Biology subjects

Issad, T.

Publications and source records attributed to Issad, T..

4 recordsLinked to original sources

bioRxiv2024

Small molecule modulation of insulin receptor-insulin like growth factor-1 receptor heterodimers in human endothelial cells

ObjectivesThe insulin receptor (IR) and insulin like growth factor-1 receptor (IGF-1R) are heterodimers consisting of two extracellular -subunits and two transmembrane {beta} -subunits. Insulin {beta} and insulin like growth factor-1 {beta} hemi-receptors can heterodimerize to form hybrids composed of one IR {beta} and one IGF-1R {beta}. The function of hybrids in the endothelium is unclear. We sought insight by developing a small molecule capable of reducing hybrid formation in endothelial cells. MethodsWe performed a high-throughput small molecule screening, based on a homology model of hybrid structure. Endothelial cells were studied using western blotting and qPCR to determine the effects of small molecules that reduced hybrid formation. ResultsOur studies unveil a first-in-class quinoline-containing heterocyclic small molecule that reduces hybrids by >50% in human umbilical vein endothelial cells (HUVECs) with no effects on insulin or insulin like growth factor-1 receptors. This small molecule reduced expression of the negative regulatory p85 subunit of phosphatidylinositol 3-kinase, increased basal phosphorylation of the downstream target Akt and enhanced insulin/insulin-like growth factor-1 and shear stress-induced serine phosphorylation of Akt. In primary saphenous vein endothelial cells (SVEC) from patients with type 2 diabetes mellitus undergoing coronary artery bypass (CABG) surgery, hybrid receptor expression was greater than in patients without type 2 diabetes mellitus. The small molecule significantly reduced hybrid expression in SVEC from patients with type 2 diabetes mellitus. ConclusionsWe identified a small molecule that decreases the formation of IR: IGF-1R hybrid receptors in human endothelial cells, without significant impact on the overall expression of IR or IGF-1R. In HUVECs, reduction of IR: IGF-1R hybrid receptors leads to an increase in insulin-induced serine phosphorylation of the critical downstream signalling kinase, Akt. The underpinning mechanism appears, at least in part to involve the attenuation of the adverse effect of IR: IGF-1R hybrid receptors on PI3-kinase signalling. HighlightsO_LIWe have discovered a small molecule (HI) that inhibits insulin receptor/IGF-1 receptor hybrid formation. C_LIO_LIHI reveals previously unrecognised actions of insulin receptor/IGF-1 receptor hybrids distinct to insulin and IGF-1 receptors in endothelial cells. C_LIO_LITreatment of endothelial cells with HI enhances activity of the downstream signalling kinase Akt due to inhibitory regulation via PI3-K. C_LI

cell biology↗

bioRxiv2024

Using site-directed mutagenesis to further the understanding of insulin receptor-insulin like growth factor-1 receptor heterodimer structure.

Type 2 diabetes is characterised by the disruption of insulin and insulin-like growth factor (IGF) signalling. The key hubs of these signalling cascades - the Insulin receptor (IR) and Insulin-like growth factor 1 receptor (IGF1R) - are known to form functional IR-IGF1R hybrid receptors which are insulin resistant. However, the mechanisms underpinning IR-IGF1R hybrid formation are not fully understood, hindering the ability to modulate this for future therapies targeting this receptor. To pinpoint suitable sites for intervention, computational hotspot prediction was utilised to identify promising epitopes for targeting with point mutagenesis. Specific IGF1R point mutations F450A, R391A and D555A show reduced affinity of the hybrid receptor in a BRET based donor-saturation assay, confirming hybrid formation could be modulated at this interface. These data provide the basis for rational design of more effective hybrid receptor modulators, supporting the prospect of identifying a small molecule that specifically interacts with this target.

molecular biology↗

bioRxiv2021

Short OGA is targeted to the mitochondria and regulates mitochondrial reactive oxygen species level

O-GlcNAcylation is a reversible post-translational modification involved the regulation of cytosolic, nuclear and mitochondrial proteins. Only two enzymes, OGT and OGA, control attachment and removal of O-GlcNAc on proteins, respectively. Whereas a variant OGT (mOGT) has been proposed as the main isoform that O-GlcNAcylates proteins in mitochondria, identification of a mitochondrial OGA has not been performed yet. Two splice variants of OGA (short and long isoforms) have been described previously. In this work, using cell fractionation experiments, we show that short-OGA is preferentially recovered in mitochondria-enriched fractions from HEK-293T cells as well as mouse embryonic fibroblasts. Moreover, fluorescent microscopy imaging confirmed that GFP-tagged short-OGA is addressed to mitochondria. In addition, using a BRET-based mitochondrial O-GlcNAcylation biosensor, we show that co-transfection of short-OGA markedly reduced O-GlcNAcylation of the biosensor, whereas long-OGA had no significant effect. Finally, using genetically encoded or chemical fluorescent mitochondrial probes, we showed that short-OGA overexpression increases mitochondrial ROS levels, whereas long-OGA had no significant effect. Together, our work reveals that the short-OGA isoform is targeted to the mitochondria where it regulates ROS homoeostasis.

cell biology↗

bioRxiv2020

LPS induces GFAT2 expression to promote O-GlcNAcylation and attenuate inflammation in macrophages

O-GlcNAc glycosylation is a reversible post-translational modification that regulates the activity of intracellular proteins according to glucose availability and its metabolism through the hexosamine biosynthesis pathway (HBP). This modification has been involved in the regulation of various immune cell types, including macrophages. However, little is known concerning the mechanisms that regulate protein O-GlcNAcylation level in these cells. In the present work, we demonstrate that LPS treatment induces a marked increase in protein O-GlcNAcylation in RAW264.7 cells, bone-marrow-derived and peritoneal mouse macrophages, as well as human monocyte-derived macrophages. Targeted deletion of OGT in macrophages resulted in an increased effect of LPS on NOS2 expression and cytokine production, suggesting that O-GlcNAcylation may restrain inflammatory processes induced by TLR4 activation. The effect of LPS on protein O-GlcNAcylation in macrophages was associated with an increased expression and activity of glutamine fructose 6-phosphate amido-transferase (GFAT), the enzyme that catalyzes the rate-limiting step of the HBP. More specifically, we observed that LPS rapidly and potently stimulated GFAT2 isoform mRNA and protein expression. Genetic or pharmacological inhibition of FoxO1 impaired LPS effect on GFAT2 expression, suggesting a FoxO1-dependent mechanism. We conclude that GFAT2 should be considered as a new TLR4-inducible gene involved in regulation of protein O-GlcNAcylation, that permits to limit exacerbated inflammation upon macrophage activation.

biochemistry↗