Mechanism of Tet(O)-mediated tetracycline resistance

Tet(O) is an elongation factor-like protein which confers resistance to the protein synthesis inhibitor tetracycline by promoting the release of the drug from its inhibitory site on the ribosome. Here we investigated the interaction of Tet(O) with the elongating ribosome and show, using dimethyl sulfate (DMS) probing and binding assays, that it interacts preferentially with the post-translocational ribosome. Furthermore, using an XTP-dependent mutant of Tet(O), we demonstrated that Tet(O) induces conformational rearrangements within the ribosome which can be detected by EF-Tu, and manifested as a stimulation in the GTPase activity of this elongation factor. As such, these conformational changes probably involve the ribosomal GTPase-associated center and, accordingly, Tet(O) alters the DMS modification pattern of the L11 region. Additionally, tetracycline binding is associated with an E(a) of 58 kJ/mol. These results suggest a model where both Tet(O) and tetracycline induce a conformational change in functionally opposite directions and the Tet(O)-induced conformation persists after it has left the ribosome; this prevents rebinding of the drug while allowing productive A-site occupation by a ternary complex in the presence of tetracycline.     

Tet(M)-promoted release of tetracycline from ribosomes is GTP dependent.

Tet(M) protein, which displays homology to elongation factor G (EF-G), interacts with the protein biosynthetic machinery to render this process resistant to tetracycline in vivo and in vitro. To clarify the basis of the resistance mechanism, the effects of Tet(M) on several reactions which occur during protein synthesis were examined. The mechanism of action of Tet(M) has been clarified by two observations. The protein relieves tetracycline inhibition of factor-dependent tRNA binding and dramatically reduces the affinity of ribosomes for tetracycline when GTP is present. This reduction in drug affinity appears to be due to a large increase in the rate of tetracycline dissociation. Addition of Tet(M) to ribosome-tetracycline complexes results in displacement of bound drug. And, while Tet(M) and EF-G GTPase activities are tetracycline resistant, the two proteins differ in their sensitivities to fusidic acid, with the latter activity inhibited by the drug. Furthermore, while Tet(M) protects translation from tetracycline inhibition in a defined system, it is unable to substitute for either EF-G or elongation factor Tu.     

Insertional inactivation of a Tet(K)/Tet(L) like transporter does not eliminate tetracycline resistance in Bacillus cereus

Bacillus cereus ATCC 10987 and ATCC 14579 can be induced to high levels of resistance to tetracycline. The chromosomal B. cereus gene bctl encodes a transmembrane protein with homology to Gram-positive tetracycline efflux proteins and relation to other members of the major facilitator superfamily of transport proteins. A mutant strain containing an insertionally inactivated bctl gene did not show impaired tetracycline resistance. No additional altered phenotype was observed in the mutant. Accumulation studies suggested that the resistance mechanism involves a reduced sensitivity to intracellular tetracycline.     

The tetracycline resistance protein Tet(o) perturbs the conformation of the ribosomal decoding centre

Tet(o) is an elongation factor-like protein found in clinical isolates of Campylobacter jejuni that confers resistance to the protein-synthesis inhibitor tetracycline. Tet(o) interacts with the 70S ribosome and promotes the release of bound tetracycline, however, as shown here, it does not form the same functional interaction with the 30S subunit. Chemical probing demonstrates that Tet(o) changes the reactivity of the 16S rRNA to dimethyl sulphate (DMS). These changes cluster within the decoding site, where C1214 is protected and A1408 is enhanced to DMS reactivity. C1214 is close to, but does not overlap, the primary tetracycline-binding site, whereas A1408 is in a region distinct from the Tet(o) binding site visualized by cryo-EM, indicating that Tet(o) induces long-range rearrangements that may mediate tetracycline resistance. Tetracycline enhances C1054 to DMS modification but this enhancement is inhibited in the presence of Tet(o) unlike the tetracycline-dependent protection of A892 which is unaffected by Tet(o). C1054 is part of the primary binding site of tetracycline and A892 is part of the secondary binding site. Therefore, the results for the first time demonstrate that the primary tetracycline binding site is correlated with tetracycline's inhibitory effect on protein synthesis.     

Cyclooxygenase-2 activity is necessary for the angiogenic properties of oncostatin M.

Macrophages play a major role in angiogenesis. We recently reported that oncostatin M (OSM), a cytokine of the interleukin (IL)-6 family secreted by macrophages, has a potent angiogenic activity on human microvascular endothelial cells (HMEC-1), but has no effect on macrovascular cells (human umbilical vein endothelial cells (HUVECs)). In this work, we show that in HMEC-1, OSM (0.5-2.5 ng/ml), leukemia inhibitory factor (LIF) (25 ng/ml), bFGF (25 ng/ml) and IL-1beta (5 ng/ml) induced production of cyclooxygenase (COX)-2. In contrast, in HUVECs, neither OSM nor LIF induced COX-2 mRNA, suggesting that COX-2 might be implicated in the angiogenic activity of OSM. This was confirmed by the inhibiting effect on OSM-induced HMEC-1 proliferation of specific COX-2 inhibitors. In vivo studies confirmed this findings. We conclude that induction of COX-2 by OSM is necessary for its angiogenic activity, but is not sufficient since IL-1beta, which also induces COX-2 in HMEC-1, has only a poor proliferative effect.     

Rme1 is necessary for Mi-1-mediated resistance and acts early in the resistance pathway

The tomato gene Mi-1 confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphid, and whitefly. Using genetic screens, we have isolated a mutant, rme1 (resistance to Meloidogyne spp.), compromised in resistance to M. javanica and potato aphid. Here, we show that the rme1 mutant is also compromised in resistance to M. incognita, M. arenaria, and whitefly. In addition, using an Agrobacterium-mediated transient assay in leaves to express constitutive gain-of-function mutant Pto(L205D), we demonstrated that the rme1 mutation is not compromised in Pto-mediated hypersensitive response. Moreover, the mutation in rme1 does not result in increased virulence of pathogenic Pseudomonas syringae or Mi-1-virulent M. incognita. Using a chimeric Mi-1 construct, Mi-DS4, which confers constitutive cell death phenotype and A. rhizogenes root transformation, we showed that the Mi-1-mediated cell death pathway is intact in this mutant. Our results indicate that Rme1 is required for Mi-1-mediated resistance and acts either at the same step in the signal transduction pathway as Mi-1 or upstream of Mi-1.     

Isoprenylation modification is required for HIPP1-mediated powdery mildew resistance in wheat

Powdery mildew (Pm), caused by Blumeria graminis f.sp. tritici (Bgt), is one of the most important wheat diseases. Heavy-metal-associated isoprenylated plant protein (HIPP1) has been proved playing important roles in response to biotic and a biotic stress. In present study, we proved HIPP1-V from Haynalidia villosa is a positive regulator in Pm resistance. HIPP1-V was rapidly induced by Bgt. Transiently or stably heterologous overexpressing HIPP1-V in wheat suppressed the haustorium formation and enhanced Pm resistance. HIPP1-V isoprenylation was critical for plasma membrane (PM) localization, interaction with E3-ligase CMPG1-V and function in Pm resistance. Bgt infection recruited the isoprenylated HIPP1-V and CMPG1s on PM; blocking the HIPP1 isoprenylation reduced such recruitment and compromised the resistance of OE-CMPG1-V and OE-HIPP1-V. Overexpressing HIPP1-VC^148G could not enhance Pm resistance. These indicated the Pm resistance was dependent on HIPP1-V's isoprenylation. DGEs related to the ROS and SA pathways were remarkably enriched in OE-HIPP1-V, revealing their involvement in Pm resistance. Our results provide evidence on the important role of protein isoprenylation in plant defense.     

Purification and characterization of Tet(M), a protein that renders ribosomes resistant to tetracycline

The tet(M) tetracycline resistance gene has been found in a wide variety of clinically important bacteria. It has been shown previously (Burdett, V. (1986) J. Bacteriol. 165, 564-569) that the tet(M) gene product mediates resistance at the level of protein synthesis as judged by in vitro assay. Using this assay, large amounts of protein were purified from an Escherichia coli overproducer expressing the gene under control of a T7 promoter. The purified activity consists of a single polypeptide of molecular weight 68,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was confirmed to be the tet(M) gene product by amino-terminal sequence analysis. Purified Tet(M) has an associated ribosome-dependent GTPase with the specific activity being similar to that of the corresponding activity associated with elongation factor G. Since Tet(M) also displays substantial homology to elongation factor G throughout its length, Tet(M) may function as an analog of this elongation factor.     

Localization of the ribosomal protection protein Tet(O) on the ribosome and the mechanism of tetracycline resistance

Tet(O) belongs to a class of ribosomal protection proteins that mediate tetracycline resistance. It is a G protein that shows significant sequence similarity to elongation factor EF-G. Here we present a cryo-electron microscopic reconstruction, at 16 A resolution, of its complex with the E. coli 70S ribosome. Tet(O) was bound in the presence of a noncleavable GTP analog to programmed ribosomal complexes carrying fMet-tRNA in the P site. Tet(O) is directly visible as a mass close to the A-site region, similar in shape and binding position to EF-G. However, there are important differences. One of them is the different location of the tip of domain IV, which in the Tet(O) case, does not overlap with the ribosomal A site but is directly adjacent to the primary tetracycline binding site. Our findings give insights into the mechanism of tetracycline resistance.     

Tyrosine kinase activity may be necessary but is not sufficient for c-erbB1-mediated tissue-specific tumorigenicity.

Expression of mutant avian c-erbB1 genes results in tissue-specific transformation in chickens. Site-directed mutagenesis was used to generate kinase-defective mutants of several tissue-specific v-erbB transforming mutants by replacement of the ATP-binding lysine residue in the kinase domain with an arginine residue. These kinase-defective v-erbB mutants were analyzed for their in vitro and in vivo transforming potentials. Specifically, kinase-defective mutants of erythroleukemogenic, hemangioma-inducing, and sarcomagenic v-erbB genes were assessed for their oncogenic potential. In vitro transformation potential was assessed by soft-agar colony formation in primary cultures of chick embryo fibroblasts (CEF). In vivo transformation potential was determined by infection of 1-day-old line 0 chicks with concentrated recombinant retrovirus and then monitoring of birds for tumor formation. These transformation assays demonstrate that kinase activity is absolutely essential for transformation by tissue-specific transforming mutants of the avian c-erbB1 gene. Since all of the tissue-specific v-erbB mutants characterized to date exhibit tyrosine kinase activity in vitro but do not transform all tissues in which they are expressed, we conclude that v-erbB-associated tyrosine kinase activity may be necessary but is not sufficient to induce tumor formation.     

Cholesterol modification is necessary for controlled planar long-range activity of Hedgehog in Drosophila epithelia

The Hedgehog morphogen is a major developmental regulator that acts at short and long range to direct cell fate decisions in invertebrate and vertebrate tissues. Hedgehog is the only known metazoan protein to possess a covalently linked cholesterol moiety. Although the role of the cholesterol group of Hedgehog remains unclear, it has been suggested to be dispensable for the its long-range activity in Drosophila. Here, we provide data in three different epithelia - ventral and dorsal embryonic ectoderm, and larval imaginal disc tissue - showing that cholesterol modification is in fact necessary for the controlled long-range activity of Drosophila Hedgehog. We provide an explanation for the discrepancy between our results and previous reports by showing that unmodified Hh can act at long range, albeit in an uncontrolled manner, only when expressed in squamous cells. Our data show that cholesterol modification controls long-range Hh activity at multiple levels. First, cholesterol increases the affinity of Hh for the plasma membrane, and consequently enhances its apparent intrinsic activity, both in vitro and in vivo. In addition, multimerisation of active Hh requires the presence of cholesterol. These multimers are correlated with the assembly of Hh into apically located, large punctate structures present in active Hh gradients in vivo. By comparing the activity of cholesterol-modified Hh in columnar epithelial cells and peripodial squamous cells, we show that epithelial cells provide the machinery necessary for the controlled planar movement of Hh, thereby preventing the unrestricted spreading of the protein within the three-dimensional space of the epithelium. We conclude that, as in vertebrates, cholesterol modification is essential for controlled long-range Hh signalling in Drosophila.     

NAT10-mediated ac4C tRNA modification promotes EGFR mRNA translation and gefitinib resistance in cancer

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N-glycosylation is necessary for enzymatic activity of a beetle (Apriona germari) cellulase

We previously reported that the beta-1,4-endoglucanase (EGase) belonging to glycoside hydrolase family 45 cloned from the mulberry longicorn beetle, Apriona germari (Ag-EGase I), is composed of 237 amino acid residues and has a potential N-glycosylation site at 97-100 amino acid residues (NSTF). We here describe the N-glycosylation and its role for enzymatic activity of the Ag-EGase I. The N-glycosylation of Ag-EGase I was revealed by the treatment of tunicamycin to the recombinant virus-infected insect Sf9 cells and by endoglycosidase F to the purified recombinant Ag-EGase I, demonstrating that the carbohydrate moieties are not necessary for secretion but essential for Ag-EGase I enzyme activity. To further elucidate the functional role of the N-glycosylation in Ag-EGase I, we have assayed the cellulase enzyme activity in Thr99Gln mutant. Lack of N-glycosylation in Ag-EGase I showed no substantial enzyme activity. This result demonstrates that N-glycosylation at site 97-100 amino acid residues (NSTF) is essential for enzyme activity.     

Binding Interaction between Tet(M) and the Ribosome: Requirements for Binding

Tet(M) protein interacts with the protein biosynthesis machinery to render this process resistant to tetracycline by a mechanism which involves release of the antibiotic from the ribosome in a reaction dependent on GTP hydrolysis. To clarify this resistance mechanism further, the interaction of Tet(M) with the ribosome has been examined by using a gel filtration assay with radioactively labelled Tet(M) protein. The presence of GTP and 5′-guanylyl imido diphosphate, but not GDP, promoted Tet(M)-ribosome complex formation. Furthermore, thiostrepton, which inhibits the activities of elongation factor G (EF-G) and EF-Tu by binding to the ribosome, blocks stable Tet(M)-ribosome complex formation. Direct competition experiments show that Tet(M) and EF-G bind to overlapping sites on the ribosome.     

IL-3-mediated TNF production is necessary for mast cell development

Mouse mast cell development and survival are largely controlled by the cytokines IL-3 and stem cell factor (SCF). We have found that IL-3 stimulation of bone marrow cells induces the production of TNF via a PI3K- and MAPK kinase/ERK-dependent pathway. Specifically, Mac-1-positive cells were responsible for TNF production, which peaked on days 7-10 of culture and decreased rapidly thereafter. The importance of IL-3-induced TNF secretion was demonstrated by the failure of TNF-deficient bone marrow cells to survive for >3 wk when cultured in IL-3 and SCF, a defect that was reversed by the addition of soluble TNF. The development of human mast cells from bone marrow progenitors was similarly hampered by the addition of TNF-blocking Abs. Cell death was due to apoptosis, which occurred with changes in mitochondrial membrane potential and caspase activation. Apoptosis appeared to be due to loss of IL-3 signaling, because TNF-deficient cells were less responsive than their wild-type counterparts to IL-3-mediated survival. In vitro cultured mast cells from TNF-deficient mice also demonstrated reduced expression of the high affinity IgE receptor, which was restored to normal levels by the addition of soluble TNF. Finally, TNF-deficient mice demonstrated a 50% reduction in peritoneal mast cell numbers, indicating that TNF is an important mast cell survival factor both in vitro and in vivo.     

Protein phosphatase activity is necessary for myofibrillogenesis

During myofibrillogenesis, myosin light-chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin II, enabling patterned assembly of myosin thick filaments. A protein phosphatase (PP) has been shown to mediate RLC dephosphorylation in adult smooth and striated muscle. A role for PP activity in regulating myofibrillogenesis during embryonic development, however, has not been investigated. Tautomycin (TM) was used to inhibit both PP1 and PP2A activities, whereas okadaic acid (OA) and fostriecin (FOS) were used to inhibit PP2A. TM affected both actin and myosin assembly at 5nM; the IC50 value was 20 and 8.5nM, respectively. In contrast, OA applied at 10 times above its reported Ki for PP2A caused no significant disruption. There was also no disruption when FOS was applied at a concentration 30 times above its reported Ki for PP2A. Thus, our results suggest a primary role for PP1 isoforms during myofibrillogenesis. Although rho kinase (RK) regulates PP activity in embryonic smooth and cardiac muscle, application of the RK inhibitor Y27632 did not affect actin or myosin assembly in skeletal myocytes. Collectively, our pharmacological results suggest that PP1 is involved in dynamic regulation of RLC phosphorylation. To specifically test involvement of the myosin-targeted isoform (PP1M), we used a morpholino antisense approach to knock down the myosin targeting (M) subunit of PP1. Embryos injected with morpholino targeted to the 110-kDa M targeting subunit had fewer somites, and myosin organization was significantly perturbed. The combined pharmacological and molecular results suggest a dynamic equilibrium between MLCK and PP1M activities is required for proper myofibrillogenesis.     

Twelve-transmembrane-segment (TMS) version (DeltaTMS VII-VIII) of the 14-TMS Tet(L) antibiotic resistance protein retains monovalent cation transport modes but lacks tetracycline efflux capacity

A "Tet(L)-12" version of Tet(L), a tetracycline efflux protein with 14 transmembrane segments (TMS), was constructed by deletion of two central TMS. Tet(L)-12 catalyzed Na+/H+ antiport and antiport with K+ as a coupling ion as well as or better than wild-type Tet(L) but exhibited no tetracycline-Me2+/H+ antiport in Escherichia coli vesicles.