The coordination of cell growth during fission yeast mating requires Ras1-GTP hydrolysis

The spatial and temporal control of polarity is fundamental to the survival of all organisms. Cells define their polarity using highly conserved mechanisms that frequently rely upon the action of small GTPases, such as Ras and Cdc42. Schizosaccharomyces pombe is an ideal system with which to study the control of cell polarity since it grows from defined tips using Cdc42-mediated actin remodeling. Here we have investigated the importance of Ras1-GTPase activity for the coordination of polarized cell growth during fission yeast mating. Following pheromone stimulation, Ras1 regulates both a MAPK cascade and the activity of Cdc42 to enable uni-directional cell growth towards a potential mating partner. Like all GTPases, when bound to GTP, Ras1 adopts an active conformation returning to an inactive state upon GTP-hydrolysis, a process accelerated through interaction with negative regulators such as GAPs. Here we show that, at low levels of pheromone stimulation, loss of negative regulation of Ras1 increases signal transduction via the MAPK cascade. However, at the higher concentrations observed during mating, hyperactive Ras1 mutations promote cell death. We demonstrate that these cells die due to their failure to coordinate active Cdc42 into a single growth zone resulting in disorganized actin deposition and unsustainable elongation from multiple tips. These results provide a striking demonstration that the deactivation stage of Ras signaling is fundamentally important in modulating cell polarity

The Repertoire of Heterotrimeric G Proteins and RGS Proteins in Ciona intestinalis

BACKGROUND:Heterotrimeric G proteins and regulators of G protein signaling (RGS) proteins are key downstream interacting partners in the G protein coupled receptor (GPCR) signaling pathway. The highly versatile GPCR transmembrane signaling system is a consequence of the coupling of a diverse set of receptors to downstream partners that include multiple subforms of G proteins and regulatory proteins including RGS proteins, among others. While the GPCR repertoire of Ciona intestinalis, representing the basal chordate is known, the repertoire of the heterotrimeric G proteins and RGS proteins is unknown. METHODOLOGY/PRINCIPAL FINDINGS:In the present study, we performed an in-silico genome-wide search of C. intestinalis for its complement of G proteins and RGS proteins. The identification of several one-to-one orthologs of human G proteins at the levels of families, subfamilies and types and of homologs of the human RGS proteins suggests an evolutionarily conserved structure function relationship of the GPCR signaling mechanism in the chordates. CONCLUSIONS:The C. intestinalis genome encodes a highly conserved, albeit, limited repertoire of the heterotrimeric G protein complexes with the size of subunit types comparable with that in lower eukaryotes

A Computational Study of Elongation Factor G (EFG) Duplicated Genes: Diverged Nature Underlying the Innovation on the Same Structural Template

BACKGROUND: Elongation factor G (EFG) is a core translational protein that catalyzes the elongation and recycling phases of translation. A more complex picture of EFG's evolution and function than previously accepted is emerging from analyzes of heterogeneous EFG family members. Whereas the gene duplication is postulated to be a prominent factor creating functional novelty, the striking divergence between EFG paralogs can be interpreted in terms of innovation in gene function. METHODOLOGY/PRINCIPAL FINDINGS: We present a computational study of the EFG protein family to cover the role of gene duplication in the evolution of protein function. Using phylogenetic methods, genome context conservation and insertion/deletion (indel) analysis we demonstrate that the EFG gene copies form four subfamilies: EFG I, spdEFG1, spdEFG2, and EFG II. These ancient gene families differ by their indispensability, degree of divergence and number of indels. We show the distribution of EFG subfamilies and describe evidences for lateral gene transfer and recent duplications. Extended studies of the EFG II subfamily concern its diverged nature. Remarkably, EFG II appears to be a widely distributed and a much-diversified subfamily whose subdivisions correlate with phylum or class borders. The EFG II subfamily specific characteristics are low conservation of the GTPase domain, domains II and III; absence of the trGTPase specific G2 consensus motif "RGITI"; and twelve conserved positions common to the whole subfamily. The EFG II specific functional changes could be related to changes in the properties of nucleotide binding and hydrolysis and strengthened ionic interactions between EFG II and the ribosome, particularly between parts of the decoding site and loop I of domain IV. CONCLUSIONS/SIGNIFICANCE: Our work, for the first time, comprehensively identifies and describes EFG subfamilies and improves our understanding of the function and evolution of EFG duplicated genes

X-ray Structures of the Signal Recognition Particle Receptor Reveal Targeting Cycle Intermediates

The signal recognition particle (SRP) and its conjugate receptor (SR) mediate cotranslational targeting of a subclass of proteins destined for secretion to the endoplasmic reticulum membrane in eukaryotes or to the plasma membrane in prokaryotes. Conserved active site residues in the GTPase domains of both SRP and SR mediate discrete conformational changes during formation and dissociation of the SRP·SR complex. Here, we describe structures of the prokaryotic SR, FtsY, as an apo protein and in two different complexes with a non-hydrolysable GTP analog (GMPPNP). These structures reveal intermediate conformations of FtsY containing GMPPNP and explain how the conserved active site residues position the nucleotide into a non-catalytic conformation. The basis for the lower specificity of binding of nucleotide in FtsY prior to heterodimerization with the SRP conjugate Ffh is also shown. We propose that these structural changes represent discrete conformational states assumed by FtsY during targeting complex formation and dissociation

G Protein Activation without a GEF in the Plant Kingdom

Animal heterotrimeric G proteins are activated by guanine nucleotide exchange factors (GEF), typically seven transmembrane receptors that trigger GDP release and subsequent GTP binding. In contrast, the Arabidopsis thaliana G protein (AtGPA1) rapidly activates itself without a GEF and is instead regulated by a seven transmembrane Regulator of G protein Signaling (7TM-RGS) protein that promotes GTP hydrolysis to reset the inactive (GDP-bound) state. It is not known if this unusual activation is a major and constraining part of the evolutionary history of G signaling in eukaryotes. In particular, it is not known if this is an ancestral form or if this mechanism is maintained, and therefore constrained, within the plant kingdom. To determine if this mode of signal regulation is conserved throughout the plant kingdom, we analyzed available plant genomes for G protein signaling components, and we purified individually the plant components encoded in an informative set of plant genomes in order to determine their activation properties in vitro. While the subunits of the heterotrimeric G protein complex are encoded in vascular plant genomes, the 7TM-RGS genes were lost in all investigated grasses. Despite the absence of a Gα-inactivating protein in grasses, all vascular plant Gα proteins examined rapidly released GDP without a receptor and slowly hydrolyzed GTP, indicating that these Gα are self-activating. We showed further that a single amino acid substitution found naturally in grass Gα proteins reduced the Gα-RGS interaction, and this amino acid substitution occurred before the loss of the RGS gene in the grass lineage. Like grasses, non-vascular plants also appear to lack RGS proteins. However, unlike grasses, one representative non-vascular plant Gα showed rapid GTP hydrolysis, likely compensating for the loss of the RGS gene. Our findings, the loss of a regulatory gene and the retention of the “self-activating” trait, indicate the existence of divergent Gα regulatory mechanisms in the plant kingdom. In the grasses, purifying selection on the regulatory gene was lost after the physical decoupling of the RGS protein and its cognate Gα partner. More broadly these findings show extreme divergence in Gα activation and regulation that played a critical role in the evolution of G protein signaling pathways

An ancient family of SelB elongation factor-like proteins with a broad but disjunct distribution across archaea

<p>Abstract</p> <p>Background</p> <p>SelB is the dedicated elongation factor for delivery of selenocysteinyl-tRNA to the ribosome. In archaea, only a subset of methanogens utilizes selenocysteine and encodes archaeal SelB (aSelB). A SelB-like (aSelBL) homolog has previously been identified in an archaeon that does not encode selenosysteine, and has been proposed to be a pyrrolysyl-tRNA-specific elongation factor (EF-Pyl). However, elongation factor EF-Tu is capable of binding archaeal Pyl-tRNA in bacteria, suggesting the archaeal ortholog EF1A may also be capable of delivering Pyl-tRNA to the ribosome without the need of a specialized factor.</p> <p>Results</p> <p>We have phylogenetically characterized the aSelB and aSelBL families in archaea. We find the distribution of aSelBL to be wider than both selenocysteine and pyrrolysine usage. The aSelBLs also lack the carboxy terminal domain usually involved in recognition of the selenocysteine insertion sequence in the target mRNA. While most aSelBL-encoding archaea are methanogenic Euryarchaea, we also find aSelBL representatives in Sulfolobales and Thermoproteales of Crenarchaea, and in the recently identified phylum Thaumarchaea, suggesting that aSelBL evolution has involved horizontal gene transfer and/or parallel loss. Severe disruption of the GTPase domain suggests that some family members may employ a hitherto unknown mechanism of nucleotide hydrolysis, or have lost their GTPase ability altogether. However, patterns of sequence conservation indicate that aSelBL is still capable of binding the ribosome and aminoacyl-tRNA.</p> <p>Conclusions</p> <p>Although it is closely related to SelB, aSelBL appears unlikely to either bind selenocysteinyl-tRNA or function as a classical GTP hydrolyzing elongation factor. We propose that following duplication of aSelB, the resultant aSelBL was recruited for binding another aminoacyl-tRNA. In bacteria, aminoacylation with selenocysteine is essential for efficient thermodynamic coupling of SelB binding to tRNA and GTP. Therefore, change in tRNA specificity of aSelBL could have disrupted its GTPase cycle, leading to relaxation of selective pressure on the GTPase domain and explaining its apparent degradation. While the specific role of aSelBL is yet to be experimentally tested, its broad phylogenetic distribution, surpassing that of aSelB, indicates its importance.</p

RNAi Methodologies for the Functional Study of Signaling Molecules

RNA interference (RNAi) was investigated with the aim of achieving gene silencing with diverse RNAi platforms that include small interfering RNA (siRNA), short hairpin RNA (shRNA) and antisense oligonucleotides (ASO). Different versions of each system were used to silence the expression of specific subunits of the heterotrimeric signal transducing G-proteins, G alpha i2 and G beta 2, in the RAW 264.7 murine macrophage cell line. The specificity of the different RNA interference (RNAi) platforms was assessed by DNA microarray analysis. Reliable RNAi methodologies against the genes of interest were then developed and applied to functional studies of signaling networks. This study demonstrates a successful knockdown of target genes and shows the potential of RNAi for use in functional studies of signaling molecules

Modeling of Human Prokineticin Receptors: Interactions with Novel Small-Molecule Binders and Potential Off-Target Drugs

The Prokineticin receptor (PKR) 1 and 2 subtypes are novel members of family A GPCRs, which exhibit an unusually high degree of sequence similarity. Prokineticins (PKs), their cognate ligands, are small secreted proteins of ∼80 amino acids; however, non-peptidic low-molecular weight antagonists have also been identified. PKs and their receptors play important roles under various physiological conditions such as maintaining circadian rhythm and pain perception, as well as regulating angiogenesis and modulating immunity. Identifying binding sites for known antagonists and for additional potential binders will facilitate studying and regulating these novel receptors. Blocking PKRs may serve as a therapeutic tool for various diseases, including acute pain, inflammation and cancer.Ligand-based pharmacophore models were derived from known antagonists, and virtual screening performed on the DrugBank dataset identified potential human PKR (hPKR) ligands with novel scaffolds. Interestingly, these included several HIV protease inhibitors for which endothelial cell dysfunction is a documented side effect. Our results suggest that the side effects might be due to inhibition of the PKR signaling pathway. Docking of known binders to a 3D homology model of hPKR1 is in agreement with the well-established canonical TM-bundle binding site of family A GPCRs. Furthermore, the docking results highlight residues that may form specific contacts with the ligands. These contacts provide structural explanation for the importance of several chemical features that were obtained from the structure-activity analysis of known binders. With the exception of a single loop residue that might be perused in the future for obtaining subtype-specific regulation, the results suggest an identical TM-bundle binding site for hPKR1 and hPKR2. In addition, analysis of the intracellular regions highlights variable regions that may provide subtype specificity