Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform

Recombinant spider silks are of interest but the multimodal and aggregation-prone nature of them is a limitation. Here, the authors report on a miniature spidroin based on the N-terminal domain which forms a hydrogel at 37 degrees C which allows for ease of production and fusion protein modification to generate functional biomaterials.Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 degrees C. The gelation is caused by NT alpha-helix to beta-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties. Our findings demonstrate that recombinant NT and fusion proteins give high expression yields and bestow attractive properties to hydrogels, e.g., transparency, cross-linker free gelation and straightforward immobilization of active proteins at high density

Orphan GPR116 mediates the insulin sensitizing effects of the hepatokine FNDC4 in adipose tissue

The proper functional interaction between different tissues represents a key component in systemic metabolic control. Indeed, disruption of endocrine inter-tissue communication is a hallmark of severe metabolic dysfunction in obesity and diabetes. Here, we show that the FNDC4-GPR116, liver-white adipose tissue endocrine axis controls glucose homeostasis. We found that the liver primarily controlled the circulating levels of soluble FNDC4 (sFNDC4) and lowering of the hepatokine FNDC4 led to prediabetes in mice. Further, we identified the orphan adhesion GPCR GPR116 as a receptor of sFNDC4 in the white adipose tissue. Upon direct and high affinity binding of sFNDC4 to GPR116, sFNDC4 promoted insulin signaling and insulin-mediated glucose uptake in white adipocytes. Indeed, supplementation with FcsFNDC4 in prediabetic mice improved glucose tolerance and inflammatory markers in a white-adipocyte selective and GPR116-dependent manner. Of note, the sFNDC4-GPR116, liver-adipose tissue axis was dampened in (pre) diabetic human patients. Thus our findings will now allow for harnessing this endocrine circuit for alternative therapeutic strategies in obesity-related pre-diabetes. The soluble bioactive form of the transmembrane protein fibronectin type III domain containing 4 (sFNDC4) has anti-inflammatory effects and improves insulin sensitivity. Here the authors show that liver derived sFNDC4 signals through adipose tissue GPCR GPR116 to promote insulin-mediated glucose uptake.Peer reviewe

P-TEFb, the Super Elongation Complex and Mediator Regulate a Subset of Non-paused Genes during Early <i>Drosophila</i> Embryo Development

<div><p>Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early <i>Drosophila</i> embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene <i>Serendipity-α</i>. We also demonstrate that another P-TEFb regulated gene, <i>terminus</i>, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing <i>in vivo</i> evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3’ end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in <i>Drosophila</i> embryos.</p></div

The expression of many patterning genes is relatively normal in P-TEFb embryos.

<p>Wild-type embryos (<b>A, C, E, G, I, K</b>) and embryos depleted of maternal Cdk9 (<b>B, D, F, H, J, L</b>) were hybridized with probes detecting <i>nanos (nos)</i>, <i>polar granule component (pgc)</i>, <i>tailless (tll)</i>, <i>even-skipped (eve)</i>, <i>fushi-tarazu (ftz)</i>, and <i>rhomboid (rho)</i> RNA. Relatively normal expression patterns were observed in Cdk9 embryos. (<b>M</b>) Expression of <i>tll</i>, <i>eve</i>, <i>ftz</i>, and <i>rho</i> in 2–4h P-TEFb embryos quantified by RT-qPCR (n = 4–6). Relative expression was normalized to a mean of four reference genes as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004971#pgen.1004971.g003" target="_blank">Fig. 3</a>. *indicates P<0.05, two-tailed paired Student’s t-test.</p

P-TEFb embryos show cellularization defects and lose cells in the embryo posterior.

<p>(<b>A</b>) Differential interference contrast (DIC) microscopy reveals a posterior loss of cells that appear prior to gastrulation in P-TEFb depleted embryos. Cellularizing P-TEFb embryos look normal (ii and iii), whereas cellularized embryos are missing cells in the posterior (v and vi). Compare to wild-type (wt) embryos in (i) and (iv). (<b>B, C</b>) Confocal images of wild-type (Bi, Ci, iii, v) and P-TEFb embryos immunostained with anti-phosphotyrosine (pTYR) antibody that marks the plasma membrane (green) and anti-Histone 4 marking the nuclei (red). Cells are lost from the posterior in P-TEFb embryos (Bii and iii). In addition, cell shape changes as well as multi-nucleated cells are evident in Cdk9 depleted embryos (Cii), the nuclei do not elongate as in wild-type (compare Ciii with iv), and some nuclei are present outside the basal side of cells in Cdk9 embryos (yellow arrowheads in C iv and vi). In cellularized P-TEFb embryos, mesodermal cells are not properly arranged (Bii, iii, and C vi). Embryos were generated as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004971#pgen.1004971.g001" target="_blank">Fig. 1</a>, and are oriented with anterior to the left and dorsal side up in A and B.</p

Pol II occupancy is reduced at genes affected by P-TEFb depletion.

<p>(<b>A</b>) Confocal images of wild-type embryos (i, iii) and embryos depleted of maternal Cdk9 (ii, iv) stained with an antibody recognizing phosphorylated Pol II CTD Ser2 (Ser2-P, Abcam ab5095). In pre-cellular embryos, an elevated Ser2-P signal was observed in the cytoplasm in Cdk9 embryos (ii) compared to wild-type (i), indicating that the maternal contribution of Ser2 phosphorylated Pol II was increased in Cdk9 embryos. In cellularizing embryos, less Ser2-P was detected in nuclei of Cdk9 embryos (iv) compared to wild-type nuclei (iii). (<b>B</b>) Western blot with extracts from 0–5h old embryos show a decrease in Ser2-P in embryos depleted of maternal Cdk9 or CycT. The monoclonal antibody 8WG16 recognizing the Pol II CTD was used as a loading control. The ratio of Ser2-P to CTD signal was quantified from 3 biological replicates. (<b>C-F</b>) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) of 2–4h wild-type or Cdk9 embryo extracts using antibodies recognizing the Pol II CTD, Pol II Ser2 phosphorylation, and Pol II Ser5 phosphorylation. (<b>C)</b> Pol II occupancy plotted as CTD enrichment relative the intergenic locus IG2c. Less Pol II associates with <i>Sry-α</i> and <i>CG7271</i> in Cdk9 embryos. (<b>D</b>) Less Ser2-P per CTD was observed in Cdk9 embryos compared to wild-type. (<b>E</b>) The Ser5-P/CTD ratio was comparable in wild-type and Cdk9 embryos. (<b>F</b>) The ratio of Pol II Ser-5 signal at the 5’ end versus the 3’ end. No increase at the 5’ end was detected in Cdk9 embryos. Error bars show standard error of the mean (n = 3–5). * indicates P<0.05, two-tailed unpaired Student´s t-test (calculations in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004971#pgen.1004971.s009" target="_blank">S4 Table</a>).</p

The Mediator subunits MED20 and MED22 phenocopy P-TEFb in early embryos.

<p>(<b>A</b>) DIC microscopy of cellularized wild-type (i) and maternally depleted MED20 (ii) or MED22 (iii) embryos show lack of cells in the posterior upon Mediator knock-down. (<b>B</b>) <i>In situ</i> hybridization shows that expression of <i>Sry-α</i> (ii) and <i>term</i> (iv) is compromised, whereas <i>slam</i> (vi) expression is unaffected in embryos depleted of maternal MED22. Wild-type embryos (i, iii, v) are shown for comparison.</p

The P-TEFb and SEC-regulated gene <i>terminus (term)</i> is essential in early embryos.

<p>(<b>A</b>) <i>In situ</i> hybridization showing <i>term/CG7271</i> expression in wild-type during different embryo stages (i-vi). The transcripts become concentrated to the posterior in cellularized embryos (iv and v) (<b>B</b>) Expression of <i>term/CG7271</i> is severely reduced in embryos depleted of maternal Cdk9 (ii) compared to wild-type (i), and rescued by the miRNA-resistant transgene in Cdk9 embryos (iii). Greatly diminished <i>term/CG7271</i> levels are also observed in embryos depleted of maternal CycT (iv) or dEll (v), and in embryos from <i>lilli</i> germline clones (vi). (<b>C</b>) Knockdown of zygotic <i>term</i> by crossing TubGal4 females with Term shmiRNA males (ii), eliminating Term and CG7271 by deletion in embryos derived from the deficiency <i>Df(3L)BSC416</i> (iii), or over-expressing Term by crossing TubGal4 females with UAS-<i>term</i> males (iv) results in severe morphological defects in early embryos, including a failure to form a cellular blastoderm. A wild-type cellularizing embryo (i) is shown for comparison.</p

P-TEFb and the Super Elongation Complex (SEC) display similar phenotypes.

<p>(<b>A</b>) RNA <i>in situ</i> hybridization using a digoxigenin-labeled <i>Serendipity-α (Sry-α)</i> probe demonstrates decreased levels of <i>Sry-α</i>mRNA in embryos depleted of maternal Cdk9 (ii) or Cyclin T (iii) compared to wild-type (i). (<b>B</b>) DIC micrographs of wild-type (wt, i), or embryos derived from females with germline clones of the SEC component dAFF4/Lilliputian (<i>lilli -/-</i>, ii) or females expressing TubGal4 and a shmiRNA targeting the SEC subunit dEll (dELLi, iii) show the same posterior phenotype as in P-TEFb embryos. A close up of the same embryos is shown below (iv-vi). (<b>C</b>) Expression of the cellularization genes <i>bottleneck</i> (<i>bnk</i>, i, ii), <i>slow-as-molasses</i> (<i>slam</i>, iii, iv), and <i>nullo</i> (v, vi) is comparable between wild-type and embryos depleted of maternal Cdk9. (<b>D</b>) Quantification of cellularization gene expression by RT-qPCR. Columns show average values in 2–4h embryos with S.E.M. (n = 5–6) and control values were set to 100%. Relative expression was normalized to a mean of four reference genes (<i>beta-tubulin</i>, <i>GAPDH</i>, <i>RpL32</i>, and <i>28SrRNA</i>). *indicates P<0.05, two-tailed paired Student’s t-test.</p

Liquid-Liquid Phase Separation Primes Spider Silk Proteins for Fiber Formation via a Conditional Sticker Domain

Many protein condensates can convert to fibrillar aggregates, but the underlying mechanisms are unclear. Liquid-liquid phase separation (LLPS) of spider silk proteins, spidroins, suggests a regulatory switch between both states. Here, we combine microscopy and native mass spectrometry to investigate the influence of protein sequence, ions, and regulatory domains on spidroin LLPS. We find that salting out-effects drive LLPS via low-affinity stickers in the repeat domains. Interestingly, conditions that enable LLPS simultaneously cause dissociation of the dimeric C-terminal domain (CTD), priming it for aggregation. Since the CTD enhances LLPS of spidroins but is also required for their conversion into amyloid-like fibers, we expand the stickers and spacers-model of phase separation with the concept of folded domains as conditional stickers that represent regulatory units