Identification and characterization of peptide: N- glycanase from Dictyostelium discoideum

Gosain, Anuradha; Lohia, Rakhee; Shrivastava, Anju; Saran, Shweta

doi:10.1186/1471-2091-13-9

Cited by 29 publications

(35 citation statements)

References 35 publications

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“…Cell culture, growth and development D. discoideum, Ax2 (axenic strain) cells (unicellular) were grown and developed according to Gosain et al, (2012). The medium was supplemented with antibiotics as indicated.…”

Section: Methodsmentioning

confidence: 99%

“…In situ hybridization: It was carried by the method of Gosain et al, (2012). Probes were obtained by in vitro transcription of exonic region after cloning in commercially available pBluescriptII phagemid vector.…”

Section: Rna Detection By In Situ Hybridization and Rt-pcrmentioning

confidence: 99%

“…RT-PCR: This was carried out by the method described in Gosain et al, (2012). RT-PCR reactions were performed using gene specific primer set2 for odc and set3 for ig7 (internal control) (Table1).…”

Section: Rna Detection By In Situ Hybridization and Rt-pcrmentioning

confidence: 99%

“…p[act15]:odc-Eyfp: Ddodc gene was PCR amplified using primer set4 (Table1) from genomic DNA and constitutively expressed under actin15 as a fusion protein with Eyfp at C-terminal (Gosain et al, 2012). p[odc]:ubi-LacZ: 1104bp odc intergenic region from genomic DNA was PCR amplified using primer set5 (Table1).…”

Section: Plasmid Construction and Transformationmentioning

confidence: 99%

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Cloning, expression and characterization of the ornithine decarboxylase gene from Dictyostelium discoideum

Kumar¹,

Rafia²,

Saran³

2014

Int. J. Dev. Biol.

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Ornithine decarboxylase (ODC) is a rate limiting enzyme in polyamine synthesis that decarboxylates ornithine to form the diamine putrescine. We report here the isolation, expression and characterization of a homolog of ODC from Dictyostelium discoideum. DdODC is conserved and shows sequence and structural homology with that from human. Both ODC transcript and protein are expressed at all stages of development and show high expression in prestalk/stalk cells. It is cytosolic and predominantly perinuclear in localization. Both overexpression of DdODC and putrescine treatment resulted in inhibition of cell proliferation. KEY WORDS: D. discoideum, ODC expression, putrescinePolyamines are ubiquitously present from bacteria to mammals Tabor 1985, Janne 2004) and regulate cell growth, proliferation, differentiation and cell death. The fast proliferating prokaryotes mostly contain putrescine and spermidine (Tabor and Tabor 1976) while the slow proliferating eukaryotes predominantly have spermidine and spermine (Fillingame 1975). A decrease in intracellular polyamines occurs during aging (Minois et al., 2011) but it still remains unclear whether this is a cause or consequence of aging. Addition of exogenous spermidine can extend lifespan in various animal models through epigenetic modifications, induction of autophagy and suppression of necrosis (Eisenberg et al., 2009).Ornithine decarboxylase (ODC, EC 4.1.1.17) catalyses the first and the rate limiting step of polyamine biosynthesis where Lornithine is converted to putrescine (Heller et al., 1976). It is strictly regulated during development (Heby and Persson 1990) and is involved in synthesis of spermidine and spermine. Decarboxylation of ornithine by ODC is the only pathway for de novo synthesis of polyamines in mammals and higher eukaryotes. The ability to strictly regulate cellular polyamine levels within a very narrow range is critical since extreme levels of polyamines (high or low) result in damaging effects, both on life of the cell and organism as a whole (Pegg and McCann 1982).This study was undertaken to understand the role of putrescine, which is the most abundant polyamine found in Dictyostelium discoideum (Saran 1998 Abbreviations used in this paper: DdODC, Dictyostelium discoideum ODC; ODC, ornithine decarboxylase.grow and divide mitotically but upon starvation and in response to the chemoattractant, cAMP, they enter the developmental phase to ultimately form a fruiting body consisting of dead vacuolated stalk cells and viable spores. Since ODC is responsible for the formation of putrescine we characterized this enzyme. Harris and North (1982) did not find any significant ODC activity during development but Campagne and Lowik (1985) have reported high activity at the slug stage. Our earlier report (Saran 1998) has shown high putrescine levels during slug stage. This is the first report on molecular cloning, expression and characterization of a putative ODC-like sequence from D. discoideum. We found it to be structurally conserved and predomina...

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Section: Methodsmentioning

confidence: 99%

Section: Rna Detection By In Situ Hybridization and Rt-pcrmentioning

confidence: 99%

Section: Rna Detection By In Situ Hybridization and Rt-pcrmentioning

confidence: 99%

Section: Plasmid Construction and Transformationmentioning

confidence: 99%

See 2 more Smart Citations

Cloning, expression and characterization of the ornithine decarboxylase gene from Dictyostelium discoideum

Kumar¹,

Rafia²,

Saran³

2014

Int. J. Dev. Biol.

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“…Des articles apparus récemment dans la littérature décrivent cependant que la délétion du gène PNG1 dans des organismes pluricellulaires entraîne l'apparition de défauts morphogéniques. Chez Dictyostelium discoideum, PNG1 est en effet indispensable à la formation d'agrégats cellulaires pendant le développement multicellulaire [28]. Des mutants de délétion du gène PNG1 chez Drosophila melanogaster montrent un arrêt ou un ralentissement du développement et une stérilité [29].…”

Section: Png1 Joue Un Rôle Dans La Mise En Place De La Polarité Celluunclassified

Nouvelles fonctions de la peptideN-glycanase indépendantes de sa capacité de déglycosylation

2014

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> La première fonction qui a été attribuée à la peptide N-glycanase (PNGase) est de déglycosyler les N-glycosylprotéines mal repliées avant leur dégradation par le protéasome. Il s'avère cependant que l'inhibition de cette enzyme modifie peu le taux de dégradation de ces N-glycosylprotéines. Des données récentes montrent que cette enzyme a également la capacité de moduler la morphogenèse de façon indépendante de sa fonction de déglycosylation. La caractérisation du premier déficit en PNGase devrait apporter une meilleure compréhension du rôle de cette enzyme multifonctionnelle dans la physiologie humaine. < stockage lysosomal la plus fréquente, et elle se manifeste par une accumulation de petits glycopeptides dans les lysosomes. Les symptômes cliniques sont un retard psychomoteur progressif, ainsi que des anomalies du squelette et du visage. De très nombreuses mutations du gène AGA codant pour cette hydrolase lysosomale ont été caractéri-sées [2]. La première description d'un patient porteur d'un déficit en PNGase ne date, elle, que de 2012 [3]. Les symptômes de ce patient peuvent être la conséquence d'un dysfonctionnement, soit de l'activité d'hydrolyse de la chaîne N-glycane, soit de fonctions nouvellement décrites qui sont indépendantes de l'activité de déglycosylation de l'enzyme. Dans cette revue, seront donc successivement exposées les données conséquentes concernant le rôle de la PNGase dans la dégradation des N-glycosylprotéines mal repliées, puis les données plus restreintes concernant les autres fonctions potentielles de cette enzyme. Principales caractéristiques de la PNGaseUne activité PNGase s'exerçant sur une N-glycosylprotéine produit de façon concomitante un oligosaccharide libre (fOS pour free oligosaccharide) et la conversion du résidu asparagine porteur de la chaîne N-glycane en résidu aspartate (Asp) ( Figure 1A). Bien que des activités PNGase aient été trouvées depuis de nombreuses années chez les plantes et les bactéries, la découverte d'activités PNGase cytosoliques chez les mammifères [4], puis chez la levure Saccharomyces Inserm U773, centre de recherche Bichat Beaujon CRB3,

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Overexpression of TOR (target of rapamycin) inhibits cell proliferation in Dictyostelium discoideum

Swer

Mishra

Lohia

et al. 2015

J. Basic Microbiol.

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TOR (target of rapamycin) protein kinase acts as a central controller of cell growth and development of an organism. Present study was undertaken to find the expression pattern and role of TOR during growth and development of Dictyostelium discoideum. Failures to generate either knockout and/or knockdown mutants indicate that interference with its levels led to cellular defects. Thus, the effects of TOR (DDB_G0281569) overexpression specifically, cells expressing Dd(Δ211-TOR)-Eyfp mutant was analyzed. Elevated expression of (Δ211-TOR)-Eyfp reduced both cell size and cell proliferation. DdTOR was found to be closer to fungus. mRNA level of TOR was found maximally in the freshly starved/aggregate cells that gradually declined. This was also strengthened by the expression patterns observed by in situ and the analysis of β-galactosidase reporter driven by the putative TOR promoter. The TOR protein was found to be highest at the aggregate stage. The fusion protein, (Δ211-TOR)-Eyfp was localized to the cell membrane, cytosol, and the nucleus. We suggest, DdTOR to be an essential protein and high TOR expression inhibits cell proliferation.

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Identification and characterization of peptide: N- glycanase from Dictyostelium discoideum

Cited by 29 publications

References 35 publications

Cloning, expression and characterization of the ornithine decarboxylase gene from Dictyostelium discoideum

Cloning, expression and characterization of the ornithine decarboxylase gene from Dictyostelium discoideum

Nouvelles fonctions de la peptideN-glycanase indépendantes de sa capacité de déglycosylation

Overexpression of TOR (target of rapamycin) inhibits cell proliferation in Dictyostelium discoideum

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