Stress and developmental responses in Arabidopsis thaliana: regulation through the transcription factor-interacting protein RCD1

Plants constantly face adverse environmental conditions, such as drought or extreme temperatures that threaten their survival. They demonstrate astonishing metabolic flexibility in overcoming these challenges and one of the key responses to stresses is changes in gene expression leading to alterations in cellular functions. This is brought about by an intricate network of transcription factors and associated regulatory proteins. Protein-protein interactions and post-translational modifications are important steps in this control system along with carefully regulated degradation of signaling proteins. This work concentrates on the RADICAL-INDUCED CELL DEATH1 (RCD1) protein which is an important regulator of abiotic stress-related and developmental responses in Arabidopsis thaliana. Plants lacking this protein function display pleiotropic phenotypes including sensitivity to apoplastic reactive oxygen species (ROS) and salt, ultraviolet B (UV-B) and paraquat tolerance, early flowering and senescence. Additionally, the mutant plants overproduce nitric oxide, have alterations in their responses to several plant hormones and perturbations in gene expression profiles. The RCD1 gene is transcriptionally unresponsive to environmental signals and the regulation of the protein function is likely to happen post-translationally. RCD1 belongs to a small protein family and, together with its closest homolog SRO1, contains three distinguishable domains: In the N-terminus, there is a WWE domain followed by a poly(ADP-ribose) polymerase-like domain which, despite sequence conservation, does not seem to be functional. The C-terminus of RCD1 contains a novel domain called RST. It is present in RCD1-like proteins throughout the plant kingdom and is able to mediate physical interactions with multiple transcription factors. In conclusion, RCD1 is a key point of signal integration that links ROS-mediated cues to transcriptional regulation by yet unidentified means, which are likely to include post-translational mechanisms. The identification of RCD1-interacting transcription factors, most of whose functions are still unknown, opens new avenues for studies on plant stress as well as developmental responses.

Plasmids and aromatic degradation in Sphingomonas for bioremediation : Aromatic ring cleavage genes in soil and rhizosphere

Microbial degradation pathways play a key role in the detoxification and the mineralization of polyaromatic hydrocarbons (PAHs), which are widespread pollutants in soil and constituents of petroleum hydrocarbons. In microbiology the aromatic degradation pathways are traditionally studied from single bacterial strains with capacity to degrade certain pollutant. In soil the degradation of aromatics is performed by a diverse community of micro-organisms. The aim of this thesis was to study biodegradation on different levels starting from a versatile aromatic degrader Sphingobium sp. HV3 and its megaplasmid, extending to revelation of diversity of key catabolic enzymes in the environment and finally studying birch rhizoremediation in PAH-polluted soil. To understand biodegradation of aromatics on bacterial species level, the aromatic degradation capacity of Sphingobium sp. HV3 and the role of the plasmid pSKY4, was studied. Toluene, m-xylene, biphenyl, fluorene, phenanthrene were detected as carbon and energy sources of the HV3 strain. Tn5 transposon mutagenesis linked the degradation capacity of toluene, m-xylene, biphenyl and naphthalene to the pSKY4 plasmid and qPCR expression analysis showed that plasmid extradiol dioxygenases genes (bphC and xylE) are inducted by phenanthrene, m-xylene and biphenyl whereas the 2,4-dichlorophenoxyacetic acid herbicide induced the chlorocatechol 1,2-dioxygenase gene (tfdC) from the ortho-pathway. A method to study upper meta-pathway extradiol dioxygenase gene diversity in soil was developed. The extradiol dioxygenases catalyse cleavage of the aromatic ring between a hydroxylated carbon and an adjacent non-hydroxylated carbon (meta-cleavage). A high diversity of extradiol dioxygenases were detected from polluted soils. The detected extradiol dioxygenases showed sequence similarity to known catabolic genes of Alpha-, Beta-, and Gammaproteobacteria. Five groups of extradiol dioxygenases contained sequences with no close homologues in the database, representing novel genes. In rhizoremediation experiment with birch (Betula pendula) treatment specific changes of extradiol dioxygenase communities were shown. PAH pollution changed the bulk soil extradiol dioxygenase community structure and birch rhizosphere contained a more diverse extradiol dioxygenase community than the bulk soil showing a rhizosphere effect. The degradation of pyrene in soil was enhanced with birch seedlings compared to soil without birch. The complete 280,923 kb nucleotide sequence of pSKY4 plasmid was determined. The open reading frames of pSKY4 were divided into putative conjugative transfer, aromatic degradation, replication/maintaining and transposition/integration function-encoding proteins. Aromatic degradation orfs shared high similarity to corresponding genes in pNL1, a plasmid from the deep subsurface strain Novosphingobium aromaticivorans F199. The plasmid backbones were considerably more divergent with lower similarity, which suggests that the aromatic pathway has functioned as a plasmid independent mobile genetic element. The functional diversity of microbial communities in soil is still largely unknown. Several novel clusters of extradiol dioxygenases representing catabolic bacteria, whose function, biodegradation pathways and phylogenetic position is not known were amplified with single primer pair from polluted soils. These extradiol dioxygenase communities were shown to change upon PAH pollution, which indicates that their hosts function in PAH biodegradation in soil. Although the degradation pathways of specific bacterial species are substantially better depicted than pathways in situ, the evolution of degradation pathways for the xenobiotic compounds is largely unknown. The pSKY4 plasmid contains aromatic degradation genes in putative mobile genetic element causing flexibility/instability to the pathway. The localisation of the aromatic biodegradation pathway in mobile genetic elements suggests that gene transfer and rearrangements are a competetive advantage for Sphingomonas bacteria in the environment.

The C-terminal domain is sufficient for endonuclease activity of Neisseria gonorrhoeae MutL

The mutL gene of Neisseria gonorrhoeae has been cloned and the gene product purified. We have found that the homodimeric N. gonorrhoeae MutL (NgoL) protein displays an endonuclease activity that incises covalently closed circular DNA in the presence of Mn2+, Mg2+ or Ca2+ ions, unlike human MutL alpha which shows endonuclease activity only in the presence of Mn2+. We report in the present paper that the C-terminal domain of N. gonorrhoeae MutL (NgoL-CTD) consisting of amino acids 460-658 exhibits Mn2+-dependent endonuclease activity. Sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments show NgoL-CTD to be a dimer. The probable endonucleolytic active site is localized to a metal-binding motif, DMHAX(2)EX(4)E, and the nicking endonuclease activity is dependent on the integrity of this motif. By in vitro comparison of wild-type and it mutant NgoL-CTD protein, we show that the latter protein exhibits highly reduced endonuclease activity. We therefore suggest that the mode of excision initiation in DNA mismatch repair may be different in organisms that lack MutH protein, but have MutL proteins that harbour the D[M/Q]HAX(2)EX(4)E motif.

Formation of an unusual copper(II) complex from the degradation of a novel tricopper(II) carbohydrazone complex

An unusual copper(II) complex [Cu(L-1a)(2)Cl-2] CH3OH center dot H2O center dot H3O+Cl- (1a) was isolated from a solution of a novel tricopper(II) complex [Cu-3(HL1)Cl-2]Cl-3 center dot 2H(2)O (1) in methanol. where L-1a is 3-(2-pyridyl)triazolo [1,5-a]-pyridine, and characterized with single crystal X-ray diffraction study. The tricopper(II) complex of potential ligand 1,5-bis(di-2-pyridyl ketone) carbohydrazone (H2L1) was synthesized and physicochemically characterized, while the formation of the complex la was followed by time-dependant monitoring of the UV-visible spectra. which reveals degradation of ligand backbone as intensity loss of bands corresponding to O -> Cu(II) charge transfer.

Farnesyltransferase Inhibitors Reduce Ras Activation and Ameliorate Acetaminophen-Induced Liver Injury in Mice

Hepatotoxicity due to overdose of the analgesic and antipyretic acetaminophen (A-PAIP) is a major cause of liver failure in adults. To better understand the contributions of different signaling pathways, the expression and role of Ras activation was evaluated after oral dosing of mice with APAP (400-500 mg/kg). Ras-guanosine triphosphate (GTP) is induced early and in an oxidative stress-dependent manner. The functional role of Ras activation was studied by a single intraperitoneal injection of the neutral sphingomyelinase and farnesyltransferase inhibitor (FTI) manumycin A (I mg/kg), which lowers induction of Ras-GTP and serum amounts of alanine aminotransferase (ALT). APAP dosing decreases hepatic glutathione amounts, which are not affected by manumycin A treatment. However, APAP-induced activation of c-Jun N-terminal kinase, which plays an important role, is reduced by manumycin A. Also, APAP-induced mitochondrial reactive oxygen species are reduced by manumycin A at a later time point during liver injury. Importantly, the induction of genes involved in the inflammatory response (including iNos, gp91phox, and Fasl) and serum amounts of proinflammatory cytokines interferon-gamma (IFN gamma) and tumor necrosis factor alpha, which increase greatly with APAP challenge, are suppressed with manumycin A. The FTI ctivity of manumycin A is most likely involved in reducing APAP-induced liver injury, because a specific neutral sphingomyelinase inhibitor, GW4869 (I mg/kg), did not show any hepatoprotective effect. Notably, a structurally distinct FTI, gliotoxin (I mg/kg), also inhibits Ras activation and reduces serum amounts of ALT and IFN-gamma after APAP dosing. Finally, histological analysis confirmed the hepatoprotective effect f manumycin A and gliotoxin during APAP-induced liver damage. Conclusion: This study identifies a key role for Ras activation and demonstrates the therapeutic efficacy of FTIs during APAP-induced liver injury.

Structure and function of GDNF receptor alpha splice variants

The growth factors of the glial cell line-derived neurotrophic factor (GDNF) family consisting of GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN), are involved in the development, differentiation and maintenance of many types of neurons. They also have important functions outside the nervous system in the development of kidney, testis and thyroid gland. Each of these GFLs preferentially binds to one of the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptors α (GFRα). GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The GFLs in the complex with their cognate GFRα receptors all bind to and signal through the receptor tyrosine kinase RET. Alternative splicing of the mouse GFRα4 gene yields three splice isoforms. These had been described as putative GPI-anchored, transmembrane and soluble forms. My goal was to characterise the function of the different forms of mouse GFRα4. I firstly found that the putative GPI-anchored GFRα4 (GFRα4-GPI) is glycosylated, membrane-bound, GPI-anchored and interacts with PSPN and RET. We also showed that mouse GFRα4-GPI mediates PSPN-induced phosphorylation of RET, promotes PSPN-dependent neuronal differentiation of the rat pheochromocytoma cell line PC6-3 and PSPN-dependent survival of cerebellar granule neurons (CGN). However, although this receptor can mediate PSPN-signalling and activate RET, GFRα4-GPI does not recruit RET into lipid rafts. The recruitment of RET into lipid rafts has previously been thought to be a crucial event for GDNF- and GFL-mediated signalling via RET. I secondly demonstrated that the putative transmembrane GFRα4 (GFRα4-TM) is indeed a real transmembrane GFRα4 protein. Although it has a weak binding capacity for PSPN, it can not mediate PSPN-dependent phosphorylation of RET, neuronal differentiation or survival. These data show that GFRα4-TM is inactive as a receptor for PSPN. Surprisingly, GFRα4-TM can negatively regulate PSPN-mediated signalling via GFRα4-GPI. GFRα4-TM interacts with GFRα4-GPI and blocks PSPN-induced phosphorylation of RET, neuronal differentiation as well as survival. Taken together, our data show that GFRα4-TM may act as a dominant negative inhibitor of PSPN-mediated signaling. The most exciting part of my work was the finding that the putative soluble GFRα4 (GFRα4-sol) can form homodimers and function as an agonist of the RET receptor. In the absence of PSPN, GFRα4-sol can promote the phosphorylation of RET, trigger the activation of the PI-3K/AKT pathway, induce neuronal differentiation and support the survival of CGN. Our findings are in line with a recent publication showing the GFRα4-sol might contribute to the inherited cancer syndrome multiple endocrine neoplasia type 2. Our data provide an explanation to how GFRα4-sol may cause or modify the disease. Mammalian GFRα4 receptors all lack the first Cys-rich domain which is present in other GFRα receptors. In the final part of my work I have studied the function of this particular domain. I created a truncated GFRα1 construct lacking the first Cys-rich domain. Using binding assays in both cellular and cell-free systems, phosphorylation assays with RET, as well as neurite outgrowth assays, we found that the first Cys-rich domain contributes to an optimal function of GFRα1, by stabilizing the interaction between GDNF and GFRα1.