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Evaluation of N8-propargyl spermidine and spermine as substrates of deoxyhypusine synthase in the in vitro hypusination of eIF5A and RIPK1

ABSTRACT: eIF5A is a well-established protein substrate of deoxyhypusine synthase (DHS), which also requires spermidine as a small-molecule substrate. At the end of 2024, a publication claimed that RIPK1 is an additional DHS substrate and that N8-propargyl spermidine can also serve as a DHS substrate. To evaluate these claims, eIF5A and RIPK1 were subjected to in vitro hypusination in the presence of DHS, deoxyhypusine hydroxylase (DOHH; the enzyme that hydroxylates the deoxyhypusine residue generated by DHS), and either N8-propargyl spermidine or spermidine. For reactions containing N8-propargyl spermidine, the samples were split: one portion was directly digested with trypsin, whereas the other was subjected to CuAAC ligation with azido-PEG3-biotin prior to tryptic digestion. Following LC-MS/MS analysis and MaxQuant processing, the data were searched for peptides bearing hypusine, deoxyhypusine, and their N-propargylated analogs. In samples that underwent CuAAC ligation, additional searches were performed for PEG3-biotin-triazole-modified N8-propargyl-spermidine-derived hypusine and deoxyhypusine, as well as for cysteine-N8-propargyl spermidine thio-triazole adducts.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Remigiusz Serwa

LAB HEAD: Remigiusz Serwa

PROVIDER: PXD071577 | Pride | 2026-03-13

REPOSITORIES: Pride

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			Action	DRS
	PK-click_s-1.raw	Raw
	PK-click_s-2.raw	Raw
	PK-click_s-3.raw	Raw
	PK2901-s-5.raw	Raw
	PK2901-s-7.raw	Raw

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Evaluation of N8-propargyl spermidine as a substrate of deoxyhypusine synthase in cellulo

Project description:Two complementary experiments were performed to evaluate whether N8-propargyl spermidine is a substrate of deoxyhypusine synthase in cells. HEK293 and mouse embryonic fibroblast (MEF) cells were incubated with either N8-propargyl spermidine or spermidine, lysed, and subjected to CuAAC ligation with azido-PEG3-biotin. In the MEF experiment, biotinylated proteins were enriched on NeutrAvidin agarose resin, digested on-bead with trypsin, and, following TMT labeling and LC-MS/MS analysis, the data were processed in MaxQuant and analyzed for intensity enrichment in samples derived from cells treated with N8-propargyl spermidine versus spermidine. In the HEK293 experiment, proteins were digested with trypsin, biotinylated peptides were enriched on NeutrAvidin agarose resin, and after LC-MS/MS analysis and MaxQuant processing, the peptides were examined for the presence of N8-propargyl spermidine thio-triazole adducts on cysteine residues.

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Project description:Deoxyhypusine synthase (DHPS) utilizes the polyamine spermidine to catalyze the hypusine modification of the mRNA translation factor eIF5A and promotes oncogenesis through poorly-defined mechanisms. Because germline deletion of Dhps is embryonically lethal, its role in normal postnatal cellular function in vivo remains unknown. We generated a mouse model that allows for inducible, postnatal deletion of Dhps specifically in postnatal islet β cells, which function to maintain glucose homeostasis. Removal of Dhps did not have an effect under normal physiologic conditions. However, upon development of insulin resistance, which induces β-cell proliferation, Dhps deletion caused alterations in proteins required for mRNA translation, reduced production of the cell cycle molecule Cyclin D2, impaired β-cell proliferation, and overt diabetes. We found that hypusine biosynthesis was downstream of protein kinase C-ζ and was required for c-Myc-induced proliferation. Our studies reveal a requirement for DHPS in β cells to link polyamines to mRNA translation to effect facultative cellular proliferation and glucose homeostasis.

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