Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748.stats.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748.gg_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748.pr_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748.protein_description.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748.pg_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748.unique_genes_matrix.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748.log.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748.manifest.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-1_ctrl_1.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-8_SweInd_2.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-3_ctrl_3.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-7_SweInd_1.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-2_ctrl_2.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-6_WT_3.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-5_WT_2.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-4_WT_1.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-9_SweInd_3.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-4_WT_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-1_ctrl_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-3_ctrl_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-7_SweInd_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-6_WT_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-2_ctrl_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-8_SweInd_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-9_SweInd_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748-5_WT_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748_SL.parquetftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748.auto.pipelineftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD053992/KK2748.parquetprimaryOK200hide0729.bri@niigata-u.ac.jpGodfried DougnonMass SpectrometryChemical cross-linking coupled with mass spectrometry proteomicsBottom-up proteomicsData-independent acquisitionNucleusExtracellular wasteNeurodegenerationAlzheimer's diseaseAmyloid precursor proteinhttps://www.ebi.ac.uk/pride/archive/projects/PXD053992Hela CellFor immunoprecipitation of FLAG-tagged APP, HeLa cells cultured in a 10 cm dish were transfected with plasmids expressing either empty vector, APP WT or APP Swe/Ind tagged with 3x FLAG. One day after transfection, the medium was changed to DMEM containing 5 µM etoposide, and the cells were cultured for another 24 hrs. Di(N-succinimidyl) 3,3'-dithiodipropionate (DSP, Tokyo Chemical Industry, Tokyo, Japan)-crosslinking immunoprecipitation (IP) was performed according to a previously described method with some modifications. Samples were prepared in triplicate. In brief, the cells were rinsed twice with prewarmed PBS and cross-linked with 0.1 mM DSP in PBS at 37°C for 30 min in a CO2 incubator. The cells were rinsed once with PBS, incubated in STOP solution (20 mM Tris-HCl, pH 7.4, in PBS) at RT for 15 min and rinsed twice with ice-cold PBS. Next, 750 μL of ice-cold IP buffer (25 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1% Triton X-100, and 5% glycerol with cOmplete Mini EDTA-free (Merck, Darmstadt, Germany)) was added, and the cells were scraped off with a scraper, transferred to 1.5 mL microtubes and incubated on ice for 10 min to complete cell lysis. The lysates were centrifuged at 20,000 × g for 10 min at 4°C. The protein concentration of the supernatant was measured using a BCA protein assay (Thermo Fisher Scientific) and adjusted to 2 mg/ml with IP buffer. A total of 2 mg of lysate (1 ml) was incubated with 10 µl of anti-FLAG M2 magnetic beads (Merck), which were prewashed twice with IP buffer, at 4°C on a rotating wheel for 2 hr. A DynaMag-2 magnet (Thermo Fisher Scientific) was used to collect the beads. The beads were then washed three times with 1 ml of IP buffer followed by once with 50 mM Tris-HCl (pH 8.0). The samples were digested on beads with trypsin (500 ng of trypsin platinum, Promega, Madison, WI, USA) in 100 µl of 50 mM Tris-HCl (pH 8.0) containing 2% lauryl maltose neopentyl glycol and 7.5 mM CaCl2 at 37°C for 15 hrs. The digested products were then reduced and alkylated with 10 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 40 mM chloroacetamide. After acidification with 5% trifluoracetic acid (TFA), the samples were desalted using a reversed-phase spin column (GL-Tip SDB, GL Science, Tokyo, Japan) followed evaporation by centrifuge and resuspension in 0.1% trifluoroacetic acid (TFA)-0.01% decyl maltose neopentyl glycol (DMNG). For LC‒MS/MS analysis, the peptides were fractionated on a 75 μm × 12 cm nanoLC column packed with C18 particles (3 µm; Nikkyo Technos Co., Ltd., Tokyo, Japan) by elution with a linear gradient of 6% to 75% solvent B over 70 min at a flow rate of 200 nl/min (solvent A = 0.1% formic acid; solvent B = 0.1% formic acid in 80% acetonitrile) with the use of an UltiMate 3000 RSLCnano LC System (Thermo Fisher Scientific).PrideNot availableThe Q Exactive HF-X (Thermo Fisher Scientific) mass spectrometer was operated in data-independent acquisition mode. MS1 spectra were collected in the range of 495–745 m/z at a resolution of 30,000 using an automated gain control (AGC) target value of 3 × 106 ions with a maximum injection time of 55 msec. MS2 spectra were collected in the range of > 200 m/z at a resolution of 30,000 using an AGC target value of 3 × 106 ions and automatic maximum ion injection times. Sixty data-independent acquisition (DIA) windows with an isolation width of 4 Da ranged from 500 to 740 m/z. The normalized collision energy was set to 23%. All DIA raw data were processed with DIA-NN (ver. 1.9.1)54 using library-free search mode against human UniProt sequences (https://www.uniprot.org/; accessed 7 March 2023). The enzyme specificity was set to “trypsin”. The precursor m/z range was set from m/z 490 to 75. Carbamidomethylation of cysteine was set as a fixed modification. Proteins for which both Precursor FDR and Protein FDR were below 1% were identified and quantified. Results were analysed using Perseus (https://maxquant.net/perseus/) and proteins with quantitative values for at least two of the triplicates in at least one group were extracted. Enrichment analysis of mass spectrometry data was performed using the WEB-based Gene SeT AnaLysis Toolkit (https://www.webgestalt.org) with the setting of Over-Representation Analysis using “cellular component” of gene ontology as the database.ProteomicsHideaki MatsuiPARTIALDepartment of Neuroscience of Disease, Brain Research Institute, Niigata UniversityHomo Sapiens (human)10.1073/PNAS.2524190123dougnong@bri.niigata-u.ac.jpNiigata UniversityJapanfalseA protective role for APP in nuclear waste clearance via lysosomal exocytosisAmyloid precursor protein (APP) is widely known for its role in Alzheimer’s disease (AD) pathogenesis through its proteolytic processing into amyloid-β peptides. However, its physiological function remains incompletely understood. Here, we uncover a protective role for full-length APP in facilitating the disposal of nuclear-derived debris under genotoxic stress. In both cultured cells and in vivo mouse models, loss of APP leads to nuclear waste accumulation, increased inflammation, and cell death, whereas APP overexpression mitigates these effects. Mechanistically, we show that APP supports the extracellular release of nuclear material through lysosomal exocytosis. APP mutants associated with familial AD fail to mediate this process. Consistently, human AD brain tissue exhibits abnormal nuclear morphology, accumulation of nuclear waste in the cytoplasm, and reduced APP levels per neuron. These findings highlight a conserved cellular mechanism by which APP contributes to nuclear and cellular homeostasis, and suggest that impaired nuclear waste clearance may represent an underappreciated contributor to neurodegeneration.2026-06-112024-07-17PXD053992NEWT:6945NEWT:3555NEWT:2NEWT:157546NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:544496NEWT:2042546NEWT:45351NEWT:43179NEWT:4513NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:1211601NEWT:876138NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:1351NEWT:1438992NEWT:2649997NEWT:272563NEWT:224326NCBITaxon:79857NEWT:95648NEWT:3885NEWT:3888NEWT:1589NEWT:135622NEWT:6915NEWT:3649NEWT:101510NEWT:3880NEWT:272559NEWT:3641NEWT:383379NEWT:10029NEWT:1000589NEWT:85962NEWT:317447NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:197NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:1270NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:287NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:1148NEWT:580240NEWT:294128NEWT:11676NEWT:55571NEWT:100226NEWT:4530NEWT:4896NEWT:75058NEWT:13616NEWT:1094343NEWT:296543NEWT:1773NEWT:1895NEWT:1182590NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NCBITaxon:1313NEWT:43330NEWT:408169NEWT:44544NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:990119NEWT:408172NEWT:408170NEWT:493760NEWT:260710NEWT:257313NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:106592NEWT:536231NEWT:1432138NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:9598NEWT:8030NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:432359NEWT:46835NEWT:2711NEWT:376686NEWT:95486NEWT:9103NEWT:29159NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:3635NEWT:5811NEWT:235443NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:169963NEWT:499175NEWT:476272NEWT:3747NEWT:367830NEWT:178616NEWT:410289NEWT:373153NEWT:352472NEWT:360094NEWT:470NEWT:1313NEWT:411469NEWT:84023NEWT:559292NCBITaxon:5811NEWT:411464NEWT:411460NEWT:2762NEWT:1174673NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:28038NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:327159NEWT:178876NEWT:327160NEWT:573NEWT:9031NEWT:7091NEWT:241368NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:5180NEWT:256737NEWT:115104NEWT:1081927NEWT:1238993NEWT:67825NEWT:185579NEWT:13076NEWT:1249668NEWT:317NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:415540NEWT:4081NEWT:554NEWT:98334NEWT:426428NEWT:7574NEWT:7215NEWT:575412NEWT:29204NEWT:2172103NEWT:507601NCBITaxon:6157NEWT:746360NEWT:6239NEWT:470150NEWT:216257NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:435590NEWT:1902NEWT:160488NEWT:28104NCBITaxon:2NEWT:985076NEWT:1215323NEWT:52641NEWT:7038NEWT:6192NEWT:28532NCBITaxon:38727NEWT:2829NEWT:216599NEWT:216595NEWT:243230NEWT:8355NEWT:9685NEWT:7029NEWT:1080772NEWT:1283300NEWT:6183NEWT:6063NEWT:334747NEWT:61235NEWT:6289NEWT:436486NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:170187NEWT:260707NCBITaxon:6191NEWT:1836NEWT:185431NEWT:29760NEWT:260704NEWT:703612NEWT:260705NEWT:80863NEWT:2697049NCBITaxon:6073NEWT:884204NEWT:6279NEWT:1123869NEWT:9544NEWT:7370NEWT:83906NEWT:607699NEWT:6282NEWT:208964NEWT:1134506NEWT:575584NEWT:38783NEWT:8727NEWT:4006NEWT:8726NEWT:6426NEWT:6669NEWT:10090NEWT:4120NEWT:51515NEWT:5693NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:5334NEWT:37334NEWT:382352NCBITaxon:10359NEWT:242619NEWT:632957NEWT:373995NEWT:5689NEWT:544404NEWT:9925NEWT:8839NEWT:4232NEWT:2758385NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:714NEWT:421932NEWT:196627NEWT:5691NEWT:48479NEWT:627025NEWT:1097677NEWT:61674NEWT:1117957NEWT:9913NEWT:4100NEWT:1076NEWT:6763NEWT:803NEWT:29722NEWT:380394NEWT:1692259NEWT:180066NEWT:135588NEWT:1843183NEWT:58002NEWT:326423NEWT:4577NEWT:1416333NEWT:5664NEWT:2157NEWT:418985NEWT:146479NEWT:1911079NEWT:264203NEWT:1480154NEWT:1274414NEWT:27606NEWT:59202NEWT:9975NEWT:9612NEWT:38865NEWT:51953NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:36329NEWT:1147787NCBITaxon:3044782NEWT:72407NEWT:349741NEWT:9605NEWT:9606NEWT:157295NEWT:641501NEWT:7668NEWT:915099NEWT:74940NEWT:9721NEWT:137221NEWT:1450511NEWT:1143193NEWT:411901NEWT:9838NEWT:105884NCBITaxon:9615NEWT:58334NEWT:1193501NEWT:3055NEWT:6326NEWT:6689NEWT:5476NEWT:1274432NEWT:1274426NEWT:70448NEWT:9825NEWT:1274423NEWT:393765NEWT:698936NEWT:39946NEWT:9823NEWT:9940NEWT:521001NEWT:39947NEWT:1274420NEWT:578458https://orcid.org/0000-0003-1383-5691