33 research outputs found
O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease.
Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation
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CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy.
Ovarian cancer and triple-negative breast cancer are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins called 'don't eat me' signals-including CD471, programmed cell death ligand 1 (PD-L1)2 and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M)3. Monoclonal antibodies that antagonize the interaction of 'don't eat me' signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers4,5. However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown 'don't eat me' signals. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a promising target for cancer immunotherapy. We demonstrate a role for tumour-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which is expressed by tumour-associated macrophages. We find that many tumours overexpress CD24 and that tumour-associated macrophages express high levels of Siglec-10. Genetic ablation of either CD24 or Siglec-10, as well as blockade of the CD24-Siglec-10 interaction using monoclonal antibodies, robustly augment the phagocytosis of all CD24-expressing human tumours that we tested. Genetic ablation and therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumour growth in vivo and an increase in survival time. These data reveal CD24 as a highly expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24 blockade in cancer immunotherapy
MFGE8 links absorption of dietary fatty acids with catabolism of enterocyte lipid stores through HNF4γ-dependent transcription of CES enzymes
Enterocytes modulate the extent of postprandial lipemia by storing dietary fats in cytoplasmic lipid droplets (cLDs). We have previously shown that the integrin ligand MFGE8 links absorption of dietary fats with activation of triglyceride (TG) hydrolases that catabolize cLDs for chylomicron production. Here, we identify CES1D as the key hydrolase downstream of the MFGE8-αvβ5 integrin pathway that regulates catabolism of diet-derived cLDs. Mfge8 knockout (KO) enterocytes have reduced CES1D transcript and protein levels and reduced protein levels of the transcription factor HNF4γ. Both Ces1d and Hnf4γ KO mice have decreased enterocyte TG hydrolase activity coupled with retention of TG in cLDs. Mechanistically, MFGE8-dependent fatty acid uptake through CD36 stabilizes HNF4γ protein level; HNF4γ then increases Ces1d transcription. Our work identifies a regulatory network that regulates the severity of postprandial lipemia by linking dietary fat absorption with protein stabilization of a transcription factor that increases expression of hydrolases responsible for catabolizing diet-derived cLDs
Chemoproteomics reveals Toll-like receptor fatty acylation
Partial funding for Open Access provided by The Ohio State University Open Access Fund.Background: Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity.
This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular
interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification
and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to
identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells.
Results: A total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been
previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new
proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was
uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2
S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased
inflammatory response to microbial ligands.
Conclusions: This work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell
type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. Spalmitoylation
of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue
for modulation of TLR2 inflammatory activity.This work was supported by funding from the NIH/NIAID (grant R00AI095348 to J.S.Y.), the NIH/NIGMS (R01GM087544 to HCH), and the Ohio State University Public Health Preparedness for Infectious Diseases (PHPID) program. NMC is supported by the Ohio State University Systems and Integrative Biology Training Program (NIH/NIGMS grant T32GM068412). BWZ is a fellow of the National Science Foundation Graduate Research Fellowship Program (DGE-0937362)
Identification of O-GlcNAc Modification Targets in Mouse Retinal Pericytes: Implication of p53 in Pathogenesis of Diabetic Retinopathy
Abstract 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 b-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
Changes in Metabolic Chemical Reporter Structure Yield a Selective Probe of O-GlcNAc Modification
International audienceMetabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain bioorthogonal functionalities and enable the direct visualization and identification of glycoproteins from living cells. Each MCR was initially thought to report on specific types of glycosylation. We and others have demonstrated that several MCRs are metabolically transformed and enter multiple glycosylation pathways. Therefore, the development of selective MCRs remains a key unmet goal. We demonstrate here that 6-azido-6-deoxy-N-acetyl-glucosamine (6AzGlcNAc) is a specific MCR for O-GlcNAcylated proteins. Biochemical analysis and comparative proteomics with 6AzGlcNAc, N-azidoacetyl-glucosamine (GlcNAz), and N-azidoacetyl-galactosamine (GalNAz) revealed that 6AzGlcNAc exclusively labels intracellular proteins, while GlcNAz and GalNAz are incorporated into a combination of intracellular and extracellular/lumenal glycoproteins. Notably, 6AzGlcNAc cannot be biosynthetically transformed into the corresponding UDP sugar-donor by the canonical salvage-pathway that requires phosphorylation at the 6-hydroxyl. In vitro experiments showed that 6AzGlcNAc can bypass this roadblock through direct phosphorylation of its 1-hydroxyl by the enzyme phosphoacetylglucosamine mutase (AGM1). Taken together, 6AzGlcNAc enables the specific analysis of O-GlcNAcylated proteins, and these results suggest that specific MCRs for other types of glycosylation can be developed. Additionally, our data demonstrate that cells are equipped with a somewhat unappreciated metabolic flexibility with important implications for the biosynthesis of natural and unnatural carbohydrates
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