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Optimization of Protein UFMylation Modification Method and Its Application in Substrate Identification in Human Cells

ABSTRACT: In this study, we generated the UFSP1KO/UFSP2KO HEK293T cell line and validated it as the most suitable cellular model for the screening, identification, and confirmation of UFMylated substrate proteins. Furthermore, exogenous expression of the E3 ligase components UFL1 and DDRGK1 significantly enhances protein UFMylation levels, enabling the large-scale enrichment of UFMylated substrates.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Zhaopeng Lv

LAB HEAD: Qian Liang

PROVIDER: PXD070408 | Pride | 2026-03-30

REPOSITORIES: Pride

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			Action	DRS
	20250430_122431_G151DP-VG-KC.sne	Other
	20250430_122431_G151DP-VG-KC_Report.tsv	Tabular
	Astral_20250429_ES906_DIA_30min_G151DP_DKO-C2.raw	Raw
	checksum.txt	Txt

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LC-MS/MS analysis of ribosome UFMylation in vitro

Project description:UFMylation, is a Ubiquitin-like modification occurring on RPL26, a ribosomal subunit, upon ribosome stalling at the ER membrane. This modification is achieved by a pool of enzymes commonly referred to as E1-activating, E2-conjugating and E3-ligase enzymes. The UFM1 E3 ligase comprises of three proteins: UFL1, UFBP1 and CDK5RAP3, referred to as UFM1 Ribosome E3 Ligase (UREL) complex. While UFL1 and UFBP1 are sufficient for E3 ligase activity in vitro, CDK5RAP3 is indispensable for ribosome UFMylation in vivo. In this work, we characterize in vitro UFMylation reaction of purified 60S ribosomes in the presence and absence of CDK5RAP3 using LC-MS/MS. Specifically, we analyse sites of UFMylation, linkage type and quantify monoUFMylation and diUFMylation on RPL26. Interestingly, we find UFMylation to occur on a specific lysine residue, K134, in the presence of UFL1/UFBP1 alone and in complex with CDK5RAP3. In addition, we also provide quantitative information on relative abundances of RPL26 mono- and di-UFMylation under these conditions.

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Project description:Ufmylation (UFM1 modification) is a newly identified ubiquitin-like modification system involved in numerous cellular processes. However, the regulatory mechanisms and biological functions of this modification remain mostly unknown. We have recently reported that Ufmylation family genes have frequent somatic copy number alterations in human cancer including melanoma, suggesting involvement of Ufmylation in skin function and disease. UFL1 is the only known Ufmylation E3-like ligase. In this study, we generated the skin-specific Ufl1 knockout mice and show that ablation of Ufl1 caused epidermal thickening, pigmentation and shortened life span. RNA-Seq analysis indicated that Ufl1 deletion resulted in upregulation of the genes involved in melanin biosynthesis. Mechanistically, we found that Endothelin-1 (ET-1) is a novel substrate of Ufmylation and this modification regulates ET-1 stability, and thereby deletion of Ufl1 upregulates the expression and secretion of ET-1, which in turn results in up-regulation of genes in melanin biosynthesis and skin pigmentation. Our findings establish the role of Ufl1 in skin pigmentation through Ufmylation modification of ET-1 and provide opportunities for therapeutic intervention of skin diseases.

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