Proteomic analysis identifies novel binding partners of BAP1

Our plain-English summary of Proteomic analysis identifies novel binding partners of BAP1, by R. Baas, F. J van der Wal, O.B. Bleijerveld, et al (PloS One, 2021).

The study

• Authors: R. Baas, F. J van der Wal, O.B. Bleijerveld, H. van Attikum, T.K. Sixma
• Journal: PloS One
• Published: 2021;16(9):e0257688
• Cited by: 11 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

BRCA1-associated protein 1 (BAP1) is a tumor suppressor and its loss can result in mesothelioma, uveal and cutaneous melanoma, clear cell renal cell carcinoma and bladder cancer. BAP1 is a deubiquitinating enzyme of the UCH class that has been implicated in various cellular processes like cell growth, cell cycle progression, ferroptosis, DNA damage response and ER metabolic stress response. ASXL proteins activate BAP1 by forming the polycomb repressive deubiquitinase (PR-DUB) complex which acts on H2AK119ub1. Besides the ASXL proteins, BAP1 is known to interact with an established set of additional proteins. Here, we identify novel BAP1 interacting proteins in the cytoplasm by expressing GFP-tagged BAP1 in an endogenous BAP1 deficient cell line using affinity purification followed by mass spectrometry (AP-MS) analysis. Among these novel interacting proteins are Histone acetyltransferase 1 (HAT1) and all subunits of the heptameric coat protein complex I (COPI) that is involved in vesicle formation and protein cargo binding and sorting. We validate that the HAT1 and COPI interactions occur at endogenous levels but find that this interaction with COPI is not mediated through the C-terminal KxKxx cargo sorting signals of the COPI complex.

In plain English

This was a laboratory proteomics study of the deubiquitinating enzyme BAP1, which the authors describe in their introduction as a tumour suppressor whose loss has been implicated in mesothelioma, uveal and cutaneous melanoma, clear cell renal cell carcinoma and bladder cancer; the work itself involved cultured human cells, not people or asbestos. The authors expressed GFP-tagged BAP1 in HeLa FRT cells, including a CRISPR-generated BAP1 knockout line, separated cytoplasmic and nuclear extracts, and identified binding partners by affinity purification followed by mass spectrometry. Known partners such as ASXL1/2, FOXK1/2, HCFC1, OGT and UBE2O were recovered, with KDM1B and MBD6 in the nuclear fraction. Cytoplasmic partners also included histone acetyltransferase 1 (HAT1), which the authors note had been seen with BAP1 before but never validated, and all seven subunits of the COPI coatomer complex, three of which the authors say had not been observed in BAP1 AP-MS experiments before. Both interactions were confirmed with endogenous proteins by immunoprecipitation, HAT1 also in U2OS cells. Mutating the C-terminal KAKRQ motif of BAP1 to SASRQ did not change COPA binding, so the authors concluded the COPI interaction is not mediated by this cargo-sorting motif.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: laboratory cell-culture proteomics experiment (affinity purification followed by mass spectrometry, with immunoprecipitation/immunoblot validation)
• Who was studied: Cultured human cell lines: HeLa FRT wild-type cells, CRISPR-generated HeLa FRT BAP1 knockout clones (#4C5 and #4D2, with 4D2 used further), and U2OS cells; no human participants
• Exposure: Not an exposure study; cells were engineered to express GFP-tagged full-length BAP1, BAP1 2-670, BAP1 delta-671-710, BAP1 C91S, the SASRQ C-terminal tail mutant, GFP-COPE, or GFP alone (N-stop)
• Main results:
  • In cytoplasmic extracts GFP-BAP1 selectively pulled down ASXL1/2, FOXK1/2, HCFC1, OGT and UBE2O; in the nuclear fraction KDM1B and MBD6 were also found
  • COPA, COPB2 and ARCN1 were seen near the edge of the significance curve in the cytoplasmic fraction and had not been observed in BAP1 AP-MS experiments before
  • All seven COPI subunits were enriched as interacting proteins in the cytoplasmic fraction of full-length BAP1 expressed in the BAP1-deficient background
  • HAT1 had previously been seen interacting with BAP1 but, according to the authors, had never been validated
  • HAT1 co-immunoprecipitated with GFP-BAP1 and with the catalytic-dead C91S mutant, and with endogenous BAP1 in U2OS cells
  • Endogenous COPA co-immunoprecipitated with endogenous BAP1 in HeLa FRT parental lysate with all BAP1 antibodies but not with normal IgG; only a small fraction of total COPA bound BAP1
  • Mutating the BAP1 C-terminal KAKRQ motif to SASRQ gave no difference in COPA co-immunoprecipitation compared with wild-type BAP1
  • In the BAP1 knockout clones 4C5 and 4D2, BAP1 expression was lost and global H2AK119ub1 levels were elevated 3-fold
  • In the cytoplasmic fraction of the BAP1 2-670 truncation mutant most interactions, including ASXL and UBE2O, were lost, while HCFC1/2, FOXK1/2 and OGT remained
• The authors' conclusion: The authors concluded that they identified novel cytoplasmic BAP1 interactors, enabled by removing endogenous BAP1, and that BAP1 partner binding differs by cellular location; the COPI interaction was validated on endogenous protein but is not mediated by the C-terminal KxKxx motif of BAP1
• Limitations the authors state:
  • The authors stated that the mechanism of BAP1 binding to COPI is not resolved and further experiments are needed
  • The authors stated that the shift in COPA band mass on immunoblot could not be explained and needs further experiments
  • The authors noted that the absolute and relative amount of COPI interacting with BAP1 in the AP-MS was low
  • The authors noted that HeLa cells tolerate BAP1 deletion unlike other cell lines, so functional data must be examined carefully

Topics

Medical terms in this study, explained in our Lexicon: BAP1, Mesothelioma, Carcinoma.

Related studies

• BAP1 regulates IP3R3-mediated Ca2+ flux to mitochondria suppressing cell transformation — A. Bononi, C. Giorgi, et al. (2017)
• Loss of BAP1 function leads to EZH2-dependent transformation — L.M. LaFave, W. Béguelin, et al. (2015)
• Proteomic Profiling of Alveolar Macrophages Identifies Loss of Lysosomal Content as an Indicator of Nanofiber-Induced Frustrated Phagocytosis — T. Stobernack, A. Vennemann, et al. (2026)
• Tumours associated with BAP1 mutations — R. Murali, T. Wiesner, et al. (2013)
• Different prognostic roles of tumor suppressor gene BAP1 in cancer: A systematic review with meta-analysis — C. Luchini, N. Veronese, et al. (2016)
• The spectrum of tumors harboring BAP1 gene alterations — Y. Laitman, J. Newberg, et al. (2021)

Browse more research in PloS One, or return to medical research.