Decoding RAS isoform and codon-specific signalling

RAS proteins are key signalling hubs that are oncogenically mutated in 30% of all cancer cases. Three genes encode almost identical isoforms that are ubiquitously expressed, but are not functionally redundant. The network responses associated with each isoform and individual oncogenic mutations remain to be fully characterized. In the present article, we review recent data defining the differences between the RAS isoforms and their most commonly mutated codons and discuss the underlying mechanisms

Proteomics Reveal a Concerted Upregulation of Methionine Metabolic Pathway Enzymes, and Downregulation of Carbonic Anhydrase-III, in Betaine Supplemented Ethanol-Fed Rats

We employed a proteomic profiling strategy to examine the effects of ethanol and betaine diet supplementation on major liver protein level changes. Male Wistar rats were fed control, ethanol or betaine supplemented diets for 4 weeks. Livers were removed and liver cytosolic proteins resolved by onedimensional and two-dimensional separation techniques. Significant upregulation of betaine homocysteine methyltransferase-1, methionine adenosyl transferase-1, and glycine N-methyltransferase were the most visually prominent protein changes observed in livers of rats fed the betaine supplemented ethanol diet. We hypothesise that this concerted upregulation of these methionine metabolic pathway enzymes is the protective mechanism by which betaine restores a normal metabolic ratio of liver S-adenosylmethionine to S-adenosylhomocysteine. Ethanol also induced significant downregulation of carbonic anhydrase- III protein levels which was not restored by betaine supplementation. Carbonic anhydrase-III can function to resist oxidative stress, and we therefore hypothesise that carbonic anhydrase-III protein levels compromised by ethanol consumption, contribute to ethanol-induced redox stress

Isoaspartate, Carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury

We had previously shown that alcohol consumption can induce cellular isoaspartate protein damage via an impairment of the activity of protein isoaspartyl methyltransferase (PIMT), an enzyme that triggers repair of isoaspartate protein damage. To further investigate the mechanism of isoaspartate accumulation, hepatocytes cultured from control or 4-week ethanol-fed rats were incubated in vitro with tubercidin or adenosine. Both these agents, known to elevate intracellular S-adenosylhomocysteine levels, increased cellular isoaspartate damage over that recorded following ethanol consumption in vivo. Increased isoaspartate damage was attenuated by treatment with betaine. To characterize isoaspartate-damaged proteins that accumulate after ethanol administration, rat liver cytosolic proteins were methylated using exogenous PIMT and 3H-S- adenosylmethionine and proteins resolved by gel electrophoresis. Three major protein bands of ~75-80 kDa, ~95-100 kDa, and ~155-160 kDa were identified by autoradiography. Column chromatography used to enrich isoaspartate-damaged proteins indicated that damaged proteins from ethanol-fed rats were similar to those that accrued in the livers of PIMT knockout (KO) mice. Carbamoyl phosphate synthase-1 (CPS-1) was partially purified and identified as the ~160kDa protein target of PIMT in ethanol-fed rats and in PIMT KO mice. Analysis of the liver proteome of 4-week ethanol-fed rats and PIMT KO mice demonstrated elevated cytosolic CPS-1 and betaine homocysteine S-methyltransferase-1 when compared to their respective controls, and a significant reduction of carbonic anhydrase-III (CA-III) evident only in ethanol-fed rats. Ethanol feeding of rats for 8 weeks resulted in a larger (~2.3-fold) increase in CPS-1 levels compared to 4- week ethanol feeding indicating that CPS-1 accumulation correlated with the duration of ethanol consumption. Collectively, our results suggest that elevated isoaspartate and CPS-1, and reduced CA-III levels could serve as biomarkers of hepatocellular injury

Postnatal Changes in the Expression Pattern of the Imprinted Signalling Protein XLαs Underlie the Changing Phenotype of Deficient Mice

The alternatively spliced trimeric G-protein subunit XLαs, which is involved in cAMP signalling, is encoded by the Gnasxl transcript of the imprinted Gnas locus. XLαs deficient mice show neonatal feeding problems, leanness, inertia and a high mortality rate. Mutants that survive to weaning age develop into healthy and fertile adults, which remain lean despite elevated food intake. The adult metabolic phenotype can be attributed to increased energy expenditure, which appears to be caused by elevated sympathetic nervous system activity. To better understand the changing phenotype of Gnasxl deficient mice, we compared XLαs expression in neonatal versus adult tissues, analysed its co-localisation with neural markers and characterised changes in the nutrient-sensing mTOR1-S6K pathway in the hypothalamus. Using a newly generated conditional Gnasxl lacZ gene trap line and immunohistochemistry we identified various types of muscle, including smooth muscle cells of blood vessels, as the major peripheral sites of expression in neonates. Expression in all muscle tissues was silenced in adults. While Gnasxl expression in the central nervous system was also developmentally silenced in some midbrain nuclei, it was upregulated in the preoptic area, the medial amygdala, several hypothalamic nuclei (e.g. arcuate, dorsomedial, lateral and paraventricular nuclei) and the nucleus of the solitary tract. Furthermore, expression was detected in the ventral medulla as well as in motoneurons and a subset of sympathetic preganglionic neurons of the spinal cord. In the arcuate nucleus of Gnasxl-deficient mice we found reduced activity of the nutrient sensing mTOR1-S6K signalling pathway, which concurs with their metabolic status. The expression in these brain regions and the hypermetabolic phenotype of adult Gnasxl-deficient mice imply an inhibitory function of XLαs in energy expenditure and sympathetic outflow. By contrast, the neonatal phenotype of mutant mice appears to be due to a transient role of XLαs in muscle tissues

Decoding RAS isoform and codon-specific signalling

Abstract RAS proteins are key signalling hubs that are oncogenically mutated in 30 % of all cancer cases. Three genes encode almost identical isoforms that are ubiquitously expressed, but are not functionally redundant. The network responses associated with each isoform and individual oncogenic mutations remain to be fully characterized. In the present article, we review recent data defining the differences between the RAS isoforms and their most commonly mutated codons and discuss the underlying mechanisms

Generation of a conditional gene trap mouse line for

<p> (<b>A</b>) The targeting construct comprises the <i>Gnas</i> exon 2 splice acceptor (Ex2SA) fused in frame to LacZ-pA, an frt-flanked <i>neo</i>^r cassette and pairs of loxP/lox2272 sites assembled in head-to-head orientation. The gene trap cassette was inserted in the non-functional (antisense) orientation at the rarely used A20 exon position (see D–F). Cre recombination results in inversion at compatible lox sites followed by excision between head-to-tail sites, which results in a stably integrated gene trap that attracts splicing from <i>Gnasxl</i> exon 1. The resulting XL domain – β-Galactosidase (XL-βGal) fusion protein lacks XLαs function, but retains β-Galactosidase activity. Relative positions of probes and restriction sites used in Southern-blots are shown, as are PCR primers (arrows) used in genotyping. (<b>B</b>) Southern blots showing a correctly targeted ES-cell clone (<i>Spe</i>I: WT = 16.1 kbp, targeted = 20.4 kbp; <i>Afl</i>II: targeted = 13.7 kbp; <i>Mfe</i>I: targeted = 8.8 kb). (<b>C</b>) Genotyping PCRs for deletion of the <i>neo</i>^r cassette via <i>Flpe</i> mice (left), and for Cre recombinase mediated inversion of the gene trap (right). The Flpe / Cre status of the samples is given above the lanes. For primer locations see (A). (<b>D</b>) Scheme indicating splicing of the rarely used A20 exon in full-length <i>Gnasxl</i> and neural-specific <i>XLN1</i> transcripts. Arrows indicate primers used in (E). (<b>E</b>) RT-PCR from wild-type brain using a common <i>Gnasxl</i> exon 1 primer combined with reverse primers in exon 5 (full-length <i>Gnasxl</i>) or exon N1 (<i>XLN1</i>). Exon A20-containing products (size increase: 95 bp) are indicated by asterisks above the respective bands and are hardly detectable in full-length transcripts (XL lanes), but are more prominent in <i>XLN1</i> transcripts (N1 lanes). (<b>F</b>) Inclusion of the A20 exon results in a frame shift and termination codon in exon 2. The translated <i>Gnasxl</i>-A20 sequence from (E) is shown: <i>Gnasxl</i> exon 1 sequence (not highlighted), exon A20 sequence (highlighted black), exon 2 sequence (highlighted grey).</p

Isoform-specific Ras signaling is growth factor dependent

From Crossref via Jisc Publications RouterHistory: ppub 2019-04-15, issued 2019-04-15HRAS, NRAS, and KRAS isoforms are almost identical proteins that are ubiquitously expressed and activate a common set of effectors. In vivo studies have revealed that they are not biologically redundant; however, the isoform specificity of Ras signaling remains poorly understood. Using a novel panel of isogenic SW48 cell lines endogenously expressing wild-type or G12V-mutated activated Ras isoforms, we have performed a detailed characterization of endogenous isoform-specific mutant Ras signaling. We find that despite displaying significant Ras activation, the downstream outputs of oncogenic Ras mutants are minimal in the absence of growth factor inputs. The lack of mutant KRAS-induced effector activation observed in SW48 cells appears to be representative of a broad panel of colon cancer cell lines harboring mutant KRAS. For MAP kinase pathway activation in KRAS-mutant cells, the requirement for coincident growth factor stimulation occurs at an early point in the Raf activation cycle. Finally, we find that Ras isoform-specific signaling was highly context dependent and did not conform to the dogma derived from ectopic expression studies

XLαs co-localisation with hypothalamic neuropeptides and assessment of arcuate nucleus mTOR1-S6K activity in <i>Gnasxl</i>^m+/p− mice.

<p>(<b>A</b>) A representative image of co-localisation of XLαs (green) with Orexin A (red) in the DMH/LH is shown (i) and quantification of co-localised cell body staining is provided on the right (ii). (<b>B</b>) XLαs (purple) and MCH (brown) are expressed in separate neuron populations in the DMH/LH. (<b>C, D</b>) Immunofluorescence staining for XLαs (C) and <i>in situ</i> hybridisation for CRH (D) in neighbouring hypothalamic sections indicate separate expression domains in peripheral and central subdivisions of the PVH, respectively. (<b>E</b>) Co-localisation of pS6, a substrate and marker of mTOR1-S6K activity, in XLαs-positive neurons of wild-type Arc tissue. A representative image (i) and quantification (ii) of a series of sections from two mice (age 12 weeks) is shown. ∼32% of XLαs-positive neurons show pS6 signals under <i>ad libitum</i> normal chow fed conditions. (<b>F–H</b>) Reduced number of pS6 positive neurons in the Arc of <i>Gnasxl</i>^m+/p− mice (aged 12 weeks on normal chow diet). Representative images of pS6 immunofluorescence of wild-type (F) and <i>Gnasxl</i>^m+/p− (G) Arc sections are shown, and a quantification is provided in (H), (n = 6; p = 0.036). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029753#s2" target="_blank">materials and methods</a> for details of the quantification method.</p

Expression of <i>Gnasxl</i> in the adult brain.

<p>(<b>A–C</b>) XGal staining of <i>CMV-Cre</i>/+; +/<i>XLlacZGT</i> sagittal brain vibratome section (thickness: 500 µm) indicating XL-βGal fusion protein activity in the hypothalamus (A) and medulla ((B) midsagittal, (C) parasagittal). Note the reduced expression levels in the 7N compared to the neonatal stage (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029753#pone-0029753-g003" target="_blank">Figure 3G</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029753#pone.0029753-Plagge2" target="_blank">[30]</a>). (<b>D–J</b>) Immunohistochemistry for XLαs on coronal wild-type brain sections in caudal (medulla) to rostral (preoptic area) order. Changes from the neonatal pattern include downregulation of expression in the 12N (D i), silencing in the LDTg of the pons (F), novel expression in the nucleus tractus solitarius (D) and medial amygdala (G), and more widespread expression in several hypothalamic nuclei (G, H) and the preoptic area (I, J). Note that in (F) the darkly stained area indicates the neuronal cell bodies of the LC, while the surrounding lighter stained area represents the dense network of neurites emerging from the LC. Higher magnification images of the region show no XLαs signals in the neurons of the LDTg. 5N – motor trigeminal nucleus, 7N – facial nucleus, 12N – hypoglossal nucleus, A14 – A14 dopamine cells, ac – anterior commissure, ADP – anterodorsal preoptic nucleus, Amb – ambiguus nucleus, Amy – medial amygdaloid nucleus, Arc – arcuate nucleus, BAC – bed nucleus of the anterior commissure, BSTM – bed nucleus of the stria terminalis (medial parts), DMH – dorsomedial hypothalamic nucleus, Gi – gigantocellular reticular nucleus, LC – locus coeruleus, LDTg – laterodorsal tegmental nucleus, LH – lateral hypothalamic area, LSV – lateral septal nucleus (ventral part), NTS – nucleus tractus solitarius, PreOp – preoptic area, PVH – paraventricular hypothalamic nucleus, RPa – raphe pallidus, SCh – suprachiamatic nucleus, SubC – subcoeruleus nucleus.</p

Spinal cord expression of <i>Gnasxl</i>.

<p>(<b>A–C</b>) Immunohistochemistry of neonatal (P1) thoracic spinal cords from <i>Nestin-Cre/+</i>; +/<i>XLlacZGT</i> mice using an anti-βGalactosidase antibody (red, A and C), or from wild-type mice using an anti-XLαs antibody (purple DAB/Ni staining, B). Transverse (A, B) and sagittal (C) sections are shown. <i>Gnasxl</i> expression is detected in scattered neurons of the intermediolateral region as well as in the ventrolateral, motoneuron containing area. (<b>D–G</b>) Co-staining for XL-βGal fusion protein (red) and Choline acetyltransferase (ChAT; green) on sagittal sections from neonatal (D, E) and adult (F, G) spinal cords. ChAT marks cholinergic sympathetic preganglionic neurons of the intermediolateral layer as well as ventrolateral motoneurons. While XLαs and ChAT are co-expressed in the majority of motoneurons, they are only occasionally co-localised in neurons of the intermediolateral layer (white arrows in (E–G)). Epifluorescent (D, F, G) and confocal (E) images are shown. (G) Shows a magnification of the area indicated in (F). Note the plasma membrane association of XL-βGal fusion protein, due to palmitoylation of the XL domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029753#pone.0029753-Ugur1" target="_blank">[97]</a>. Neonatal tissues in (D, E) were obtained from <i>Nestin-Cre</i>/+; +/<i>XLlacZGT</i> offspring, while adult samples in (F, G) were derived from <i>CMV-Cre</i>/+; +/<i>XLlacZGT</i> mice.</p