Drosophila neprilysins control insulin signaling and food intake via cleavage of regulatory peptides

  1. Benjamin Hallier
  2. Ronja Schiemann
  3. Eva Cordes
  4. Jessica Vitos-Faleato
  5. Stefan Walter
  6. Jürgen J Heinisch
  7. Anders Malmendal
  8. Achim Paululat
  9. Heiko Meyer  Is a corresponding author
  1. University of Osnabrück, Germany
  2. Institute for Research in Biomedicine, Spain
  3. University of Copenhagen, Denmark
7 figures and 1 table

Figures

Modulating nep4 expression affects life span and body size.

(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 …

https://doi.org/10.7554/eLife.19430.003
Figure 1—source data 1

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.

https://doi.org/10.7554/eLife.19430.004
Figure 1—source data 2

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.

https://doi.org/10.7554/eLife.19430.005
Figure 2 with 1 supplement
Muscle-specific modulation of nep4 expression affects the metabolite composition in transgenic third instar larvae.

(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 …

https://doi.org/10.7554/eLife.19430.006
Figure 2—source data 1

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.

https://doi.org/10.7554/eLife.19430.007
Figure 2—figure supplement 1
NMR-spectra of glucose and fructose.

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 …

https://doi.org/10.7554/eLife.19430.008
Figure 3 with 1 supplement
Muscle-specific modulation of nep4 expression affects food intake and dilp expression in transgenic third instar larvae.

(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…

https://doi.org/10.7554/eLife.19430.009
Figure 3—source data 1

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.

https://doi.org/10.7554/eLife.19430.010
Figure 3—figure supplement 1
Glial cell-specific modulation of nep4 expression affects dilp expression in transgenic third instar larvae.

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 …

https://doi.org/10.7554/eLife.19430.011
Nep4 localizes to the surface of muscle cells.

(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 …

https://doi.org/10.7554/eLife.19430.012
Nep4 is expressed in glial cells and neurons in the central nervous system.

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 …

https://doi.org/10.7554/eLife.19430.013
Nep4 localizes to the surface of insulin-producing cells.

(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 …

https://doi.org/10.7554/eLife.19430.014
Figure 7 with 1 supplement
Nep4 catalyzes the hydrolysis of peptides that regulate dilp expression or feeding behavior.

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 …

https://doi.org/10.7554/eLife.19430.015
Figure 7—figure supplement 1
Heterologously expressed Nep4B can be purified to homogeneity.

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 …

https://doi.org/10.7554/eLife.19430.016

Tables

Table 1

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.

https://doi.org/10.7554/eLife.19430.017

Name

Sequence

Mass (Da)

Δ(Da)

Sequence of cleavage products

Mass (Da)

Δ(Da)

Cleavage position

Allatostatin A1

VERYAFGLa4

953.5

−0.0676

VERYAFG

VERYAF

840.4

783.4

−0.0893

−0.0898

G/L

F/G

Allatostatin A2

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

Allatostatin A3

SRPYSFGLa1, 4

924.5

−0.0523

YSFGLa

584.3

−0.0241

P/Y

Allatostatin A4

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

AKH

QLTFSPDWa1, 2, 3, 4

992.5

0.0051

TFSPDWa

FSPDWa

750.3

649.3

−0.0360

−0.0473

L/T

T/F

Corazonin

QTFQYSRGWTNa1, 2, 3, 4, 5

1385.6

−0.0582

FQYSRGWTNa

QTFQYSRG

1156.5

985.5

−0.0319

−0.0743

T/F

G/W

DH31

TVDFGLARGYSGTQ-EAKHRMGLAAANFA-GGPan.d.

3149.5

−0.0814

YSGTQEAKHRMG

TVDFGLARG

1363.6

934.5

−0.1761

−0.0198

G/Y; G/L

G/Y

Drosulfakinin 1

FDDYGHMRFa1, 4, 5

1185.5

−0.0572

FDDYGHMR

1039.4

−0.1147

R/F

Drosulfakinin 2

GGDDQFDDYGHMRFa1, 4, 5

1657.7

−0.0298

GGDDQFDDYGHMR

FDDYGHMRFa

1511.6

1185.5

−0.1201

−0.0711

R/F

Q/F

Leucokinin

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

sNPF11-11

AQRSPSLRLRFa2, 3, 4

1328.8

−0.0520

AQRSPSLRL

1026.6

−0.0962

L/R

sNPF14-11/ sNPF212-19

SPSLRLRFa1, 2, 3, 4, 5

973.6

−0.0859

SPSLRLR

LRLRFa

827.5

702.5

−0.1543

−0.1451

R/F

S/L

Tachykinin 1

APTSSFIGMRa1, 4

1064.5

−0.0579

APTSSFIG

FIGMRa

778.4

621.3

−0.0434

−0.0706

G/M

S/F

Tachykinin 2

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

Tachykinin 4

APVNSFVGMRa1, 4, 5

1075.6

−0.0742

APVNSFVG

789.4

−0.0314

G/M

Tachykinin 5

APNGFLGMRa1, 5

960.5

0.0231

FLGMRa

621.3

−0.0666

G/F

Hugin

SVPFKPRLa1, 2, 3, 4, 5

941.6

−0.0776

NPF

SNSRPPRKNDVNTMA-DAYKFLQDLDTYYGD-RARVRFan.d.

4278.2

0.50

Proctolin

RYLPTn.d.

648.4

−0.0841

sNPF3

KPQRLRWa5

981.6

−0.05

sNPF4

KPMRLRWa5

984.6

−0.05

Tachykinin 3

APTGFTGMRa1

935.5

−0.0733

Tachykinin 6

AALSDSYDLRGKQQR-

FADFNSKFVAVRan.d.

3087.6

−0.1694

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