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1.
Adv Exp Med Biol ; 1184: 207-216, 2019.
Article in English | MEDLINE | ID: mdl-32096040

ABSTRACT

Myotonic dystrophies (DM) are rare inherited neuromuscular disorders linked to microsatellite unstable expansions in non-coding regions of ubiquitously expressed genes. The DMPK and ZNF9/CNBP genes which mutations are responsible for DM1 and DM2 respectively. DM are multisystemic disorders with brain affection and cognitive deficits. Brain lesions consisting of neurofibrillary tangles are often observed in DM1 and DM2 brain. Neurofibrillary tangles (NFT) made of aggregates of hyper and abnormally phosphorylated isoforms of Tau proteins are neuropathological lesions common to more than 20 neurological disorders globally referred to as Tauopathies. Although NFT are observed in DM1 and DM2 brain, the question of whether DM1 and DM2 are Tauopathies remains a matter of debate. In the present review, several pathophysiological processes including, missplicing, nucleocytoplasmic transport disruption, RAN translation which are common mechanisms implicated in neurodegenerative diseases will be described. Together, these processes including the missplicing of Tau are providing evidence that DM1 and DM2 are not solely muscular diseases but that their brain affection component share many similarities with Tauopathies and other neurodegenerative diseases. Understanding DM1 and DM2 pathophysiology is therefore valuable to more globally understand other neurodegenerative diseases such as Tauopathies but also frontotemporal lobar neurodegeneration and amyotrophic lateral sclerosis.


Subject(s)
Gain of Function Mutation , Myotonic Dystrophy/genetics , Myotonic Dystrophy/metabolism , RNA/metabolism , Tauopathies/genetics , Tauopathies/metabolism , Humans , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , tau Proteins/chemistry , tau Proteins/genetics , tau Proteins/metabolism
2.
Biol Reprod ; 96(1): 93-106, 2017 01 01.
Article in English | MEDLINE | ID: mdl-28395323

ABSTRACT

Sperm motility notably depends on the structural integrity of the flagellum and the regulation of microtubule dynamics. Although researchers have started to use "omics" techniques to characterize the human sperm's molecular landscape, the constituents responsible for the assembly, organization, and dynamics of the flagellum microtubule have yet to be fully defined. In this study, we defined a core set of 116 gene products associated with the human sperm microtubulome (including products potentially involved in abnormal ciliary phenotypes and male infertility disorders). To this end, we designed and applied an integrated genomics workflow and combined relevant proteomics, transcriptomics, and interactomics datasets to reconstruct a dynamic interactome map. By further integrating phenotypic information, we defined a disease-interaction network; this enabled us to highlight a number of novel factors potentially associated with altered sperm motility and male fertility. Lastly, we experimentally validated the expression pattern of two candidate genes (CUL3 and DCDC2C) that had never previously been associated with male germline differentiation. Our analysis suggested that CUL3 and DCDC2C's products have important roles in the sperm flagellum. Taken as a whole, our results demonstrate that an integrated genomics strategy can highlight relevant molecular factors in specific sperm components. This approach could be easily extended by including other "omics" data (from asthenozoospermic men, for example) and identifying other critical proteins from the human sperm microtubulome.


Subject(s)
Microtubules/metabolism , Protein Interaction Maps , Spermatozoa/metabolism , Cullin Proteins/metabolism , Flagella/metabolism , Genomics , Humans , Male , Meiosis , Microtubule-Associated Proteins/metabolism , Proteome
3.
Mol Neurodegener ; 10: 28, 2015 Jul 14.
Article in English | MEDLINE | ID: mdl-26170022

ABSTRACT

The number of known pathologies involving deregulated Tau expression/metabolism is increasing. Indeed, in addition to tauopathies, which comprise approximately 30 diseases characterized by neuronal aggregation of hyperphosphorylated Tau in brain neurons, this protein has also been associated with various other pathologies such as cancer, inclusion body myositis, and microdeletion/microduplication syndromes, suggesting its possible function in peripheral tissues. In addition to Tau aggregation, Tau deregulation can occur at the expression and/or splicing levels, as has been clearly demonstrated in some of these pathologies. Here, we aim to review current knowledge regarding the regulation of human MAPT gene expression at the DNA and RNA levels to provide a better understanding of its possible deregulation. Several aspects, including repeated motifs, CpG island/methylation, and haplotypes at the DNA level, as well as the key regions involved in mRNA expression and stability and the splicing patterns of different mRNA isoforms at the RNA level, will be discussed.


Subject(s)
Gene Expression Regulation , tau Proteins/genetics , 3' Untranslated Regions/genetics , 5' Untranslated Regions/genetics , Brain/embryology , Brain/growth & development , Brain/metabolism , CpG Islands , DNA/genetics , DNA Methylation , DNA Transposable Elements , Fetus/metabolism , Haplotypes , Humans , Microsatellite Repeats , Neurons/metabolism , Promoter Regions, Genetic , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , RNA Splicing , RNA Stability , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Regulatory Sequences, Nucleic Acid , Tauopathies/genetics , Tauopathies/metabolism , Transcription Factors/metabolism , Transcription Initiation Site , tau Proteins/biosynthesis
4.
PLoS One ; 9(9): e107324, 2014.
Article in English | MEDLINE | ID: mdl-25211016

ABSTRACT

With the goal of identifying splicing alterations in myotonic dystrophy 1 (DM1) tissues that may yield insights into targets or mechanisms, we have surveyed mis-splicing events in three systems using a RT-PCR screening and validation platform. First, a transgenic mouse model expressing CUG-repeats identified splicing alterations shared with other mouse models of DM1. Second, using cell cultures from human embryonic muscle, we noted that DM1-associated splicing alterations were significantly enriched in cytoskeleton (e.g. SORBS1, TACC2, TTN, ACTN1 and DMD) and channel (e.g. KCND3 and TRPM4) genes. Third, of the splicing alterations occurring in adult DM1 tissues, one produced a dominant negative variant of the splicing regulator RBFOX1. Notably, half of the splicing events controlled by MBNL1 were co-regulated by RBFOX1, and several events in this category were mis-spliced in DM1 tissues. Our results suggest that reduced RBFOX1 activity in DM1 tissues may amplify several of the splicing alterations caused by the deficiency in MBNL1.


Subject(s)
Muscle, Skeletal/metabolism , Myotonic Dystrophy/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/physiology , Adult , Alternative Splicing , Animals , Cell Line , Female , Humans , Male , Mice, Inbred C57BL , Mice, Transgenic , Middle Aged , Myoblasts/metabolism , Myotonic Dystrophy/genetics , RNA Splicing Factors , RNA-Binding Proteins/metabolism
6.
Front Mol Neurosci ; 6: 57, 2014 Jan 09.
Article in English | MEDLINE | ID: mdl-24409116

ABSTRACT

Myotonic dystrophy (DM) of type 1 and 2 (DM1 and DM2) are inherited autosomal dominant diseases caused by dynamic and unstable expanded microsatellite sequences (CTG and CCTG, respectively) in the non-coding regions of the genes DMPK and ZNF9, respectively. These mutations result in the intranuclear accumulation of mutated transcripts and the mis-splicing of numerous transcripts. This so-called RNA gain of toxic function is the main feature of an emerging group of pathologies known as RNAopathies. Interestingly, in addition to these RNA inclusions, called foci, the presence of neurofibrillary tangles (NFT) in patient brains also distinguishes DM as a tauopathy. Tauopathies are a group of nearly 30 neurodegenerative diseases that are characterized by intraneuronal protein aggregates of the microtubule-associated protein Tau (MAPT) in patient brains. Furthermore, a number of neurodegenerative diseases involve the dysregulation of splicing regulating factors and have been characterized as spliceopathies. Thus, myotonic dystrophies are pathologies resulting from the interplay among RNAopathy, spliceopathy, and tauopathy. This review will describe how these processes contribute to neurodegeneration. We will first focus on the tauopathy associated with DM1, including clinical symptoms, brain histology, and molecular mechanisms. We will also discuss the features of DM1 that are shared by other tauopathies and, consequently, might participate in the development of a tauopathy. Moreover, we will discuss the determinants common to both RNAopathies and spliceopathies that could interfere with tau-related neurodegeneration.

7.
Neurobiol Aging ; 34(3): 757-69, 2013 Mar.
Article in English | MEDLINE | ID: mdl-22926167

ABSTRACT

A prerequisite to dephosphorylation at Ser-Pro or Thr-Pro motifs is the isomerization of the imidic peptide bond preceding the proline. The peptidyl-prolyl cis/trans isomerase named Pin1 catalyzes this mechanism. Through isomerization, Pin1 regulates the function of a growing number of targets including the microtubule-associated tau protein and is supposed to be deregulated Alzheimer's disease (AD). Using proteomics, we showed that Pin1 is posttranslationally modified on more than 5 residues, comprising phosphorylation, N-acetylation, and oxidation. Although Pin1 expression remained constant, Pin1 posttranslational two-dimensional pattern was modified by tau overexpression in a tau-inducible neuroblastoma cell line, in our THY-Tau22 mouse model of tauopathy as well as in AD. Interestingly, in all of these systems, Pin1 modifications were very similar. In AD brain tissue when compared with control, Pin1 is hyperphosphorylated at serine 16 and found in the most insoluble hyperphosphorylated tau fraction of AD brain tissue. Furthermore, in all tau pathology conditions, acetylation of Pin1 may also contribute to the differences observed. In conclusion, Pin1 displays several posttranslational modifications, which are specific in tauopathies and may be useful as biomarker.


Subject(s)
Brain/metabolism , Peptidylprolyl Isomerase/metabolism , Protein Processing, Post-Translational/physiology , Tauopathies/metabolism , tau Proteins/metabolism , Acetylation , Adult , Aged , Aged, 80 and over , Alzheimer Disease/metabolism , Animals , Biomarkers/metabolism , Cell Line, Tumor , Disease Models, Animal , Female , Humans , Male , Mice , Mice, Transgenic , Middle Aged , NIMA-Interacting Peptidylprolyl Isomerase , Oxidation-Reduction , Phosphorylation/physiology , Proline/metabolism , Proteome , Serine/metabolism
8.
J Biol Chem ; 286(18): 16435-46, 2011 May 06.
Article in English | MEDLINE | ID: mdl-21454535

ABSTRACT

Muscleblind-like-1 (MBNL1) is a splicing regulatory factor controlling the fetal-to-adult alternative splicing transitions during vertebrate muscle development. Its capture by nuclear CUG expansions is one major cause for type 1 myotonic dystrophy (DM1). Alternative splicing produces MBNL1 isoforms that differ by the presence or absence of the exonic regions 3, 5, and 7. To understand better their respective roles and the consequences of the deregulation of their expression in DM1, here we studied the respective roles of MBNL1 alternative and constitutive exons. By combining genetics, molecular and cellular approaches, we found that (i) the exon 5 and 6 regions are both needed to control the nuclear localization of MBNL1; (ii) the exon 3 region strongly enhances the affinity of MBNL1 for its pre-mRNA target sites; (iii) the exon 3 and 6 regions are both required for the splicing regulatory activity, and this function is not enhanced by an exclusive nuclear localization of MBNL1; and finally (iv) the exon 7 region enhances MBNL1-MBNL1 dimerization properties. Consequently, the abnormally high inclusion of the exon 5 and 7 regions in DM1 is expected to enhance the potential of MBNL1 of being sequestered with nuclear CUG expansions, which provides new insight into DM1 pathophysiology.


Subject(s)
Alternative Splicing , Cell Nucleus/metabolism , Exons , Protein Multimerization , RNA-Binding Proteins/metabolism , Active Transport, Cell Nucleus/genetics , Cell Nucleus/genetics , Cell Nucleus/pathology , HeLa Cells , Humans , Myotonic Dystrophy/genetics , Myotonic Dystrophy/metabolism , Myotonic Dystrophy/pathology , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA-Binding Proteins/genetics , Trinucleotide Repeat Expansion
9.
Therapie ; 65(5): 401-7, 2010.
Article in French | MEDLINE | ID: mdl-21144474

ABSTRACT

Alzheimer's disease is a neurodegenerative disorder characterized by neuropathological lesions: amyloid deposits and neurofibrillary degeneration. However, the links between these two brain hallmarks are still poorly understood. Until now, mainly amyloid pathology has been targeted un many clinical trials without any success. Both new therapeutic strategies and diagnosis improvement are needed.


Subject(s)
Alzheimer Disease/physiopathology , Amyloid beta-Peptides/metabolism , Neurofibrillary Tangles/pathology , Alzheimer Disease/diagnosis , Alzheimer Disease/therapy , Amyloid beta-Protein Precursor/metabolism , Animals , Humans
10.
Glycoconj J ; 27(1): 99-114, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19768537

ABSTRACT

The second human beta-galactoside alpha-2,6-sialyltransferase (hST6Gal II) differs from hST6Gal I, the first member of ST6Gal family, in substrate specificity and tissue expression pattern. While ST6GAL1 gene is expressed in almost all human tissues, ST6GAL2 shows a restricted tissue-specific pattern of expression, mostly expressed in embryonic and adult brain. In order to understand the mechanisms involved in the transcriptional regulation of ST6GAL2, we first characterized the transcription start sites (TSS) in SH-SY5Y neuroblastoma cells. 5' RACE experiments revealed multiple TSS located on three first alternative 5' exons, termed EX, EY and EZ, which are unusually close on the genomic sequence and are all located more than 42 kbp upstream of the first common coding exon. Using Taqman duplex Q-PCR, we showed that the ST6GAL2 transcripts initiated by EX or EY are mainly expressed in both brain-related cell lines and human cerebral cortex, testifying for the use of a similar transcriptional regulation in vivo. Furthermore, we also showed for the first time hST6Gal II protein expression in the different lobes of the human cortex. Luciferase reporter assays allowed us to define two sequences upstream EX and EY with a high and moderate promoter activity, respectively. Bioinformatics analysis and site-directed mutagenesis showed that NF-kappaB and NRSF are likely to act as transcriptional repressors, whereas neuronal-related development factors Sox5, Puralpha and Olf1, are likely to act as transcriptional activators of ST6GAL2. This suggests that ST6GAL2 transcription could be potentially activated for specific neuronal functions.


Subject(s)
Cerebral Cortex/enzymology , Gene Expression Regulation, Enzymologic , Neurons/enzymology , Sialyltransferases/genetics , Transcription, Genetic , 5' Untranslated Regions/genetics , Base Pairing/genetics , Base Sequence , Binding Sites , Cell Line, Tumor , Computational Biology , Enzyme Assays , Humans , Luciferases/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/metabolism , Transfection
11.
FEBS Lett ; 583(4): 675-9, 2009 Feb 18.
Article in English | MEDLINE | ID: mdl-19166838

ABSTRACT

Among the different mechanisms underlying the etiopathogenesis of myotonic dystrophy type 1 (DM1), a backward reprogramming to a foetal splicing machinery is an interesting hypothesis. To address this possibility, Tau splicing, which is regulated during development and modified in DM1, was analyzed. Indeed, a preferential expression of the foetal Tau isoform, instead of the six normally found, is observed in adult DM1 brains. By using two cell lines, we show here that the cis-regulating elements necessary to generate the unique foetal Tau isoform are dispensable to reproduce the trans-dominant effect induced by DM1 mutation on Tau exon 2 inclusion. Our results suggest that the mis-splicing of Tau in DM1 is resulting from a disease-associated mechanism.


Subject(s)
Alternative Splicing , Fetus/metabolism , Myotonic Dystrophy , tau Proteins/metabolism , 3' Untranslated Regions , Adult , Cell Line, Tumor , Exons , Fetus/pathology , Humans , Myotonic Dystrophy/genetics , Myotonic Dystrophy/metabolism , Myotonic Dystrophy/pathology , Plasmids , Protein Isoforms/genetics , Protein Isoforms/metabolism , Transfection , tau Proteins/genetics
12.
Expert Rev Proteomics ; 5(2): 207-24, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18466052

ABSTRACT

Microtubule-associated Tau proteins belong to a family of factors that polymerize tubulin dimers and stabilize microtubules. Tau is strongly expressed in neurons, localized in the axon and is essential for neuronal plasticity and network. From the very beginning of Tau discovery, proteomics methods have been essential to the knowledge of Tau biochemistry and biology. In this review, we have summarized the main contributions of several proteomic methods in the understanding of Tau, including expression, post-translational modifications and structure, in both physiological and pathophysiological aspects. Finally, recent advances in proteomics technology are essential to develop further therapeutic targets and early predictive and discriminative diagnostic assays for Alzheimer's disease and related disorders.


Subject(s)
Alzheimer Disease/etiology , Nervous System Diseases/etiology , tau Proteins/genetics , Alzheimer Disease/diagnosis , Humans , Nervous System Diseases/diagnosis , Protein Isoforms , Protein Processing, Post-Translational , tau Proteins/chemistry , tau Proteins/physiology
13.
Biomark Med ; 2(4): 363-84, 2008 Aug.
Article in English | MEDLINE | ID: mdl-20477391

ABSTRACT

The microtubule-associated protein Tau is mainly expressed in neurons of the CNS and is crucial in axonal maintenance and axonal transport. The rationale for Tau as a biomarker of neurodegenerative diseases is that it is a major component of abnormal intraneuronal aggregates observed in numerous tauopathies, including Alzheimer's disease. The molecular diversity of Tau is very useful when analyzing it in the brain or in the peripheral fluids. Immunohistochemical and biochemical characterization of Tau aggregates in the brain allows the postmortem classification and differential diagnosis of tauopathies. As peripheral biomarkers of Alzheimer's disease in the cerebrospinal fluid, Tau proteins are now validated for diagnosis and predictive purposes. For the future, the detailed characterization of Tau in the brain and in peripheral fluids will lead to novel promising biomarkers for differential diagnosis of dementia and monitoring of therapeutics.

14.
J Neurosci Res ; 84(4): 852-9, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16862542

ABSTRACT

Altered splicing of transcripts, including the insulin receptor (IR) and the cardiac troponin (cTNT), is a key feature of myotonic dystrophy type I (DM1). CELF and MBNL splicing factor members regulate the splicing of those transcripts. We have previously described an alteration of Tau exon 2 splicing in DM1 brain, resulting in the favored exclusion of exon 2. However, the factors required for alternative splicing of Tau exon 2 remain undetermined. Here we report a decreased expression of CELF family member and MBNL transcripts in DM1 brains as assessed by RT-PCR. By using cellular models with a control- or DM1-like splicing pattern of Tau transcripts, we demonstrate that ETR-3 promotes selectively the exclusion of Tau exon 2. These results together with the analysis of Tau exon 6 and IR exon 11 splicing in brain, muscle, and cell models suggest that DM1 splicing alteration of several transcripts involves various factors.


Subject(s)
Alternative Splicing/physiology , Exons , Myotonic Dystrophy/genetics , Nerve Tissue Proteins/physiology , RNA-Binding Proteins/physiology , tau Proteins/metabolism , Blotting, Western/methods , Brain/metabolism , CELF Proteins , Cell Line, Tumor , Electrophoresis, Polyacrylamide Gel/methods , Exons/physiology , Humans , Middle Aged , Muscle, Skeletal/metabolism , Myotonic Dystrophy/metabolism , Neuroblastoma , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Transfection/methods , tau Proteins/genetics
15.
Biochim Biophys Acta ; 1762(4): 460-7, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16487687

ABSTRACT

Alternative splicing is altered in myotonic dystrophy of type 1 (DM1), a syndrome caused by an increase of CTG triplet repeats in the 3' untranslated region of the myotonic dystrophy protein kinase gene. Previously, we reported the preferential skipping of Tau exon 2 in DM1 brains. In this study, we analyze the alternative splicing of Tau exon 6 which can be inserted in three different forms (c, p and d) depending on the 3' splice site used. In fact, inclusion of exon 6c decreases in DM1 brains compared to control brains whereas inclusion of 6d increases. Alteration of exon 6 splicing was not observed in DM1 muscle although this exon was inserted in RNAs from normal muscle and DM1 splicing alterations were first described in this organ. In contrast, alteration of exon 2 of Tau mRNA was observed in both muscle and brain. However, co-transfections of a minigene containing exon 6 with CELF or MBNL1 cDNAs, two splicing factor families suspected to be involved in DM1, showed that they influence exon 6 splicing. Altogether, these results show the importance of determining all the exons and organs targeted by mis-splicing to determine the dysregulation mechanisms of mis-splicing in DM1.


Subject(s)
Alternative Splicing , Brain/metabolism , Exons , Myotonic Dystrophy/genetics , Nerve Tissue Proteins/genetics , CCAAT-Enhancer-Binding Protein-delta/metabolism , Cell Line , Humans , Middle Aged , Muscle, Skeletal/metabolism , Myotonic Dystrophy/metabolism , Myotonin-Protein Kinase , Nerve Tissue Proteins/metabolism , Neuroglia/metabolism , Neurons/metabolism , Protein Serine-Threonine Kinases/metabolism , RNA-Binding Proteins/metabolism , tau Proteins
16.
Biochim Biophys Acta ; 1619(2): 167-76, 2003 Jan 20.
Article in English | MEDLINE | ID: mdl-12527113

ABSTRACT

Both phosphorylation and O-GlcNAc glycosylation posttranslationally modify microtubule-associated Tau proteins. Whereas the hyperphosphorylation of these proteins that occurs in Alzheimer's disease is well characterized, little is known about the O-GlcNAc glycosylation. The present study demonstrates that a balance exists between phosphorylation and O-GlcNAc glycosylation of Tau proteins, and furthermore that a dysfunction of this balance correlates with reduced nuclear localization. The affinity of Tau proteins for WGA lectin, together with evidence from [3H]-galactose transfer and analysis of beta-eliminated products, demonstrated the presence of O-GlcNAc residues on both cytosolic and nuclear Tau proteins. In addition, our data indicated the existence of a balance between phosphorylation and O-GlcNAc glycosylation events. Indeed, as demonstrated by 2D-electrophoresis and Western blotting, O-GlcNAc residues were mainly located on the less phosphorylated Tau 441 variants, whereas the more phosphorylated forms were devoid of O-GlcNAc residues. Furthermore, the Tau protein hyperphosphorylation induced by cellular okadaic acid treatment was correlated with reduced incorporation of O-GlcNAc residues into Tau proteins and with diminished Tau transfer into the nucleus. Hence, this paper establishes a direct relationship between O-GlcNAc glycosylation, phosphorylation and cellular localization of Tau proteins.


Subject(s)
Cell Nucleus/metabolism , tau Proteins/metabolism , Blotting, Western , Cytosol/metabolism , Electrophoresis, Gel, Two-Dimensional , Enzyme Inhibitors/pharmacology , Glycosylation/drug effects , Humans , N-Acetylglucosaminyltransferases/antagonists & inhibitors , N-Acetylglucosaminyltransferases/metabolism , Okadaic Acid/pharmacology , Phosphorylation , Protein Isoforms/metabolism , Tumor Cells, Cultured , tau Proteins/biosynthesis , tau Proteins/genetics
18.
J Cell Biochem ; 85(1): 208-18, 2002.
Article in English | MEDLINE | ID: mdl-11891864

ABSTRACT

Many transcription factors are demonstrated as being glycosylated with O-N-acetylglucosamine (GlcNAc) residue in transfected insect cell lines, but rarely in the original cells. For the first time, we demonstrate the O-GlcNAc modification of the p48/p46 Pax-6 gene (a developmental control gene involved in the eye morphogenesis) products in the quail neuroretina (QNR). In conjunction with a systematic PNGase F treatment, we used wheat germ agglutinin (WGA) binding, in vitro labeling with bovine galactosyltransferase, and labeling of cultured QNR with [14C]GlcNH2. Glycosylated forms of Pax-6 proteins were found in the nucleus of the neuroretina cells. WGA-selected Pax-6 proteins produced in the reticulocyte lysate were able to bind a DNA target, as well as to the unglycosylated form. The O-GlcNAc may, however, modulate protein interactions, mainly with other factors involved in the transcription process. Characterization of products released after reductive alkaline treatment of the proteins clearly demonstrates that N-acetylglucosamine is directly linked to serine or threonine residues. Examination of Pax-6 primary sequence allowed us to determine potential O-GlcNAc attachment sites. Most of these expected glycosylation sites appear to be located on the two DNA binding domains and on the carboxyterminal transactivation domain, while experimental evidence taken from WGA-selected proteins experiment points in favor of a main localization on the paired-box domain.


Subject(s)
Acetylglucosamine/metabolism , DNA-Binding Proteins/metabolism , Homeodomain Proteins/metabolism , Retina/cytology , Amino Acid Sequence , Animals , Carbon Radioisotopes , Cells, Cultured , DNA/metabolism , Eye Proteins , Galactosyltransferases/metabolism , Glycosides/analysis , Glycosylation , Molecular Sequence Data , Nuclear Proteins/metabolism , PAX6 Transcription Factor , Paired Box Transcription Factors , Quail , Repressor Proteins , Retina/metabolism , Wheat Germ Agglutinins/metabolism
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