Dataset Information

Hexosamine Pathway Disruption by GFPT1 Loss Drives Coordinated Defects in Glycosylation, Autophagy, and Trafficking

ABSTRACT: Gfpt1, the rate limiting enzyme of the hexosamine biosynthetic pathway (HBP), provides the UDP-GlcNAc required for protein glycosylation. Biallelic mutations in GFPT1 cause congenital myasthenic syndromes (GFPT1-CMS), yet the molecular mechanisms linking impaired glycosylation to skeletal muscle dysfunction remain incompletely understood. Here, we combine cellular models of inducible Gfpt1 knockdown and a muscle-specific Gfpt1 knockout mouse (Gfpt1Tm1d/Tm1d) with whole-cell proteomics, immunoblot studies and secretomics to define glycosylation-dependent defects in intracellular trafficking, ER stress signaling and autophagy. Global proteomic profiling of Gfpt1-deficient myoblasts revealed marked downregulation of protein trafficking pathways and impaired secretion of key muscle cargo proteins, including Srgn. Loss of GFPT1 reduced both high–molecular weight glycosylated SRGN and its core protein, accompanied by intracellular retention and decreased secretion. These trafficking defects coincided with robust activation of the unfolded protein response (UPR), evidenced by increased Xbp1 expression and accumulation of spliced Xbp1s across pharmacologic, cellular, and mouse models of GFPT1 deficiency. Converging evidence from proteomics, immunoblotting, and immunofluorescence demonstrated impaired autophagy, including increased LC3 II accumulation, elevated p62/SQSTM1 levels, and enhanced p62 positive puncta in both myoblasts and Gfpt1-deficient skeletal muscle. Soluble/insoluble fractionation further confirmed p62 accumulation, indicating defective autophagic flux and buildup of aggregated cargo. Together, these findings identify a glycosylation dependent failure in protein trafficking that triggers ER stress, UPR activation, and autophagy impairment in Gfpt1-deficient skeletal muscle. This mechanistic cascade provides a unifying explanation for muscle pathology in GFPT1 associated congenital myasthenic syndromes and suggests that restoring glycosylation or improving proteostasis may represent viable therapeutic approaches.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Andreas Hentschel

LAB HEAD: Prof. Dr. Albert Sickmann

PROVIDER: PXD079191 | Pride | 2026-08-04

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20250514_100355_aH_C2C12_secretome_sh6FPH_Holland_Ottawa.sne	Other
	Sample_Matrix_supplement.xlsx	Xlsx
	Std-QExactive01-07238_C2C12_Scr_secretomics_Pellet_N-1.msf	Msf
	Std-QExactive01-07238_C2C12_Scr_secretomics_Pellet_N-1.raw	Raw
	Std-QExactive01-07239_C2C12_Scr_secretomics_Pellet_N-2.raw	Raw

Items per page:

1 - 5 of 22

Similar Datasets

Project description:Huntington's disease (HD) is a dominantly inherited genetic disease caused by mutant huntingtin (htt) protein with expanded polyglutamine tracts. A neuropathological hallmark of HD is the presence of neuronal inclusions of mutant htt. p62 is an important regulatory protein in selective autophagy, a process by which aggregated proteins are degraded, and it is associated with several neurodegenerative disorders including HD. Here we investigated the effect of p62 depletion in three HD model mice: R6/2, HD190QG and HD120QG mice. We found that loss of p62 in these models led to longer lifespans and reduced nuclear inclusions, although cytoplasmic inclusions increased with polyglutamine length. In mouse embryonic fibroblasts (MEFs) with or without p62, mutant htt with a nuclear localization signal (NLS) showed no difference in nuclear inclusion between the two MEF types. In the case of mutant htt without NLS, however, p62 depletion increased cytoplasmic inclusions. Furthermore, to examine the effect of impaired autophagy in HD model mice, we crossed R6/2 mice with Atg5 conditional knockout mice. These mice also showed decreased nuclear inclusions and increased cytoplasmic inclusions, similar to HD mice lacking p62. These data suggest that the genetic ablation of p62 in HD model mice enhances cytoplasmic inclusion formation by interrupting autophagic clearance of polyQ inclusions. This reduces polyQ nuclear influx and paradoxically ameliorates disease phenotypes by decreasing toxic nuclear inclusions. Gene expression profiles were analyzed to examine the effects of p62 depletion in mouse with or without mutant huntingtin exon 1 To examine the effect of p62 depletion on the transcriptome of Huntington's disease model mice, we crossed p62 knockout mice with HD model mice. We extracted total RNA from the striatum of these mice at 8 weeks and used for a microaaray analysis. The samples are HD transgenic mice with p62 knockout (HD_p62KO), HD mice with normal p62 (HD_p62WT), non-HD-transgenic mice with p62 knockout (NT_p62KO), and non-HD-transgenic mice with normal p62 (NT_p62WT).

Dataset Information

Hexosamine Pathway Disruption by GFPT1 Loss Drives Coordinated Defects in Glycosylation, Autophagy, and Trafficking

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets