DOI: 10.5061/dryad.82pr5td
title: Drosophila carboxypeptidase D (SILVER) is a key enzyme in neuropeptide processing required to maintain locomotor activity levels and survival rate

description: Neuropeptides are processed from larger preproproteins by a dedicated set
 of enzymes. The molecular and biochemical mechanisms underlying
 preproprotein processing and the functional importance of processing
 enzymes are well‐characterised in mammals, but little studied outside this
 group. In contrast to mammals, Drosophila melanogaster lacks a gene for
 carboxypeptidase E (CPE), a key enzyme for mammalian peptide processing.
 By combining peptidomics and neurogenetics, we addressed the role of
 carboxypeptidase D (dCPD) in global neuropeptide processing and selected
 peptide‐regulated behaviours in Drosophila. We found that a deficiency in
 dCPD results in C‐terminally extended peptides across the peptidome,
 suggesting that dCPD took over CPE function in the fruit fly. dCPD is
 widely expressed throughout the nervous system, including peptidergic
 neurons in the mushroom body and neuroendocrine cells expressing
 adipokinetic hormone. Conditional hypomorphic mutation in the
 dCPD‐encoding gene silver in the larva causes lethality, and leads to
 deficits in starvation‐induced hyperactivity and appetitive gustatory
 preference, as well as to reduced viability and activity levels in adults.
 A phylogenomic analysis suggests that loss of CPE is not common to
 insects, but only occurred in Hymenoptera and Diptera. Our results show
 that dCPD is a key enzyme for neuropeptide processing and
 peptide‐regulated behaviour in Drosophila. dCPD thus appears as a suitable
 target to genetically shut down total neuropeptide production in
 peptidergic neurons. The persistent occurrence of CPD in insect genomes
 may point to important further CPD functions beyond neuropeptide
 processing which cannot be fulfilled by CPE.
description: <pre class="western"> 1) MALDI-TOF data is contained in
 the zipped folder &lt;MALDI_data.zip&gt;. The included t2d files
 contains the spectra including MS/MS fragmentation spectra. # gives the
 spot number (e.g. A2), description and analysis is found in the Excel file
 &lt;MALDI_rawdata_analysis_total.xlsx&gt;: The final analysis used
 for Table 1, Fig. 2 and Fig S2 is contained in the Excel file
 &lt;MALDI-finalanalysis_forfigure.xlsx&gt;   Methods: MALDI-TOF
 mass spectra were acquired in positive ion mode on an Applied Biosystems
 4800 Plus MALDI-TOF/TOF mass spectrometer (Applied Biosystems/ MDS Sciex).
 Samples were analysed in positive reflector mode within a mass window of
 850–3,000 Da. Some mass peaks were additionally fragmented in MS/MS mode
 using PSD. Laser power was adjusted manually to provide optimal
 signal-to-noise ratio. Raw data were analysed using Data Explorer 4.10
 software (Applied Biosystems/MDS Sciex). 2) LC-MS/MS data (Orbitrap) The
 final analysis is contained in the Excel file
 &lt;LC_MS_finalanalysis.xlsx&gt;. Originally, we made 3 separate
 runs of separate samples for both wildtype and mutants. However, one run
 for the mutants was ignored since only very few peptides were found,
 possibly due to a poor extraction. Thus, n=3 for wildtype, n=2 for
 mutants. All runs (including the discarded one) are included in the raw
 data set from the Orbitrap Fusion &lt;processing mutants
 drome_version8_0&gt; which is provided as a zipped folder (.zip).
  This file is in a format for Peaks Studio 8.5 which can be downloaded for
 free as a viewer:  http://www.bioinfor.com/download-peaks-studio/ Methods:
 NanoLC-MS/MS analyses were performed on an Orbitrap Fusion (Thermo
 Scientific) equipped with a PicoView Ion Source (New Objective) and
 coupled to an EASY-nLC 1000 (Thermo Scientific). Data analysis was
 performed with PEAKS Studio 8.5 (Bioinformatics Solutions Inc.; Zhang et
 al., 2012). 3) Behavioural data The data underlying the behavioural
 experiments are contained in the zipped folder
 &lt;behavioural_data.zip&gt;. Raw data of larval tracking are
 given as Microsoft Excel files (.xlsx), named after the respective Figure
 5A to D, or Fig. 6.  Please note that the x-axis in Fig. 6 a-c shows time
 in seconds, not minutes as is incorrectly stated.   4) Microscopy data
 Several preparations were made to prepare Fig. 4, using two crosses and
 anti-GFP and anti-Brp (nc82) staining: -   y1w*P{w+mWhs=GawB}svrPG33/FM7h
 crossed to 10xUAS-IVS-myr::GFP  Folder
 &lt;141219_L3_PG33GFP_brpCy5.zip&gt; contains the compressed
 stacks in .lif format, readable with Leica software or Fiji/ImageJ. -  
 y1w*P{w+mWhs=GawB}svrPG33/FM7h crossed to UAS-stinger Folder
 &lt;150116_L3_PG33stingerGFP.zip&gt; contains the compressed
 stacks in .lif format, readable with Leica software or Fiji/ImageJ.
 </pre>
description: For methods, please refer to the paper