(A) Lethality assay. The percentages (%) of animals of a specific stage that did not develop into the next stage are shown. While muscle-specific overexpression of Nep4A (mef2-Gal4 x UAS-Nep4A) led …
Lethality assay.
Depicted are the percentages of animals of a specific stage that do not develop into the next stage. valuesmarked in bold indicate statistically significant deviations from the respective controls.
Size and weight measurements.
3rd instar larvae where grouped into genotype-specific cohorts of 10 individuals. The weights of at least five cohorts per genotype were averaged to calculate the mean weight of one respective larva. P values marked in bold indicate statistically significant deviations from the respective controls.
For size measurements, animals where photographed on scale paper and individual lengths were calculated with Adobe Photoshop. P values marked in bold indicate statistically significant deviations from the respective controls.
(A) Score plot based on genotype-specific NMR spectra. PCA score plot showing the scores of six biological replicates for each genotype. Principal component analysis (PCA) was applied to identify …
Chemical shifts and detected changes of significantly affected metabolites.
Significant changes are based on correlations with predictive scores from cross validated OPLS-DA models (Q2 = 0.95 and 0.74, respectively). A cutoff value for R2 corresponding to p<0.05 with Bonferroni correction for an assumed number of 100 metabolites was used.
The depicted spectra are specific to either glucose or fructose. Signals with contributions from both sugars are excluded. The individual spectra indicate increased levels of both monosaccharides in …
(A) The genotype-specific rates of food intake are depicted as percentages (%) relative to the intake in control specimens (mef2-Gal4 x w1118) after 40 min of feeding, which was set to 100%. While ne…
Feeding assay.
Depicted are dye intensities (no. of detected pixels) within the intestines of animals of the indicated genotype. At least six individuals per genotype and time point were analyzed. P values marked in bold indicate statistically significant deviations from the respective controls.
Changes in the expression of dilp genes are depicted as percentages (%) relative to the expression in control specimens (repo-Gal4 x w1118), which was set to 100%. Glial cell-specific overexpression …
(A) Nep4 protein was labeled with a monospecific antibody (red). In addition to membranes continuous with the nuclear membrane (arrowheads), Nep4 accumulated at the surface of body wall muscles …
nep4 expression was visualized using a reporter construct that drives nuclear GFP (nGFP) expression in a nep4-specific manner (nep4 > nGFP, green). Reversed polarity protein was labeled with a …
(A–C) nep4 expression was assessed using a reporter line that drives nuclear mCherry expression in a nep4-specific manner (nep4 > mCherry, red). dilp2 expression was visualized using a reporter …
Base peak all MS chromatograms of analyzed peptides. The respective sequences of unprocessed full-length peptides (bold) and of identified Nep4-specific cleavage products are indicated. Unlabeled …
Coomassie-stained SDS-polyacrylamide gel of Ni-NTA elution fractions. Nep4B (approx. 113 kDa) was efficiently purified from whole cell lysates of nep4B-transfected SF21 cells (arrow). In identically …
Nep4 hydrolyzes peptides that regulate dilp expression or food intake.
Candidate peptides were analyzed for Nep4-specific cleavage. The individual molecular masses of full length peptides and cleavage products are depicted as the monoisotopic value. Cleavage positions and deviations from the respective theoretical masses (∆) are shown separately. Cleaved peptides are highlighted in blue, and non-cleaved peptides are depicted in red. Superscripts indicate the studies that biochemically characterized the respective peptides (1(Baggerman et al., 2005), 2(Wegener et al., 2006), 3(Wegener and Gorbashov, 2008), 4(Predel et al., 2004), 5(Yew et al., 2009)). n.d. indicates ‘not detected’, thus the respective sequences represent genomic data based predictions.
Name | Sequence | Mass (Da) | Δ(Da) | Sequence of cleavage products | Mass (Da) | Δ(Da) | Cleavage position |
---|---|---|---|---|---|---|---|
| VERYAFGLa4 | 953.5 | −0.0676 | VERYAFG VERYAF | 840.4 783.4 | −0.0893 −0.0898 | G/L F/G |
| LPVYNFGLa5 | 920.5 | −0.0205 | LPVYNFG LPVYNF LPVYN | 808.4 751.4 604.3 | −0.0492 −0.0148 −0.0223 | G/L F/G N/F |
| SRPYSFGLa1, 4 | 924.5 | −0.0523 | YSFGLa | 584.3 | −0.0241 | P/Y |
| TTRPQPFNFGLa1, 4, 5 | 1275.7 | −0.0629 | TTRPQPFNFG TTRPQPFN FNFGLa | 1163.6 959.5 595.3 | −0.0850 −0.0790 −0.0301 | G/L N/F P/F |
| QLTFSPDWa1, 2, 3, 4 | 992.5 | 0.0051 | TFSPDWa FSPDWa | 750.3 649.3 | −0.0360 −0.0473 | L/T T/F |
| QTFQYSRGWTNa1, 2, 3, 4, 5 | 1385.6 | −0.0582 | FQYSRGWTNa QTFQYSRG | 1156.5 985.5 | −0.0319 −0.0743 | T/F G/W |
TVDFGLARGYSGTQ-EAKHRMGLAAANFA-GGPan.d. | 3149.5 | −0.0814 | YSGTQEAKHRMG TVDFGLARG | 1363.6 934.5 | −0.1761 −0.0198 | G/Y; G/L G/Y | |
| FDDYGHMRFa1, 4, 5 | 1185.5 | −0.0572 | FDDYGHMR | 1039.4 | −0.1147 | R/F |
| GGDDQFDDYGHMRFa1, 4, 5 | 1657.7 | −0.0298 | GGDDQFDDYGHMR FDDYGHMRFa | 1511.6 1185.5 | −0.1201 −0.0711 | R/F Q/F |
| NSVVLGKKQRFHSWGa1, 3, 4, 5 | 1741.0 | −0.0905 | NSVVLGKKQRFHS NSVVLGKKQRFH NSVVLGKKQR FHSWGa | 1498.3 1411.8 1127.7 631.3 | −0.1474 −0.1094 −0.1121 −0.0100 | S/W H/S R/F R/F |
AQRSPSLRLRFa2, 3, 4 | 1328.8 | −0.0520 | AQRSPSLRL | 1026.6 | −0.0962 | L/R | |
SPSLRLRFa1, 2, 3, 4, 5 | 973.6 | −0.0859 | SPSLRLR LRLRFa | 827.5 702.5 | −0.1543 −0.1451 | R/F S/L | |
| APTSSFIGMRa1, 4 | 1064.5 | −0.0579 | APTSSFIG FIGMRa | 778.4 621.3 | −0.0434 −0.0706 | G/M S/F |
| APLAFVGLRa1, 5 | 941.6 | −0.0396 | LAFVGLRa APLAFVG FVGLRa APLAF | 773.5 673.4 589.4 517.3 | −0.0858 −0.0202 −0.0686 −0.0183 | P/L G/L A/F F/V |
| APVNSFVGMRa1, 4, 5 | 1075.6 | −0.0742 | APVNSFVG | 789.4 | −0.0314 | G/M |
| APNGFLGMRa1, 5 | 960.5 | 0.0231 | FLGMRa | 621.3 | −0.0666 | G/F |
| SVPFKPRLa1, 2, 3, 4, 5 | 941.6 | −0.0776 | ||||
| SNSRPPRKNDVNTMA-DAYKFLQDLDTYYGD-RARVRFan.d. | 4278.2 | 0.50 | ||||
| RYLPTn.d. | 648.4 | −0.0841 | ||||
| KPQRLRWa5 | 981.6 | −0.05 | ||||
| KPMRLRWa5 | 984.6 | −0.05 | ||||
| APTGFTGMRa1 | 935.5 | −0.0733 | ||||
| AALSDSYDLRGKQQR- FADFNSKFVAVRan.d. | 3087.6 | −0.1694 |