Dataset Information

Proteomic profiling of the oncogenic septin 9 reveals isoform-specific interactions in breast cancer cells

ABSTRACT: Septins are a family of multimeric GTP-binding proteins, which are abnormally expressed in cancer. Septin 9 (SEPT9) is an essential and ubiquitously expressed septin with multiple isoforms, which have differential expression patterns and effects in breast cancer cells. It is unknown, however, if SEPT9 isoforms associate with different molecular networks and functions. Here, we performed a proteomic screen in MCF-7 breast cancer cells to identify the interactome of GFP-SEPT9 isoforms 1, 4 and 5, which vary in the length of their N-terminus. While all three isoforms associated with SEPT2 and SEPT7, the truncated SEPT9_i4 and SEPT9_i5 interacted with septins of the SEPT6 group more promiscuously than SEPT9_i1, which bound predominately SEPT8. Spatial mapping and functional clustering of non-septin partners showed isoform-specific differences in interactions with proteins of distinct subcellular organelles (nuclei, centrosomes, cilia) and functions (signaling, degradation). Notably, the interactome of the full length SEPT9_i1 was more enriched in cytoskeletal regulators, while the truncated SEPT9_i4 and SEPT9_i5 exhibited preferential and isoform-specific interactions with nuclear and signaling factors as well as ubiquitinating enzymes. These data provide evidence for isoform-specific interactions, which arise from truncations in the N-terminal extensions of SEPT9, and point to novel roles in the pathogenesis of breast cancer.

INSTRUMENT(S): LTQ

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Louis Devlin

LAB HEAD: ELIAS T SPILIOTIS, PHD

PROVIDER: PXD012977 | Pride | 2022-10-13

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Injection 1_SEPT9_19Jul2016_version10_0.pride.mgf.gz	Mgf
	Injection 2_SEPT9_20Jul2016_version8_5.pride.mgf.gz	Mgf
	Injection1_MCF7_SEPT9_6_160719144238.mzxml	Mzxml
	Injection1_SEPT9_19Jul2016_version10_0.mgf	Mgf
	Injection2_MCF7_SEPT9_6_160720133510.mzxml	Mzxml

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Proteomic profiling of the oncogenic septin 9 reveals isoform-specific interactions in breast cancer cells.

Devlin Louis L Okletey Joshua J Perkins George G Bowen Jonathan R JR Nakos Konstantinos K Montagna Cristina C Spiliotis Elias T ET

Proteomics 20210831 19

Septins are a family of multimeric GTP-binding proteins, which are abnormally expressed in cancer. Septin 9 (SEPT9) is an essential and ubiquitously expressed septin with multiple isoforms, which have differential expression patterns and effects in breast cancer cells. It is unknown, however, if SEPT9 isoforms associate with different molecular networks and functions. Here, we performed a proteomic screen in MCF-7 breast cancer cells to identify the interactome of GFP-SEPT9 isoforms 1, 4 and 5, ...[more]

PMID: 34409731

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Project description:Our research addresses the difficult question of how to dissect a process happening in multiple dimensions inside the cell. In order to exploit all the new information obtained by proteomics methods, it is necessary to integrate it into the process of dynamic remodeling of structures. Our research is relevant to this general area of endeavor where it concerns cytoskeleton. The cytoskeleton consists of actin, microtubules, septins, and intermediate filaments and, in most cells, is anchored to an extracellular matrix. Each cell has a unique arrangement of this network and readjusts it from time to time. To investigate the regulation of these reorganizations, we identified interactors from extracts of four cultured lines representing basal cells from the airway epithelium. Methods: After immunoprecipitation with an antibody against keratin 17, samples were processed by liquid chromatography and tandem mass spectrometry. Samples not undergoing antibody-mediated capture were processed in parallel. Results: The main keratins of basal cells, namely Krt14 (type I) and Krt5 (type II), constituted 67% of the total keratin recovered. Several other intermediate filament proteins, nestin, lamin-B1, and prelamin A/C, were present but not enriched upon immunoprecipitation. Although the class of armadillo-repeat proteins was represented by beta-catenin1 and plakoglobin, other desmosome plaque constituents were absent. Large cytolinkers were represented by the spectraplakin, microtubule-actin cross-linking factor (Macf1), which was enriched by immunoprecipitation, and the plakin, plectin, which was not enriched. Subunits of actin filaments and microtubules, along with numerous proteins associated with them, were recovered in both immunoprecipitated samples and those lacking the capture step. Coefficients of determination were computed based on abundance. The actin-associated proteins, alpha-spectrin and brain-specific angiogenesis inhibitor (Baiaip2l), were modestly correlated with keratin abundance but highly correlated with one another and with the keratin-binding protein, annexin A2. This interaction network resembled the pedestal formed by pathogenic Escherichia coli. Microtubule-associated proteins, dynamin 1-like protein and cytoplasmic dynein 1 heavy chain (Dync1h1), were enriched by immunoprecipitation, suggesting association with keratins, whereas kinesin-1 heavy chain and microtubule-associated protein retinitis pigmentosa 1 (EB1), were not enriched. Dync1h1 abundance was negatively correlated with that of all the septins, suggesting resemblance to a known antagonistic septin-dynein 1 relationship on microtubules. Conclusions: The cell lines showed remarkable uniformity with respect to the candidates interacting with cytoskeleton. The alpha-spectrin-Baiap2l network may link actin filaments to keratin precursor particles. A smaller interaction network centered on Dync1h1 was negatively correlated with all spectrin-Baiap2l constituents, suggesting that it and its binding partners are excluded from the pedestal-like domain.