Biochemical, Structural and Cellular Studies on IIGP1, a member of the p47 Family of GTPases

My thesis work focuses on the biochemical, structural and cellular characterisation of IIGP1, a member of the p47 family of GTPases. The p47 family of GTPases are induced transcriptionally from very low resting levels in mouse cells by interferons and are implicated in cell autonomous resistance to intracellular pathogens. A vast subset of genes are regulated by IFNs and the mechanistic details of only a few have been described. Therefore, to understand the function and features of this family of GTPases, an in depth study on IIGP1, a member of this family is investigated. Recombinant IIGP1 was expressed in (FROL and purified to homogeneity, and a detailed biochemical characterisation of IIGP1 was carried out. IIGP1 is a GTPase with low affinity for nucleotides (micromolar range) and a low GTPase activity. The GTPase activity is concentration dependent and functional interaction between IIGP1 molecules occur in a nucleotide dependent manner. IIGP1 shares micromolar nucleotide affinities, and oligomerisation-dependent hydrolytic activity with the 67 kDa GTPase hGBP1 (induced by type I and type II interferons), with the antiviral Mx proteins (type1 interferon induced) and with the paradigm of the self-activating large GTPases, the dynamins. Besides, IIGP1 differs by having a high affinity for GDP and low GTPase activity. The crystal structure of IIGP1 has a N- terminal helical domain followed by a typical G- domain fold and C- terminal helical regions. IIGP1 structure is a representative structure for the p47 family of GTPases. This conclusion arises firstly, from secondary structure analysis of other members of the family which are very similar to IIGP-1 and conform to the expectation derived from the crystal structure. Secondly, from the conservation of several pairs of distant residues in other p47 family members which interact to stabilise the IIGP-1 structure. This conservation could not be explained unless the structures of the other p47 GTPases are very similar to IIGP-1. Structural analysis of IIGP1 with respect to the sequence comparison with the members show several conserved regions, forming the core regions. However large deviations in the primary structure between the members, especially at the N- and C- termini explains their non- redundancy as indicated by their distinct subcellular locations, and resistance to specific pathogens. Cellular characterisation of IIGP1 was investigated in order to understand the features of IIGP1 in the cell. Previous studies on IIGP1 has shown the association of IIGP1 with the endoplasmic reticulum. Since the primary sequence does not indicate signal sequences, or ER retention or retreival signals, IIGP1 is rather peripherally associated with the ER. Cellular fractionation studies reveal differential distribution of the protein, present mostly in the membrane bound form and partly in the soluble form. Membrane attachment is dependent on myristoyl modification, although not solely. There is evidence for ionic interactions which could allow the association of IIGP1 to the membranes, and another pool of the protein is independent of the above two modifications for membrane attachment. This could be accomplished by the C- terminal CaaX like motif for IIGP1 (CLRN) which has not been tested so far. IIGP1 could be immunoprecipitated with D 165 serum, as well as with monoclonal antibodies. Co-precipitation of three putative proteins was acheived, but the identity of these molecules remains to be unknown. IIGP1 is a monomer in the nucleotide-free state in solution, and also in the presence of GDP, but cyrstallised as a dimer with or without GDP. In the presence of nucleotide triphosphates, IIGP1 forms higher oligomers. The importance of the dimer on the properties of IIGP1 was investigated by interfering with the dimer interfaces. The interface mutants have no defects in nucleotide binding but have abrogated cooperativity unlike the wild type, suggesting the functional importance of the dimer. However, the oligomerisation properties of the interface mutants has not been analysed. The data described here formulates a platform for further analysis on IIGP1 function and provides essential parameters to understand the molecular mechanism by which IIGP1 participates in this complex resistance programme. The characterisation of IIGP1 gives an understanding to the behaviour and properties of the protein LQ YLWUR and LQ YLYR

Effect of nitrogen, phosphorus and potassium levels on growth and yield of turmeric (Curcuma longa L.) in the hill zone of Karnataka

A nutritional trial on turmeric (Curcuma longa) variety CO-1 conducted for three seasons at Regional Research Station, Mudigere, Karnataka indicated that a nutrient level of 120 : 60 : 120 kg NPK/ha is optimum for growth and yield of the crop in the hill zone of Karnataka. &nbsp

Effect of nitrogen, phosphorus and potassium levels on growth and yield of turmeric (Curcuma longa L.) in the hill zone of Karnataka

A nutritional trial on turmeric (Curcuma longa) variety CO-1 conducted for three seasons at Regional Research Station, Mudigere, Karnataka indicated that a nutrient level of 120 : 60 : 120 kg NPK/ha is optimum for growth and yield of the crop in the hill zone of Karnataka. &nbsp

Effect of nitrogen, phosphorus and potassium levels on growth and yield of turmeric (Curcuma longa L.) in the hill zone of Karnataka

A nutritional trial on turmeric (Curcuma longa) variety CO-1 conducted for three seasons at Regional Research Station, Mudigere, Karnataka indicated that a nutrient level of 120 : 60 : 120 kg NPK/ha is optimum for growth and yield of the crop in the hill zone of Karnataka. &nbsp

The activation mechanism of Irga6, an interferon-inducible GTPase contributing to mouse resistance against Toxoplasma gondii

Background: The interferon-inducible immunity-related GTPases (IRG proteins/p47 GTPases) are a distinctive family of GTPases that function as powerful cell-autonomous resistance factors. The IRG protein, Irga6 (IIGP1), participates in the disruption of the vacuolar membrane surrounding the intracellular parasite, Toxoplasma gondii, through which it communicates with its cellular hosts. Some aspects of the protein's behaviour have suggested a dynamin-like molecular mode of action, in that the energy released by GTP hydrolysis is transduced into mechanical work that results in deformation and ultimately rupture of the vacuolar membrane. Results: Irga6 forms GTP-dependent oligomers in vitro and thereby activates hydrolysis of the GTP substrate. In this study we define the catalytic G-domain interface by mutagenesis and present a structural model, of how GTP hydrolysis is activated in Irga6 complexes, based on the substrate-twinning reaction mechanism of the signal recognition particle (SRP) and its receptor (SRalpha). In conformity with this model, we show that the bound nucleotide is part of the catalytic interface and that the 3'hydroxyl of the GTP ribose bound to each subunit is essential for trans-activation of hydrolysis of the GTP bound to the other subunit. We show that both positive and negative regulatory interactions between IRG proteins occur via the catalytic interface. Furthermore, mutations that disrupt the catalytic interface also prevent Irga6 from accumulating on the parasitophorous vacuole membrane of T. gondii, showing that GTP-dependent Irga6 activation is an essential component of the resistance mechanism. Conclusions: The catalytic interface of Irga6 defined in the present experiments can probably be used as a paradigm for the nucleotide-dependent interactions of all members of the large family of IRG GTPases, both activating and regulatory. Understanding the activation mechanism of Irga6 will help to explain the mechanism by which IRG proteins exercise their resistance function. We find no support from sequence or G-domain structure for the idea that IRG proteins and the SRP GTPases have a common phylogenetic origin. It therefore seems probable, if surprising, that the substrate-assisted catalytic mechanism has been independently evolved in the two protein families

Differential Distribution of Retinal Ca2+/Calmodulin-Dependent Kinase II (CaMKII) Isoforms Indicates CaMKII-β and -δ as Specific Elements of Electrical Synapses Made of Connexin36 (Cx36)

AII amacrine cells are essential interneurons of the primary rod pathway and transmit rod-driven signals to ON cone bipolar cells to enable scotopic vision. Gap junctions made of connexin36 (Cx36) mediate electrical coupling among AII cells and between AII cells and ON cone bipolar cells. These gap junctions underlie a remarkable degree of plasticity and are modulated by different signaling cascades. In particular, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been characterized as an important regulator of Cx36, capable of potentiating electrical coupling in AII cells. However, it is unclear which CaMKII isoform mediates this effect. To obtain a more detailed understanding of the isoform composition of CaMKII at retinal gap junctions, we analyzed the retinal distribution of all four CaMKII isoforms using confocal microscopy. These experiments revealed a differential distribution of CaMKII isoforms: CaMKII-α was strongly expressed in starburst amacrine cells, which are known to lack electrical coupling. CaMKII-β was abundant in OFF bipolar cells, which form electrical synapses in the outer and the inner retina. CaMKII-γ was diffusely distributed across the entire retina and could not be assigned to a specific cell type. CaMKII-δ labeling was evident in bipolar and AII amacrine cells, which contain the majority of Cx36-immunoreactive puncta in the inner retina. We double-labeled retinas for Cx36 and the four CaMKII isoforms and revealed that the composition of the CaMKII enzyme differs between gap junctions in the outer and the inner retina: in the outer retina, only CaMKII-β colocalized with Cx36-containing gap junctions, whereas in the inner retina, CaMKII-β and -δ colocalized with Cx36. This finding suggests that gap junctions in the inner and the outer retina may be regulated differently although they both contain the same connexin. Taken together, our study identifies CaMKII-β and -δ as Cx36-specific regulators in the mouse retina with CaMKII-δ regulating the primary rod pathway

Developmental Acquisition of a Rapid Calcium-Regulated Vesicle Supply Allows Sustained High Rates of Exocytosis in Auditory Hair Cells

Auditory hair cells (HCs) have the remarkable property to indefinitely sustain high rates of synaptic vesicle release during ongoing sound stimulation. The mechanisms of vesicle supply that allow such indefatigable exocytosis at the ribbon active zone remain largely unknown. To address this issue, we characterized the kinetics of vesicle recruitment and release in developing chick auditory HCs. Experiments were done using the intact chick basilar papilla from E10 (embryonic day 10) to P2 (two days post-hatch) by monitoring changes in membrane capacitance and Ca2+ currents during various voltage stimulations. Compared to immature pre-hearing HCs (E10-E12), mature post-hearing HCs (E18-P2) can steadily mobilize a larger readily releasable pool (RRP) of vesicles with faster kinetics and higher Ca2+ efficiency. As assessed by varying the inter-pulse interval of a 100 ms paired-pulse depolarization protocol, the kinetics of RRP replenishment were found much faster in mature HCs. Unlike mature HCs, exocytosis in immature HCs showed large depression during repetitive stimulations. Remarkably, when the intracellular concentration of EGTA was raised from 0.5 to 2 mM, the paired-pulse depression level remained unchanged in immature HCs but was drastically increased in mature HCs, indicating that the Ca2+ sensitivity of the vesicle replenishment process increases during maturation. Concomitantly, the immunoreactivity of the calcium sensor otoferlin and the number of ribbons at the HC plasma membrane largely increased, reaching a maximum level at E18-P2. Our results suggest that the efficient Ca2+-dependent vesicle release and supply in mature HCs essentially rely on the concomitant engagement of synaptic ribbons and otoferlin at the plasma membrane

Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii

GTPases of the mouse IRG protein family, mediators of resistance against Toxoplasma gondii in the mouse, are inactivated by a polymorphic kinase of the parasite, resulting in enhanced parasite virulence