Viral capsid mobility: A dynamic conduit for inactivation

Mass spectrometry and fluorescent probes have provided direct evidence that alkylating agents permeate the protein capsid of naked viruses and chemically inactivate the nucleic acid. N-acetyl-aziridine and a fluorescent alkylating agent, dansyl sulfonate aziridine, inactivated three different viruses, flock house virus, human rhinovirus-14, and foot and mouth disease virus. Mass spectral studies as well as fluorescent probes showed that alkylation of the genome was the mechanism of inactivation. Because particle integrity was not affected by selective alkylation (as shown by electron microscopy and sucrose gradient experiments), it was reasoned that the dynamic nature of the viral capsid acts as a conduit to the interior of the particle. Potential applications include fluorescent labeling for imaging viral genomes in living cells, the sterilization of blood products, vaccine development, and viral inactivation in vivo.

Inactivation of menin, a Smad3-interacting protein, blocks transforming growth factor type β signaling

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by endocrine tumors of parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene encodes a nuclear protein called menin. In MEN1 carriers inactivating mutations give rise to a truncated product consistent with menin acting as a tumor suppressor gene. However, the role of menin in tumorigenesis and its physiological functions are not known. Here, we show that menin inactivation by antisense RNA antagonizes transforming growth factor type β-mediated cell growth inhibition. Menin interacts with Smad3, and antisense menin suppresses transforming growth factor type β-induced and Smad3-induced transcriptional activity by inhibiting Smad3/4-DNA binding at specific transcriptional regulatory sites. These results implicate a mechanism of tumorigenesis by menin inactivation.

A Conserved Acidic Motif in the N-Terminal Domain of Nitrate Reductase Is Necessary for the Inactivation of the Enzyme in the Dark by Phosphorylation and 14-3-3 Binding1

It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611–621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II.

Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts.

Inflammatory processes in chronic rejection remain a serious clinical problem in organ transplantation. Activated cellular infiltrate produces high levels of both superoxide and nitric oxide. These reactive oxygen species interact to form peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. We identified enhanced immunostaining for nitrotyrosine localized to tubular epithelium of chronically rejected human renal allografts. Western blot analysis of rejected tissue demonstrated that tyrosine nitration was restricted to a few specific polypeptides. Immunoprecipitation and amino acid sequencing techniques identified manganese superoxide dismutase, the major antioxidant enzyme in mitochondria, as one of the targets of tyrosine nitration. Total manganese superoxide dismutase protein was increased in rejected kidney, particularly in the tubular epithelium; however, enzymatic activity was significantly decreased. Exposure of recombinant human manganese superoxide dismutase to peroxynitrite resulted in a dose-dependent (IC50 = 10 microM) decrease in enzymatic activity and concomitant increase in tyrosine nitration. Collectively, these observations suggest a role for peroxynitrite during development and progression of chronic rejection in human renal allografts. In addition, inactivation of manganese superoxide dismutase by peroxynitrite may represent a general mechanism that progressively increases the production of peroxynitrite, leading to irreversible oxidative injury to mitochondria.

Locust adipokinetic hormones: carrier-independent transport and differential inactivation at physiological concentrations during rest and flight.

Since concomitant release of structurally related peptide hormones with apparently similar functions seems to be a general concept in endocrinology, we have studied the dynamics of the lifetime of the three known adipokinetic hormones (AKHs) of the migratory locust, which control flight-directed mobilization of carbohydrate and lipid from fat body stores. Although the structure of the first member of the AKHs has been known for 20 years, until now, reliable data on their inactivation and removal from the hemolymph are lacking, because measurement requires AKHs with high specific radioactivity. Employing tritiated AKHs with high specific radioactivity, obtained by catalytic reduction with tritium gas of the dehydroLeu2 analogues of the AKHs synthesized by the solid-phase procedure, studies with physiological doses of as low as 1.0 pmol per locust could be conducted. The AKHs appear to be transported in the hemolymph in their free forms and not associated with a carrier protein, despite their strong hydrophobicity. Application of AKHs in their free form in in vivo and in vitro studies therefore now has been justified. We have studied the degradation of the three AKHs during rest and flight. The first cleavage step by an endopeptidase is crucial, since the resulting degradation products lack any adipokinetic activity. Half-lives for AKH-I, -II and -III were 51, 40, and 5 min, respectively, for rest conditions and 35, 37, and 3 min, respectively, during flight. The rapid and differential degradation of structurally related hormones leads to changes in the ratio in which they are released and therefore will have important consequences for concerted hormone action at the level of the target organ or organs, suggesting that each of the known AKHs may play its own biological role in the overall syndrome of insect flight.

Photo-induced inactivation of viruses: adsorption of methylene blue, thionine, and thiopyronine on Qbeta bacteriophage.

The adsorption of cationic organic dyes (methylene blue, thionine, and thiopyronine) on Qbeta bacteriophage was studied by UV-visible and fluorescence spectroscopy. The dyes have shown a strong affinity to the virus and some have been used as sensitizers for photo-induced inactivation of virus. In the methylene blue concentration range of 0.1-5 microM and at high ratios of dye to virus (greater than 1000 dye molecules per virion), the dyes bind as aggregates on the virus. Aggregation lowers the efficiency of photoinactivation because of self-quenching of the dye. At lower ratios of dye to virus (lower than 500 dye molecules per virion), the dye binds to the virus as a monomer. Fluorescence polarization and time-resolved studies of the fluorescence support the conclusions based on fluorescence quenching. Increasing the ionic strength (adding NaCl) dissociates bound dye aggregates on the virus and releases monomeric dye into the bulk solution.

Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378.

Leukotriene A4 (LTA4) hydrolase [(7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7, 9,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the potent chemotactic agent leukotriene B4 (LTB4). LTA4 hydrolase/aminopeptidase is suicide inactivated during catalysis via an apparently mechanism-based irreversible binding of LTA4 to the protein in a 1:1 stoichiometry. Previously, we have identified a henicosapeptide, encompassing residues Leu-365 to Lys-385 in human LTA4 hydrolase, which contains a site involved in the covalent binding of LTA4 to the native enzyme. To investigate the role of Tyr-378, a potential candidate for this binding site, we exchanged Tyr for Phe or Gln in two separate mutants. In addition, each of two adjacent and potentially reactive residues, Ser-379 and Ser-380, were exchanged for Ala. The mutated enzymes were expressed as (His)6-tagged fusion proteins in Escherichia coli, purified to apparent homogeneity, and characterized. Enzyme activity determinations and differential peptide mapping, before and after repeated exposure to LTA4, revealed that wild-type enzyme and the mutants [S379A] and [S380A]LTA4hydrolase were equally susceptible to suicide inactivation whereas the mutants in position 378 were no longer inactivated or covalently modified by LTA4. Furthermore, in [Y378F]LTA4 hydrolase, the value of kcat for epoxide hydrolysis was increased 2.5-fold over that of the wild-type enzyme. Thus, by a single-point mutation in LTA4 hydrolase, catalysis and covalent modification/inactivation have been dissociated, yielding an enzyme with increased turnover and resistance to mechanism-based inactivation.

Irreversible inactivation of interleukin 2 in a pump-based delivery environment.

The physical stability of pharmaceutical proteins in delivery environments is a critical determinant of biological potency and treatment efficacy, and yet it is often taken for granted. We studied both the bioactivity and physical stability of interleukin 2 upon delivery via continuous infusion. We found that the biological activity of the delivered protein was dramatically reduced by approximately 90% after a 24-hr infusion program. Only a portion of these losses could be attributed to direct protein deposition on the delivery surfaces. Analysis of delivered protein by size exclusion chromatography gave no indication of insulin-like, surface-induced aggregation phenomena. Examination of the secondary and tertiary structure of both adsorbed and delivered protein via Fourier-transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopy indicated that transient surface association of interleukin 2 with the catheter tubing resulted in profound, irreversible structural changes that were responsible for the majority of the biological activity losses.

Viral cross talk: intracellular inactivation of the hepatitis B virus during an unrelated viral infection of the liver.

Hepatitis B virus (HBV) infection is thought to be controlled by virus-specific cytotoxic T lymphocytes (CTL). We have recently shown that HBV-specific CTL can abolish HBV replication noncytopathically in the liver of transgenic mice by secreting tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) after antigen recognition. We now demonstrate that hepatocellular HBV replication is also abolished noncytopathically during lymphocytic choriomeningitis virus (LCMV) infection, and we show that this process is mediated by TNF-alpha and IFN-alpha/beta produced by LCMV-infected hepatic macrophages. These results confirm the ability of these inflammatory cytokines to abolish HBV replication; they elucidate the mechanism likely to be responsible for clearance of HBV in chronically infected patients who become superinfected by other hepatotropic viruses; they suggest that pharmacological activation of intrahepatic macrophages may have therapeutic value in chronic HBV infection; and they raise the possibility that conceptually similar events may be operative in other viral infections as well.

Internal cleavage of the inhibitory 7B2 carboxyl-terminal peptide by PC2: a potential mechanism for its inactivation.

The neuroendocrine protein 7B2 contains two domains, a 21-kDa protein required for prohormone convertase 2 (PC2) maturation and a carboxyl-terminal (CT) peptide that inhibits PC2 at nanomolar concentrations. To determine how the inhibition of PC2 is terminated, we studied the metabolic fate of the 7B2 CT peptide in RinPE-7B2, AtT-20/PC2-7B2, and alphaTC1-6 cells. Extracts obtained from cells labeled for 6 h with [3H]valine were subjected to immunoprecipitation using an antibody raised against the extreme carboxyl terminus of r7B2, and immunoprecipitated peptides were separated by gel filtration. All three cell lines yielded two distinct peaks at about 3.5 kDa and 1.5 kDa, corresponding to the CT peptide and a smaller fragment consistent with cleavage at an interior Lys-Lys site. These results were corroborated using a newly developed RIA against the carboxyl terminus of the CT peptide which showed that the intact CT peptide represented only about half of the stored CT peptide immunoreactivity, with the remainder present as the 1.5-kDa peptide. Both peptides could be released upon phorbol 12-myristate 13-acetate stimulation. We investigated the possibility that PC2 itself could be responsible for this cleavage by performing in vitro experiments. When 125I-labeled CT peptide was incubated with purified recombinant PC2, a smaller peptide was generated. Analysis of CT peptide derivatives for their inhibitory potency revealed that CT peptide 1-18 (containing Lys-Lys at the carboxyl terminus) represented a potent inhibitor, but that peptide 1-16 was inactive. Inclusion of carboxypeptidase E (CPE) in the reaction greatly diminished the inhibitory potency of the CT peptide against PC2, in line with the notion that the CT peptide cleavage product is not inhibitory after the removal of terminal lysines by CPE. In summary, our data support the idea that PC2 cleaves the 7B2 CT peptide at its internal Lys-Lys site within secretory granules; deactivation of the cleavage product is then accomplished by CPE, thus providing an efficient mechanism for intracellular inactivation of the CT peptide.

Targeted gene inactivation in petunia by PCR-based selection of transposon insertion mutants.

Establishment of loss-of-function phenotypes is often a key step in determining the biological function of a gene. We describe a procedure to obtain mutant petunia plants in which a specific gene with known sequence is inactivated by the transposable element dTph1. Leaves are collected from batches of 1000 plants with highly active dTph1 elements, pooled according to a three-dimensional matrix, and screened by PCR using a transposon- and a gene-specific primer. In this way individual plants with a dTph1 insertion can be identified by analysis of about 30 PCRs. We found insertion alleles for various genes at a frequency of about 1 in 1000 plants. The plant population can be preserved by selfing all the plants, so that it can be screened for insertions in many genes over a prolonged period.

Leukotriene A4 hydrolase: mapping of a henicosapeptide involved in mechanism-based inactivation.

Leukotriene A4 (LTA4) hydrolase [7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7,9 ,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme which converts LTA4 into the chemotactic agent leukotriene B4 (LTB4). Suicide inactivation, a typical feature of LTA4 hydrolase/aminopeptidase, occurs via an irreversible, apparently mechanism-based, covalent binding of LTA4 to the protein in a 1:1 stoichiometry. Differential lysine-specific peptide mapping of unmodified and suicide-inactivated LTA4 hydrolase has been used to identify a henicosapeptide, encompassing the amino acid residues 365-385 of human LTA4 hydrolase, which is involved in the binding of LTA4, LTA4 methyl ester, and LTA4 ethyl ester to the native enzyme. A modified form of this peptide, generated by lysine-specific digestion of LTA4 hydrolase inactivated by LTA4 ethyl ester, could be isolated for complete Edman degradation. The sequence analysis revealed a gap at position 14, which shows that binding of the leukotriene epoxide had occurred via Tyr-378 in LTA4 hydrolase. Inactivation of the epoxide hydrolase and the aminopeptidase activity was accompanied by a proportionate modification of the peptide. Furthermore, both enzyme inactivation and peptide modification could be prevented by preincubation of LTA4 hydrolase with the competitive inhibitor bestatin, which demonstrates that the henicosapeptide contains functional elements of the active site(s). It may now be possible to clarify the molecular mechanisms underlying suicide inactivation and epoxide hydrolysis by site-directed mutagenesis combined with structural analysis of the lipid molecule, covalently bound to the peptide.

Inactivation of simian immunodeficiency virus by hydrostatic pressure.

The inactivation of the simian immunodeficiency viruses SIVmac251 and SIVagm by pressures of 150 and 250 MPa was determined. The extent of inactivation depended on the time that the virus was subjected to compression as well as the level of the pressure and at 150 Mpa reached 5 log10 dilution units after approximately 10 hr. The inactivations, which were uniformly carried out at room temperature, were independent of the concentration of the virus. Possible applications of pressure inactivation for molecular biological and clinical use are discussed.

Interaction with the recombination hot spot chi in vivo converts the RecBCD enzyme of Escherichia coli into a chi-independent recombinase by inactivation of the RecD subunit.

The RecBCD enzyme of Escherichia coli promotes recombination preferentially at chi nucleotide sequences and has in vivo helicase and strong duplex DNA exonuclease (exoV) activities. The enzyme without the RecD subunit, as in a recD null mutant, promotes recombination efficiently but independently of chi and has no nucleolytic activity. Employing phage lambda red gam crosses, phage T4 2- survival measurements, and exoV assays, it is shown that E. coli cells in which RecBCD has extensive opportunity to interact with linear chi-containing DNA (produced by rolling circle replication of a plasmid with chi or by bleomycin-induced fragmentation of the cellular chromosome) acquire the phenotype of a recD mutant and maintain this for approximately 2 h. It is concluded that RecBCD is converted into RecBC during interaction with chi by irreversible inactivation of RecD. After conversion, the enzyme is released and initiates recombination on other DNA molecules in a chi-independent fashion. Overexpression of recD+ (from a plasmid) prevented the phenotypic change and providing RecD after the change restored chi-stimulated recombination. The observed recA+ dependence of the downregulation of exoV could explain the previously noted "reckless" DNA degradation of recA mutants. It is proposed that chi sites are regulatory elements for the RecBCD to RecBC switch and thereby function as cis- and trans-acting stimulators of RecBC-dependent recombination.