Quantitative proteomics identifies altered O‐GlcNAcylation of structural, synaptic and memory‐associated proteins in Alzheimer's disease

Wang, Sheng; Yang, Feng; Petyuk, Vladislav; Shukla, Anil; Monroe, Matthew E.; Gritsenko, Marina; Rodland, Karin D.; Smith, Richard; Qian, Weijun; Chen, Gong; Liu, Tao

doi:10.1002/path.4929

Cited by 110 publications

(134 citation statements)

References 59 publications

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“…Consistent with the high levels of modification we observed in our pulldown, recent proteomics experiments using the same chemoenzymatic strategy have identified many different endogenous MYPT1 O-GlcNAcylation sites [42][43][44] localized around two different regions of the protein (Figure 5d). The most N-terminal of these areas, with modification at Ser379 and Thr381, is located near the portion of MYPT1 responsible for binding the phosphatase catalytic subunit PP1cδ and its substrate, phosphorylated MLC [45][46][47] .…”

Section: Mypt1 Is a Heavily And Dynamically O-glcnacylated Proteinsupporting

confidence: 89%

MYPT1 O-GlcNAc modification controls the sensitivity of fibroblasts to sphingosine-1-phosphate mediated cellular contraction

Pedowitz

Batt

Darabedian

et al. 2019

Preprint

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Many intracellular proteins can be modified by N-acetylglucosamine, a posttranslational modification known as O-GlcNAc.Because this modification is found on serine and threonine side-chains, O-GlcNAc has the potential to dynamically regulate cellular signaling pathways through interplay with phosphorylation. Here, we discover and characterize one such pathway.First, we find that O-GlcNAcylation levels control the sensitivity of fibroblasts to actin contraction induced by the signaling lipid sphingosine-1-phosphate (S1P). In subsequent mechanistic investigations, we find that S1P agonizes the S1PR2 receptor, resulting in activation of the Rho/Rho kinase signaling pathway. This pathway typically culminates in the phosphorylation of myosin light chain (MLC), resulting in myosin activation and cellular contraction. We find that O-GlcNAcylation of the phosphatase subunit MYPT1 inhibits this pathway by blocking MYPT1 phosphorylation, maintaining its activity and causing the dephosphorylation of MLC. Therefore, MYTP1 O-GlcNAcylation levels function as a rheostat to regulate the sensitivity of cells to S1P-mediated cellular contraction. Our findings have important implications for the role of in fibroblast motility and differentiation, as well as several other signaling pathways that use MLC/MYTP1 to control actin contraction in various tissues.

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Section: Mypt1 Is a Heavily And Dynamically O-glcnacylated Proteinsupporting

confidence: 89%

MYPT1 O-GlcNAc modification controls the sensitivity of fibroblasts to sphingosine-1-phosphate mediated cellular contraction

Pedowitz

Batt

Darabedian

et al. 2019

Preprint

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“…Three studies show decreases in synaptic proteins or pathways involved in synaptic function, which could have been explained by synapse loss 30, 31, 32 . To our knowledge only one previous study examined the effects of APOE on synaptic proteins 33 . This study used whole tissue homogenates from AD and control subjects for proteomics but focused their analysis on a group of 191 proteins that had previously been detected in synaptosome fractions of healthy subjects.…”

Section: Discussionmentioning

confidence: 99%

Comparative profiling of the synaptic proteome from Alzheimer’s disease patients with focus on the APOE genotype

Hesse

Hurtado

Jackson

et al. 2019

Preprint

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Highlights 42• Proteomic analysis of synapses isolated from Alzheimer's disease and control subject 43 brains identifies over 5,500 proteins in human synapses. 44• In silico analysis reveals region-specific decreases in proteins involved in synaptic 45 and mitochondrial function and increases in proteins involved in neuroimmune 46 signaling and intracellular signaling in AD. 47• The apolipoprotein E4 risk gene is associated with exacerbated changes in synaptic 48 proteins in AD. 49 50 Abstract 51Degeneration of synapses in Alzheimer's disease (AD) strongly correlates with cognitive 52 decline, and synaptic pathology contributes to disease pathophysiology. We recently 53 discovered that the strongest genetic risk factor for sporadic AD, apolipoprotein E epsilon 4 54 (APOE4), exacerbates synapse loss and synaptic accumulation of oligomeric amyloid beta in 55 human AD brain. To begin to understand the molecular cascades involved in synapse loss in 56 AD and how this is mediated by APOE, and to generate a resource of knowledge of changes 57 in the synaptic proteome in AD, we conducted a proteomic screen and systematic in-silico 58 analysis of synaptoneurosome preparations from temporal and occipital cortices of human 59 AD and control subjects with known APOE gene status. Our analysis identified over 5,500 60 proteins in human synaptoneurosomes and highlighted disease, brain region, and APOE-61 associated changes in multiple molecular pathways including a decreased abundance in AD 62 of proteins important for synaptic and mitochondrial function and an increased abundance of 63 proteins involved in neuroimmune interactions and intracellular signaling. 64 65

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“…In contrast to most PTMs, O-GlcNAc is installed and removed by only two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which modify over 3,000 protein substrates ( Figure 1) (11)(12)(13)(14)(15). O-GlcNAc is critical to cellular function as deletion of OGT in mice is embryonic lethal (16), deletion of OGA leads to perinatal death (17), and the conditional deletion of OGT in numerous cell types leads to senescence and apoptosis (18).…”

Section: Introductionmentioning

confidence: 99%

Selective protein O-GlcNAcylation in cells by a proximity-directed O-GlcNAc transferase

Ramirez

Aonbangkhen

et al. 2019

Preprint

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O-Linked N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Yet, the study of post-translational modifications like O-GlcNAc has been limited by the lack of strategies to induce O-GlcNAcylation on a target protein in cells. Here, we report a generalizable genetic strategy to induce O-GlcNAc to specific target proteins in cells using a nanobody as a proximitydirecting agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT(4), a truncated form of OGT, yielded a nanobody-OGT(4) construct that selectively delivered O-GlcNAc to the target protein (e.g., JunB, cJun, Nup62) and reduced alteration of global O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the selective increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein. Finally, we demonstrate the ability to selectively target endogenous ⍺-synuclein for glycosylation in HEK293T cells. Thus, the use of nanobodies to redirect OGT substrate selection is a versatile strategy to induce glycosylation of desired target proteins in cells that will facilitate discovery of O-GlcNAc functions and provide a mechanism to engineer O-GlcNAc signaling. The proximity-directed OGT approach for protein-selective O-GlcNAcylation is readily translated to additional protein targets and nanobodies that may constitute a generalizable strategy to control post-translational modifications in cells. Significance StatementNature uses post-translational modifications (PTMs) like glycosylation as a mechanism to alter protein signaling and function. However, the study of these modified proteins in cells is confined to loss-of-function strategies, such as mutagenic elimination of the modification site. Here, we report a generalizable strategy for induction of O-GlcNAc to a protein target in cells. The O-GlcNAc modification is installed by O-GlcNAc transferase (OGT) to thousands of nucleocytoplasmic proteins. Fusion of a nanobody to OGT enables the selective increase of O-GlcNAc levels on a series of target proteins. The described approach will facilitate direct studies of O-GlcNAc and its regulatory enzymes and drive new approaches to engineer protein signaling via a strategy that may be conceptually translatable to additional PTMs.

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Quantitative proteomics identifies altered O‐GlcNAcylation of structural, synaptic and memory‐associated proteins in Alzheimer's disease

Cited by 110 publications

References 59 publications

MYPT1 O-GlcNAc modification controls the sensitivity of fibroblasts to sphingosine-1-phosphate mediated cellular contraction

MYPT1 O-GlcNAc modification controls the sensitivity of fibroblasts to sphingosine-1-phosphate mediated cellular contraction

Comparative profiling of the synaptic proteome from Alzheimer’s disease patients with focus on the APOE genotype

Selective protein O-GlcNAcylation in cells by a proximity-directed O-GlcNAc transferase

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