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"labhead_mail":["gimi@cancer.dk"],"submitter":["Giacomo Milletti"],"technology_type":["Mass Spectrometry","Top-down proteomics"],"software":[""],"submitter_keywords":["Ambra","Dna","P21"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD053083"],"tissue":["Cell Culture"],"sample_protocol":["All the experiments have been performed in a labeling free setting. Samples were processed using iST-KIT (Preomics). Instruments for LC MS/MS analysis consisted of a NanoLC 1200 coupled via a nano-electrospray ionization source to the quadrupole-based Q Exactive HF benchtop mass spectrometer. Peptide separation was carried out according to their hydrophobicity on a home-made chromatographic column, 75m ID, 8Um tip, 350mm bed packed with Reprosil-PUR, C18-AQ, 1.9m particle size, 120 Angstrom pore size, using a binary buffer system consisting of solution A: 0.1% formic acid and B: 80% acetonitrile, 0.1% formic acid. Runs of 120 min after loading were used for proteome samples (DDA), with a constant flow rate of 300nl/min. Runs of 60 min after loading were used for proteome samples (DIA), with a constant flow rate of 300nl/min. Q Exactive HF settings: MS spectra were acquired using 3E6 as an AGC target, a maximal injection time of 20ms and a 120,000 resolution at 200m/z. The mass spectrometer operated in a data dependent Top20 mode with sub sequent acquisition of higher-energy collisional dissociation (HCD) fragmentation MS/MS spectra of the top 20 most intense peaks. Resolution, for MS/MS spectra, was set to 15,000 at 200m/z, AGC target to 1E5, max injection time to 20ms and the isolation window to 1.6Th. The intensity threshold was set at 2.0 E4 and Dynamic exclusion at 30 seconds."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["DIA acquired RAW files were processed using DIANN or Spectronaut. For protein assignment, spectra were correlated with the UniProt human database (v. 2023) including a list of common contaminants. Searches were performed with tryptic specifications and default settings for mass tolerances for MS and MS/MS spectra. Carbamidomethyl at cysteine residues was set as a fixed modification, while oxidations at methionine, acetylation at the N-terminus were defined as variable modifications. The minimal peptide length was set to seven amino acids, and the false discovery rate for proteins and peptide-spectrum matches to 1%. The match-between-run feature with a time window of 1 min was used. For further analysis, the Perseus software (1.6.2.3) was used and first filtered for contaminants and reverse entries as well as proteins that were only identified by a modified peptide. The LFQ Ratios were logarithmized, grouped and filtered for min. valid number (min. 3 in at least one group). Missing values have been replaced by random numbers that are drawn from a normal distribution. Two sample t-test was performed using FDR=0.05. Probability values (p) < 0.05 were considered statistically significant. To identify significant enriched GO terms, we utilized the 1D enrichment tool in Perseus. Data visualization was done in the statistical environment R. MS analyses of three independent samples for each experiment were performed. Peptides with Log2 Difference ≥ 1 and -Log p-value > 1.3 were considered significantly enriched."],"omics_type":["Proteomics"],"labhead":["Milletti Giacomo"],"instrument_platform":[""],"labhead_affiliation":["Danish Cancer Institute"],"submission_type":["PARTIAL"],"species":["Homo Sapiens (human)"],"submitter_mail":["gimi@cancer.dk"],"publication":["42339621 Milletti G, Cadeddu G, Adelantado-Rubio A, Ferraina C, Keuper K, Munk SHN, De Stefanis C, Quacquarini D, Rossi S, Mastronuzzi A, Locatelli F, Grumati P, Cianfanelli V, Nazio F, Bartek J, Maya-Mendoza A, Cecconi F. Ubiquitin-dependent degradation of p27Kip1 and p21Waf1/Cip1 by AMBRA1 ensures G1 and S phase progression and limits replication stress. Nucleic Acids Res. 2026 54(12):gkag595 10.1093/nar/gkag595"],"submitter_affiliation":["Danish Cancer Institute"],"submitter_country":["Denmark"],"pubmed_abstract":["The cell cycle orchestrates the events that lead to cell replication and division. AMBRA1 interacts with the E3 ubiquitin ligase CRL4DDB1 complex to regulate the stability of D-type cyclins, key regulators of the G1-S phase transition. However, whether AMBRA1 has a role in the S phase remains to be elucidated. Here, we show that AMBRA1 affects the turnover of p21Waf1/Cip1 and p27Kip1 by coupling the CRL4DDB1 complex to these proteins. In the absence of AMBRA1, the increased stability of p21Waf1/Cip1, rather than p27Kip1, resulted in the accumulation of replication stress. Mechanistically, the excess of p21Waf1/Cip1 during the S phase resulted in more PCNA-bound p21Waf1/Cip1, negatively affecting the binding of FEN1 to PCNA, which left under-replicated DNA. Consequently, AMBRA1-depleted cells are sensitive to FEN1 inhibition. Aberrantly low levels of AMBRA1 occurring in Sonic Hedgehog-type medulloblastomas correlate with high levels of p21Waf1/Cip1 and a worse prognosis. Finally, AMBRA1 and p21 levels may serve as biomarkers for patient stratification and treatment in medulloblastoma."],"pubmed_title":["Ubiquitin-dependent degradation of p27Kip1 and p21Waf1/Cip1 by AMBRA1 ensures G1 and S phase progression and limits replication stress."],"pubmed_authors":["Milletti Giacomo G, Cadeddu Giulia G, Adelantado-Rubio Alba A, Ferraina Caterina C, Keuper Kristina K, Munk Sebastian Howen Nesgaard SHN, De Stefanis Cristiano C, Quacquarini Denise D, Rossi Sabrina S, Mastronuzzi Angela A, Locatelli Franco F, Grumati Paolo P, Cianfanelli Valentina V, Nazio Francesca F, Bartek Jiri J, Maya-Mendoza Apolinar A, Cecconi Francesco F"],"additional_accession":[]},"is_claimable":false,"name":"Ubiquitin-dependent degradation of p21Waf1/Cip1 is mediated by AMBRA1 to limit DNA replication stress","description":"The cell cycle orchestrates the events that lead to cell replication and division. AMBRA1 is a scaffold factor that interacts with the E3 ubiquitin ligase DDB1-CRL4 complex to regulate the stability of Cyclin D-type proteins, key regulators of the G1-S phase transition. However, the role of AMBRA1 in regulating the stability of other cell-cycle-related proteins is far from being fully characterized. Here we demonstrate that AMBRA1 affects the turnover of p21Waf1/Cip1 and p27Kip1, by coupling the DDB1-CRL4 complex directly to these proteins mainly independent of Cyclin D. In the absence of AMBRA1, the increased stability of p21Waf1/Cip1, rather than p27Kip1, resulted in the accumulation of replication stress by leaving under-replicated DNA during the S phase. We also found that cells with a low expression of AMBRA1 and high p21Waf1/Cip1 are sensitive to drugs that interfere with the lagging-strand DNA synthesis. Of clinical relevance, low levels of AMBRA1 and high levels of p21Waf1/Cip1 Sonic-Hedgehog medulloblastoma correlate with a worse prognosis, suggesting that tumors with low levels of AMBRA1 can be a criterion for patient stratification and treatment.","dates":{"publication":"2026-06-29","submission":"2024-06-13"},"accession":"PXD053083","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NEWT:625351","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:1073882","NEWT:6819","NEWT:475174","NEWT:111205","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:1590","NEWT:10034","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:1589","NEWT:135622","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:36630","NEWT:383379","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:111225","NEWT:7719","NEWT:868565","NEWT:135674","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:160621","NEWT:212790","NEWT:110368","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:1912919","NEWT:410658","NEWT:44688","NEWT:44689","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:44685","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:410661","NEWT:1140","NCBITaxon:2157","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NEWT:270643","NEWT:2065263","NEWT:177437","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:56636","NEWT:2709072","NEWT:2499525","NEWT:2853422","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:294607","NEWT:1502","NEWT:128017","NEWT:376686","NEWT:237610","NEWT:95486","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:108458","NEWT:5936","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:1255228","NEWT:410289","NEWT:373153","NEWT:1640278","NEWT:472","NEWT:940825","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:5911","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:215402","NEWT:1547","NEWT:906914","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:2903","NEWT:185579","NEWT:13076","NEWT:207340","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:550","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:36185","NEWT:1054211","NEWT:1225786","NEWT:771870","NEWT:51338","NEWT:575412","NEWT:28112","NEWT:6493","NEWT:10832","NEWT:6494","NEWT:6491","NEWT:507601","NEWT:520","NEWT:186441","NEWT:643680","NEWT:214092","NCBITaxon:6157","NEWT:13095","NEWT:162425","NEWT:104105","NEWT:216257","NEWT:9986","NEWT:8654","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:5147","NEWT:28108","NEWT:28104","NEWT:407821","NEWT:568703","NCBITaxon:2","NEWT:568708","NEWT:1613","NEWT:986","NEWT:52641","NEWT:28532","NEWT:353152","NEWT:298176","NEWT:353153","NEWT:227377","NEWT:1774284","NEWT:40674","NEWT:1194669","NEWT:51329","NEWT:443906","NEWT:519","NEWT:2510939","NEWT:7394","NEWT:6063","NEWT:1328388","NEWT:105487","NEWT:1548728","NEWT:667127","NEWT:9557","NEWT:377586","NEWT:300641","NEWT:39655","NEWT:9554","NEWT:38323","NEWT:475932","NEWT:256318","NEWT:206411","NCBITaxon:6191","NEWT:229533","NEWT:214053","NEWT:28550","NEWT:80863","NEWT:90675","NEWT:52639","NEWT:52638","NEWT:57075","NEWT:1666905","NEWT:8697","NEWT:8695","NEWT:884204","NEWT:510516","NEWT:1045775","NEWT:1123869","NEWT:9544","NEWT:9545","NEWT:979","NEWT:7370","NEWT:83906","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