Proteomic changes in response to crystal formation in<i>Drosophila</i>Malpighian tubules

Chung, Vera Y.; Konietzny, Rebecca; Charles, P. David; Kessler, Benedikt M.; Fischer, Román; Turney, Benjamin

doi:10.1080/19336934.2016.1171947

Cited by 12 publications

(6 citation statements)

References 30 publications

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“…Dried tryptic peptides were reconstituted in 12 µL of LC-MS grade water containing 2% acetonitrile and 0.1% trifluoroacetic acid. Samples were subsequently analyzed by nano-LC_MS/MS using a Dionex Ultimate 3000 UPLC coupled to a QExactive instrument (Thermo Scientific, Bremen, Germany) as described previously [60][61][62] . Peptides were online desalted on a PepMapC18 column (300 µm x 5 mm, 5 µm particle size, Thermo Fischer) for 1 min at a flow rate of 20 µL/min and separated on a directly coupled nEASY column (PepMap C18, 75 µm x 500 mm, 2 µm particle, Thermo…”

Section: Mass Spectrometrymentioning

confidence: 99%

SPRTN Protease and Checkpoint Kinase 1 Cross-Activation Loop Safeguards DNA Replication

Halder

Torrecilla

Burkhalter³

et al. 2018

Preprint

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The SPRTN metalloprotease is essential for DNA-protein crosslink (DPC) repair and DNA replication in vertebrate cells. Cells deficient in SPRTN protease activity exhibit severe DPC-induced replication stress and genome instability, manifesting as premature ageing and liver cancer in humans and mice. Strikingly, SPRTN-deficient cells also show a severe G2/M checkpoint defect and fail to activate a robust checkpoint kinase 1 (CHK1) signalling cascade normally triggered in response to replication stress. Here, we show that SPRTN activates the CHK1 signalling cascade during physiological (steady-state) DNA replication by proteolysis-dependent eviction of CHK1 from chromatin. The N-terminal CHK1 fragments cleaved by SPRTN protease still possess kinase activity and phosphorylate SPRTN. This CHK1-dependent phosphorylation stimulates SPRTN recruitment to chromatin to further promote CHK1 eviction from chromatin, DPC removal and unperturbed DNA replication. Our data suggest that a SPRTN-CHK1 cross-activation loop is essential for steady-state DNA replication and protection from DNA replication stress, a major source of genome instability in diseases that cause premature ageing and cancer. In addition, we disclose a mechanism of CHK1 activation during physiological DNA replication, when long stretches of single stranded (ss) DNA that are induced by genotoxic stress and activate canonical and robust CHK1 signalling are not present.

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Section: Mass Spectrometrymentioning

confidence: 99%

SPRTN Protease and Checkpoint Kinase 1 Cross-Activation Loop Safeguards DNA Replication

Halder

Torrecilla

Burkhalter³

et al. 2018

Preprint

Self Cite

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“…However, the amounts of yeast are difficult to obtain both technically and financially ( 6 ) and consequently experimental procedures were changed either toward feeding yeast paste or dried yeast ( 5 , 45 ) or reduced yeast content in solid medium ( 6 ). Despite remaining costly ( 46 ), labeling ranged from 93.7% to 97.7%, and it had been shown that the reason for not achieving complete labeling has been yeast and not fly metabolism ( 5 ). Furthermore, as yeast had to be labeled first, comparable to Escherichia coli labeling in Caenorhabditis elegans -SILAC ( 47 ), amino acids are exposed to potential metabolic modifications in yeast cells.…”

Section: Discussionmentioning

confidence: 99%

Stable Isotope Labeling of Amino Acids in Flies (SILAF) Reveals Differential Phosphorylation of Mitochondrial Proteins Upon Loss of OXPHOS Subunits

Schober

Atanassov

Moore

et al. 2021

Molecular & Cellular Proteomics

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SILAF recipe47 ingredients Lysine-0 or Lysine-6 Protein digest with Lys-C >99% heavy lysine incorporation in larvae at day 6 Mitochondrial phosphoproteomics upon a genetic insult Quantitative total fly phosphoproteomics Highlights• Adaptation of a fully defined, holidic food source for fruit fly SILAC (SILAF) • More than 99% labeling efficiency with lysine-6 within one larval generation • One milligram of fully lysine-6 labeled peptides for less than $15 • 14,000 Drosophila phosphorylated sites with 1500 occupancy values • Mitochondrial DmLRPPRC1 loss increases phosphorylation of NDUFB10 and NDUFA4

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“…However, the amounts of yeast are difficult to obtain both technically and financially (8) and consequently experimental procedures were changed either towards feeding yeast paste or dried yeast (7, 13) or reduced yeast content in solid medium (8). Despite remaining costly (26), labelling ranged from 93.7% to 97.7% and it had been shown that the reason for not achieving complete labelling has been yeast and not fly metabolism (7). Furthermore, as yeast had to be labelled first, comparable to E. coli labelling in C. elegans -SILAC (27), amino acids are exposed to potential metabolic modifications in yeast cells.…”

Section: Discussionmentioning

confidence: 99%

Versatile proteome labelling in fruit flies with SILAF

Atanassov

Moore

et al. 2019

Preprint

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23Drosophila melanogaster has been a working horse of genetics and cell biology for more than a 24 century. However, proteomic-based methods have been limited due to technical obstacles, 25 especially the lack of reliable labelling methods. Here, we advanced a chemically defined food 26 source into stable-isotope labelling of amino acids in flies (SILAF). It allows for the rapid generation 27 of a large number of flies with full incorporation of lysine-6. SILAF followed by fractionation and 28 enrichment gave proteomic insights at a depth of 5,966 proteins and 7,496 phosphorylation sites, 29 which substantiated metabolic regulation on enzymatic level. Furthermore, the label can be traced 30 and predicts protein turnover rates during different developmental stages. The ease and versatility 31 of the method actuates the fruit fly as an appealing model in proteomic and post-translational 32 modification studies and it enlarges potential metabolic applications based on heavy amino acid 33 diets. 34Schober et al., 3 of 32 relying on accurate quantification of peptides, e.g. post-translational modification scans have not 50 been feasible in Drosophila yet. Furthermore, the somewhat unpredictable yeast metabolism does 51 not allow precise tracing of amino acid labels in order to reliably predict protein turnover rates and 52 complicates the introduction of several labels or additive substrates at the same time. Here, we 53 present an alternative, direct labelling approach by employing a fully defined chemical food source 54 that contains heavy amino acids isotopes, which addresses several key concerns of stable-isotope 55 labelling in fruit flies. 56 RESULTS 57To overcome the difficulties associated with traditional yeast-based food in SILAC, we used a 58 holidic food source in this study containing 47 ingredients (9, 10), including defined amounts of 59 eight essential amino acids. Flies grown on this holidic food are viable and fertile with a comparable 60 lifespan, although they do present with a prolonged developmental larvae stage, pupating after 61 approximately nine instead of five days after egg laying (dae) at 25°C (9, 10). 62 SILAF enables full amino acid labelling 63In its more popular application in cell culture, SILAC relies on labelling with both "heavy" 13 C 6 -lysine 64 and 13 C 6 15 N 4 -arginine followed by digestion with trypsin, which cuts C-terminally of both amino 65 acids. However, previous results in both cells and the fly suggested that arginine can be 66 metabolised into other amino acids (8, 11). In agreement with this, growing larvae on holidic food 67 containing heavy-lysine and -arginine, followed by protein identification using liquid 68 chromatography-tandem mass spectrometry (LC-MS/MS), identified a number of heavy [ 13 C 5 15 N 1 ] 69 proline-containing peptides ( Supplementary Fig. 1a, b). In addition, we observed a decrease in the 70 average intensity of the heavy fraction in a peptide mixture from heavy and light holidic food 71 indicating a "loss" of the heavy label ( Supplementary ...

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Proteomic changes in response to crystal formation inDrosophilaMalpighian tubules

Cited by 12 publications

References 30 publications

SPRTN Protease and Checkpoint Kinase 1 Cross-Activation Loop Safeguards DNA Replication

SPRTN Protease and Checkpoint Kinase 1 Cross-Activation Loop Safeguards DNA Replication

Stable Isotope Labeling of Amino Acids in Flies (SILAF) Reveals Differential Phosphorylation of Mitochondrial Proteins Upon Loss of OXPHOS Subunits

Versatile proteome labelling in fruit flies with SILAF

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