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Proximity labeling via MLF2-APEX2 fusion

ABSTRACT: Proximity labeling approach to identify protein inside nuclear envelope blebs arising in Torsin-deficient HeLa cells. WT and TorsinKO cells were engineered to express MLF2-APEX2 fusion. APEX reaction was carried out via 1 mM H2O2 for 1 minute in the presence of biotin phenol. Biotinylated protein were captured via streptavidin beads. Each APEX reaction was accompanied by an untreated (no H2O2) control.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Sarah Prophet

LAB HEAD: Christian Schlieker

PROVIDER: PXD036262 | Pride | 2022-08-25

REPOSITORIES: Pride

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			Action	DRS
	5.13.19_APEX_MLF2.mzid.gz	Mzid
	5.13.19_APEX_MLF2.mzid_OTF19-2037-40_Rampello_KO_APEX_1-4_merged_biotin-tyramide__OTF19-2037_Rampello_KO_APEX_1.MGF	Mzid
	5.13.19_APEX_MLF2.mzid_OTF19-2037-40_Rampello_KO_APEX_1-4_merged_biotin-tyramide__OTF19-2038_Rampello_KO_APEX_2.MGF	Mzid
	5.13.19_APEX_MLF2.mzid_OTF19-2037-40_Rampello_KO_APEX_1-4_merged_biotin-tyramide__OTF19-2039_Rampello_KO_APEX_3.MGF	Mzid
	5.13.19_APEX_MLF2.mzid_OTF19-2037-40_Rampello_KO_APEX_1-4_merged_biotin-tyramide__OTF19-2040_Rampello_KO_APEX_4.MGF	Mzid

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Project description:Poly(ADP-ribose) Polymerase 1 (PARP-1) is a nuclear enzyme that catalyse the transfer of ADP-ribose units from NAD+ to several target proteins involved in cellular stress responses. Using the cell line WRL68, we have previously shown that the activation of PARP-1 induced by oxidative stress after H2O2 treatment lead to depletion of cellular NAD+ and ATP, which promote cell death. In this work, we used LC-MS/MS based phosphoproteomics to show that the oxidative damage induced by H2O2 treatment modified the phosphorylation of several proteins in WRL68 cells, most of them related to transcription process. Interestingly, genetic or pharmacological inhibition of PARP-1 reduced the phosphorylation of YAP1, a transcriptional coactivator involved in cell survival, in WRL68 cells after H2O2 treatment. Inhibition of PARP-1 increased cell viability after oxidative treatment and diminished the degradation of YAP1; the elimination of YAP1 by gene silencing abrogated the protective effect of PARP-1 inhibition, indicating that YAP1 is important for the survival of WRL68 cells after oxidative damage. In the same way, the supplementation of NAD+, substrate of PARP-1, on WRL68 cells suffering oxidative damage reduced the phosphorylation of YAP1, suggesting that the loss of cellular NAD+ caused by PARP-1 activation after oxidative treatment is responsible for the phosphorylation of YAP1. Finally, PARP-1 silencing after oxidative treatment diminished the activation of AMPK, a master regulator of cell metabolism implicated in the restoration of energy balance during metabolic stress. Since NAD+ supplementation reduced the phosphorylation of some AMPK substrates in WRL68 cells after oxidative treatment, we hypothesize that the loss of cellular NAD+ after PARP-1 activation may induce a metabolic stress that activates AMPK. In summary, we showed a new crucial role of PARP-1 in the response to oxidative stress in WRL68 cells; PARP-1 activation reduces the cell viability by promoting the phosphorylation and degradation of YAP1, which is mediated by the energy stress caused by PARP-1 activation.

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Proximity labeling via MLF2-APEX2 fusion

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