Pattern changes of pituitary peptides in rat after salt-loading as detected by means of direct, semiquantitative mass spectrometric profiling

We have established a differential peptide display method, based on a mass spectrometric technique, to detect peptides that show semiquantitative changes in the neurointermediate lobe (NIL) of individual rats subjected to salt-loading. We employed matrix-assisted laser desorption/ionization mass spectrometry, using a single-reference peptide in combination with careful scanning of the whole crystal rim of the matrix-analyte preparation, to detect in a semiquantitative manner the molecular ions present in the unfractionated NIL homogenate. Comparison of the mass spectra generated from NIL homogenates of salt-loaded and control rats revealed a selective and significant decrease in the intensities of several molecular ion species of the NIL homogenates from salt-loaded rats. These ion species, which have masses that correspond to the masses of oxytocin, vasopressin, neurophysins, and an unidentified putative peptide, were subsequently chemically characterized. We confirmed that the decreased molecular ion species are peptides derived exclusively from propressophysin and prooxyphysin (i.e., oxytocin, vasopressin, and various neurophysins). The putative peptide is carboxyl-terminal glycopeptide. The carbohydrate moiety of the latter peptide was determined by electrospray tandem MS as bisected biantennary Hex3HexNAc5Fuc. This posttranslational modification accounts for the mass difference between the predicted mass of the peptide based on cDNA studies and the measured mass of the mature peptide.

Sorting by diffusion: An asymmetric obstacle course for continuous molecular separation

A separation technique employing a microfabricated sieve has been demonstrated by observing the motion of DNA molecules of different size. The sieve consists of a two-dimensional lattice of obstacles whose asymmetric disposition rectifies the Brownian motion of molecules driven through the device, causing them to follow paths that depend on their diffusion coefficient. A nominal 6% resolution by length of DNA molecules in the size range 15–30 kbp may be achieved in a 4-inch (10-cm) silicon wafer. The advantage of this method is that samples can be loaded and sorted continuously, in contrast to the batch mode commonly used in gel electrophoresis.

Carcinogen-induced loss of heterozygosity at the Aprt locus in somatic cells of the mouse

Genetic events leading to the loss of heterozygosity (LOH) have been shown to play a crucial role in the development of cancer. However, LOH events do not occur only in genetically unstable cancer cells but also have been detected in normal somatic cells of mouse and man. Mice, in which one of the alleles for adenine phosphoribosyltransferase (Aprt) has been disrupted by gene targeting, were used to investigate the potency of carcinogens to induce LOH in vivo. After 7,12-dimethyl-1,2-benz[a]anthracene (DMBA) exposure, a 3-fold stronger mutagenic response was detected at the autosomal Aprt gene than at the X chromosomal hypoxantine-guanine phosphoribosyltransferase (Hprt) gene in splenic T-lymphocytes. Allele-specific PCR analysis showed that the normal, nontargeted Aprt allele was lost in 70% of the DMBA-induced Aprt mutants. Fluorescence in situ hybridization analysis demonstrated that the targeted allele had become duplicated in almost all DMBA-induced mutants that displayed LOH at Aprt. These results indicate that the main mechanisms by which DMBA caused LOH were mitotic recombination or chromosome loss and duplication but not deletion. However, after treatment with the alkylating agent N-ethyl-N-nitrosourea, Aprt had a similar mutagenic response to Hprt while the majority (90%) of N-ethyl-N-nitrosourea-induced Aprt mutants had retained both alleles. Unexpectedly, irradiation with x-rays, which induce primarily large deletions, resulted in a significant increase of the mutant frequency at Hprt but not at Aprt. This in vivo study clearly indicates that, in normal somatic cells, carcinogen exposure can result in the induction of LOH events that are compatible with cell survival and may represent an initiating event in tumorigenesis.

Polar residues drive association of polyleucine transmembrane helices

Although many polar residues are directly involved in transmembrane protein functions, the extent to which they contribute to more general structural features is still unclear. Previous studies have demonstrated that asparagine residues can drive transmembrane helix association through interhelical hydrogen bonding [Choma, C., Gratkowski, H., Lear, J. D. & DeGrado, W. F. (2000) Nat. Struct. Biol. 7, 161–166; and Zhou, F. X., Cocco, M. J., Russ, W. P., Brunger, A. T. & Engelman, D. M. (2000) Nat. Struct. Biol. 7, 154–160]. We have studied the ability of other polar residues to promote helix association in detergent micelles and in biological membranes. Our results show that polyleucine sequences with Asn, Asp, Gln, Glu, and His, residues capable of being simultaneously hydrogen bond donors and acceptors, form homo- or heterooligomers. In contrast, polyleucine sequences with Ser, Thr, and Tyr do not associate more than the polyleucine sequence alone. The results therefore provide experimental evidence that interactions between polar residues in the helices of transmembrane proteins may serve to provide structural stability and oligomerization specificity. Furthermore, such interactions can allow structural flexibility required for the function of some membrane proteins.

Oxidative Turnover of Soybean Root Glutamine Synthetase. In Vitro and in Vivo Studies1

Glutamine synthetase (GS) is the key enzyme in ammonia assimilation and catalyzes the ATP-dependent condensation of NH3 with glutamate to produce glutamine. GS in plants is an octameric enzyme. Recent work from our laboratory suggests that GS activity in plants may be regulated at the level of protein turnover (S.J. Temple, T.J. Knight, P.J. Unkefer, C. Sengupta-Gopalan [1993] Mol Gen Genet 236: 315–325; S.J. Temple, S. Kunjibettu, D. Roche, C. Sengupta-Gopalan [1996] Plant Physiol 112: 1723–1733; S.J. Temple, C. Sengupta-Gopalan [1997] In C.H. Foyer, W.P. Quick, eds, A Molecular Approach to Primary Metabolism in Higher Plants. Taylor & Francis, London, pp 155–177). Oxidative modification of GS has been implicated as the first step in the turnover of GS in bacteria. By incubating soybean (Glycine max) root extract enriched in GS in a metal-catalyzed oxidation system to produce the ·OH radical, we have shown that GS is oxidized and that oxidized GS is inactive and more susceptible to degradation than nonoxidized GS. Histidine and cysteine protect GS from metal-catalyzed inactivation, indicating that oxidation modifies the GS active site and that cysteine and histidine residues are the site of modification. Similarly, ATP and particularly ATP/glutamate give the enzyme the greatest protection against oxidative inactivation. The roots of plants fed ammonium nitrate showed a 3-fold increase in the level of GS polypeptides and activity compared with plants not fed ammonium nitrate but without a corresponding increase in the GS transcript level. This would suggest either translational or posttranslational control of GS levels.

Modulation of growth factor receptor function by isoform heterodimerization.

Activation of prolactin (PRL)-dependent signaling occurs as the result of ligand-induced dimerization of receptor (PRLr). Although three PRLr isoforms (short, intermediate, and long) have been characterized and are variably coexpressed in PRL-responsive tissues, the functional effects of ligand-induced PRLr isoform heterodimerization have not been examined. To determine whether heterodimeric PRLr complexes were capable of ligand-induced signaling and cellular proliferation, chimeras consisting of the extracellular domain of either the alpha or beta subunit of human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFr) and the intracellular domain of the rat intermediate or short PRLr isoforms (PRLr-I or PRLr-S) were synthesized. Because high affinity binding of GM-CSF is mediated by the extracellular domain of one alpha and beta GM-CSFr pair, use of GM-CSFr/PRLr chimera specifically directed the dimerization of the PRLr intracellular domains within ligand-receptor complexes. Stable transfection of these constructs into the Ba/F3 line was demonstrated by Northern blot and immunoprecipitation analyses. Flow cytometry revealed specific binding of a phycoerythrin-conjugated human GM-CSF to the transfectants, confirming cell surface expression of the chimeric receptors. When tested for their ability to proliferate in response to GM-CSF, only chimeric transfectants expressing GM-CSFr/PRLr-I homodimers demonstrated significant [3H]thymidine incorporation. GM-CSF stimulation of transfectants expressing either GM-CSFr/PRLr-S homodimers or GM-CSFr/PRLr-S+1 heterodimers failed to induce proliferation. Consistent with these data, the GM-CSF-induced activation of two phosphotyrosine kinases, Jak2 and Fyn, was observed only in homodimeric GM-CSFr/PRLr-I transfectants. These results show that the PRLr-S functions as a dominant negative isoform, down-regulating both signaling and proliferation mediated by the receptor complex. Thus, structural motifs necessary for Jak2 and Fyn activation within the carboxy terminus of the PRLr-I, absent in the PRLr-S, are required in each member of the dimeric PRLr complex.

The ability to associate with activation domains in vitro is not required for the TATA box-binding protein to support activated transcription in vivo.

The TATA box-binding protein (TBP) interacts in vitro with the activation domains of many viral and cellular transcription factors and has been proposed to be a direct target for transcriptional activators. We have examined the functional relevance of activator-TBP association in vitro to transcriptional activation in vivo. We show that alanine substitution mutations in a single loop of TBP can disrupt its association in vitro with the activation domains of the herpes simplex virus activator VP16 and of the human tumor suppressor protein p53; these mutations do not, however, disrupt the transcriptional response of TBP to either activation domain in vivo. Moreover, we show that a region of VP16 distinct from its activation domain can also tightly associate with TBP in vitro, but fails to activate transcription in vivo. These data suggest that the ability of TBP to interact with activation domains in vitro is not directly relevant to its ability to support activated transcription in vivo.

Molecular characterization of PsbW, a nuclear-encoded component of the photosystem II reaction center complex in spinach.

We describe the isolation and characterization of cDNAs encoding the precursor polypeptide of the 6.1-kDa polypeptide associated with the reaction center core of the photosystem II complex from spinach. PsbW, the gene encoding this polypeptide, is present in a single copy per haploid genome. The mature polypeptide with 54 amino acid residues is characterized by a hydrophobic transmembrane segment, and, although an intrinsic membrane protein, it carries a bipartite transit peptide of 83 amino acid residues which directs the N terminus of the mature protein into the chloroplast lumen. Thylakoid integration of this polypeptide does not require a delta pH across the membrane, nor is it azide-sensitive, suggesting that the polypeptide chain inserts spontaneously in an as yet unknown way. The PsbW mRNA levels are light regulated. Similar to cytochrome b559 and PsbS, but different from the chlorophyll-complexing polypeptides D1, D2, CP43, and CP47 of photosystem II, PsbW is present in etiolated spinach seedlings.

Phosphorylation of the human leukemia inhibitory factor (LIF) receptor by mitogen-activated protein kinase and the regulation of LIF receptor function by heterologous receptor activation.

We used a bacterially expressed fusion protein containing the entire cytoplasmic domain of the human leukemia inhibitory factor (LIF) receptor to study its phosphorylation in response to LIF stimulation. The dose- and time-dependent relationships for phosphorylation of this construct in extracts of LIF-stimulated 3T3-L1 cells were superimposable with those for the stimulation of mitogen-activated protein kinase (MAPK). Indeed, phosphorylation of the cytoplasmic domain of the low-affinity LIF receptor alpha-subunit (LIFR) in Mono Q-fractionated, LIF-stimulated 3T3-L1 extracts occurred only in those fractions containing activated MAPK; Ser-1044 served as the major phosphorylation site in the human LIFR for MAPK both in agonist-stimulated 3T3-L1 lysates and by recombinant extracellular signal-regulated kinase 2 in vitro. Expression in rat H-35 hepatoma cells of LIFR or chimeric granulocyte-colony-stimulating factor receptor (G-CSFR)-LIFR mutants lacking Ser-1044 failed to affect cytokine-stimulated expression of a reporter gene under the control of the beta-fibrinogen gene promoter but eliminated the insulin-induced attenuation of cytokine-stimulated gene expression. Thus, our results identify the human LIFR as a substrate for MAPK and suggest a mechanism of heterologous receptor regulation of LIFR signaling occurring at Ser-1044.

Translational termination efficiency in mammals is influenced by the base following the stop codon.

The base following stop codons in mammalian genes is strongly biased, suggesting that it might be important for the termination event. This proposal has been tested experimentally both in vivo by using the human type I iodothyronine deiodinase mRNA and the recoding event at the internal UGA codon and in vitro by measuring the ability of each of the 12 possible 4-base stop signals to direct the eukaryotic polypeptide release factor to release a model peptide, formylmethionine, from the ribosome. The internal UGA in the deiodinase mRNA is used as a codon for incorporation of selenocysteine into the protein. Changing the base following this UGA codon affected the ratio of termination to selenocysteine incorporation in vivo at this codon: 1:3 (C or U) and 3:1 (A or G). These UGAN sequences have the same order of efficiency of termination as was found with the in vitro termination assay (4th base: A approximately G >> C approximately U). The efficiency of in vitro termination varied in the same manner over a 70-fold range for the UAAN series and over an 8-fold range for the UGAN and UAGN series. There is a correlation between the strength of the signals and how frequently they occur at natural termination sites. Together these data suggest that the base following the stop codon influences translational termination efficiency as part of a larger termination signal in the expression of mammalian genes.

Metallothionein protects against the cytotoxic and DNA-damaging effects of nitric oxide.

In inflammatory states, nitric oxide (.NO) may be synthesized from precursor L-arginine via inducible .NO synthase (iNOS) in large amounts for prolonged periods of time. When .NO acts as an effector molecule under these conditions, it may be toxic to cells by inhibition of iron-containing enzymes or initiation of DNA single-strand breaks. In contrast to molecular targets of .NO, considerably less is known regarding mechanisms by which cells become resistant to .NO. Metallothionein (MT), the major protein thiol induced in cells exposed to cytokines and bacterial products, is capable of forming iron-dinitrosyl thiolates in vitro. Therefore, we tested the hypothesis that overexpression of MT reduces the sensitivity of NIH 3T3 cells to the .NO donor, S-nitrosoacetylpenicillamine (SNAP), and to .NO released from cells (NIH 3T3-DFG-iNOS) after infection with a retroviral vector expressing human iNOS gene. There was a 4-fold increase in MT in cells transfected with the mouse MT-1 gene (NIH 3T3/MT) compared to cells transfected with the promoter-free inverted gene (NIH 3T3/TM). NIH 3T3/MT cells were more resistant than NIH 3T3/TM cells to the cytotoxic effects of SNAP (0.1-1.0 mM) or .NO released from NIH 3T3-DFG-iNOS cells. A brief (1 h) exposure to 10 mM SNAP caused DNA single-strand breaks that were 9-fold greater in NIH 3T3/TM compared to NIH 3T3/MT cells. Electron paramagnetic resonance spectroscopy of NIH 3T3 cells revealed a greater peak at g = 2.04 (e.g., iron-dinitrosyl complex) in NIH 3T3/MT than NIH 3T3/TM cells. These data are consistent with a role for cytoplasmic MT in interacting with .NO and reducing .NO-induced cyto- and nuclear toxicity.

Human autoantibody recognition of DNA.

Combinatorial IgG Fab phage display libraries prepared from a systemic lupus erythematosus (SLE) donor and a healthy donor were affinity selected against human placental DNA. Human monoclonal antibody Fab fragments specific for DNA were isolated from both libraries, although Fabs of the highest affinity were isolated only from the lupus library. Generally, apparent affinities of the Fabs for human placental DNA, purified double-stranded DNA, and denatured DNA were approximately equivalent. Surface plasmon resonance indicated Fab binding constants for a double-stranded oligodeoxynucleotide of 0.2-1.3 x 10(8) M-1. The higher-affinity Fabs, as ranked by binding to human placental DNA or to the oligonucleotide probe, tested positive in the Crithidia luciliae assay commonly used in the diagnosis of SLE, and interestingly the genes encoding the heavy-chain variable regions of these antibodies displayed evidence of only minimal somatic hypermutation. The heavy chains of the SLE Fabs were characterized by a predominance of basic residues toward the N terminus of complementarity-determining region 3 (CDR3). The crucial role of heavy-chain CDR3 (HCDR3) in high-affinity DNA recognition was suggested by the creation of DNA binding in an unrelated antibody by HCDR3 transplantation from SLE antibodies. We propose that high-affinity DNA-binding antibodies can arise in SLE without extensive somatic hypermutation in the variable-region genes because of the expression of inappropriate HCDR3s.