Tucker, M. R. | Search bioRxiv

bioRxiv2020

A structural model of a Ras-Raf signalosome

The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the MAPK pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras-effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric, GTP-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1, and accessory proteins (Galectin-3 and 14-3-3{sigma}) on the helical assembly. The resulting Ras-Raf signalosome model offers an explanation for a large body of data on MAPK signaling.

molecular biology↗

bioRxiv2020

GWAS reveals the genetic complexity of fructan accumulation patterns in barley grain

We profiled the grain oligosaccharide content of 154 two-row spring barley genotypes and quantified 27 compounds, mainly fructans, that exhibited differential abundance. Clustering revealed two major profile groups where the ‘high’ set contained greater amounts of sugar monomers, sucrose and overall fructans, but lower fructosylraffinose. GWAS identified a significant association for the variability of two fructan types; neoseries-DP7 and inulin-DP9 which showed increased strength when a compound-ratio GWAS was applied. Gene models within this region included five fructan biosynthesis genes, of which three (fructan:fructan 1-fructosyltransferase, sucrose:sucrose 1-fructosyltransferase, and sucrose:fructan 6-fructosyltransferase) have already been described. The remaining two, 6(G)-fructosyltransferase and vacuolar invertase1 have not previously been linked to fructan biosynthesis in barley and showed expression patterns distinct from those of the other three genes, including exclusive expression of 6(G)-fructosyltransferase in outer grain tissues at the storage phase. From exome capture data several SNPs related to inulin- and neoseries-type fructan variability were identified in fructan:fructan 1-fructosyltransferase and 6(G)-fructosyltransferase genes Co-expression analyses uncovered potential regulators of fructan biosynthesis including transcription factors. Our results provide evidence for the distinct biosynthesis of neoseries-type fructans during barley grain maturation plus new gene candidates likely involved in the differential biosynthesis of the various fructan types.Highlight Grain fructan profiles in barley are more complex than previously expected and variations in a diversity panel relate to a genomic region where fructan biosynthesis genes cluster.Abbreviations1-FFTfructan:fructan 1-fructosyltransferase1-SSTsucrose:sucrose 1-fructosyltransferase6-SFTsucrose:fructan 6-fructosyltransferase6G-FFT6(G)-fructosyltransferaseDAPdays after pollinationDPdegree of polymerisationDMdry matterELSDevaporative light scattering detectionFDRfalse discovery rateFOSfructooligosaccharidesFPKMfragments per kilobase, per million mapped readsGWAgenome wide associationGWASgenome wide association studyHAIhours after imbibitionHPAEC–PADhigh pH anion exchange chromatography with pulsed amperometric detectionHPLChigh performance liquid chromatographyKPkestopentaoseKTkestotetraoseLCliquid chromatographyLDlinkage disequilibriumLODlogarithm of oddsMAFminimum allele frequencyMSmass spectrometryNGNeural GasNSneoseries-type fructanPprobability valuePEGpolyethylene glycolQTLquantitative trait lociRFOraffinose family oligosaccharidesRTretention timeSNPsingle nucleotide polymorphismsSPEsolid phase extractionTFAtrifluoroacetic acidTPMtranscripts per millionVI-1vacuolar invertase1View Full Text

plant biology↗

bioRxiv2020

Targeted mutation of barley (1,3;1,4)-β-glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content

Barley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-{beta}-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-{beta}-glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of the Cellulose synthase-like (Csl) gene superfamily that encode known (HvCslF6 and HvCslH1) and putative (HvCslF3 and HvCslF9) (1,3;1,4)-{beta}-glucan synthases. Resultant mutations ranged from single amino acid (aa) substitutions to frameshift mutations causing premature stop codons, and led to specific differences in grain morphology, composition and (1,3;1,4)-{beta}-glucan content. (1,3;1,4)-{beta}-Glucan was absent in the grain of cslf6 knock-out lines whereas cslf9 knock-out lines had similar (1,3;1,4)-{beta}-glucan content to WT. However, cslf9 mutants showed changes in the abundance of other cell wall-related monosaccharides compared to WT. Thousand grain weight (TGW), grain length, width and surface area were altered in cslf6 knock-outs and to a lesser extent TGW in cslf9 knock-outs. cslf3 and cslh1 mutants had no effect on grain (1,3;1,4)-{beta}-glucan content. Our data indicate that multiple members of the CslF/H family fulfil important functions during grain development but, with the exception of HvCslF6, do not impact the abundance of (1,3;1,4)-{beta}-glucan in mature grain.

plant biology↗