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Detection of structure-specific interactions of Rab GTPases

ABSTRACT: Protein–protein interactions (PPIs) mediate multiple essential functions and regulatory events in living organisms. The physical interactome of a given protein can be abnormally altered in response to external and internal cues, thus modulating cell physiology and contributing to human pathologies including neurodegenerative diseases. Despite substantial efforts, current approaches cannot pinpoint structure-specific PPIs or their interaction interfaces on a proteome-wide scale. To address this limitation, we have adapted the structural proteomics method known as limited proteolysis–mass spectrometry to identify PPIs by probing protein structural alterations within cellular extracts upon treatment with a protein of interest. Here, we added Rab GTPases, namely Rab5A and Rab2A in their GTP- and GDP-bound forms to a HEK293T cellular extract and probed their structure-specific interactomes. We identified already know interactors of these proteins bassed on previously published data as well as a number of candidate interactor proteins.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Aleš Holfeld

LAB HEAD: Prof. Paola Picotti

PROVIDER: PXD048849 | Pride | 2024-02-10

REPOSITORIES: Pride

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			Action	DRS
	191108_Control_LiP_0101.raw	Raw
	191108_Control_LiP_0102.raw	Raw
	191108_Control_LiP_0103.raw	Raw
	191108_Control_LiP_0104.raw	Raw
	191108_Rab2D_LiP_0101.raw	Raw

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Project description:Genomic, proteomic, and metabolomic technologies continue to receive increasing interest from environmental toxicologists. This interest is due to the great potential of these technologies to identify detailed modes of action and to provide assistance in the evaluation of a contaminant?s risk to aquatic organisms. Our experimental model is the zebrafish (Danio rerio) exposed to reference endocrine disrupting compounds in order to investigate compound-induced changes in gene transcript profiles. Adult, female zebrafish were exposed to 0, 15, 40, and 100 ng/L of 17 alpha-ethynylestradiol (EE2) and concentration and time-dependent changes in hepatic gene expression were examined using Affymetrix GeneChip® Zebrafish Genome Microarrays. At 24, 48, and 168 hours, fish were sacrificed and liver mRNA was extracted for gene expression analysis (24 and 168 hours only). In an effort to link gene expression changes to effects on higher levels of biological organization, body and ovary weights were measured and blood was collected for measurement of plasma steroid hormones (17 beta-estradiol (E2), testosterone (T)) and vitellogenin (VTG) using ELISA. EE2 exposure significantly affected GSI, E2, T, VTG and gene expression. We observed 1575 genes that were significantly affected (up- or down-regulated by at least 1.5-fold (p ? 0.001) in a concentration-dependent manner by EE2 exposure at either 24 or 168 hours. EE2 exposure altered transcription of genes involved in steroid hormone homeostasis, cholesterol homeostasis, retinoic acid metabolism, and cell growth and proliferation. Plasma VTG was significantly increased at 24, 48, and 168 hours (p<0.05) at 40 and 100 ng/L and at 15 ng/L at 168 hours. E2 and T were significantly reduced following EE2 exposure at 48 and 168 hours. GSI was decreased in a dose-dependent manner at 168 hours. In this study, we identified genes involved in a variety of biological functions that have the potential to be used as markers of exposure to estrogenic substances. Future work will evaluate the use of these genes in zebrafish exposed to weak estrogens to determine if these genes are indicative of exposure to estrogens with varying potencies.

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Detection of structure-specific interactions of Rab GTPases

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