Browsing by Author "Swati Ghosh"

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Age-dependent decrease in the interaction of β-tubulin with estrogen receptor alpha transactivation domain in mouse brain
(2009) Swati Ghosh; M.K. Thakur
In the previous paper, we reported that four proteins of 100 kDa, 80 kDa, 68 kDa and 50 kDa from the mouse brain interacted with estrogen receptor (ER) α-transactivation domain (TAD) and 68 kDa protein showed age and sex dependent changes. Here, we describe the identification of 50 kDa protein as β-tubulin and changes in its interaction with age and sex in mouse brain. It is a microtubule-associated protein which binds to activation function (AF)-1 region of ERα-TAD and is involved in estrogen signaling. The extent of interaction of mouse ERα-TAD with β-tubulin was higher in adult female as compared to old female and adult male. However, the expression of β-tubulin showed no significant change with age and sex. Such age and sex dependent alteration in the interaction of β-tubulin might account for the estrogen-mediated brain functions during aging. © 2009 Elsevier Ireland Ltd. All rights reserved.
GST-tagged mouse estrogen receptor a-transactivation domain fusion protein is specifically degraded during its over-expression in E. coli and purification
(2010) M.K. Thakur; Swati Ghosh
Escherichia coli BL21 (DE3) is commonly used for the overproduction of fusion proteins. Using this system, we recently reported the overproduction of histidine-tagged mouse estrogen receptor (ER) α-ligand binding domain as an intact 30 kD protein and its inhibitory effect on the growth of bacteria. However, when GST-tagged mouse ERα transactivation domain (TAD) was overproduced using this system, it showed no effect on the growth of bacteria but was specifically degraded during its expression and purification. Here we report the expression of 47 kD GSTtagged mouse ERα-TAD protein, which was degraded partially and specifically into 46 and 43 kD fragments. This fusion protein was further degraded into 37, 31, 29 and 26 kD fragments during its purification by affinity chromatography. Such specific degradation of GST-tagged mouse ERα-TAD during its overproduction in E. coli and purification indicates the induction of specific protease and suggests the modification of expression system. © Springer Science+Business Media B.V. 2009.
Interaction of estrogen receptor α transactivation domain with MTA1 decreases in old mouse brain
(2009) M.K. Thakur; Swati Ghosh
We have reported earlier that estrogen receptor (ER) α- transactivation domain (TAD) interacted with four nuclear proteins of 100 kD, 80 kD, 68 kD, and 50 kD of mouse brain and identified 68 kD as p68 RNA helicase and 50 kD as β-tubulin. In this paper, we describe the identification of 80 kD nuclear protein as metastasis associated protein 1 (MTA1) and its interaction and expression in the brain of aging mice. Far-Western blotting and immunoprecipitation data revealed lower interaction of MTA1 in old than adult mice of both sexes. Furthermore, adult male showed lower expression of protein as compared to adult female. Altogether these findings suggest that age-dependent decrease in the expression of MTA1 and its interaction with ERα-TAD may influence the estrogen-mediated signaling pathway during aging of mouse brain. © 2008 Humana Press.
Interaction of estrogen receptor-α ligand binding domain with nuclear proteins of aging mouse brain
(2009) Swati Ghosh; M.K. Thakur
After the interaction of estrogen with the ligand binding domain (LBD) of mouse estrogen receptor-α (mERα) and hormone-responsive elements of target genes, many nuclear proteins are recruited to regulate the expression of specific genes. Because it is not known which brain proteins interact with LBD or whether these proteins vary with age and sex, we used pull-down assay and far Western blotting to detect five nuclear proteins of 160, 140, 87, 60, and 46 kD in the mouse brain. These interacting proteins were identified as PELP1, RIP140, PGC1α, BAF60, and ADA3, respectively. The level of PELP1, RIP140, PGC1α, and BAF60 decreased drastically in old compared with adult male mice, whereas the ADA3 level showed no significant change. PELP1, PGC1α, and BAF60 levels were lower in old male compared with female mice. Thus we report the identification and interaction of five nuclear proteins with mERα-LBD, indicating their role in estrogen signaling and brain functions during aging. © 2009 Wiley-Liss, Inc.
Interaction of estrogen receptor-α transactivation domain with nuclear proteins of mouse brain: p68 RNA helicase shows age- and sex-specific change
(2009) Swati Ghosh; M.K. Thakur
Estrogen receptor (ER)-α interacts with nuclear proteins to mediate its multiple functions in the brain. However, it is not known which proteins interact with the ERα-transactivation domain (TAD) in mouse brain and whether they change with age and sex. Therefore, we have used affinity-purified GST-tagged mouse ERα-TAD fusion protein for interaction with nuclear proteins from the mouse brain. The pull-down assay and far-Western blotting detected four nuclear proteins of 100, 80, 68, and 50 kD. We have recently identified the 80-kD protein as MTA1 and demonstrated its decrease in old age. Here we report alteration in the interaction and expression of the 68-kD protein of adult and old mice of both sexes. This protein was identified as p68 RNA helicase through NCBI database search, immunoprecipitation, and immunoblotting. Further analysis showed that the extent of its interaction was relatively lower in old mice of both sexes and in male mice of both ages compared with their counterparts. However, the expression of p68 was significantly lower in old males compared with adult males, although other groups did not show significant changes. Such age- and sex-specific interaction of p68 suggests its implication in ERα-mediated brain functions during aging. © 2008 Wiley-Liss, Inc.
Overproduction of mouse estrogen receptor alpha-ligand binding domain decreases bacterial growth
(2008) Swati Ghosh; Mahendra Kumar Thakur
Escherichia coli (E. coli) is the most widely used prokaryotic host system for the synthesis of recombinant proteins. The overproduction of recombinant proteins is sometimes lethal to the host cells. In the present study, we expressed the ligand binding domain (LBD) of mouse estrogen receptor alpha (mouse ERα) using an expression vector (pIVEX) in E. coli BL21(DE3) and examined the effect of production of this protein on bacterial growth. The expressed protein was immunologically detected as a 30 kD histidine-tagged protein in the soluble part of the bacterial lysate. The overproduction of mouse ERα-LBD, as reflected by total protein content and expression pattern, resulted in the decrease of bacterial growth. © 2007 Springer Science+Business Media B.V.
Tissue-specific expression of receptor-interacting protein in aging mouse
(2008) Swati Ghosh; M.K. Thakur
Receptor-interacting protein (RIP) is a well-characterized coregulator for nuclear receptors. Here, we report the expression of RIP as two isoforms with molecular weights of 140 kDa and 137 kDa in liver and kidney, but only as one isoform of 140 kDa in lung, adipose tissue, prostate and testis of mice. The levels of both the isoforms decreased in liver and kidney of old mice compared with adult mice. The expression of RIP140 in kidney was relatively lower in old males than females. In contrast, adipose tissue showed remarkably higher levels of RIP140 in old than adult mice of both sexes. Thus, the expression of RIP varied with the type of tissue, sex and age of mice, suggesting differences in its function as a coregulator. © 2008 American Aging Association.