A lipid anchor improves the protective effect of ectoine in inflammation.

Others and we have shown in several studies that the natural tetrahydropyrimidine ectoine protects mammalian cells and tissues against various stress factors including ischemia/reperfusion injury, UV-irradiation, and inflammation. Since little is known about the molecular mechanism of this protective effect, which was ascribed exclusively to an extracellular action of this small water-soluble molecule, we asked whether and how a hydrophobic anchor modulates the inflammation protective properties of ectoine. We therefore investigated the influence of ectoine and of its semi-synthetic derivative lauryl-ectoine on inflammation in RAW 264.7 macrophages and primary cultured rat intestinal smooth muscle (RISM) cells. Both, ectoine and lauryl-ectoine considerably decreased lipopolysaccharide (LPS)-induced interleukin (IL)- 1, IL-6, tumor necrosis factor (TNF)- α, and cyclooxygenase (COX)-2 gene expression in macrophages as well as TNF-α- induced IL-1, IL-6 and COX-2 expression in RISM cells. This reduction of inflammatory agents was accompanied on the one hand by a significant decrease of nuclear translocation of nuclear factor (NF)-κB and on the other hand by a reduction of cellular ceramide content. Interestingly, lauryl- ectoine was much more active exerting its effect at about 10-fold lower concentrations than its natural counterpart. Note that ectoine was almost completely recovered in the medium whereas lauryl-ectoine was found to be cell-associated. Together our data indicate that a lipid anchor considerably improves a possible preventive and/or therapeutic implementation of ectoine in inflammatory processes.

Important role of the tetraloop region of 4.5S RNA in SRP binding to its receptor FtsY.

Binding of Escherichia coli signal recognition particle (SRP) to its receptor, FtsY, requires the presence of 4.5S RNA, although FtsY alone does not interact with 4.5S RNA. In this study, we report that the exchange of the GGAA tetraloop sequence in domain IV of 4.5S RNA for UUCG abolishes SRP-FtsY interaction, as determined by gel retardation and membrane targeting experiments, whereas replacements with other GNRA-type tetraloops have no effect. A number of other base exchanges in the tetraloop sequence have minor or intermediate inhibitory effects. Base pair disruptions in the stem adjacent to the tetraloop or replacement of the closing C-G base pair with G-C partially restored function of the otherwise inactive UUCG mutant. Chemical probing by hydroxyl radical cleavage of 4.5S RNA variants show that replacing GGAA with UUCG in the tetraloop sequence leads to structural changes both within the tetraloop and in the adjacent stem; the latter change is reversed upon reverting the C-G closing base pair to G-C. These results show that the SRP-FtsY interaction is strongly influenced by the structure of the tetraloop region of SRP RNA, in particular the tetraloop stem, and suggest that both SRP RNA and Ffh undergo mutual structural adaptation to form SRP that is functional in the interaction with the receptor, FtsY.

Interaction of guanine nucleotides with the signal recognition particle from Escherichia coli.

The bacterial signal recognition particle (SRP) is an RNA-protein complex. In Escherichia coli, the particle consists of a 114 nt RNA, a 4.5S RNA, and a 48 kDa GTP-binding protein, Ffh. GDP-GTP exchange on, and GTP hydrolysis by, Ffh are thought to regulate SRP function in membrane targeting of translating ribosomes. In the present paper, we report the equilibrium and kinetic constants of guanine nucleotide binding to Ffh in different functional complexes. The association and dissociation rate constants of GTP/GDP binding to Ffh were measured using a fluorescent analogue of GTP/GDP, mant-GTP/GDP. For both nucleotides, association and dissociation rate constants were about 10(6) M-1 s-1 and 10 s-1, respectively. The equilibrium constants of nonmodified GTP and GDP binding to Ffh alone and in SRP, and in the complex with the ribosomes were measured by competition with mant-GDP. In all cases, the same 1-2 microM affinity for GTP and GDP was observed. Binding of both GTP and GDP to Ffh was independent of Mg2+ ions. The data suggest that, at conditions in vivo, (i) there will be rapid spontaneous GDP-GTP exchange, and (ii) the GTP-bound form of Ffh, or of SRP, will be predominant.

The Escherichia coli SRP and SecB targeting pathways converge at the translocon.

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.

Structure of 4.5S RNA in the signal recognition particle of Escherichia coli as studied by enzymatic and chemical probing.

The structure of 4.5S RNA, the Escherichia coli homologue of the signal recognition particle (SRP) RNA, alone and in the SRP complex with protein P48 (Ffh) was probed both enzymatically and chemically. The molecule is largely resistant against single strand-specific nucleases, indicating a highly base paired structure. Reactivity appears mainly in the apical tetraloop and in one of the conserved internal loops. Although some residues are found reactive toward dimethylsulphate and kethoxal in regions predicted to be unpaired by the phylogenetic secondary structure model of 4.5S RNA, generally the reactivity is low, and some residues in internal loops are not reactive at all. RNase V1 cleaves the RNA at multiple sites that coincide with predicted helices, although the cleavages show a pronounced asymmetry. The binding of protein P48 to 4.5S RNA results in a protection of residues in the apical part of the molecule homologous to eukaryotic SRP RNA (domain IV), whereas the cleavages in the conserved apical tetraloop are not protected. Hydroxyl radical treatment reveals an asymmetric pattern of backbone reactivity; in particular, the region encompassing nucleotides 60-82, i.e., the 3' part of the conserved domain IV, is protected. The data suggest that a bend in the domain IV region, most likely at the central asymmetric internal loop, is an important element of the tertiary structure of 4.5S RNA. Hyperchromicity and lead cleavage data are consistent with the model as they reveal the unfolding of a higher-order structure between 30 and 40 degrees C. Protection by protein P48 occurs in this region of the RNA and, more strongly, in the 5' part of domain IV (nt 26-50, most strongly from 35 to 49). It is likely that P48 binds to the outside of the bent form of 4.5S RNA.

The functioning of the SRP receptor FtsY in protein-targeting in E. coli is correlated with its ability to bind and hydrolyse GTP.

In this study, we have established that FtsY, the E. coli homolog of the mammalian signal recognition particle (SRP) receptor, is a GTP-binding protein which displays intrinsic GTPase activity. GTP was found to influence the protease sensitivity of FtsY indicative of a conformational change. FtsY mutated in the 4th GTP-binding consensus element displayed reduced GTP-binding and -hydrolysis which correlated with a reduced ability to interact with SRP. Overexpression of the mutant proteins had a stronger inhibitory effect on protein translocation than overexpression of wild-type FtsY. These observations suggest that in E. coli GTP is important for proper functioning of FtsY in protein-targeting.

Formation of SRP-like particle induces a conformational change in E. coli 4.5S RNA.

E. coli P48 protein is homologous to the SRP54 component of the eukaryotic signal recognition particle. In vivo, P48 is associated with 4.5S RNA which shares a homology with eukaryotic SRP RNA. To study the interaction between P48 and 4.5S RNA in vitro, we used 4.5S RNA with fluorescein coupled to the 3'-terminal ribose. Upon binding of P48, the fluorescent 4.5S RNA shows a substantial decrease in fluorescence. Fluorescence quenching as well as anisotropy measurements reveal that the effect is not due to a direct interaction of P48 with the dye. This suggests that the binding of P48 induces a conformational change in 4.5S RNA which affects the structure at the 3' end of the RNA. From equilibrium titrations with fluorescent 4.5S RNA, a dissociation constant of 0.15 microns is obtained for the RNA.protein complex. The formation of the complex is not affected by GTP binding to or hydrolysis by P48.