The search of partners of a new mammalian selenium-containing protein V (SelV) and expression it's mRNA during ontogenesis and spermatogenesis

Selenium is a biologically active trace elements, which is part of several proteins, and thus linked with the activity of many organs, tissues and systems of organism. There are 25 mammalian selenoproteins at present, one of which is SelV (Selenoprotein V). Since this protein has thioredoxin-like folding and a conserved motif (CXXU, where C is cysteine, U-selenocysteine) in its catalytic center, it belongs to the family of redox proteins, whose members are involved in redox reactions. In this paper, we show that the redox protein SelV can interact with O-linked N-acetylglucosamine transferase (OGT) and proteins belonging to the family of ASB: Asb-17, and Asb-9. The specificity of interactions SelV with OGT and Asb-9, but not with Asb-17 is confirmed by coimmunopretsipitation. In addition, expression of SelV mRNA in the later stages spermatogenesis, as well as in puberty and reproductive periods of rats is shown.     

Functional analysis of Asb-1 using genetic modification in mice

The Asbs are a family of ankyrin repeat proteins that, along with four other protein families, contain a C-terminal SOCS box motif, which was first identified in the suppressor of cytokine signaling (SOCS) proteins. While it is clear that the SOCS proteins are involved in the negative regulation of cytokine signaling, the biological roles of the other SOCS box-containing families are unknown. We have investigated Asb-1 function by generating mice that lack this protein, as well as mice that overexpress full-length or truncated Asb-1 in a wide range of tissues. Although Asb-1 is expressed in multiple organs, including the hematopoietic compartment in wild-type mice, Asb-1(-/-) mice develop normally and exhibit no anomalies of mature blood cells or their progenitors. While most organs in these mice appear normal, the testes of Asb-1(-/-) mice display a diminution of spermatogenesis with less complete filling of seminiferous tubules. In contrast, the widespread overexpression of Asb-1 in the mouse has no apparent deleterious effects.     

Ankyrin repeat and suppressors of cytokine signaling box protein asb-9 targets creatine kinase B for degradation

The suppressors of cytokine signaling (SOCS) proteins inhibit cytokine action by direct interaction with Janus kinases or activated cytokine receptors. In addition to the N-terminal and Src homology 2 domains that mediate these interactions, SOCS proteins contain a C-terminal SOCS box. DNA data base searches have identified a number of other protein families that possess a SOCS box, of which the ankyrin repeat and SOCS box-containing (Asb) proteins constitute the largest. Although it is known that the SOCS proteins are involved in the negative regulation of cytokine signaling, the biological and biochemical functions of the Asbs are largely undefined. Using a proteomics approach, we demonstrate that creatine kinase B (CKB) interacts with Asb-9 in a specific, SOCS box-independent manner. This interaction increases the polyubiquitylation of CKB and decreases total CKB levels within the cell. The targeting of CKB for degradation by Asb-9 was primarily SOCS box-dependent and suggests that Asb-9 acts as a specific ubiquitin ligase regulating levels of this evolutionarily conserved enzyme.     

SelT, SelW, SelH, and Rdx12: genomics and molecular insights into the functions of selenoproteins of a novel thioredoxin-like family

Selenium is an essential trace element in many life forms due to its occurrence as a selenocysteine (Sec) residue in selenoproteins. The majority of mammalian selenoproteins, however, have no known function. Herein, we performed extensive sequence similarity searches to define and characterize a new protein family, designated Rdx, that includes mammalian selenoproteins SelW, SelV, SelT and SelH, bacterial SelW-like proteins and cysteine-containing proteins of unknown function in all three domains of life. An additional member of this family is a mammalian cysteine-containing protein, designated Rdx12, and its fish selenoprotein orthologue. Rdx proteins are proposed to possess a thioredoxin-like fold and a conserved CxxC or CxxU (U is Sec) motif, suggesting a redox function. We cloned and characterized three mammalian members of this family, which showed distinct expression patterns in mouse tissues and different localization patterns in cells transfected with the corresponding GFP fusion proteins. By analogy to thioredoxin, Rdx proteins can use catalytic cysteine (or Sec) to form transient mixed disulfides with substrate proteins. We employed this property to identify cellular targets of Rdx proteins using affinity columns containing mutant versions of these proteins. Rdx12 was found to interact with glutathione peroxidase 1, whereas 14-3-3 protein was identified as one of the targets of mammalian SelW, suggesting a mechanism for redox regulation of the 14-3-3 family of proteins.     

ASB9 interacts with ubiquitous mitochondrial creatine kinase and inhibits mitochondrial function

Background  

The ankyrin repeat and suppressor of cytokine signalling (SOCS) box proteins (Asbs) are a large protein family implicated in diverse biological processes including regulation of proliferation and differentiation. The SOCS box of Asb proteins is important in a ubiquitination-mediated proteolysis pathway. Here, we aimed to evaluate expression and function of human Asb-9 (ASB9).     

Roles of the tetratricopeptide repeat domain in O-GlcNAc transferase targeting and protein substrate specificity

The abundant and dynamic post-translational modification of nuclear and cytosolic proteins by beta-O-linked N-acetylglucosamine (O-GlcNAc) is catalyzed by O-GlcNAc-transferase (OGT). Recently, we reported the identification of a novel family of OGT-interacting proteins (OIPs) that interact strongly with the tetratricopeptide repeat (TPR) domain of OGT (Iyer, S. P., Akimoto, Y., and Hart, G. W. (2003) J. Biol. Chem. 278, 5399-5409). Members of this family are modified by O-GlcNAc and are excellent substrates of OGT. Here, we further investigated the role of the TPR domain in the O-GlcNAcylation of OIP106, one of the members of this OIP family. Using N-terminal deletions, we first identified the region of OIP106 that binds OGT, termed the OGT-interacting domain (OID). Deletion analysis indicated that TPRs 2-6 of OGT interact with the OID of OIP106. The apparent Km of OGT for the OID of OIP106 is 3.35 microm. Unlike small peptide substrates, glycosylation of the OID was dependent upon its interaction with the first 6 TPRs of OGT. Furthermore, the isolated TPR domain of OGT competitively inhibited glycosylation of the OID protein, but did not inhibit glycosylation of a 12-amino acid casein kinase II peptide substrate, providing kinetic evidence for the role of the TPR domain as a protein substrate docking site. Additionally, both the OID of OIP106 and nucleoporin p62 competed with each other for glycosylation by OGT. These studies support the model that the catalytic subunit of OGT achieves both high specificity and a remarkable diversity of substrates by complexing with a variety of targeting proteins via its TPR protein-protein interaction domains.     

Alloxan is an inhibitor of the enzyme O-linked N-acetylglucosamine transferase

We have previously shown that diabetogenic antibiotic streptozotocin (STZ), an analog of N-acetylglucosamine (GlcNAc), inhibits the enzyme O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase) which is responsible for the removal of O-GlcNAc from proteins. Alloxan, another beta-cell toxin is a uracil analog. Since the O-GlcNAc transferase (OGT) uses UDP-GlcNAc as a substrate, we investigated whether alloxan might interfere with the process of protein O-glycosylation by blocking OGT, a very abundant enzyme in beta-cells. In isolated pancreatic islets, alloxan almost completely blocked both glucosamine-induced and STZ-induced protein O-GlcNAcylation, suggesting that alloxan indeed was inhibiting (OGT). In order to show definitively that alloxan was inhibiting OGT activity, recombinant OGT was incubated with 0-10 mM alloxan, and OGT activity was measured directly by quantitating UDP-[(3)H]-GlcNAc incorporation into the recombinant protein substrate, nucleoporin p62. Under these conditions, OGT activity was completely inhibited by 1 mM alloxan with half-maximal inhibition achieved at a concentration of 0.1 mM alloxan. Together, these data demonstrate that alloxan is an inhibitor of OGT, and as such, is the first OGT inhibitor described.     

Expression of murine Asb-9 during mouse spermatogenesis

We previously showed that Asb-4 and Asb-17 is uniquely expressed in developing male germ cells. A recent report showed that Asb-9 is specifically expressed in the kidney and testes; however, detailed expression patterns in developing germ cells have not been shown. Northern blot analysis in various tissues demonstrated that mAsb-9 was strongly expressed in the testes. Expression analysis by RT-PCR and Northern blot in developing mouse testes indicates that mAsb-9 is expressed from the fourth week after birth to adulthood, with the highest expression in round spermatids. Expression sites were further localized by in situ hybridization in the testes. Pachytene spermatocytes and spermatids expressed mAsb-9 but spermatogonia and generated spermatozoa did not. This study reveals that mAsb-9 could be a specific marker of active spermatogenesis and would be useful for studies of male germ cell development.     

Ataxin-10 interacts with O-GlcNAc transferase OGT in pancreatic beta cells

Several nuclear and cytoplasmic proteins in metazoans are modified by O-linked N-acetylglucosamine (O-GlcNAc). This modification is dynamic and reversible similar to phosphorylation and is catalyzed by the O-linked GlcNAc transferase (OGT). Hyperglycemia has been shown to increase O-GlcNAc levels in pancreatic beta cells, which appears to interfere with beta-cell function. To obtain a better understanding of the role of O-linked GlcNAc modification in beta cells, we have isolated OGT interacting proteins from a cDNA library made from the mouse insulinoma MIN6 cell line. We describe here the identification of Ataxin-10, encoded by the SCA10 (spinocerebellar ataxia type 10) gene as an OGT interacting protein. Mutations in the SCA10 gene cause progressive cerebellar ataxias and seizures. We demonstrate that SCA10 interacts with OGT in vivo and is modified by O-linked glycosylation in MIN6 cells, suggesting a novel role for the Ataxin-10 protein in pancreatic beta cells.     

Ankyrin repeat and SOCS box containing protein 4 (Asb-4) interacts with GPS1 (CSN1) and inhibits c-Jun NH2-terminal kinase activity

Asb-4 is a gene that is specifically expressed in the hypothalamic energy homeostasis-associated areas and is down-regulated in the arcuate nucleus of fasted Sprague Dawley and obese Zucker rats. It has two functional domains, the ankyrin repeat and the SOCS box. The function of Asb-4 is unclear. We used yeast two hybridization to search for protein(s) that interact with Asb-4. With Asb-4 minus its SOCS box (Asb-4/Deltasb) as a bait, we screened mouse testis and arcuate nucleus cDNA libraries and identified G-protein pathway suppressor 1 (GPS1, also known as CSN1) as an Asb-4 interacting protein. GPS1 co-immunoprecipitated with Asb-4 both in vitro and in human HEK293 cells. When Asb-4 and GPS1 were co-transfected into HEK293 cells, expression of Asb-4 reduced the protein level of GPS1. Deletion of the SOCS box (Asb4/Deltasb) did not abolish the inhibitory effect of Asb-4 on GPS1, indicating that the SOCS box was not needed for its inhibitory effect. In NIH 3T3 L1 cells, expression of GPS1 enhanced c-Jun NH2-terminal kinase (JNK) activity. Co-expression of Asb-4 with GPS1 inhibited JNK activity. Treatment of the cells with insulin (20 nM) stimulated JNK activity. Expression of GPS1 potentiated the stimulatory effect of insulin, whereas co-expression of Asb-4 along with GPS1 inhibited JNK activity. In HEK293 cells expression of GPS1 elevated phosphorylation of insulin receptor substrate 1 (IRS-1) at serine307, co-expression of Asb-4 with GPS1 reduced the IRS-1ser307 phosphorylation. The present study demonstrates that Asb-4 interacts with GPS1 and inhibits JNK activity.     

SPINDLY 与赤霉素的信号转导

SPINDLY(SPY)作为一负调节子参与GA的信号转导,34肽重复结构(TPR)与C-端区域对其正常功能都十分重要。SPY基因在植物中呈组成型表达,其蛋白主要出现在细胞核部位。SPY蛋白与动物中的氧连N-乙酰葡萄糖胺转移酶(OGT)具有广泛的同源性,两者可能有着类似的作用机制。本文主要介绍GA突变体、SPY基因、SPY蛋白及其在大麦中的同源物HvSPY的结构与功能相关方面的一些研究进展。     

Functional analysis of SPINDLY in gibberellin signaling in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) protein negatively regulates the gibberellin (GA) signaling pathway. SPY is an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) with a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPR). OGTs add a GlcNAc monosaccharide to serine/threonine residues of nuclear and cytosolic proteins. Determination of the molecular defects in 14 new spy alleles reveals that these mutations cluster in three TPRs and the C-terminal catalytic region. Phenotypic characterization of 12 spy alleles indicates that TPRs 6, 8, and 9 and the catalytic domain are crucial for GA-regulated stem elongation, floral induction, and fertility. TPRs 8 and 9 and the catalytic region are also important for modulating trichome morphology and inflorescence phyllotaxy. Consistent with a role for SPY in embryo development, several alleles affect seedling cotyledon number. These results suggest that three of the TPRs and the OGT activity in SPY are required for its function in GA signal transduction. We also examined the effect of spy mutations on another negative regulator of GA signaling, REPRESSOR OF ga1-3 (RGA). The DELLA motif in RGA is essential for GA-induced proteolysis of RGA, and deletion of this motif (as in rga-delta17) causes a GA-insensitive dwarf phenotype. Here, we demonstrate that spy partially suppresses the rga-delta17 phenotype but does not reduce rga-delta17 or RGA protein levels or alter RGA nuclear localization. We propose that SPY may function as a negative regulator of GA response by increasing the activity of RGA, and presumably other DELLA proteins, by GlcNAc modification.     

Glucose-induced expression of MIP-1 genes requires O-GlcNAc transferase in monocytes

O-glycosylation has emerged as an important modification of nuclear proteins, and it appears to be involved in gene regulation. Recently, we have shown that one of the histone methyl transferases (MLL5) is activated through O-glycosylation by O-GlcNAc transferase (OGT). Addition of this monosaccharide is essential for forming a functional complex. However, in spite of the abundance of OGT in the nucleus, the impact of nuclear O-glycosylation by OGT remains largely unclear. To address this issue, the present study was undertaken to test the impact of nuclear O-glycosylation in a monocytic cell line, THP-1. Using a cytokine array, MIP-1α and -1β genes were found to be regulated by nuclear O-glycosylation. Biochemical purification of the OGT interactants from THP-1 revealed that OGT is an associating partner for distinct co-regulatory complexes. OGT recruitment and protein O-glycosylation were observed at the MIP-1α gene promoter; however, the known OGT partner (HCF-1) was absent when the MIP-1α gene promoter was not activated. From these findings, we suggest that OGT could be a co-regulatory subunit shared by functionally distinct complexes supporting epigenetic regulation.     

O-GlcNAc Transferase Regulates Mitotic Chromatin Dynamics

Mitosis must faithfully divide the genome such that each progeny inherits the same genetic material. DNA condensation is crucial in ensuring that chromosomes are correctly attached to the mitotic spindle for segregation, preventing DNA breaks or constrictions from the contractile ring. Histones form an octameric complex of basic proteins important in regulating DNA organization and accessibility. Histone post-translational modifications are altered during mitosis, although the roles of these post-translational modifications remain poorly characterized. Here, we report that N-acetylglucosamine (O-GlcNAc) transferase (OGT), the enzyme catalyzing the addition of O-GlcNAc moieties to nuclear and cytoplasmic proteins at serine and threonine residues, regulates some aspects of mitotic chromatin dynamics. OGT protein amounts decrease during M phase. Modest overexpression of OGT alters mitotic histone post-translational modifications at Lys-9, Ser-10, Arg-17, and Lys-27 of histone H3. Overexpression of OGT also prevents mitotic phosphorylation of coactivator-associated arginine methyltransferase 1 (CARM1) and prevents its correct cellular localization during mitosis. Moreover, OGT overexpression results in an increase in abnormal chromosomal bridge formation. Together, these results show that regulating the amount of OGT during mitosis is important in ensuring correct chromosomal segregation during mitosis.     

Ataxin-10 interacts with O-linked beta-N-acetylglucosamine transferase in the brain

Modification by O-GlcNAc involves a growing number of eucaryotic nuclear and cytosolic proteins. Glycosylation of intracellular proteins is a dynamic process that in several cases competes with and acts as a reciprocal modification system to phosphorylation. O-Linked beta-N-acetylglucosamine transferase (OGT) levels are highest in the brain, and neurodegenerative disorders such as Alzheimer disease have been shown to involve abnormally phosphorylated key proteins, probably as a result of hypoglycosylation. Here, we show that the neurodegenerative disease protein ataxin-10 (Atx-10) is associated with cytoplasmic OGT p110 in the brain. In PC12 cells and pancreas, this association is competed by the shorter OGT p78 splice form, which is down-regulated in brain. Overexpression of Atx-10 in PC12 cells resulted in the reconstitution of the Atx-10-OGT p110 complex and enhanced intracellular glycosylation activity. Moreover, in an in vitro enzyme assay using PC12 cell extracts, Atx-10 increased OGT activity 2-fold. These data indicate that Atx-10 might be essential for the maintenance of a critical intracellular glycosylation level and homeostasis in the brain.     

AMP-activated protein kinase and p38 MAPK activate O-GlcNAcylation of neuronal proteins during glucose deprivation

We have demonstrated previously that a wide array of stress signals induces O-GlcNAc transferase (OGT) expression and increases O-GlcNAcylation of many intracellular proteins, a response that is critical for cell survival. Here, we describe a mechanism by which glucose deprivation induces OGT expression and activity in Neuro-2a neuroblastoma cells. Glucose deprivation increases OGT mRNA and protein expression in an AMP-activated protein kinase-dependent manner, whereas OGT enzymatic activity is regulated in a p38 MAPK-dependent manner. OGT is not phosphorylated by p38, but rather it interacts directly with p38 through its C terminus; this interaction increases with p38 activation during glucose deprivation. Surprisingly, the catalytic activity of OGT, as measured toward peptide substrates, is not altered by glucose deprivation. Instead, p38 regulates OGT activity within the cell by recruiting it to specific targets, including neurofilament H. Neurofilament H is O-GlcNAcylated during glucose deprivation in a p38-dependent manner. Interestingly, neurofilament H solubility is increased by glucose deprivation in an O-GlcNAc-dependent manner, suggesting that O-GlcNAcylation of neurofilament H regulates its disassembly from filaments. Not only do these data help to reveal how OGT is regulated by stress, but these findings also describe a possible mechanism by which defective brain glucose metabolism, as found in aging and ischemia, may directly affect axonal structure.