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STIM1 Translocation to The Nucleus Protects Cells from DNA Damage

ABSTRACT: DNA damage represents a challenge for cells as this damage must be eliminated to preserve cell viability and the transmission of genetic information. To reduce or eliminate unscheduled chemical modifications in genomic DNA, an extensive signaling network, known as the DNA damage response (DDR) pathway, ensures this repair. In this work, and by means of a proteomic analysis aimed at the study of the STIM1 protein interactome, we have found that STIM1 is closely related to the protection from endogenous DNA damage, replicative stress, as well as in the response to interstrand crosslinks (ICLs). Here we show that STIM1 has a nuclear localization signal (NLS) that mediates its translocation to the nucleus, and that this translocation and the association of STIM1 to chromatin is enhanced in response to mitomycin C (MMC), an ICL-inducing agent. Consequently, STIM1-deficient cell lines show a higher level of basal DNA damage, replicative stress, and a higher sensitivity to MMC. We show that STIM1 normalizes FANCD2 protein levels in the nucleus, explaining the increased sensitivity to MMC in STIM1-KO cells. Our results show for the first time a nuclear function for the endoplasmic reticulum protein STIM1 and increase the number of genes involved in DNA repair.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Disease Free

SUBMITTER: Sergio Lilla

LAB HEAD: Sara Rossana Zanivan

PROVIDER: PXD041181 | Pride | 2023-12-22

REPOSITORIES: pride

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			Action	DRS
	20191129_SEL_HF_SA_FRR_EXT_211119_Nuclear_GFP-CT-STIM1_004.raw	Raw
	20191129_SEL_HF_SA_FRR_EXT_211119_Nuclear_GFP-CT-STIM1_005.raw	Raw
	20191129_SEL_HF_SA_FRR_EXT_211119_Nuclear_GFP-CT-STIM1_006.raw	Raw
	20191129_SEL_HF_SA_FRR_EXT_211119_Nuclear_GFP_001.raw	Raw
	20191129_SEL_HF_SA_FRR_EXT_211119_Nuclear_GFP_002.raw	Raw

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Project description:The in vivo labeling technique Split-Biotin identification (Split-BioID, De Munter et al., 2017, Schopp et al., 2017, Cho et al., 2020) was used to capture the common microenvironment of Bre5 and the ribosomal protein Rps2 (uS5). Biotinylated proteins were enriched from cell lysates using affinity purification. After SDS-PAGE, proteins were digested in-gel with trypsin and the resulting peptides were analyzed by liquid chromatography-mass spectrometry (LC-MS) for identification and relative quantification against controls. An aliquot of the cell lysate that was used for the affinity purification was taken as an input control and analyzed as well by LC-MS to account for possible differences in the input abundance of enriched proteins. Stable isotope labeling with amino acids in cell culture (SILAC) was used for relative quantification of proteins according to the 2nSILAC approach (Dannenmaier et al., 2018). To evaluate the labeling efficiency, an aliquot of each culture was harvested separately prior to the pooling of the cells for the Split-BioID experiment. Cell lysates from these samples were used for SDS-PAGE, and a part of each gel lane was used for an in-gel digest of proteins. These peptides were also analyzed with LC-MS and the percentages of SILAC-labeled peptides (based on MSMS counts) were calculated. The captured proteins belong to a common cellular microenvironment of Bre5 and Rps2. References: Cho, K.F., Branon, T.C., Rajeev, S., Svinkina, T., Udeshi, N.D., Thoudam, T., Kwak, C., Rhee, H.W., Lee, I.K., Carr, S.A., Ting, A.Y. (2020). Split-TurboID enables contact-dependent proximity labeling in cells. Proc Natl Acad Sci U S A. 117, 12143-12154. Dannenmaier, S., Stiller, S.B., Morgenstern, M., Lübbert, P., Oeljeklaus, S., Wiedemann, N., Warscheid, B. (2018). Complete Native Stable Isotope Labeling by Amino Acids of Saccharomyces cerevisiae for Global Proteomic Analysis. Anal Chem 90, 10501-10509. De Munter, S., Görnemann, J., Derua, R., Lesage, B., Qian, J., Heroes, E., Waelkens, E., Van Eynde, A., Beullens, M., Bollen, M. (2017). Split-BioID: a proximity biotinylation assay for dimerization-dependent protein interactions. FEBS Lett 591, 415-424. Schopp, I.M., Amaya Ramirez, C.C., Debeljak, J., Kreibich, E., Skribbe, M., Wild, K., Béthune, J. (2017). Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes. Nat Commun 8,15690.

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STIM1 Translocation to The Nucleus Protects Cells from DNA Damage

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