An intermediate filament-associated protein, p50, recognized by monoclonal antibodies.

Monoclonal antibodies (mAB) were raised to be used as probes to identify cytoplasmic components associated with intermediate filaments (IF). Four hybridomas (B27, B76, B78, and B100) secreting mAB were generated by fusing mouse myeloma cells with the spleen cells of mice immunized intraperitoneally with Triton-high salt insoluble materials from BHK-21 cells. This insoluble material consists mostly of IF, a small number of microfilaments, and some polyribosomes. Biochemical studies show that the Triton-insoluble materials contain many proteins, including vimentin (decamin) and desmin. Immunofluorescence microscopy of BHK-21 cells stained with the four mAB showed that these mAB decorate the IF in a dotted pattern. Double staining with polyclonal antibody to vimentin confirmed the reactivity of the mAB with the IF. These mAB also stained the vimentin-containing filament system in a variety of other cells including epithelial cells (PTK1 and HeLa) and cells of astroglial origin. Histological studies showed that mAB-B100 stained many types of tissue including epidermis, smooth muscle, and subdermis pericytes, but not the white matter nor the gray matter of the cerebellum and spinal cord. Immunoelectron microscopy with colloidal gold has shown that the mAB-B100 decorated the IF in clusters or aggregates around proteinaceous materials associated with the filaments. Results of immunoprecipitation indicate that mAB-B100 reacted with a protein of 50,000 daltons. These findings suggest that the mAB-B100 we have developed recognizes one of the many components of what appears to be an integrated cytoskeletal structure connected with intermediate filaments.

Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein.

The transforming protein of Rous sarcoma virus, p60src, was shown to be acylated at its amino terminus with the long-chain fatty acid myristic acid by isolation of a tryptic peptide with the following structure: myristylglycylserylseryllysine. The occurrence of this unusual posttranslational modification in the cyclic adenosine monophosphate-dependent protein kinase and in several transforming protein kinases of mammalian retroviruses suggests that myristylation of the amino terminal glycyl residue may be critical for the function of certain proteins related to cell transformation and growth control.

N-terminal deletions in Rous sarcoma virus p60src: effects on tyrosine kinase and biological activities and on recombination in tissue culture with the cellular src gene.

We have constructed deletions within the region of cloned Rous sarcoma virus DNA coding for the N-terminal 30 kilodaltons of p60src. Infectious virus was recovered after transfection. Deletions of amino acids 15 to 149, 15 to 169, or 149 to 169 attenuated but did not abolish transforming activity, as assayed by focus formation and anchorage-independent growth. These deletions also had only slight effects on the tyrosine kinase activity of the mutant src protein. Deletion of amino acids 169 to 264 or 15 to 264 completely abolished transforming activity, and src kinase activity was reduced at least 10-fold. However, these mutant viruses generated low levels of transforming virus by recombination with the cellular src gene. The results suggest that as well as previously identified functional domains for p60src myristylation and membrane binding (amino acids 1 to 14) and tyrosine kinase activity (amino acids 250 to 526), additional N-terminal sequences (particularly amino acids 82 to 169) can influence the transforming activity of the src protein.

Processing of p60v-src to its myristylated membrane-bound form.

p60src of wild-type Rous sarcoma virus is myristylated at its N-terminal glycine residue. We have shown previously that this myristylation is necessary for p60src membrane association and for cell transformation by using src mutants with alterations within the N-terminal 30 kilodaltons of p60src. In this study we analyzed the process of p60src myristylation in wild type- and mutant-infected cells. All myristylated src proteins examined lack the initiator methionine, but two mutant src proteins lacking the initiator methionine are not myristylated, indicating that removal of the initiator methionine and myristylation are not obligatorily coupled. Analysis of the kinetics of myristylation and the association of p60src with cellular proteins p50 and p90 indicated that myristylation occurs before p60src becomes membrane associated and that transient association with p50 and p90 occurs regardless of myristylation. Myristylation is required for stable association of p60src with the plasma membrane but is not sufficient for membrane association. A mutant with an src deletion of amino acids 169 through 264 has an src protein that is myristylated but not membrane bound, remaining stably associated with p50 and p90. This mutant is transformation defective. Several N-terminal deletion mutants possessing tyrosine kinase activity have myristylated and membrane-bound src proteins but are not fully active in cell transformation, suggesting that additional N-terminal functional domains exist.

Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src.

We have previously found that Rous sarcoma virus variants in which the viral src (v-src) gene is replaced by the cellular src (c-src) gene have no transforming activity. In this study, we analyzed the basis for the inability of the p60c-src overproduced by these variants to transform cells. Phosphorylations of tyrosine residues in total cell protein or in cellular 34K protein are known to be markedly enhanced upon infection with wild-type Rous sarcoma virus. We found that these tyrosine phosphorylations were only slightly increased in the c-src-containing virus-infected cells, whereas both levels were significantly increased by infection with wild-type Rous sarcoma virus, or transforming mutant viruses which are derived from c-src-containing viruses by spontaneous mutation. Phosphorylation at tyrosine 416 of p60 itself was also extremely low in overproduced p60c-src and high in p60s of transforming mutant viruses. In immunoprecipitates with monoclonal antibody, the overproduced p60c-src had much lower casein tyrosine kinase activity than did p60v-src. We previously showed that p60 myristylation and plasma membrane localization may be required for cell transformation. p60c-src was similar to transforming p60s in these properties. These results strongly suggest that the low level of tyrosine phosphorylation by overproduced p60c-src accounts for its inability to transform cells.

A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation.

We have constructed mutants by using linker insertion followed by deletion in the region of cloned Rous sarcoma virus DNA coding for the N-terminal 9 kilodaltons of the src protein. Previous work implicated this region in the membrane association of the protein. The mutations had little effect on src tyrosine kinase activity. Substitution of a tri- or tetrapeptide for amino acids 15 to 27, 15 to 49, or 15 to 81 had little effect on the in vitro transforming capacity of the virus. Like wild-type p60src, the src proteins of these mutants associated with plasma membranes and were labeled with [3H]myristic acid. In contrast, a mutant whose src protein had the dipeptide Asp-Leu substituted for amino acids 2 to 81 and a mutant with the tripeptide Asp-Leu-Gly substituted for amino acids 2 to 15 were transformation defective, and the mutant proteins did not associate with membranes and were not labeled with [3H]myristic acid. These results suggest that amino acids 2 to 15 serve as an attachment site for myristic acid and as a membrane anchor. Since deletions including this region prevent transformation, and since tyrosine kinase activity is not diminished by the deletions, these results imply that target recognition is impaired by mutations altering the very N terminus, perhaps through their effect on membrane association.

Size-variant pp60src proteins of recovered avian sarcoma viruses interact with adhesion plaques as peripheral membrane proteins: effects on cell transformation.

We have shown previously that the membrane association of the src proteins of recovered avian sarcoma viruses (rASVs) 1702 (56 kilodaltons) and 157 (62.5 kilodaltons), whose size variations occur within 8 kilodaltons of the amino terminus, is salt sensitive and that, in isotonic salt, these src proteins fractionate as soluble cytoplasmic proteins. In contrast, wild-type Rous sarcoma virus pp60src behaves as an integral plasma membrane protein in cellular fractionation studies and shows prominent membrane interaction by immunofluorescence microscopy. In this study we have examined the distribution of these size-variant src proteins between free and complexed forms, their subcellular localization by immunofluorescence microscopy, and their ability to effect several transformation-related cell properties. Glycerol gradient sedimentation of extracts from cells infected either with rASV 1702 or rASV 157 showed that soluble src proteins of these viruses were distributed between free and complexed forms as has been demonstrated for wild-type Rous sarcoma virus pp60src. Pulse-chase studies with rASV pp60src showed that, like wild-type Rous sarcoma virus pp60src, it was transiently found in a complexed form. Indirect immunofluorescence showed that size-variant pp60src proteins are localized in adhesion plaques and regions of cell-to-cell contact in rASV 1702- or 157-infected cells. This result is in contrast with the generalized localization of pp60src in plasma membranes of control rASV-infected cells which produce pp60src. Chicken embryo fibroblasts infected by rASVs 1702 and 157 display a partial-transformation phenotype with respect to (i) transformation-related morphology, (ii) cell surface membrane changes, and (iii) retained extracellular fibronectin. It is possible that the induction of a partial-transformation phenotype may be the result of the unique interaction of the src proteins encoded by these viruses with restricted areas of the plasma membrane.

Reduced synthesis of pp60src and expression of the transformation-related phenotype in interferon-treated Rous sarcoma virus-transformed rat cells.

Treatment of Rous sarcoma virus-transformed rat cells with rat interferon-alpha (specific activity, 10(6) U/mg of protein) for 24 h caused a 50% reduction in intracellular pp60src-associated protein kinase activity. Staphylococcus aureus V8 protease digestion of pp60src, derived from 32P-labeled monolayer cultures incubated with or without interferon, revealed no differences either in the phosphopeptide pattern or in the phosphoserine-phosphotyrosine ratio. However, [3H]leucine pulse-labeling experiments showed that the synthesis of pp60src was reduced by 42 to 48%, relative to the level of bulk protein synthesis, in the interferon-treated cultures. Rat interferon-alpha also reduced the growth rate of Rous sarcoma virus-transformed rat cells in a dose-dependent manner over a 72-h period. The decrease in growth rate was accompanied by increases in the thickness and number of actin fibers per cell and by a decline in intracellular tyrosine phosphorylation by pp60src. The results suggest that interferon can inhibit the expression of the transformation-related phenotype by selectively reducing the synthesis of the Rous sarcoma virus transforming gene product. However, the interferon effects on the cytoskeletal organization and proliferation of Rous sarcoma virus-transformed cells may be due at least in part to the predominance of interferon-induced phenotypic changes over those caused by pp60src.

Circular dichroism studies of the binding of mammalian and non-mammalian cytochromes c to cytochrome c oxidase, cytochrome c peroxidase, and polyanions.

The effects of binding of Candida krusei, Drosophila melanogaster, horse, human, and rat cytochromes c to beef cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC 1.9.3.1) and yeast cytochrome c peroxidase (ferricytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) on their circular dichroism spectra were determined. The binding to cytochrome oxidase results in a positive increase in the ellipticities of the positive and negative Cotton effects at 404 nm and 417 nm of cytochrome c. The horse, human, and rat cytochromes c display less of an increase in the ellipticity of the positive Cotton effect at 404 nm, but more of a positive change in the negative Cotton effect at 417 nm than the C. krusei or D. melanogaster proteins. Interaction with yeast cytochrome c peroxidase elicits only a positive change in the ellipticity of the positive Cotton effect at 404 nm. No significant change is observed in the negative Cotton effect at 417 nm. Rat cytochrome c variants with a phenylalanine in place of tyrosine-67 and/or an alanine in place of proline-30 all display circular dichroism spectral changes upon binding to cytochrome c oxidase or cytochrome c peroxidase identical to those of the unaltered protein. The increase in ellipticity at 404 nm upon binding occurs even though replacement of tyrosine-67 results in the loss of the positive Cotton effect at this position. Polyglutamate and phosvitin complexes of cytochrome c show changes in the circular dichroism spectrum similar to those observed with cytochrome c peroxidase. However, the magnitudes of the spectral changes were considerably less. A model is proposed in which the main cause of the circular dichroism spectral changes observed upon complexation arise from the exclusion of solvent from the exposed front heme edge. According to this model, the exclusion of solvent changes the relative asymmetry of the environment of the electronic transitions of the heme prosthetic group of cytochrome c, resulting in observed circular dichroic effects.

Interaction of cytochrome c with cytochrome c oxidase: an understanding of the high- to low-affinity transition.

The steady-state kinetics of high- and low-affinity electron transfer reactions between various cytochromes c and cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) preparations were studied spectrophotometrically and polarographically. The dissociation constants for the binding of the first and second molecules of horse cytochrome c (I = 15 mM) are 5.10(-8) M and 1.10(-5) M, respectively, close to the spectrophotometric Km values and consistent with the controlled binding model for the interaction between cytochrome c and cytochrome oxidase (Speck, S.H., Dye, D. and Margoliash, E. (1984) Proc. Natl. Acad. Sci. USA 81, 346-351) which postulates that the binding of a second molecule of cytochrome c weakens that of the first, resulting in low-affinity kinetics. While the Km of the polarographically assayed high-affinity reaction is comparable to that observed spectrophotometrically, the low-affinity Km is over an order of magnitude smaller and cannot be attributed to the binding of a second molecule of cytochrome c. Increasing the viscosity has no effect on the Vmax of the low-affinity reaction assayed polarographically, but increases the Km. Thus, the transition from high- to low-affinity kinetics is dependent on the frequency of productive collisions, as expected for a hysteresis model ascribing the transition to the trapping of the oxidase in a primed state for turnover. At ionic strengths above 150 mM, the rate of cytochrome c oxidation decreases without any correlation to the calculated net charge of the cytochrome c, indicating rate-limiting rearrangement of the two proteins in proximity to each other.

A chemical modification of cytochrome-c lysines leading to changes in heme iron ligation.

Although 13 lysines of horse cytochrome c are invariant, and three more are extremely conserved, the modification of their side-chain epsilon-amino groups by beta-thiopropionylation caused important changes in protein properties for only three of them; lysines 72,73 and 79. Optical spectroscopy, electron and nuclear paramagnetic resonance, electron spin echo envelope modulation, and molecular weight studies, as well as the unique features of their reaction with cytochrome-c oxidase, indicate that in the oxidized state the modification of these lysines resulted in equilibria between two different states of iron ligation: the native state, in which the metal is coordinated by the methionine-80 sulfur, and a new state in which this ligand is displaced by the sulfhydryl groups of the elongated side chains. The reduction potentials of the TP Lys-72 and the TP Lys-79 derivatives were 201 and 196 millivolt, respectively, indicating that the equilibria favored the sulfhydryl ligated state by 1.5 and 1.7 kcal/mol, respectively. In the ferric state, the protein modified at lysine 72 remained stable as a monomer, but that modified at lysine 73 dimerized rapidly through disulfide bond formation, while the TP Lys-79 cytochrome c dimerized with a half-time of approx. 3 h, both recovering the native-like iron ligation. By contrast, in the ferrous state the monomeric state and the native ligation were preserved in all cases, indicating that the affinity of the cytochrome-c ferrous iron for the methionine-80 sulfur is particularly strong. The dimerized derivatives lost most, but not all, of the capability of the native protein for electron transfer from ascorbate-TMPD to cytochrome-c oxidase.

Comparative biological responses to human Sonic, Indian, and Desert hedgehog.

A comprehensive comparison of Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehog biological activities has not previously been undertaken. To test whether the three higher vertebrate Hh proteins have distinct biological properties, we compared recombinant forms of the N-terminal domains of human Shh, Ihh, and Dhh in a variety of cell-based and tissue explant assays in which their activities could be assessed at a range of concentrations. While we observed that the proteins were similar in their affinities for the Hh-binding proteins; Patched (Ptc) and Hedgehog-interacting protein (Hip), and were equipotent in their ability to induce Islet-1 in chick neural plate explant; there were dramatic differences in their potencies in several other assays. Most dramatic were the Hh-dependent responses of C3H10T1/2 cells, where relative potencies ranged from 80nM for Shh, to 500nM for Ihh, to >5microM for Dhh. Similar trends in potency were seen in the ability of the three Hh proteins to induce differentiation of chondrocytes in embryonic mouse limbs, and to induce the expression of nodal in the lateral plate mesoderm of early chick embryos. However, in a chick embryo digit duplication assay used to measure polarizing activity, Ihh was the least active, and Dhh was almost as potent as Shh. These findings suggest that a mechanism for fine-tuning the biological actions of Shh, Ihh, and Dhh, exists beyond the simple temporal and spatial control of their expression domains within the developing and adult organism.

Regular articles: conditional disruption of hedgehog signaling pathway defines its critical role in hair development and regeneration.

Members of the vertebrate hedgehog family (Sonic, Indian, and Desert) have been shown to be essential for the development of various organ systems, including neural, somite, limb, skeletal, and for male gonad morphogenesis. Sonic hedgehog and its cognate receptor Patched are expressed in the epithelial and/or mesenchymal cell components of the hair follicle. Recent studies have demonstrated an essential role for this pathway in hair development in the skin of Sonic hedgehog null embryos. We have further explored the role of the hedgehog pathway using anti-hedgehog blocking monoclonal antibodies to treat pregnant mice at different stages of gestation and have generated viable offspring that lack body coat hair. Histologic analysis revealed the presence of ectodermal placode and primodium of dermal papilla in these mice, yet the subsequent hair shaft formation was inhibited. In contrast, the vibrissae (whisker) development appears to be unaffected upon anti-hedgehog blocking monoclonal antibody treatment. Strikingly, inhibition of body coat hair morphogenesis also was observed in mice treated postnatally with anti-hedgehog monoclonal antibody during the growing (anagen) phase of the hair cycle. The hairless phenotype was reversible upon suspension of monoclonal antibody treatment. Taken together, our results underscore a direct role of the Sonic hedgehog signaling pathway in embryonic hair follicle development as well as in subsequent hair cycles in young and adult mice. Our system of generating an inducible and reversible hairless phenotype by anti-hedgehog monoclonal antibody treatment will be valuable for studying the regulation and mechanism of hair regeneration.

Recombinant antibodies: a new reagent for biological agent detection.

Antibodies are critical reagents used in several biodetection platforms for the identification of biological agents. Recent advances in phage display technology allow isolation of high affinity recombinant antibody fragments (Fabs) that may bind unique epitopes of biological threat agents. The versatility of the selection process lends itself to efficient screening methodologies and can increase the number of antigen binding clones that can be isolated. Pilot scale biomanufacturing can then be used for the economical production of these immunoglobulin reagents in bacterial fermentation systems, and expression vectors with hexahistidine tags can be used to simplify downstream purification. One such Fab reagent directed against botulinum neurotoxin A/B has been shown to be sensitive in a variety of assay formats including surface plasmon resonance (SPR), flow cytometry, enzyme linked immunosorbent assay (ELISA), and hand-held immunochromatographic assay. Recombinant antibodies can provide another source of high quality detection reagents in our arsenal to identify or detect pathogens in environmental samples.

NH2-terminal sequences of two src proteins that cause aberrant transformation.

Two isolates of recovered avian sarcoma viruses (rASVs), rASV157 and rASV1702, transform cells in culture, but have greatly reduced in vivo tumorigenicity. The src proteins of rASV157 and rASV1702 have alterations within their NH2 termini, are not myristoylated, and have an altered subcellular localization. We have molecularly cloned and determined the nucleotide sequences of the src genes of rASV157 and rASV1702. We found that their src proteins have unusual NH2 termini: the rASV157 src protein NH2 terminus consists of 30 amino acids of the env signal peptide attached to Ser-6 of the src sequence, while the rASV1702 src protein NH2 terminus consists of 45 amino acids of the env signal peptide attached to Ala-76 of the src sequence. Expression of recombinant Rous sarcoma virus constructs containing the molecularly cloned rASV src genes produced src proteins with the same properties as those of the parental viruses. Our results suggest that the NH2-terminal structures are responsible for many unusual properties of the mutant src proteins.

Positional isotopic equivalence of nitrogen in N2O produced by the denitrifying bacterium Pseudomonas stutzeri. Indirect evidence for a nitroxyl pathway.

During the concomitant reduction of [15N]nitrite and 14NO, the denitrifying bacterium Pseudomonas stutzeri produced considerable amounts of isotopically mixed N2O (14,15N2O) but did not isotopically intermix the nitrite and NO pools (Garber, E. A. E., and Hollocher, T. C. (1981) J. Biol. Chem. 256, 5459-5465). It was determined that the mass spectrometric abundance of 14N15NO was about equal to that of 15N14NO in 14,15N2O by examination of the abundances of 14NO+, 15NO+, and 15N2+ that arose from the fragmentation of N2O+ species in the mass spectrometer. This positional isotopic equivalence requires that N2O arose from [15N]nitrite and 14NO by a process in which loss of oxygen occurred with equal probability from 15N and 14N precursors and suggests that at some point the precursors were identical if monomeric or effectively symmetrical if dimeric. One pathway, which is consistent with the available data and for which there is chemical precedence, is the reduction of nitrite and NO to nitroxyl (NOH or HNO), dimerization of free nitroxyl to a dinitrogen intermediate of short half-life, and dehydration of this intermediate to form N2O.

CD154 variant lacking tumor necrosis factor homologous domain inhibits cell surface expression of wild-type protein.

X-linked hyper-IgM (XHIM) syndrome is an immunological disorder resulting from mutations in the CD154 gene. Some mutations occur in splicing sites and result in transcripts encoding wild-type and mutant proteins. These mutants lack the tumor necrosis factor homologous (TNFH) domain and consequently fail to trimerize. Given that the TNFH domain is responsible for trimerization, one may predict that the TNFH mutant can not participate in the assembly of wild-type CD154. Thus, it was puzzling why these patients exhibit XHIM phenotype, presumably resulting from a lack of functional CD154. One possibility is that the TNFH mutant exhibits a dominant negative effect over the wild-type protein. To investigate this, we coexpressed the wild-type protein and a TNFH mutant and examined the biochemical and functional properties of the resulting CD154 products. We demonstrate that despite the lack of the TNFH domain, the TNFH mutant can associate with the wild-type protein. Furthermore, such an association compromises the ability of the wild-type protein to mature onto the cell surface. These results provide a mechanism for the defect of CD154 in XHIM patients producing both wild-type and TNFH variants and suggest that besides the TNFH domain, the stalk region participates in the assembly of CD154 trimers.

15N-tracer and NMR studies on the pathway of denitrification. Evidence against trioxodinitrate but for nitroxyl as an intermediate.

The effects of four species of denitrifying bacteria on the conversion of [15N]nitrite to trioxodinitrate (HN2O3-) and N2O and of trioxodinitrate to N2O were studied. For all species, the N2O produced in the presence of [15N]nitrite and trioxodinitrate was isotopically randomized throughout the period of incubation and was not composed at the outset predominantly of 14N2O or 14N2O plus 15N2O. The N2O produced was also heavily enriched in 15N at times when the trioxodinitrate pool was only weakly enriched in 15N. By 15N NMR, the N(2) position, but not the N(1) position, of trioxodinitrate was found to become progressively labeled with 15N during incubation with [15N]nitrite. These results argue that (a) the N-N bond of trioxodinitrate is not preserved in its conversion to N2O, (b) trioxodinitrate can be neither a free nor enzyme-bound intermediate in denitrifying bacteria, and (c) the pathways from nitrite and trioxodinitrate involve a common mononitrogen intermediate. The conclusion that this intermediate is probably nitroxyl (HNO), at least with Paracoccus denitrificans and Pseudomonas stutzeri, provides indirect evidence that N-N bond formation in denitrification can occur through the dimerization of nitroxyl.

Membrane association of the transforming protein of avian sarcoma virus UR2 and mutants temperature sensitive for cellular transformation and protein kinase activity.

The localization of the transforming protein P68gag-ros of avian sarcoma virus UR2, which has a hydrophobic region at the N terminus of its ros-specific tyrosine kinase-encoding sequence, was examined by subcellular fractionation. P68 behaved as an integral membrane protein associated with the plasma membrane of transformed cells. P68 became membrane associated very rapidly in its biogenesis. Three temperature-sensitive mutants of UR2 were isolated and characterized. Cells infected with the mutants were temperature sensitive for morphological alteration and colony formation. The mutant P68 proteins were membrane associated in mutant-infected cells regardless of the temperature but were active as protein kinases only at the permissive temperature. The results suggest that P68 is a membrane-associated protein whose kinase activity plays a crucial role in UR2-mediated cell transformation.