The role of salt bridge networks in the stability of the yeast hexose transporter 1.

BACKGROUND: The yeast S. cerevisiae preferably metabolizes glucose through aerobic glycolysis. Glucose transport is facilitated by multiple hexose transporters (Hxts), and their expression and activity are tightly regulated by multiple mechanisms. However, detailed structural and functional analyses of Hxts remain limited, largely due to the lack of crystal structure. METHODS: Homology modeling was used to build a 3D structural model for the yeast glucose transporter Hxt1 and investigate the effects of site directed mutations on Hxt1 stability and glucose transport activity. RESULTS: The conserved salt bridge-forming residues observed in the human Glut4 and the yeast glucose receptor Rgt2 were identified within and between the two 6-transmembrane spanning segments of Hxt1. Most of the RGT2 mutations that disrupt the salt bridge networks were known to cause constitutive signal generation, whereas the corresponding substitutions in HXT1 were shown to decrease Hxt1 stability. While substitutions of the two residues in the salt bridge 2 in Glut4-E329Q and E393D-were reported to abolish glucose transport, the equivalent substitutions in Hxt1 (D382Q and E454D) did not affect Hxt1 glucose transport activity. CONCLUSIONS: Substitutions of equivalent salt bridge-forming residues in Hxt1, Rgt2, and Glut4 are predicted to lock them in an inward-facing conformation but lead to different functional consequences. GENERAL SIGNIFICANCE: The salt bridge networks in yeast and human glucose transporters and yeast glucose receptors may play different roles in maintaining their structural and functional integrity.

Bone Morphogenetic Protein-4 Impairs Retinal Endothelial Cell Barrier, a Potential Role in Diabetic Retinopathy.

Bone Morphogenetic Protein 4 (BMP4) is a secreted growth factor of the Transforming Growth Factor beta (TGFß) superfamily. The goal of this study was to test whether BMP4 contributes to the pathogenesis of diabetic retinopathy (DR). Immunofluorescence of BMP4 and the vascular marker isolectin-B4 was conducted on retinal sections of diabetic and non-diabetic human and experimental mice. We used Akita mice as a model for type-1 diabetes. Proteins were extracted from the retina of postmortem human eyes and 6-month diabetic Akita mice and age-matched control. BMP4 levels were measured by Western blot (WB). Human retinal endothelial cells (HRECs) were used as an in vitro model. HRECs were treated with BMP4 (50 ng/mL) for 48 h. The levels of phospho-smad 1/5/9 and phospho-p38 were measured by WB. BMP4-treated and control HRECs were also immunostained with anti-Zo-1. We also used electric cell-substrate impedance sensing (ECIS) to calculate the transcellular electrical resistance (TER) under BMP4 treatment in the presence and absence of noggin (200 ng/mL), LDN193189 (200 nM), LDN212854 (200 nM) or inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2; SU5416, 10 µM), p38 (SB202190, 10 µM), ERK (U0126, 10 µM) and ER stress (Phenylbutyric acid or PBA, 30 µmol/L). The impact of BMP4 on matrix metalloproteinases (MMP2 and MMP9) was also evaluated using specific ELISA kits. Immunofluorescence of human and mouse eyes showed increased BMP4 immunoreactivity, mainly localized in the retinal vessels of diabetic humans and mice compared to the control. Western blots of retinal proteins showed a significant increase in BMP4 expression in diabetic humans and mice compared to the control groups (p < 0.05). HRECs treated with BMP4 showed a marked increase in phospho-smad 1/5/9 (p = 0.039) and phospho-p38 (p = 0.013). Immunofluorescence of Zo-1 showed that BMP4-treated cells exhibited significant barrier disruption. ECIS also showed a marked decrease in TER of HRECs by BMP4 treatment compared to vehicle-treated HRECs (p < 0.001). Noggin, LDN193189, LDN212854, and inhibitors of p38 and VEGFR2 significantly mitigated the effects of BMP4 on the TER of HRECs. Our finding provides important insights regarding the role of BMP4 as a potential player in retinal endothelial cell dysfunction in diabetic retinopathy and could be a novel target to preserve the blood-retinal barrier during diabetes.