Antipeptide antiserum identifies a widely distributed cellular tyrosine kinase related to but distinct from the c-fps/fes-encoded protein.

We raised antibodies directed against a synthetic peptide representing an amino acid sequence of the conserved kinase domain of the transforming protein of Fujinami sarcoma virus (FSV) (P140). The antiserum obtained specifically recognized FSV-P140 and its cellular homolog and in addition, it recognized a new cellular protein of 94,000 daltons (NCP94) in avian and mammalian cells. NCP94 was found to be associated with a cyclic nucleotide-independent protein kinase activity that was specific for tyrosine residues. Although NCP94 and FSV-P140 share antigenic determinants, NCP94 is not a cellular homolog of FSV-P140: NCP94 and the previously identified c-fps/fes product were different in their tryptic fingerprints and in their tissue specificities. Thus, the function of NCP94 in normal cells is probably different than that of the c-fps/fes product. NCP94 was expressed in every tissue and cell line that was examined. In chickens, NCP94 levels were highest during embryonic development and NCP94 expression was high in gizzard, brain, and spleen throughout embryonic and adult life. The universal expression of NCP94 suggests that this protein may be involved in an essential function of normal cells. NCP94 may be a new cellular tyrosine kinase of the src gene family.     

Identification in chickens of an evolutionarily conserved cellular ets-2 gene (c-ets-2) encoding nuclear proteins related to the products of the c-ets proto-oncogene.

In chicken cells, we previously identified a set of proteins (p58-64) structurally related to, but distinct from, the products encoded by the c-ets proto-oncogene. We report here the isolation and nucleotide sequence of a cDNA encoding nuclear products of mol. wt 58, 60, 62 and 64 kd, indistinguishable from those detected in chicken cells. The p60 and p64 species appear to represent phosphorylated versions on serine and threonine residues of p58 and p62. The homology of p58-64 to other ets-related proteins, including the v-ets encoded domain of the transforming protein of avian leukemia virus E26 and p54c-ets, the translation product of the chicken (Ck) c-ets gene, is confined to two regions of 175 and 96 amino acid residues localized respectively at the carboxy-terminal domain and close to the amino-terminal domain of these molecules. This cDNA corresponds to a gene localized in a locus distinct from that of c-ets which is transcribed as a 4.0-kb RNA species in most chicken tissues. We also identified the human (Hu) c-ets-2-encoded products as two proteins of 60 and 62 kd, highly related to chicken p58-64. This, together with the fact that the amino acid sequence of the cDNA encoding p58-64 is 95% identical to the reported partial sequence of a Hu-c-ets-2 cDNA, indicates that p58-64 are the translation products of the Ck-c-ets-2 gene.     

Identification of cellular proteins binding to the scrapie prion protein

The scrapie prion protein (PrPSc) is an abnormal isoform of the cellular protein PrPc. PrPSc is found only in animals with scrapie or other prion diseases. The invariable association of PrPSc with infectivity suggests that PrPSc is a component of the infectious particle. In this study, we report the identification of two proteins from hamster brain of 45 and 110 kDa (denoted PrP ligands Pli 45 and Pli 110) which were able to bind to PrP 27-30, the protease-resistant core of PrPSc on ligand blots. Pli 45 and Pli 110 also bound PrPC. Both Pli's had isoelectric points of approximately 5. The dissociation rate constant of the Pli 45/PrP 27-30 complex was 3 x 10(-6) s-1. Amino acid and protein sequence analyses were performed on purified Pli 45. Both the composition and the sequence were almost identical with those predicted for mouse glial fibrillary acidic protein (GFAP). Furthermore, antibodies to Pli 45 reacted with recombinant GFAP. The identification of proteins which interact with the PrP isoforms in normal and diseased brain may provide new insights into the function of PrPC and into the molecular mechanisms underlying prion diseases.     

Identification and cellular distribution of distinct proteins forming human GM-CSF receptor.

Two proteins forming the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF)1 were identified and characterized. One with apparent Mr of about 80,000 was defined as alpha-chain and has Kd of 0.7-2.8 nM. The other binding molecule with apparent Mr of about 135,000 was defined as beta-chain and is related to the high-affinity binding with Kd of 10-40 pM. The binding kinetic studies confirmed that the 125I-GM-CSF associated slower to and dissociated more rapidly from the alpha-chain than the beta-chain. The alpha-chain is expressed not only on hemopoietic cells but also on full-term placental tissues, choriocarcinoma cells, and other solid tumor cells. In contrast, the distribution of the beta-chain is restricted on hemopoietic cells. The alpha-chain probably corresponds to the low-affinity GM-CSF receptor whose cDNA has been cloned and sequenced.     

Cloning of ovocalyxin-36, a novel chicken eggshell protein related to lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and plunc family proteins

The avian eggshell is a composite biomaterial composed of noncalcifying eggshell membranes and the overlying calcified shell matrix. The shell is deposited in a uterine fluid where the concentration of different protein species varies at different stages of its formation. The role of avian eggshell proteins during shell formation remains poorly understood, and we have sought to identify and characterize the individual components in order to gain insight into their function during elaboration of the eggshell. In this study, we have used direct sequencing, immunochemistry, expression screening, and EST data base mining to clone and characterize a 1995-bp full-length cDNA sequence corresponding to a novel chicken eggshell protein that we have named Ovocalyxin-36 (OCX-36). Ovocalyxin-36 protein was only detected in the regions of the oviduct where egg-shell formation takes place; uterine OCX-36 message was strongly up-regulated during eggshell calcification. OCX-36 localized to the calcified eggshell predominantly in the inner part of the shell, and to the shell membranes. BlastN data base searching indicates that there is no mammalian version of OCX-36; however, the protein sequence is 20-25% homologous to proteins associated with the innate immune response as follows: lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and Plunc family proteins. Moreover, the genomic organization of these proteins and OCX-36 appears to be highly conserved. These observations suggest that OCX-36 is a novel and specific chicken eggshell protein related to the superfamily of lipopolysaccharide-binding proteins/bactericidal permeability-increasing proteins and Plunc proteins. OCX-36 may therefore participate in natural defense mechanisms that keep the egg free of pathogens.     

A yeast protein related to a mammalian Ras-binding protein, Vps9p, is required for localization of vacuolar proteins.

In the yeast Saccharomyces cerevisiae, mutations in vacuolar protein sorting (VPS) genes result in secretion of proteins normally localized to the vacuole. Characterization of the VPS pathway has provided considerable insight into mechanisms of protein sorting and vesicle-mediated intracellular transport. We have cloned VPS9 by complementation of the vacuolar protein sorting defect of vps9 cells, characterized its gene product, and investigated its role in vacuolar protein sorting. Cells with a vps9 disruption exhibit severe vacuolar protein sorting defects and a temperature-sensitive growth defect at 38 degrees C. Electron microscopic examination of delta vps9 cells revealed the appearance of novel reticular membrane structures as well as an accumulation of 40- to 50-nm-diameter vesicles, suggesting that Vps9p may be required for the consumption of transport vesicles containing vacuolar protein precursors. A temperature-conditional allele of vps9 was constructed and used to investigate the function of Vps9p. Immediately upon shifting of temperature-conditional vps9 cells to the nonpermissive temperature, newly synthesized carboxypeptidase Y was secreted, indicating that Vps9p function is directly required in the VPS pathway. Antibodies raised against Vps9p immunoprecipitate a rare 52-kDa protein that fractionates with cytosolic proteins following cell lysis and centrifugation. Analysis of the VPS9 DNA sequence predicts that Vps9p is related to human proteins that bind Ras and negatively regulate Ras-mediated signaling. We term the related regions of Vps9p and these Ras-binding proteins a GTPase binding homology domain and suggest that it defines a family of proteins that bind monomeric GTPases. Vps9p may bind and serve as an effector of a rab GTPase, like Vps2lp, required for vacuolar protein sorting.     

Two distinct mechanisms regulate the levels of a cellular tumor antigen, p53.

The steady-state levels of p53 protein and p53 mRNA in transformed and nontransformed cells were examined to elucidate the mechanisms controlling expression of p53. mRNA levels were determined by Northern blot hybridization analysis, employing a p53-specific cDNA clone (M. Oren and A.J. Levine, Proc. Natl. Acad. Sci. U.S.A. 80:56-59, 1983), and protein levels were determined by the Western blotting technique. Analysis of p53 mRNA revealed a single polyadenylated mRNA species migrating at ca. 18S. Levels of p53 mRNA in simian virus 40-transformed cell line (SVT2) and in an homologous nontransformed cell line (3T3) were equivalent, although the steady-state levels of p53 protein were 25- to 100-fold higher in the SVT2 cells than in the 3T3 cells. A study with a non-virus-transformed cell system revealed a different result. Embryonal carcinoma cells (F9) were found to have nearly 20-fold higher levels of p53 mRNA in comparison with differentiated benign progeny cells. In this system the difference in p53 mRNA levels corresponded to the difference in p53 protein levels. Pulse-chase experiments were performed to study the half-life of p53 protein in these four types of cells. The turnover of p53 protein occurred with biphasic kinetics. In addition, it was found that protein synthesis inhibitors placed in the medium during the chase period prevented the turnover of p53 protein in transformed cells, but not in nontransformed (3T3) cells. These results provide evidence that the regulation of p53 expression in cells can occur at the level of p53 mRNA abundancy or p53 protein stability depending upon the experimental system under study, and that a regulated degradation process controls the turnover of p53 protein.     

Purification of p100, a protein antigenically related to the signal transducing G proteins Gt and Gi. Evidence for an adaptin-like protein.

A 100-kDa protein, termed p100, cross-reacts with antisera raised against a synthetic peptide corresponding to the carboxyl-terminal decapeptide of the alpha-subunit of the retinal G protein Gt. p100 is abundantly expressed in liver and, on subcellular fractionation of rat liver homogenates, is distributed between the cytosolic and microsome fractions (Traub, L. M., Evans, W. H., and Sagi-Eisenberg, R. (1990) Biochem. J. 272, 453-458; Udrisar, D., and Rodbell, M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6321-6325). We have now purified p100 to near-homogeneity from rat liver microsomes. The protein was purified approximately 500-fold by ATP extraction followed by a series of four chromatographic steps. Similar to partially purified p100, on two-dimensional electrophoresis, the final preparation contained a major series of five immunoreactive 100-kDa charge isoforms. Partial amino terminus amino acid sequencing of the purified protein revealed that p100 is a previously unidentified protein. Further analysis of the soluble form of p100 showed the protein migrated with an apparent molecular weight of approximately 110,000 on gel filtration, indicating that the soluble protein occurs as a monomeric polypeptide. The soluble form of p100 was also partially purified from rat liver cytosol and amino acid sequencing yielded the same amino-terminal sequence as obtained from the microsome-associated form. The amino-terminal sequence of p100 exhibits significant similarity to the deduced amino-terminal amino acid sequences of both alpha- and gamma-adaptins. Using the amino-terminal sequence from p100, we have raised antipeptide polyclonal antisera. The antisera reacted specifically with the purified 100-kDa protein on immunoblots. With the purified protein and specific antisera now available, it will be possible to explore the physiological role of p100.     

Identification of chURP, a nuclear calmodulin-binding protein related to hnRNP-U.

In a screen for myosin-like proteins in embryonic chicken brain, we have identified a novel nuclear protein structurally related to hnRNP-U (heterogeneous nuclear ribonuclear protein U). We have called this protein chURP, for chicken U-related protein. In this screen, chURP was immunoreactive with two myosin antibodies and, in common with the unconventional myosins, bound calmodulin in vitro in both the presence and absence of calcium ions. Determination of 757 amino acids of the chURP sequence revealed that it shares 41% amino acid identity with human and rat hnRNP-U, although chURP and hnRNP-U appear not to be orthologous proteins. ChURP is ubiquitously expressed in the nuclei of all chick tissues and, as one of a growing number of calmodulin-binding proteins to be identified in the nucleus, further highlights the potential of calmodulin as a regulator of nuclear metabolism.     

Identification of the first human gyrovirus, a virus related to chicken anemia virus

We have identified in a skin swab sample from a healthy donor a new virus that we have named human gyrovirus (HGyV) because of its similarity to the chicken anemia virus (CAV), the only previously known member of the Gyrovirus genus. In particular, this virus encodes a homolog of the CAV apoptin, a protein that selectively induces apoptosis in cancer cells. By PCR screening, HGyV was found in 5 of 115 other nonlesional skin specimens but in 0 of 92 bronchoalveolar lavages or nasopharyngeal aspirates and in 0 of 92 fecal samples.     

Identification and molecular characterisation of a biotin-binding protein distinct from avidin of chicken egg white and comparison with yolk biotin-binding protein

By immunological and biochemical methods a biotin-binding protein, distinct from avidin, has been shown to be present in chicken egg white. This vitamin-binding protein (Mr 67,000) bound [14C]biotin, displayed thermally induced biotin exchange reaction and exhibited gross immunological cross-reactivity with the purified yolk biotin-binding protein. In vitro labelling of soluble proteins with radioactive amino acids in the oviduct tissue explants from estrogenised chicks revealed that approx. 2% of the total radioactive proteins was immunoprecipitated with anti-yolk biotin-binding protein antibodies. The protein could be purified to homogeneity by employing ion-exchange chromatography on DEAE-cellulose and biotin-AH Sepharose affinity chromatography. The purified protein specifically bound [14C]biotin, and exhibited complete immunological homology with the yolk biotin-binding protein but not with avidin. Its electrophoretic mobility (at pH 8.3), acidic nature, biotin-binding characteristics, immunological cross-reactivity and tryptic peptide maps were very similar to that of yolk biotin-binding protein, and not avidin.     

Identification of a human cDNA encoding a novel protein structurally related to the yeast membrane-associated metalloprotease, Ste24p

Recently, a novel membrane-associated metalloprotease, designated Ste24p, has been identified in Saccharomyces cerevisiae [K. Fujimura-Kamada, F.J. Nouvet, S. Michaelis, J. Cell Biol. 27 (1997) 271-285]. We cloned a human brain cDNA encoding a protein homologous to Ste24p (designated Hs Ste24p). The predicted 475-amino acid product of its open reading frame exhibited 62% similarity to Ste24p, and contained a zinc metalloprotease motif (HEXXH) and multiple predicted membrane spans. Northern blot analysis showed that this gene was expressed in most tissues. Immunofluorescence analysis of epitope-tagged Hs Ste24p constructs suggested that it is localized in the ER and possibly also in the Golgi compartment. A search of the expression sequence tag database identified a fragment of DNA encoding a segment homologous to the segment of Hs Ste24p containing the HEXXH motif in insects and nematodes. Thus, Hs Ste24p could be a member of a new family of Ste24p-like membrane-associated metalloproteases which are widely conserved in eukaryotes.     

Identification of cooperative folding units in a set of native proteins.

Cooperative unfolding penalties are calculated by statistically evaluating an ensemble of denatured states derived from native structures. The ensemble of denatured states is determined by dividing the native protein into short contiguous segments and defining all possible combinations of native, i.e., interacting, and non-native, i.e., non-interacting, segments. We use a novel knowledge-based scoring function, derived from a set of non-homologous proteins in the Protein Data Bank, to describe the interactions among residues. This procedure is used for the structural identification of cooperative folding cores for four globular proteins: bovine pancreatic trypsin inhibitor, horse heart cytochrome c, French bean plastocyanin, and staphylococcal nuclease. The theoretical folding units are shown to correspond to regions that exhibit enhanced stability against denaturation as determined from experimental hydrogen exchange protection factors. Using a sequence similarity score for related sequences, we show that, in addition to residues necessary for enzymatic function, those amino acids comprising structurally important folding cores are also preferentially conserved during evolution. This implies that the identified folding cores may be part of an array of fundamental structural folding units.     

Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go.

A guanine-nucleotide-binding protein (G-protein) was purified from cholate extracts of bovine brain membranes by sequential DEAE-Sephacel, Ultrogel AcA-34, heptylamine-Sepharose and Sephadex G-150 chromatography. Guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S])-binding activity copurified with a 25,000 Da peptide and a 35,000-36,000 Da protein doublet. Neither pertussis toxin nor cholera toxin catalysed the ADP-ribosylation of a protein associated with the GTP[35S]-binding activity. Photoaffinity labelling of the purified protein with 8-azido[gamma-32P]GTP indicated that the GTP-binding site resides on the 25,000 Da protein. The 35,000-36,000 Da protein doublet was electrophoretically indistinguishable from the beta-subunits of other GTP-binding proteins, and the 36,000 Da protein was recognized by antiserum to oligomeric Gt. The purified protein specifically bound 17.2 nmol of GTP[35S]/mg of protein. The Kd of the binding site for radioligand was approx. 15 nM. The brain GTP-binding protein co-migrated during SDS/polyacrylamide-gel electrophoresis with a GTP-binding protein, named Gp, purified from human placenta [Evans, Brown, Fraser & Northup (1986) J. Biol. Chem. 261, 7052-7059], and cross-reacted with antiserum raised against the placental protein, but not with antiserum raised to brain Go. SDS/polyacrylamide-gel electrophoresis of the brain and placental GTP-binding proteins in the presence of Staphylococcus aureus V8 protease yielded identical peptide maps.     

Identification of the proteins related to p53-mediated radioresponse in nasopharyngeal carcinoma by proteomic analysis

Radiotherapy is the primary treatment for nasopharyngeal cancer (NPC), and p53 is closely associated with the radiosensitivity of cancer, but the molecular mechanisms of p53-mediated radioresponse in NPC remains unclear. We previously established NPC CNE2sip53 cell line with p53 knockdown and paired control cell line CNE2/pSUPER, which provides a cell model system to investigate mechanisms of p53-mediated radioresponse in NPC. In this study, we first compared the radiosensitivity of CNE2sip53 and CNE2/pSUPER by a clonogenic survival assay, cell growth assay, and Hoechst 33258 staining and flow cytometry analysis of apoptotic cells. The results showed that the radiosensitivity of CNE2sip53 was significantly lower than that of CNE2/pSUPER, indicating that p53 plays a role in mediating NPC radiosensitivity. To search for the proteins associated with the p53-mediated radioresponse in NPC, a proteomic approach was performed to identify the radioresponsive proteins in CNE2sip53 and CNE2p/SUPER, respectively, and then the difference of radioresponsive proteins in CNE2sip53 and CNE2p/SUPER was compared. As a result, 14 differential radioresponsive proteins were identified in the two cell lines, 4 proteins of which were conformed by Western blot. Among them, 9 and 5 proteins were identified solely from CNE2p/SUPER and CNE2sip53, respectively. Furthermore, protein-protein interaction analysis showed that 7 differential radioresponsive proteins identified only in CNE2p/SUPER were related to p53 protein. Our results suggest that the differential radioresponsive proteins unique to CNE2p/SUPER may be involved in p53-mediated radioresponse in NPC, which will be helpful for elucidating the mechanisms of p53-mediated NPC cellular response to radiotherapy.     

The receptor for heat shock protein 60 on macrophages is saturable, specific, and distinct from receptors for other heat shock proteins.

Previous studies have shown that human heat shock protein (hsp) 60 elicits a strong proinflammatory response in cells of the innate immune system with CD14, Toll-like receptor (TLR) 2, and TLR4 as mediators of signaling, but probably not of binding. In the present study, we directly demonstrate binding of hsp60 to the macrophage surface and find the binding receptor for hsp60 different from the previously described common receptor for several other heat shock proteins, including hsp70, hsp90, and gp96. Fluorescence-labeled human hsp60 bound to cell surfaces of the murine macrophage lines J774 A.1 and RAW264.7 and to mouse bone marrow-derived macrophages. By flow cytometry, we could demonstrate for the first time that hsp60 binding to macrophages occurred at submicromolar concentrations, is saturable, and can be competed by unlabeled hsp60, but not by unrelated proteins, thus confirming the classic characteristics of specific ligand-receptor interactions. Binding of hsp60 at 4 degrees C was followed by endocytosis at 37 degrees C. Hsp60 binding to macrophages could not be competed by excess hsp70, hsp90, or gp96, all of which share the alpha(2)-macroglobulin receptor as binding site. Hsp60 binding occurred in the absence of surface TLR4. However, no cytokine response was induced by hsp60 in TLR4-deficient macrophages. We conclude that hsp60 binds to a stereo-specific receptor on macrophages, and that different surface molecules are engaged in binding and signal transduction. Furthermore, the binding site for hsp60 is separate from the common receptor for hsp70, hsp90, and gp96, which suggests an independent role of hsp60 as danger Ag and in immunoregulation.     

Identification of N-acetylglucosamine-binding immunoglobulins in chicken egg yolk and serum distinct from the major mannose-binding immunoglobulins.

An N-acetyl-D-glucosamine (GlcNAc)-binding protein of 170 kDa has been isolated from hen serum and egg yolk. Another GlcNAc-binding protein of higher molecular mass was present only in the serum. The 170 kDa protein co-electrophoresed and co-chromatographed in gel filtration with a chicken IgG, and behaved identical to chicken IgG in double immunodiffusion with goat anti-chicken gamma chain antiserum. The sugar-binding hierarchy for the serum and yolk binding proteins, determined with bovine serum albumin neoglycoproteins, was GlcNAc greater than N-acetyl-D-galactosamine greater than glucose = galactose = L-fucose greater than mannose. This hierarchy was unlike any previously reported GlcNAc-binding proteins. The larger serum binding protein component was shown to be an IgM by double immunodiffusion with goat anti-chicken mu chain antiserum. The serum and yolk GlcNAc-binding proteins comprise a unique set of sugar-binding immunoglobulins distinct from the previously reported hen serum and yolk mannose-binding proteins (Wang et al., 1986).     

Identification of a putative collagen-binding protein from chicken skeletal muscle as glycogen phosphorylase.

We have purified and generated antisera to a 95 kDa skeletal muscle protein that constitutes the largest mass fraction of gelatin-agarose binding proteins in skeletal muscle. Preliminary results indicated that this 95 kDa chicken skeletal muscle protein bound strongly to gelatin-agarose and type IV collagen-agarose, suggesting a possible function in muscle cell adhesion to collagen. However, N-terminal sequencing of proteolytic fragments of the 95 kDa protein indicates that it is the chicken skeletal muscle form of glycogen phosphorylase, the binding of which to gelatin-agarose is unlikely to be biologically relevant. Further characterization showed that the skeletal muscle form of glycogen phosphorylase is immunologically distinct from the liver and brain forms in the chicken, and suggests that, unlike mammalian skeletal muscle, chicken skeletal muscle may have two phosphorylase isoforms. Furthermore, immunolocalization data and solubility characteristics of glycogen phosphorylase in muscle extraction experiments suggest the enzyme may interact strongly with an unidentified component of the muscle cytoskeleton. Thus, this study yields a novel purification technique for skeletal muscle glycogen phosphorylase, provides new information on the distribution and isoforms of glycogen phosphorylase, and provides a caveat for using gelatin affinity chromatography as a primary step in purifying collagen-binding proteins from skeletal muscle.