phosphatase type 1. Solubilization of the membrane-associated GRP followed by gel filtration in the absence of detergent yields a 150-kDa peak of latent receptor phosphatase activity. Western blot analysis of this phosphatase reveals a likely subunit composition of AB alpha C. PP-2A of this subunit composition has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed. The subcellular distribution and substrate specificity of the GRP suggests significant differences between it and previously characterized forms of PP-2A.

Proenzyme of Manduca sexta phenol oxidase: purification, activation, substrate specificity of the active enzyme, and molecular cloning.

Phenol oxidase (PO) was isolated as a proenzyme (pro-phenol oxidase, pro-PO) from the hemolymph of Manduca sexta larvae and purified to homogeneity. Pro-PO exhibits a M(r) of 130,000 on gel filtration and two bands with an apparent M(r) of approximately 100,000 on SDS/PAGE, as well as size-exclusion HPLC. Activation of pro-PO was achieved either by specific proteolysis by a cuticular protease or by the detergent cetylpyridinium chloride at a concentration below the critical micellar concentration. A cDNA clone for M. sexta pro-PO was obtained from a larval hemocyte cDNA library. The clone encodes a polypeptide of approximately 80,000 Da that contains two copper-binding sites and shows high sequence similarity to POs, hemocyanins, and storage proteins of arthropods. The M. Sexta pro-PO, together with other arthropod pro-POs, contains a short stretch of amino acids with sequence similarity to the thiol ester region of alpha-macroglobulins and complement proteins C3 and C4.

Cell cycle-related shifts in subcellular localization of BCR: association with mitotic chromosomes and with heterochromatin.

The disruption of the BCR gene and its juxtaposition to and consequent activation of the ABL gene has been implicated as the critical molecular defect in Philadelphia chromosome-positive leukemias. The normal BCR protein is a multifunctional molecule with domains that suggest its participation in phosphokinase and GTP-binding pathways. Taken together with its localization to the cytoplasm of uncycled cells, it is therefore presumed to be involved in cytoplasmic signaling. By performing a double aphidicolin block for cell cycle synchronization, we currently demonstrate that the subcellular localization of BCR shifts from being largely cytoplasmic in interphase cells to being predominantly perichromosomal in mitosis. Furthermore, with the use of immunogold labeling and electron microscopy, association of BCR with DNA, in particular heterochromatin, can be demonstrated even in quiescent cells. Results were similar in cell lines of lymphoid or myeloid origin. These observations suggest a role for BCR in the phosphokinase interactions linked to condensed chromatin, a network previously implicated in cell cycle regulation.