The contributions of the HRas C-terminus and its lipid modifications to HRas signal transduction events and membrane binding

In the GTP bound state, Ras proteins activate multiple downstream effector proteins, the combination of which regulate cell growth, differentiation, and changes in cell shape. However, Ras proteins must undergo C-terminal lipidation before they can support biological activity. Specifically, the C-terminus of the HRas protein must be modified by a farnesyl at Cys186 and two palmitates at Cys181 and Cys184. The current paradigm suggests that palmitate is not attached without prior farnesyl addition and that these post-translational lipid modifications are only necessary for membrane association. However, emerging evidence suggests that the C-terminus and its lipid modifications may also be responsible for Ras mediated signal transduction and may represent a previously unsuspected mechanism to control either normal or malignant cell growth;Our studies with two novel mutants of HRas, ExtRas and G43:Ras, demonstrated that palmitoylation could occur in the absence of farnesylation, and begin to explore the role of palmitoylation and the C-terminus in interactions with effector proteins and membrane association. Specifically, the efficiency of membrane association via palmitoylation signals alone, either at the N-terminus (G43:HRas) or the C-terminus (ExtRas), is significantly less than that of a farnesyl, palmitate combination. These two novel HRas proteins also possess different biological activities despite the fact that they contain the same core Ras effector sequence (residues 32--40) and flanking residues which are important for interactions with all identified downstream effector proteins. Further analysis of the unusual biological activity Ext61L protein has found an imbalance in the activation of two specific Ras effector proteins which may underlie its unusual biological activity. These studies emphasize that palmitate and isoprenoid modifications have distinct structural and biological roles and point toward an unexpected participation of the HRas C-terminus in signal transduction

Mutation of Ha-Ras C Terminus Changes Effector Pathway Utilization

In PC12 cells, Ha-Ras modulates multiple effector proteins that induce neuronal differentiation. To regulate these pathways Ha-Ras must be located at the plasma membrane, a process normally requiring attachment of farnesyl and palmitate lipids to the C terminus. Ext61L, a constitutively activated and palmitoylated Ha-Ras that lacks a farnesyl group, induced neurites with more actin cytoskeletal changes and lamellipodia than were induced by farnesylated Ha-Ras61L. Ext61L-triggered neurite outgrowth was prevented easily by co-expressing inhibitory Rho, Cdc42, or p21-activated kinase but required increased amounts of inhibitory Rac. Compared with Ha-Ras61L, Ext61L caused 2-fold greater Rac GTP binding and phosphatidylinositol 3-kinase activity in membranes, a hyperactivation that explained the numerous lamellipodia and ineffectiveness of Rac(N17). In contrast, Ext61L activated B-Raf kinase and ERK phosphorylation more poorly than Ha-Ras61L. Thus, accentuated differentiation by Ext61L apparently results from heightened activation of one Ras effector (phosphatidylinositol 3-kinase) and suboptimal activation of another (B-Raf). This surprising unbalanced effector activation, without changes in the designated Ras effector domain, indicates the Ext61L C-terminal alternations are a new way to influence Ha-Ras-effector utilization and suggest a broader role of the lipidated C terminus in Ha-Ras biological functions

Leukemia-Associated Rho Guanine Nucleotide Exchange Factor Promotes G q-Coupled Activation of RhoA

Leukemia-associated Rho guanine-nucleotide exchange factor (LARG) belongs to the subfamily of Dbl homology RhoGEF proteins (including p115 RhoGEF and PDZ-RhoGEF) that possess amino-terminal regulator of G protein signaling (RGS) boxes also found within GTPase-accelerating proteins (GAPs) for heterotrimeric G protein α subunits. p115 RhoGEF stimulates the intrinsic GTP hydrolysis activity of Gα12/13 subunits and acts as an effector for G13-coupled receptors by linking receptor activation to RhoA activation. The presence of RGS box and Dbl homology domains within LARG suggests this protein may also function as a GAP toward specific Gα subunits and couple Gα activation to RhoA-mediating signaling pathways. Unlike the RGS box of p115 RhoGEF, the RGS box of LARG interacts not only with Gα12 and Gα13 but also with Gαq. In cellular coimmunoprecipitation studies, the LARG RGS box formed stable complexes with the transition state mimetic forms of Gαq, Gα12, and Gα13. Expression of the LARG RGS box diminished the transforming activity of oncogenic G protein-coupled receptors (Mas, G2A, and m1-muscarinic cholinergic) coupled to Gαq and Gα13. Activated Gαq, as well as Gα12 and Gα13, cooperated with LARG and caused synergistic activation of RhoA, suggesting that all three Gα subunits stimulate LARG-mediated activation of RhoA. Our findings suggest that the RhoA exchange factor LARG, unlike the related p115 RhoGEF and PDZ-RhoGEF proteins, can serve as an effector for Gq-coupled receptors, mediating their functional linkage to RhoA-dependent signaling pathways

Remodelling of a polypyrimidine tract-binding protein complex during apoptosis activates cellular IRESs.

Post-transcriptional control of gene expression is mediated by the interaction of RNA-binding proteins with their cognate mRNAs that specifically regulate their stability, localization and translation. mRNA-binding proteins are multifunctional and it has been proposed therefore that a combinatorial RNA-binding protein code exists that allows specific protein sub-complexes to control cytoplasmic gene expression under a range of pathophysiological conditions. We show that polypyrimidine tract-binding protein (PTB) is central to one such complex that forms in apoptotic cells. Thus, during apoptosis initiated by TNF-related apoptosis inducing ligand there is a change in the repertoire of RNA-binding proteins with which PTB interacts. We show that altering the cellular levels of PTB and its binding partners, either singly or in combination, is sufficient to directly change the rates of apoptosis with increased expression of PTB, YBX1, PSF and NONO/p54(nrb) accelerating this process. Mechanistically, we show that these proteins post-transcriptionally regulate gene expression, and therefore apoptotic rates, by interacting with and stimulating the activity of RNA elements (internal ribosome entry segments) found in mRNAs that are translated during apoptosis. Taken together, our data show that PTB function is controlled by a set of co-recruited proteins and importantly provide further evidence that it is possible to dictate cell fate by modulating cytoplasmic gene expression pathways alone

A Non-farnesylated Ha-Ras Protein Can Be Palmitoylated and Trigger Potent Differentiation and Transformation

Ha-Ras undergoes post-translational modifications (including attachment of farnesyl and palmitate) that culminate in localization of the protein to the plasma membrane. Because palmitate is not attached without prior farnesyl addition, the distinct contributions of the two lipid modifications to membrane attachment or biological activity have been difficult to examine. To test if palmitate is able to support these crucial functions on its own, novel C-terminal mutants of Ha-Ras were constructed, retaining the natural sites for palmitoylation, but replacing the C-terminal residue of the CAAX signal for prenylation with six lysines. Both the Ext61L and ExtWT proteins were modified in a dynamic fashion by palmitate, without being farnesylated; bound to membranes modestly (40% as well as native Ha-Ras); and retained appropriate GTP binding properties. Ext61L caused potent transformation of NIH 3T3 cells and, unexpectedly, an exaggerated differentiation of PC12 cells. Ext61L with the six lysines but lacking palmitates was inactive. Thus, farnesyl is not needed as a signal for palmitate attachment or removal, and a combination of transient palmitate modification and basic residues can support Ha-Ras membrane binding and two quite different biological functions. The roles of palmitate can therefore be independent of and distinct from those of farnesyl. Reciprocally, if membrane association can be sustained largely through palmitates, farnesyl is freed to interact with other proteins

On the development of an experimental rig for hydrogen micromix combustion testing

This work describes the development of a combustion rig, aimed at testing hydrogen-fuelled micromix burners for aero gas-turbines at pressures up to 15barg, inlet-air temperatures up to 600K and equivalence ratios (Φ) from leanblow- out to 0.5. It discusses the test facility used, and the design procedure of the experimental apparatus: the requirements of it, the design choices and implementation of instrumentation. Emphasis is placed on the design and manufacture of the burner. Comparison between Additive Manufacturing (AM) and micro-machining techniques for the sub-millimetre injection points shows that further research is needed in this area, to achieve adequate geometric accuracy of the injection holes economically. This rig forms a unique facility for hydrogen micromix testing, offering simultaneous measurements of NOx emissions, Flame-Transfer–Function (FTF) and flame imaging

Expression of RHOGTPase regulators in human myometrium

<p>Abstract</p> <p>Background</p> <p>RHOGTPases play a significant role in modulating myometrial contractility in uterine smooth muscle. They are regulated by at least three families of proteins, RHO guanine nucleotide exchange factors (RHOGEFs), RHOGTPase-activating proteins (RHOGAPs) and RHO guanine nucleotide inhibitors (RHOGDIs). RHOGEFs activate RHOGTPases from the inactive GDP-bound to the active GTP-bound form. RHOGAPs deactivate RHOGTPases by accelerating the intrinsic GTPase activity of the RHOGTPases, converting them from the active to the inactive form. RHOGDIs bind to GDP-bound RHOGTPases and sequester them in the cytosol, thereby inhibiting their activity. Ezrin-Radixin-Moesin (ERM) proteins regulate the cortical actin cytoskeleton, and an ERM protein, moesin (MSN), is activated by and can also activate RHOGTPases.</p> <p>Methods</p> <p>We therefore investigated the expression of various RHOGEFs, RHOGAPs, a RHOGDI and MSN in human myometrium, by semi-quantitative reverse transcription PCR, real-time fluorescence RT-PCR, western blotting and immunofluorescence microscopy. Expression of these molecules was also examined in myometrial smooth muscle cells.</p> <p>Results</p> <p>ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN mRNA and protein expression was confirmed in human myometrium at term pregnancy, at labour and in the non-pregnant state. Furthermore, their expression was detected in myometrial smooth muscle cells. It was determined that ARHGAP24 mRNA expression significantly increased at labour in comparison to the non-labour state.</p> <p>Conclusion</p> <p>This study demonstrated for the first time the expression of the RHOGTPase regulators ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24, ARHGDIA and MSN in human myometrium, at term pregnancy, at labour, in the non-pregnant state and also in myometrial smooth muscle cells. ARHGAP24 mRNA expression significantly increased at labour in comparison to the non-labouring state. Further investigation of these molecules may enable us to further our knowledge of RHOGTPase regulation in human myometrium during pregnancy and labour.</p

LOVTRAP: an optogenetic system for photoinduced protein dissociation

Here we introduce LOVTRAP, an optogenetic approach for reversible, light-induced protein dissociation. LOVTRAP is based on protein A fragments that bind to the LOV domain only in the dark, with tunable kinetics and a >150-fold change in Kd. By reversibly sequestering proteins at mitochondria, we precisely modulated the proteins’ access to the cell edge, demonstrating a naturally occurring 3 mHz cell edge oscillation driven by interactions of Vav2, Rac1 and PI3K

Finding consistent disease subnetworks across microarray datasets

<p>Abstract</p> <p>Background</p> <p>While contemporary methods of microarray analysis are excellent tools for studying individual microarray datasets, they have a tendency to produce different results from different datasets of the same disease. We aim to solve this reproducibility problem by introducing a technique (SNet). SNet provides both quantitative and descriptive analysis of microarray datasets by identifying specific connected portions of pathways that are significant. We term such portions within pathways as “subnetworks”.</p> <p>Results</p> <p>We tested SNet on independent datasets of several diseases, including childhood ALL, DMD and lung cancer. For each of these diseases, we obtained two independent microarray datasets produced by distinct labs on distinct platforms. In each case, our technique consistently produced almost the same list of significant nontrivial subnetworks from two independent sets of microarray data. The gene-level agreement of these significant subnetworks was between 51.18% to 93.01%. In contrast, when the same pairs of microarray datasets were analysed using GSEA, t-test and SAM, this percentage fell between 2.38% to 28.90% for GSEA, 49.60% tp 73.01% for t-test, and 49.96% to 81.25% for SAM. Furthermore, the genes selected using these existing methods did not form subnetworks of substantial size. Thus it is more probable that the subnetworks selected by our technique can provide the researcher with more descriptive information on the portions of the pathway actually affected by the disease.</p> <p>Conclusions</p> <p>These results clearly demonstrate that our technique generates significant subnetworks and genes that are more consistent and reproducible across datasets compared to the other popular methods available (GSEA, t-test and SAM). The large size of subnetworks which we generate indicates that they are generally more biologically significant (less likely to be spurious). In addition, we have chosen two sample subnetworks and validated them with references from biological literature. This shows that our algorithm is capable of generating descriptive biologically conclusions.</p