N-glycosylation as a eukaryotic protective mechanism against protein aggregation

Duran-Romaña, Ramon; Houben, Bert; De Vleeschouwer, Matthias; Louros, Nikolaos; Wilson, Matthew P.; Matthijs, Gert; Schymkowitz, Joost; Rousseau, Frederic

doi:10.1126/sciadv.adk8173

Cited by 7 publications

(2 citation statements)

References 88 publications

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“…ALG5 is an enzyme involved in N-glycosylation modification and may affect the function and stability of enzymes related to milk fat synthesis, FASN, and LPL . The results of RT-qPCR and western blotting showed that ALG5 expression was upregulated in the BMECs treated with STF, and the increase was most significant under stimulation with 20 μM STF (Figure A,B).…”

Section: Resultsmentioning

confidence: 96%

ALG5 Regulates STF-62247-Induced Milk Fat Synthesis via the mTOR Signaling Pathway

Meng,

Lyu,

et al. 2024

J. Agric. Food Chem.

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Milk fat content is a critical indicator of milk quality. Exploring the key regulatory genes involved in milk fat synthesis is essential for enhancing milk fat content. STF-62247 (STF), a thiazolamide compound, has the potential to bind with ALG5 and upregulate lipid droplets in fat synthesis. However, the effect of STF on the process of milk fat synthesis and whether it acts through ALG5 remains unknown. In this study, the impact of ALG5 on milk fat synthesis and its underlying mechanism were investigated using bovine mammary epithelial cells (BMECs) and mouse models through real-time PCR, western blotting, Oil Red O staining, and triglyceride analysis. Experimental findings revealed a positive correlation between STF and ALG5 with the ability to synthesize milk fat. Silencing ALG5 led to decreased expression of FASN, SREBP1, and PPARγ in BMECs, as well as reduced phosphorylation levels in the PI3K/AKT/mTOR signaling pathway. Moreover, the phosphorylation levels of the PI3K/AKT/mTOR signaling pathway were restored when ALG5 silencing was followed by the addition of STF. These results suggest that STF regulates fatty acid synthesis in BMECs by affecting the PI3K/AKT/mTOR signaling pathway through ALG5. ALG5 is possibly a new factor in milk fat synthesis.

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Section: Resultsmentioning

confidence: 96%

ALG5 Regulates STF-62247-Induced Milk Fat Synthesis via the mTOR Signaling Pathway

Meng,

Lyu,

et al. 2024

J. Agric. Food Chem.

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“…Glycosylation is critical for protein folding and various other cellular processes (Ruotsalainen et al, 2022). Additionally, N-glycosylation has recently been described to be protective against protein aggregation due to steric hindrance, with overrepresentation reported at the N terminus of aggregation prone regions (APRs) (Duran-Romaña et al, 2024). Considering glycosylation in the modelling of MfgE8 could provide a more comprehensive understanding of both inter-and intra-domain interactions, as well as accessibility to the putative medin cleavage sites.…”

Section: Discussionmentioning

confidence: 99%

Investigating Medin Cleavage Accessibility in MfgE8: Conformational Insights Derived from Molecular Dynamics Simulations and AlphaFold2 Models

Mesdaghi,

Price,

et al. 2024

Preprint

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Recent studies have indicated that the human amyloidogenic protein medin is associated with a range of vascular diseases, including aortic aneurysms, vascular dementia, and Alzheimer's disease. Medin accumulates in the vasculature with age, leading to endothelial dysfunction through oxidative and nitrative stress and inducing pro-inflammatory activation. Medin is a cleavage product from the C2 domain of MfgE8. The exact mechanism of medin production from MfgE8 is unknown, with crystal structures of homologous C2 domains suggesting that the cleavage sites are buried, requiring a conformational transition for medin production. Molecular dynamics simulations can explore a wide range of conformations, from small-scale bond rotations to large-scale changes like protein folding or ligand binding. This study employed a combination of full-atom and coarse-grained molecular dynamics simulations, along with CONCOORD- and AlphaFold2-generated models, to investigate MfgE8 conformations and their implications for medin cleavage site accessibility. The simulations revealed that MfgE8 tends to adopt a compact conformation with the RGD motif, important for cell attachment within the N-terminal domain, and the medin region in the C-terminal domain close in proximity. Formation of this compact structure is facilitated by interdomain electrostatic interactions that promote stability and in turn decrease the solvent-accessible surface area of the medin region and particularly the C-terminal medin cleavage site. This data enhances current knowledge on medin generation to propose that alterations in local environmental conditions, possibly through changes in glycosylation or other post-translational modifications are required to induce MfgE8 to unfold partially or fully: this would result in enhanced accessibility of the cleavage sites and therefore enable medin generation.

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Uncovering a Latent Bioactive Interleukin‐6 Glycoform

Liu,

Maki,

Okamoto

et al. 2024

Angewandte Chemie

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A bioinspired semisynthesis of human‐interleukin‐6 bearing N‐glycan at Asn143 (143glycosyl‐IL‐6) was performed by intentional glycosylation effects and protein folding chemistry for regioselective peptide‐backbone activation. 143Glycosyl‐IL‐6 is a genetically coded cytokine, but isolation was difficult owing to a tiny amount. IL6‐polypeptide (1‐141‐position) with an intentionally inserted cysteine at 142‐position was expressed in E. coli. The expressed polypeptide was treated with a chemical folding process to make a specific helices bundle conformation through native two‐disulfide bonds (43–49 and 72–82). Utilizing the successfully formed free‐142‐cysteine, sequential conversions using cyanation of 142‐cysteine, hydrazinolysis, and thioesterification created a long polypeptide (1–141)‐thioester. However, the resultant polypeptide‐thioester caused considerable aggregation owing to a highly hydrophobic peptide sequence. After the reduction of two‐disulfide bonds of polypeptide (1–141)‐thioester, an unprecedented hydrophilic N‐glycan tag was inserted at the resultant cysteine thiols. The N‐glycan tags greatly stabilized polypeptide‐thioester. The subsequent native chemical ligation and desulfurization successfully gave a whole 143glycosyl‐IL‐6 polypeptide (183‐amino acids). Removal of four N‐glycan tags and immediate one‐pot in vitro folding protocol efficiently produced the folded 143glycosyl‐IL‐6. The folded 143glycosyl‐IL‐6 exhibited potent cell proliferation activity. The combined studies with molecular dynamics simulation, semisynthesis, and bioassays predict the bioactive conformation of latent 143glycosyl‐IL‐6.

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N-glycosylation as a eukaryotic protective mechanism against protein aggregation

Cited by 7 publications

References 88 publications

ALG5 Regulates STF-62247-Induced Milk Fat Synthesis via the mTOR Signaling Pathway

ALG5 Regulates STF-62247-Induced Milk Fat Synthesis via the mTOR Signaling Pathway

Investigating Medin Cleavage Accessibility in MfgE8: Conformational Insights Derived from Molecular Dynamics Simulations and AlphaFold2 Models

Uncovering a Latent Bioactive Interleukin‐6 Glycoform

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