Extent of Inhibition of α‑Synuclein Aggregation in Vitro by SUMOylation Is Conjugation Site- and SUMO Isoform-Selective

α-Synuclein, the major aggregating protein in Parkinson’s disease, can be modified by the small protein SUMO, indicating a potential role in disease. However, the effects of SUMOylation on α-synuclein aggregation remain controversial due to heterogeneous nature of the proteins previously investigated. Here we used protein semisynthesis to obtain homogeneously SUMOylated α-synuclein and discovered site- and isoform-dependent effects of SUMOylation on α-synuclein aggregation. Our results indicate that SUMOylation at K102 is a better inhibitor of aggregation than corresponding modification at K96 and SUMO1 modification, a better inhibitor than SUMO3

Chemical Reporter for Visualizing Metabolic Cross-Talk between Carbohydrate Metabolism and Protein Modification

Metabolic chemical reporters have been largely used to study posttranslational modifications. Generally, it was assumed that these reporters entered one biosynthetic pathway, resulting in labeling of one type of modification. However, because they are metabolized by cells before their addition onto proteins, metabolic chemical reporters potentially provide a unique opportunity to read-out on both modifications of interest and cellular metabolism. We report here the development of a metabolic chemical reporter 1-deoxy-<i>N</i>-pentynyl glucosamine (1-deoxy-GlcNAlk). This small-molecule cannot be incorporated into glycans; however, treatment of mammalian cells results in labeling of a variety proteins and enables their visualization and identification. Competition of this labeling with sodium acetate and an acetyltransferase inhibitor suggests that 1-deoxy-GlcNAlk can enter the protein acetylation pathway. These results demonstrate that metabolic chemical reporters have the potential to isolate and potentially discover cross-talk between metabolic pathways in living cells

Chemical Reporter for Visualizing Metabolic Cross-Talk between Carbohydrate Metabolism and Protein Modification

Metabolic chemical reporters have been largely used to study posttranslational modifications. Generally, it was assumed that these reporters entered one biosynthetic pathway, resulting in labeling of one type of modification. However, because they are metabolized by cells before their addition onto proteins, metabolic chemical reporters potentially provide a unique opportunity to read-out on both modifications of interest and cellular metabolism. We report here the development of a metabolic chemical reporter 1-deoxy-<i>N</i>-pentynyl glucosamine (1-deoxy-GlcNAlk). This small-molecule cannot be incorporated into glycans; however, treatment of mammalian cells results in labeling of a variety proteins and enables their visualization and identification. Competition of this labeling with sodium acetate and an acetyltransferase inhibitor suggests that 1-deoxy-GlcNAlk can enter the protein acetylation pathway. These results demonstrate that metabolic chemical reporters have the potential to isolate and potentially discover cross-talk between metabolic pathways in living cells

The list of O-GlcNAc modified proteins involved in cellular death processes.

<p>The list of O-GlcNAc modified proteins involved in cellular death processes.</p

Identification of O-GlcNAc Modification Targets in Mouse Retinal Pericytes: Implication of p53 in Pathogenesis of Diabetic Retinopathy

<div><p>Hyperglycemia is the primary cause of the majority of diabetes complications, including diabetic retinopathy (DR). Hyperglycemic conditions have a detrimental effect on many tissues and cell types, especially the retinal vascular cells including early loss of pericytes (PC). However, the mechanisms behind this selective sensitivity of retinal PC to hyperglycemia are undefined. The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is elevated under hyperglycemic condition, and thus, may present an important molecular modification impacting the hyperglycemia-driven complications of diabetes. We have recently demonstrated that the level of O-GlcNAc modification in response to high glucose is variable in various retinal vascular cells. Retinal PC responded with the highest increase in O-GlcNAc modification compared to retinal endothelial cells and astrocytes. Here we show that these differences translated into functional changes, with an increase in apoptosis of retinal PC, not just under high glucose but also under treatment with O-GlcNAc modification inducers, PUGNAc and Thiamet-G. To gain insight into the molecular mechanisms involved, we have used click-It chemistry and LC-MS analysis and identified 431 target proteins of O-GlcNAc modification in retinal PC using an alkynyl-modified GlcNAc analog (GlcNAlk). Among the O-GlcNAc target proteins identified here 115 of them were not previously reported to be target of O-GlcNAc modification. We have identified at least 34 of these proteins with important roles in various aspects of cell death processes. Our results indicated that increased O-GlcNAc modification of p53 was associated with an increase in its protein levels in retinal PC. Together our results suggest that post-translational O-GlcNAc modification of p53 and its increased levels may contribute to selective early loss of PC during diabetes. Thus, modulation of O-GlcNAc modification may provide a novel treatment strategy to prevent the initiation and progression of DR.</p></div

Effects of high glucose and O-GlcNAc modification inducers on apoptosis of retinal vascular cells.

<p>TUNEL staining was used to detect cell apoptosis (red). The nuclei were counterstained with DAPI (blue). Violet color represents TUNEL-positive nuclei on merged photos. (A): represents retinal PC grown in 5 mM glucose medium, (B): in 25 mM glucose medium, (C): treatment with 100 nM Thiamet-G for 1 day in 5 mM glucose medium, (D): positive control, cells treated with 1 µM staurosporine (STP) for 6 h. These images are representative of images evaluated at least 1000 cells for each condition with 3 replicates (original magnification x200). (E); Bar graphs quantify apoptosis, which is expressed as percentage of apoptotic cells for each condition. Data are presented as mean ± SEM (n = 3). Mean ± SEM; ****(p≤0.001) significantly different from 5 mM glucose control.</p

Increased O-GlcNAc modification of p53 in retinal PC under different glucose conditions.

<p>Same amount of p53 was precipitated from retinal PC lysates (equivalent to 500 µg total protein) with anti-p53 antibody conjugated agarose beads. Line 1 and 2; beginning materials for 5 and 25 mM glucose conditions. Line 3 and 4; precipitated p53. Please note increased O-GlcNAc modified p53 under high glucose conditions.</p

Subcellular localization of identified O-GlcNAc proteins in retinal PC.

<p>Proteins have dual localization, indicated in overlapping areas. The complete list of identified proteins provided in Table S1 and Table S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095561#pone.0095561.s001" target="_blank">File S1</a>.</p

Functional categories of identified O-GlcNAc proteins in retinal PC.

<p>Multifunctional proteins are included in more than one functional category. The complete list of identified proteins provided in Table S1 and Table S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095561#pone.0095561.s001" target="_blank">File S1</a>.</p

Effects of high glucose and O-GlcNAc modification inducers on retinal vascular cell death and proliferation.

<p>Cells were assayed for cell death (A) and cell proliferation (B) under varying glucose concentration, with or without O-GlcNAcylation inhibitors, Don and Alloxan, and with or without O-GlcNAcylation inducers, Thiamet G (T–G) and PUGNAc. Cell viability was assessed by counting trypan blue-positive cells. Proliferation rates were determined by a MTS-based assay. High glucose conditions, or low glucose with O-GlcNAcylation inducers, significantly increased PC death (A) as well as decreased cell proliferation (B) compared to both EC and AC. Conversely, O-GlcNAcylation inhibitors neutralized the negative effects of high glucose on retinal PC. Mean ± SEM; ***(p≤0.01), and ****(p≤0.001) significantly different from 5 mM glucose control.</p