O-Glycosylation of Nuclear and Cytosolic Proteins

Self Cite

128

113

A hallmark of signal transduction is the dynamic and inducible post-translational modification of proteins. In addition to the well characterized phosphorylation of proteins, other modifications have been shown to be regulatory, including O-linked ␤-N-acetylglucosamine (O-GlcNAc). O-GlcNAc modifies serine and threonine residues on a myriad of nuclear and cytosolic proteins, and for several proteins there appears to be a reciprocal relationship between phosphorylation and O-GlcNAc modification. Here we report further evidence of this yin-yang relationship by demonstrating that O-GlcNAc transferase, the enzyme that adds O-GlcNAc to proteins, exists in stable and active complexes with the serine/ threonine phosphatases PP1␤ and PP1␥, enzymes that remove phosphate from proteins. The existence of this complex highlights the importance of understanding the dynamic relationship between O-GlcNAc and phosphate in modulating protein function in many cellular processes and disease states such as Alzheimer's disease and type II diabetes.Although there are only 21, counting selenocysteine, genomically encoded mammalian amino acids, post-translational modification results in proteins that contain more than 50 different amino acids (1). Thus, covalent modification of polypeptides plays a major role in the generation of functional polypeptides and adds an extra level of complexity in attempts to understand the proteome (2). While several protein modifications are static, a subset of post-translational modifications is both dynamic and inducible. It is the study of these "regulatory" posttranslational modifications and the enzymes that add and subtract them to proteins that comprises a significant portion of the signal transduction field (3).The most well studied and understood regulatory post-translational modification is phosphorylation (3). Accordingly, a significant body of literature has focused on the kinase superfamily, which makes up more than 2% of the proteins encoded by the human genome (4). Interestingly, in humans there appear to be only about 15 catalytic serine/threonine protein phosphatases (PPP subfamily) compared with literally hundreds of serine/threonine kinases (4). Thus, understanding how protein dephosphorylation is regulated has been an area of intense study for the last two decades (5, 6). One emerging theme is that the catalytic phosphatases have binding partners that regulate their localization and activity (7).Similar to phosphorylation, O-linked ␤-N-acetylglucosamine (O-GlcNAc) modification of nuclear and cytosolic proteins is an abundant, dynamic, and inducible post-translational modification (8 -10). However, unlike kinases, there appears to be only one O-GlcNAc transferase catalytic subunit (OGT, 1 the enzyme that adds O-GlcNAc to proteins) in mammals (11). Similar to phosphatases, it has been proposed that OGT is regulated by a multitude of binding partners as well as post-translational modification and alternative splicing (12, 13). It has been demonstrated recently that OGT interacts with sever...

Section: Ogt-pp1 a Yin-yang Complexmentioning

confidence: 79%

O-GlcNAc Transferase Is in a Functional Complex with Protein Phosphatase 1 Catalytic Subunits

Wells

Kreppel

Comer

et al. 2004

Self Cite

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113

“…One possible explanation is that sialylation may not be the direct cause of the disease. For example, the loss of GNE enzymatic activity may induce the accumulation of the substrate, UDP-GlcNAc, leading to the abnormal O-GlcNAc modification of various proteins in the cells (23). Nevertheless, this possibility may also be unlikely because the overexpression of O-GlcNAc transferase did not cause any morphological abnormality in skeletal muscles in mice (24).…”

Section: Discussionmentioning

confidence: 93%

Reduction of UDP-N-acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase Activity and Sialylation in Distal Myopathy with Rimmed Vacuoles

Noguchi

Keira

Murayama³

et al. 2004

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Distal myopathy with rimmed vacuoles is an autosomal recessive muscle disease with preferential involvement of the tibialis anterior that spares the quadriceps muscles in young adulthood. In a Japanese patient with distal myopathy with rimmed vacuoles, we identified pathogenic mutations in the gene encoding the bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase, which catalyzes the initial two steps in the biosynthesis of sialic acid. In this study, we demonstrated the relationship between the genetic mutations and enzymatic activities using an in vitro expression assay system. Furthermore, we also showed that the levels of sialic acid in muscle and primary cultured cells from DMRV patients were reduced to 60 -75% of control. The reactivities to lectins were also variable in some myofibers, suggesting that hyposialylation and abnormal glycosylation in muscles may contribute to the focal accumulations of autophagic vacuoles, amyloid deposits, or both in patient muscle tissue. The addition of ManNAc and NeuAc to primary cultured cells normalized sialylation levels, thus demonstrating the therapeutic potential of these compounds for this disease.

“…␤-O-Linked N-acetylglucosamine (O-GlcNAc) is an abundant post-translational modification occurring on serine and threonine residues. This modification has been detected on a variety of nuclear and cytoplasmic proteins involved in diverse cellular functions (25). To determine whether parkin interacts with O-linked glycosylated ␣-synuclein in H19-7 cells, co-immunoprecipitation experiments were performed by using ␣-synuclein antibodies for immunoprecipitation, followed by immunoblotting with either ␣-synuclein antibody or well characterized monoclonal antibodies for Ser-O-GlcNAc and Thr-OGlcNAc residues.…”

Section: Resultsmentioning

confidence: 99%

“…The diversity of proteins modified by O-GlcNAc implies its importance in many basic cellular and disease processes (43). For example, several proteins that are thought to be involved in Alzheimer's disease, including Tau, AP3, and the ␤-amyloid precursor protein, are modified by O-GlcNAc, and O-GlcNAc levels are perturbed in this disease state (25). In contrast, our data show that parkin directly interacts with nonglycosylated ␣-synuclein in hippocampal H19-7 cells.…”

Section: Discussionmentioning

confidence: 99%

Parkin Cleaves Intracellular α-Synuclein Inclusions via the Activation of Calpain

Kim

Sung

et al. 2003

Mutations in the ␣-synuclein and parkin genes cause heritable forms of Parkinson's disease. In the present study, we examined the possible functional relationship between the parkin and ␣-synuclein genes in a conditionally immortalized embryonic hippocampal cell (H19-7) line. Whereas transient transfection of ␣-synuclein into neuronal H19-7 cells caused the formation of its intracytoplasmic inclusions and a significant cell death, the combined overexpression of parkin restored the ␣-synucleininduced decrease in cell viability to control levels. In addition, the overexpression of parkin was found to generate selective cleavage of ␣-synuclein. Furthermore, the cytoprotective effect of parkin on ␣-synuclein-induced cell death was not inhibited in the presence of a proteasome inhibitor. Interestingly, the overexpression of parkin induced the activation of an intracellular cysteine protease, calpain, but not caspase, and the cytoprotective effect of parkin on ␣-synuclein cytotoxicity was significantly inhibited by the presence of calpain-specific inhibitors. In conclusion, our results suggest that parkin accelerates the degradation of ␣-synuclein via the activation of the nonproteasomal protease, calpain, leading to the prevention of ␣-synuclein-induced cell death in embryonic hippocampal progenitor cells.