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.

For methods, please refer to the paper

1) MALDI-TOF data is contained in the zipped folder <MALDI_data.zip>. 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 <MALDI_rawdata_analysis_total.xlsx>:

The final analysis used for Table 1, Fig. 2 and Fig S2 is contained in the Excel file
<MALDI-finalanalysis_forfigure.xlsx>  

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
<LC_MS_finalanalysis.xlsx>. 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
<processing mutants drome_version8_0> 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
<behavioural_data.zip>.

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
<141219_L3_PG33GFP_brpCy5.zip> 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 <150116_L3_PG33stingerGFP.zip>
contains the compressed stacks in .lif format, readable with Leica software or Fiji/ImageJ.