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ERO1 alpha deficiency increases N-glycosylation of a proangiogenic VEGFA by MAGT1-dependent OST complex

ABSTRACT: N-glycosylation and disulfide bond formation are two essential steps in protein folding, that both take place in the endoplasmic reticulum (ER). These two modifications can influence each other, but the exact timing, as well as the mediators of this important crosstalk, are still not completely elucidated. In previous work, we demonstrated that cells deficient in ER oxidoreductin 1-alpha (ERO1-alpha), a protein disulfide oxidase, are impaired in their ability to secrete Vascular Endothelial Growth Factor-A (VEGF121), a key regulator of vascular homeostasis in health and of metastasis in cancer. Here, to analyze the crosstalk between N-glycosylation and disulfide bond formation in newly synthesized proteins of the ER, we investigated how ERO1-alpha deficit affects glycosylation of VEGF121. We found that reduced ER redox poise imposed by ERO1 deficiency, while slowing down the intracellular formation of disulfide-bonds in VEGF121, promoted the full utilization of its single N-glycosylation consensus site, which lies close to an intra-polypeptide disulfide bridge. Unexpectedly, this hyperglycosylation impairs the kinetics of VEGF121 secretion. Unbiased mass-spectrometric data of VEGF121 immunoprecipitates followed by pathway analysis indicated a stable interaction between VEGF121 and proteins involved in the N-glycosylation in ERO1-alpha knockout, but not wild-type cells. Notably, MAGT1, a thioredoxin-containing protein and part of the post-translational oligosaccharyltransferase complex (OST), was a major hit exclusive to ERO1-deficient cells. We demonstrate that post-translational N-glycosylation VEGF121 is increased under the altered redox poise caused by ERO1 deficit: on the one hand by a reduced rate of formation of its disulfide bridges, and on the other, by the increased trapping potential of MAGT1's thioredoxin domain. These findings have implications for a variety of pathological processes involving altered redox conditions in the ER.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Michele Bianchi

LAB HEAD: Angela Bachi

PROVIDER: PXD033941 | Pride | 2022-08-29

REPOSITORIES: Pride

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			Action	DRS
	EZ_201230_KO_4.raw	Raw
	EZ_201230_KO_4_GluC_LysC_all.mzid.gz	Mzid
	EZ_201230_KO_4_GluC_LysC_all.mzid_EZ_201230_KO_4_GluC_LysC_all.MGF	Mzid
	EZ_201230_KO_5.raw	Raw
	EZ_201230_KO_5_GluC_LysC_all.mzid.gz	Mzid

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Project description:SEPN1 is a type II protein of the endoplasmic reticulum (ER) whose loss of function gives rise to a collection of debilitating autosomal recessive myopathies gathered under the umbrella term of SEPN1-related myopathy (RM). At the moment, SEPN1-RM lacks an effective pharmacological treatment; thus, the medical management of the disease is only supportive. The potentially fatal diaphragmatic weakness leading to respiratory insufficiency in patients still ambulant is the main reason for medical concerns. Thus, studies on SEPN1-RM pathogenesis are necessary to implement a targeted pharmacological therapy aimed at relieving the general muscle weakness and the more worrisome diaphragmatic dysfunction. Here, we show a physical and functional interaction between SEPN1 and the ER stress mediator ERO1 alpha (henceforth, ERO1). Both SEPN1 and ERO1 are involved in the redox regulation of proteins into the ER, although in an opposite way SEPN1 imposes a less oxidant ER poise while ERO1 a more oxidant one, conceivable with a reductase function of SEPN1 and an oxidase one of ERO1. Furthermore, both are mainly localized in a region of the ER in close contact with mitochondria termed mitochondria-associated membranes (MAMs) and their loss impacts oppositely on the short-range MAMs, thereby impinging ER-mitochondria Ca2+ dynamics, OXPHOS, and bioenergetics. We find that ERO1 depletion restores the impaired short-range MAMs due to SEPN1 loss together with mitochondrial ATP. ERO1 knockout in a mouse background of SEPN1 loss blunts ER stress and the consequent Unfolded Protein Response (UPR) while it rescues the diaphragmatic weakness by improving ER/mitochondria contacts, calcium dynamics, and OXPHOS. Importantly, treatments of SEPN1 knock out mice with the chemical chaperone tauroursodeoxycholic acid (TUDCA) mimic the results of ERO1 loss improving calcium dynamics, OXPHOS, ER/mitochondria contacts, thereby rescuing diaphragmatic weakness as well. In addition, TUDCA-treated SEPN1-RM patients-derived myoblasts show a dynamic dose-dependent increase in ATP levels under ER stress conditions suggesting improved mitochondrial function. Thus, our findings point to the ERO1 axis in the pathogenesis of SEPN1-RM thereby impinging on MAMs and mitochondria bioenergetics and recall for the efficacy of a pharmacology therapy with ad hoc ER stress/ERO1 inhibitors for SEPN1-RM.

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ERO1 alpha deficiency increases N-glycosylation of a proangiogenic VEGFA by MAGT1-dependent OST complex

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