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Charting the dynamic trophoblast plasma membrane identifies LYN as a functional regulator of syncytialization

ABSTRACT: The differentiation of placental cytotrophoblasts (CTBs) into the syncytiotrophoblast (STB) layer results in significant remodeling of the plasma membrane proteome. Here, we use a peroxidase-catalyzed proximity labeling strategy to quantitatively map the dynamic plasma membrane proteomes of CTBs and STBs. Coupled with mass spectrometry-based proteomics, we identify hundreds of plasma membrane proteins and observe quantitative changes in protein abundance throughout differentiation, including the upregulation of the plasma membrane-localized non-receptor tyrosine kinase LYN. We show that both siRNA-mediated knockdown and small molecule inhibition of LYN kinase function impairs CTB fusion and abrogates the expression of syncytialization markers, presenting a function for LYN outside of its canonical role in immunological signaling. Our results demonstrate the use of the proximity labeling platform to discover functional regulators within the plasma membrane and provide new avenues to regulate trophoblast differentiation.

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ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Placenta, Epithelial Cell

SUBMITTER: Abigail Reeves

LAB HEAD: Mia L

PROVIDER: PXD052032 | Pride | 2025-10-02

REPOSITORIES: Pride

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Project description:B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is caused by abnormal expansion of immature B cells. Genetic alterations can be identified in most of the BCP-ALL cases, however, some specific lesions including rearrangements of the mixed lineage leukemia (MLL) gene encoding lysine methyltransferase 2A (KMT2A) are associated with particularly poor prognosis. This MLL-rearranged subtype of leukemia poorly responds to conventional treatment and frequently undergoes a lymphoid-to-myeloid lineage switch in response to CD19-directed immunotherapy with consequent loss of B cell-specific targets for immunotherapy. Therefore, there is an urgent need to identify new targets for treatment of relapsed/refractory MLLr-BCP-ALL. Attractive targets may be found in a cell surfaceome, as membrane proteins are often expressed abnormally on cancer cells and are more accessible for therapeutics. Membrane proteins also play key roles in mediating external signaling and interactions with the microenvironment, thus being essential for cancer cell survival. RNA-based data may not always correlate with plasma membrane protein levels. Therefore, we performed the analysis of the BCP-ALL cell surfaceome by biotin labeling of plasma membrane proteins followed by tandem mass spectrometry (LC-MS/MS). This method requires high cell input and therefore was previously applied mainly to cell lines. However, the cell lines often do not fully represent primary samples due to genetic modifications and culture conditions. We have optimized the protocol of plasma membrane protein isolation and LC-MS/MS identification of patient-derived xenografts (PDXs) BCP-ALL cells. We confirmed the LC-MS/MS results by flow cytometry. Additionally, we compared the surfaceomes of PDXs BCP-ALL cells with BCP-ALL cell lines. We profiled six BCP-ALL PDXs and one cell line SEM, all with MLL translocations. In BCP-ALL PDXs we identified 1409 membrane-associated proteins, 945 of which were present in at least 3 samples. Tissue specificity analysis demonstrated that 93 of these proteins are specific for lymphoid cells, including already reported promising immunotherapeutic targets CD19, CD22, CD38, CD70, CD72, CD79A, and CD79B. The expression of selected proteins was confirmed by flow cytometry and compared to the expression on B-ALL cell lines with MLL translocation. Moreover, we identified an additional subset of proteins with potential therapeutic significance, including CD48, IL7R, ITGB7, LAIR1, LILRB1. Importantly, we observed profound differences between surfaceomes of BCP-ALL cell lines and PDX cells.

			Action	DRS
	220929_Abby_tmt_1_.msf	Msf
	220929_Abby_tmt_1_.mzML	Mzml
	220929_Abby_tmt_1_.mzid.gz	Mzid
	220929_Abby_tmt_1_1.raw	Raw
	220929_Abby_tmt_2_.msf	Msf

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Charting the dynamic trophoblast plasma membrane identifies LYN as a functional regulator of syncytialization

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