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DCD – a novel plant specific domain in proteins involved in development and programmed cell death
(2005)
Background: Recognition of microbial pathogens by plants triggers the hypersensitive reaction, a common form of programmed cell death in plants. These dying cells generate signals that activate the plant immune system and alarm the neighboring cells as well as the whole plant to activate defense responses to limit the spread of the pathogen. The molecular mechanisms behind the hypersensitive reaction are largely unknown except for the recognition process of pathogens. We delineate the NRP-gene in soybean, which is specifically induced during this programmed cell death and contains a novel protein domain, which is commonly found in different plant proteins.
Results: The sequence analysis of the protein, encoded by the NRP-gene from soybean, led to the identification of a novel domain, which we named DCD, because it is found in plant proteins involved in d evelopment and c ell d eath. The domain is shared by several proteins in the Arabidopsis and the rice genomes, which otherwise show a different protein architecture. Biological studies indicate a role of these proteins in phytohormone response, embryo development and programmed cell by pathogens or ozone.
Conclusion: It is tempting to speculate, that the DCD domain mediates signaling in plant development and programmed cell death and could thus be used to identify interacting proteins to gain further molecular insights into these processes.
Poster presentation: NO-sensitive guanylyl cyclases (GC) are the principal receptors for nitric oxide (NO) and convert GTP into the second messenger cGMP. We showed that GC is prone to tyrosine phosphorylation in COS1 cells overexpressing the human holoenzyme. Similar results were obtained in PC12 cells and in rat aortic tissue slices. The major phosphorylation site was mapped to position 192 in the regulatory domain of the beta1 subunit. Tyrosine phosphorylation of GC was reduced in the presence of the inhibitors PP1 and PP2 indicating that Src-like kinases are critically involved in phosphorylation. Moreover, co-immunoprecipitation experiments revealed an interaction between Src and GC. To further analyse the relevance of this posttranslational modification we generated a phospho-specific antibody raised against pTyr192. This antibody clearly distinguishes between phosphorylated and non-phosphorylated GC and may be a powerful tool to analyse the subcellular localisation of the phosphorylated enzyme.
Poster presentation: NO-sensitive guanylyl cyclases (sGCs) are cytosolic receptors for nitric oxide (NO) catalyzing the conversion of GTP to cGMP. sGCs are obligate heterodimers composed of one alpha and beta subunit each. The allosteric mechanism of sGC activation via NO is well understood, however, our knowledge about alternative mechanisms such as protein-protein interactions regulating activity, availability, translocation and expression of sGC is rather limited. In a search by the yeast two-hybrid system using the catalytic domain of the alpha1 subunit as the bait, we have identified two structurally related proteins AGAP1 [1] and MRIP2 as novel sGC interacting proteins. MRIP2 is a multi-domain protein of 75 kDa comprising a single PH and ArfGAP domain each and two ankyrin repeats. Co-immunoprecipitation experiments using COS1 cells overexpressing both proteins demonstrated the interaction of MRIP2 with both subunits of the sGC alpha1beta1. Confocal microscopical analysis showed a prominent plasma membrane staining of MRIP2. This membrane association is mediated through an N-terminal myristoylation site and through binding of its PH domain to phospholipids such as phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2). We hypothesize that MRIP2 may represent an acceptor protein for sGC that mediates recruitment of cytosolic sGC to the plasma membrane or other subcellular compartments.
Poster presentation NO-sensitive guanylyl cyclases (soluble guanylyl cyclase, sGC) are among the key regulators of intracellular cGMP concentration. The mechanisms underlying NO-mediated activation of sGC are quite well understood, however, little is known about the fine-tuning of sGC activity through alternative mechanisms such as protein phosphorylation. Several reports have demonstrated the reversible phosphorylation of sGC on serine/threonine residues, and it has been speculated, though not experimentally proven, that sGC might also be phosphorylated on tyrosine residues. Using broad-spectrum phosphatase inhibitors we were able to demonstrate tyrosine phosphorylation at Tyr192 of the beta 1 subunit of human sGC in COS1 cells. This residue forms part of a sequence segment (YEDL) representing a preferential binding site for SH2 domains of Src-like kinases. Pull-down assays and co-immunoprecipitation experiments showed that Src can indeed bind via its SH2 domain to pTyr192 of beta 1 indicating that tyrosine phosphorylation of sGC may be followed by recruitment of Src-like kinases to the phosphorylated beta 1 subunit. In support of this hypothesis, immunofluorescence studies showed a colocalization of overexpressed sGC and Src at the plasma membrane of COS1 and Hela cells. Together, our results point to an unexpected crosstalk between tyrosine kinase pathway(s) and the NO/cGMP signalling cascade which may result in translocation of the predominantly cytosolic sGC to the cytosolic face of the plasma membrane.
The NO/cGMP pathway inhibits Rap1 activation in human platelets via cGMP-dependent protein kinase I
(2005)
The NO/cGMP signalling pathway strongly inhibits agonist-induced platelet aggregation. However, the molecular mechanisms involved are not completely defined.We have studied NO/cGMP effects on the activity of Rap1, an abundant guanine-nucleotidebinding protein in platelets. Rap1-GTP levels were reduced by NO-donors and activators of NO-sensitive soluble guanylyl cyclase. Four lines of evidence suggest that NO/cGMP effects are mediated by cGMP-dependent protein kinase (cGKI): (i) Rap1 inhibition correlated with cGKI activity as measured by the phosphorylation state ofVASP, an established substrate of cGKI, (ii) 8-pCPT-cGMP, a membrane permeable cGMP-analog and activator of cGKI, completely blocked Rap1 activation, (iii) Rp- 8pCPT-cGMPS, a cGKI inhibitor, reversed NO effects and (iv) expression of cGKI in cGKI-deficient megakaryocytes inhibited Rap1 activation. NO/cGMP/cGKI effects were independent of the type of stimulus used for Rap1 activation.Thrombin-,ADPand collagen-induced formation of Rap1-GTP in platelets as well as turbulence-induced Rap1 activation in megakaryocytes were inhibited. Furthermore, cGKI inhibited ADP-induced Rap1 activation induced by the G a i -coupled P2Y12 receptor alone, i.e. independently of effects on Ca2+-signalling. From these studies we conclude that NO/cGMP inhibit Rap1 activation in human platelets and that this effect is mediated by cGKI. Since Rap1 controls the function of integrin a IIbß 3 , we propose that Rap1 inhibition might play a central role in the anti-aggregatory actions of NO/cGMP.
Background: The flavin in its FMN and FAD forms is a versatile cofactor that is involved in catalysis of most disparate types of biological reactions. These include redox reactions such as dehydrogenations, activation of dioxygen, electron transfer, bioluminescence, blue light reception, photobiochemistry (as in photolyases), redox signaling etc. Recently, hitherto unrecognized types of biological reactions have been uncovered that do not involve redox shuffles, and might involve the reduced form of the flavin as a catalyst. The present work addresses properties of reduced flavin relevant in this context. Results: N(5)-H exchange reactions of the flavin reduced form and its pH dependence were studied using the 15N-NMR-signals of 15N-enriched, reduced flavin in the pH range from 5 to 12. The chemical shifts of the N(3) and N(5) resonances are not affected to a relevant extent in this pH range. This contrasts with the multiplicity of the N(5)-resonance, which strongly depends on pH. It is a doublet between pH 8.45 and 10.25 that coalesces into a singlet at lower and higher pH values. From the line width of the 15N(5) signal the pH-dependent rate of hydrogen exchange was deduced. The multiplicity of the 15N(5) signal and the proton exchange rates are little dependent on the buffer system used. Conclusion: The exchange rates allow an estimation of the pKa value of N(5)-H deprotonation in reduced flavin to be ≥ 20. This value imposes specific constraints for mechanisms of flavoprotein catalysis based on this process. On the other hand the pK ≈ 4 for N(5)-H protonation (to form N(5)+-H2) would be consistent with a role of N(5)-H as a base.
In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase a (pol alpha) and Klenow fragment (exo-) of DNA polymerase I (Escherichia coli ). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electronwithdrawing (trifluoromethyl and dinitro). Both pol a and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol a and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol a and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol {alpha} and Klenow fragment.
Im Zuge der steigenden Bedeutung der Proteomforschung und der »Molekularisierung« der Medizin werden neue, effizientere Plattformen zur Untersuchung von Proteinen und deren Wechselwirkungen notwendig. Hier bietet die Nanotechnologie, eine Wissenschaft mit Ursprüngen in der Physik und der Halbleiterindustrie, attraktive Lösungsperspektiven. Ein Bereich der Forschung am Institut für Biochemie der Universität Frankfurt um Prof. Dr. Robert Tampé widmet sich den Aspekten der Nanotechnologie zur Entwicklung von Protein-Chips für die Proteomforschung und Erzeugung von Mustern im Kleinstformat.
Chemically modified bases are frequently used to stabilize nucleic acids, to study the driving forces for nucleic acid structure formation and to tune DNA and RNA hybridization conditions. In particular, fluorobenzene and fluorobenzimidazole base analogues can act as universal bases able to pair with any natural base and to stabilize RNA duplex formation. Although these base analogues are compatible with an A-form RNA geometry, little is known about the influence on the fine structure and conformational dynamics of RNA. In the present study, nano-second molecular dynamics (MD) simulations have been performed to characterize the dynamics of RNA duplexes containing a central 1'-deoxy-1'-(2,4-difluorophenyl)-ß-D-ribofuranose base pair or opposite to an adenine base. For comparison, RNA with a central uridine:adenine pair and a 1'-deoxy-1'-(phenyl)-ß-D-ribofuranose opposite to an adenine was also investigated. The MD simulations indicate a stable overall A-form geometry for the RNAs with base analogues. However, the presence of the base analogues caused a locally enhanced mobility of the central bases inducing mainly base pair shear and opening motions. No stable ‘base-paired’ geometry was found for the base analogue pair or the base analogue:adenine pairs, which explains in part the universal base character of these analogues. Instead, the conformational fluctuations of the base analogues lead to an enhanced accessibility of the bases in the major and minor grooves of the helix compared with a regular base pair.