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Proteomic Analysis of Diabetic Retinas

ABSTRACT: As a metabolic disease, diabetes often leads to health complications such as heart failure, nephropathy, neurological disorders, and vision loss. Diabetic retinopathy (DR) affects as many as 100 million people worldwide. The mechanism of DR is complex and known to impact both neural and vascular components in the retina. While recent advances in the field have identified major cellular signaling contributing to DR pathogenesis, little has been reported on the protein post-translational modifications (PTM) -known to define protein localization, function, and activity -in the diabetic retina overall. Protein glycosylation is the enzymatic addition of carbohydrates to proteins, which can influence many protein attributes including folding, stability, function, and subcellular localization. Olinked glycosylation is the addition of sugars to an oxygen atom in amino acids with a free oxygen atom in their side chain (i.e., threonine, serine). To date, more than 100 congenital disorders of glycosylation have been described. However, no studies have identified the retinal O-linked glycoproteome in health or disease. With a critical need to expedite the discovery of PTMomics in diabetic retinas, we identified both global changes in protein levels and the retinal O-glycoproteome of control and diabetic mice. Liquid chromatography/mass spectrometry-based glycoproteomics and high throughput screening identified proteins differentially glycosylated in the retinas of wildtype and diabetic mice. Here, we provide evidence that diabetes shifts O-glycosylation of metabolic and synaptic proteins in the retina.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Epithelial Cell, Retina

DISEASE(S): Diabetes Mellitus

SUBMITTER: james mobley

LAB HEAD: James A. Mobley

PROVIDER: PXD044269 | Pride | 2023-08-16

REPOSITORIES: Pride

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Project description:Protein glycolysation is an essential posttranslational modification in eukaryotic cells. In pathogenic yeasts, it is involved in a large number of biological processes, such as protein folding quality control, cell viability and host/pathogen relationships. A link between protein glycosylation and apoptosis was established by the analysis of the phenotypes of oligosaccharyltransferase mutants in budding yeast. However, little is known about the contribution of glycosylation modifications to the adaptive response to apoptosis inducers. The cysteine protease metacaspase Mca1p plays a key role in the apoptotic response in Candida albicans triggered by the quorum sensing molecule farnesol. We subjected wild-type and mca1-deletion strains to farnesol stress and then studied the early phase of apoptosis release in quantitative glycoproteomics and glycomics experiments on cell-free extracts essentially devoid of cell walls. We identified and characterized 62 new glycosylated peptides with their glycan composition: 17 N-glycosylated, 45 O-glycosylated, and 81 additional sites of N-glycosylation. The glycosylated proteins were predicted as located mostly in the “membrane” and “cell wall” compartments, but they were also related to the “nucleus” and the “mitochondrial” compartments. They were found to be involved in the control of protein folding, cell wall integrity and cell cycle regulation. We showed a general increase in the O-glycosylation of proteins in the mca1 deletion strain after farnesol challenge. We identified 44 new putative protein substrates of the metacaspase in the glycoprotein fraction enriched on concanavalin A. Most of these substrates are involved in protein folding or protein resolubilization and in mitochondrial functions. We show here that key Mca1p substrates, such as Cdc48p or Ssb1p, involved in degrading misfolded glycoproteins and in the protein quality control system, are themselves differentially glycosylated. We found putative substrates, such as Bgl2p, Srb1p or Ugp1p, that are involved in the biogenesis of glycans. We validated as a substrate of Mca1p the endo-beta-1,3-glucanase Bgl2p which is involved in the incorporation of newly synthetized mannoproteins in the cell wall. Our findings highlight a new role of the metacaspase in amplifying cell death processes by affecting several critical protein quality control systems through the alteration of the protein glycosylation machinery.

Project description:Diabetic retinopathy (DR), the leading cause of blindness in working-age adults, is thought to be primarily a microvascular complication of diabetes. Müller cells, the major macroglia of the retina, are important for maintaining a healthy and functional retina and play a critical role in several pathological events during DR disease progression as they are a central element of the retinal neurovascular unit. Here, we aim to improve our understanding of Müller cell-specific signaling pathways that are altered during disease progression in order to develop novel gene therapy strategies targeting Müller cells in DR. To achieve this, we used a multi-omics approach on purified Müller cells from 6-month-old control and diabetic db/db mice, including (i) RNA-seq followed by oPOSSUM-3 transcription factor (TF) binding site cluster analysis, (ii) glial chromatin landscape analysis by ATAC-seq, and (iii) Müller cell proteomics by MS/MS mass spectrometry. This led to the identification of the glucocorticoid receptor (GR, gene ID: Nr3c1) as the most promising candidate. Its transcripts and protein were most highly expressed in Müller cells and significantly reduced in cells from diabetic mice, its target gene cluster was downregulated in Müller cells from diabetic animals, and importantly, it was identified as a potential master regulator not only by oPOSSUM analysis based on differentially expressed mRNA in Müller cells, but also validated by the ATAC-seq approach. Next, we investigated the effect of GR modulation in an in vitro cortisol-stimulated retinal explant model. Cortisol not only increased GR phosphorylation levels, but also induced changes in the expression of known downstream GR target genes. Finally, we evaluated whether AAV-mediated GR overexpression in Müller cells improves retinal functionality and found moderate functional improvements in electroretinogram recordings. Although synthetic and topical glucocorticoids are widely used in ophthalmology with undeniable beneficial effects, our study provides valuable new insights into the role of GR signaling and glial alterations in the diabetic retina and thus supports the therapeutic concept of locally enhancing the GR signaling axis. However, our finding of reduced GR levels in Müller cells of the diabetic retina suggests that therapeutic approaches should aim at increasing the expression of the receptor rather than adding more ligands. This may result in optimized local efficacy with fewer systemic unwarranted side effects.

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Proteomic Analysis of Diabetic Retinas

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