Biomedical subjects

Barsanjit Mazumder

Publications and source records attributed to Barsanjit Mazumder.

10 recordsLinked to original sources

PubMed2026

Conditional eIF2A Deletion Suggests Extra-Adipose Mechanisms Underlying Metabolic Syndrome in Total-Body eIF2A Knockout Mice.

Dynamic regulation of protein synthesis is essential for metabolic homeostasis, with translation initiation playing a key role in this process. Emerging evidence strongly indicates that in addition to canonical eukaryotic initiation factors (e.g., eIF2, eIF4E) non-canonical factors, such as eukaryotic initiation factor 2A can modulate metabolic homeostasis. eIF2A is a highly conserved eukaryotic protein originally proposed to function analogously to bacterial IF2, promoting initiator Met-tRNAi recruitment to the 40S ribosomal subunit, though its precise mechanism remains debated. To investigate its organismal role, we have previously generated the total-body eIF2A knockout mouse, which revealed eIF2A functions in lipid homeostasis, glucose tolerance, insulin sensitivity, and susceptibility to metabolic syndrome. To further determine whether adipose tissue drives these phenotypes, we presently generated adipose-specific eIF2A knockout mice. Despite dysregulation of some key adipokines, including for example, adiponectin, these mice did not develop metabolic syndrome, even under high-fat diet conditions, indicating that adipose tissue specific deficiency of eIF2A is insufficient to reproduce the metabolic defects observed in total-body knockout. However, we found that eIF2A deficiency in the liver of the total body eIF2A-KO mice can independently drive metabolic syndrome components via translational control of Lpin1 (a phosphatidate phosphatase and a transcriptional coactivator) that controls hepatic lipid storage and metabolism. eIF2A deficiency in the liver leads to disruption of fatty acid oxidation and the production of ketone bodies, not observed in adipose-specific eIF2A knockout mice. Our findings suggest that systemic metabolic effects observed in the total body eIF2A-KO mice may arise from coordinated functions across multiple organs.

adipose tissue↗

PubMed2007

L13a blocks 48S assembly: role of a general initiation factor in mRNA-specific translational control.

Transcript-specific translational control restricts macrophage inflammatory gene expression. The proinflammatory cytokine interferon-gamma induces phosphorylation of ribosomal protein L13a and translocation from the 60S ribosomal subunit to the interferon-gamma-activated inhibitor of translation (GAIT) complex. This complex binds the 3'UTR of ceruloplasmin mRNA and blocks its translation. Here, we elucidate the molecular mechanism underlying repression by L13a. Translation of the GAIT element-containing reporter mRNA is sensitive to L13a-mediated silencing when driven by internal ribosome entry sites (IRESs) that require initiation factor eIF4G, but is resistant to silencing when driven by eIF4F-independent IRESs, demonstrating a critical role for eIF4G. Interaction of L13a with eIF4G blocks 43S recruitment without suppressing eIF4F complex formation. eIF4G attack, e.g., by virus, stress, or caspases, is a well-known mechanism of global inhibition of protein synthesis. However, our studies reveal a unique mechanism in which targeting of eIF4G by mRNA-bound L13a elicits transcript-specific translational repression.

Binding Sites↗

PubMed2006

Nonstructural proteins of respiratory syncytial virus suppress premature apoptosis by an NF-kappaB-dependent, interferon-independent mechanism and facilitate virus growth.

The two nonstructural (NS) proteins NS1 and NS2 of respiratory syncytial virus (RSV) are abundantly expressed in the infected cell but are not packaged in mature progeny virions. We found that both proteins were expressed early in infection, whereas the infected cells underwent apoptosis much later. Coincident with NS protein expression, a number of cellular antiapoptotic factors were expressed or activated at early stages, which included NF-kappaB and phosphorylated forms of protein kinases AKT, phosphoinositide-dependent protein kinase, and glycogen synthase kinase. Using specific short interfering RNAs (siRNAs), we achieved significant knockdown of one or both NS proteins in the infected cell, which resulted in abrogation of the antiapoptotic functions and led to early apoptosis. NS-dependent suppression of apoptosis was observed in Vero cells that are naturally devoid of type I interferons (IFN). The siRNA-based results were confirmed by the use of NS-deleted RSV mutants. Early activation of epidermal growth factor receptor (EGFR) in the RSV-infected cell did not require NS proteins. Premature apoptosis triggered by the loss of NS or by apoptosis-promoting drugs caused a severe reduction of RSV growth. Finally, recombinantly expressed NS1 and NS2, individually and together, reduced apoptosis by tumor necrosis factor alpha, suggesting an intrinsic antiapoptotic property of both. We conclude that the early-expressed nonstructural proteins of RSV boost viral replication by delaying the apoptosis of the infected cell via a novel IFN- and EGFR-independent pathway.

Animals↗

PubMed2006

Purification and characterization of a 43 kD hepatoprotective protein from the herb Cajanus indicus L.

Cajanus indicus L, a herb, is popularly known for its hepatoprotective activity. Aqueous extract of the leaves of this plant contains hepatoprotective and hepatostimulatory molecule(s). Present study was aimed to isolate, purify and characterize the active principle(s) responsible for that activity. A hepatoprotective protein molecule has been purified to homogeneity (approximately 300 fold). Homogeneous preparation of the protein was achieved by homogenization, (NH(4))(2)SO(4) precipitation, ion-exchange chromatography, gel filtration and high performance liquid chromatography. The protein purified is composed of a single polypeptide chain having an apparent molecular mass of 43 kD as determined by SDS-PAGE and gel filtration through sephadex G-75 column. The isoelectric point of the protein determined was 4.8. Loss of biological activity after heat and protease treatment confirmed that the active molecule is a protein. Peptide fragments of the protein generated by trypsin cleavage were subjected to MALDI-TOF as well as LC-MS analyses and among the various fragments, four were very prominent and used for the determination of the amino acid sequence of the hepatoprotective protein. While one of the peptide fragment revealed strong sequence homology with plastocyanin, another fragment showed some similarity with a tomato protein present in the NCBI non-redundant database. The third peptide, on the other hand, is unique as it did not show any sequence homology with any known protein in the database. The protein showed maximum hepatoprotective activity when administered at a dose of 2 mg/kg body weight for five days after CCl(4 )administration. Histopathological studies also supported the hepatoprotective nature of the protein. Along with its curative property, the protein also possesses preventive role against a number of toxin induced hepatic damages.

Animals↗

PubMed2006

Translational control of ceruloplasmin gene expression: beyond the IRE.

Translational control is a common regulatory mechanism for the expression of iron-related proteins. For example, three enzymes involved in erythrocyte development are regulated by three different control mechanisms: globin synthesis is modulated by heme-regulated translational inhibitor, erythroid 5-aminolevulinate synthase translation is inhibited by binding of the iron regulatory protein to the iron response element in the 5'-untranslated region (UTR); and 15-lipoxygenase is regulated by specific proteins binding to the 3'-UTR. Ceruloplasmin (Cp) is a multi-functional, copper protein made primarily by the liver and by activated macrophages. Cp has important roles in iron homeostasis and in inflammation. Its role in iron metabolism was originally proposed because of its ferroxidase activity and because of its ability to stimulate iron loading into apo-transferrin and iron efflux from liver. We have shown that Cp mRNA is induced by interferon (IFN)-gamma in U937 monocytic cells, but synthesis of Cp protein is halted by translational silencing. The silencing mechanism requires binding of a cytosolic inhibitor complex, IFN-Gamma-Activated Inhibitor of Translation (GAIT), to a specific GAIT element in the Cp 3'-UTR. Here, we describe our studies that define and characterize the GAIT element and elucidate the specific trans-acting proteins that bind the GAIT element. Our experiments describe a new mechanism of translational control of an iron-related protein and may shed light on the role that macrophage-derived Cp plays at the intersection of iron homeostasis and inflammation.

3' Untranslated Regions↗

PubMed2005

Regulation of macrophage ceruloplasmin gene expression: one paradigm of 3'-UTR-mediated translational control.

Ceruloplasmin (Cp) is a copper protein with important functions in iron homeostasis and in inflammation. Cp mRNA expression is induced by interferon (IFN)-g in U937 monocytic cells, but synthesis of Cp protein is halted after about 12 h by transcript-specific translational silencing. The silencing mechanism requires binding of a 4-component cytosolic inhibitor complex, IFN-gamma-activated inhibitor of translation (GAIT), to a defined structural element (GAIT element) in the Cp 3'-UTR. Translational silencing of Cp mRNA requires the essential proteins of mRNA circularization, suggesting that the translational inhibition requires end-to-end mRNA closure. These studies describe a new mechanism of translational control, and may shed light on the role that macrophage-derived Cp plays at the intersection of iron homeostasis and inflammation.

3' Untranslated Regions↗

PubMed2004

Noncanonical function of glutamyl-prolyl-tRNA synthetase: gene-specific silencing of translation.

Aminoacyl tRNA synthetases (ARS) catalyze the ligation of amino acids to cognate tRNAs. Chordate ARSs have evolved distinctive features absent from ancestral forms, including compartmentalization in a multisynthetase complex (MSC), noncatalytic peptide appendages, and ancillary functions unrelated to aminoacylation. Here, we show that glutamyl-prolyl-tRNA synthetase (GluProRS), a bifunctional ARS of the MSC, has a regulated, noncanonical activity that blocks synthesis of a specific protein. GluProRS was identified as a component of the interferon (IFN)-gamma-activated inhibitor of translation (GAIT) complex by RNA affinity chromatography using the ceruloplasmin (Cp) GAIT element as ligand. In response to IFN-gamma, GluProRS is phosphorylated and released from the MSC, binds the Cp 3'-untranslated region in an mRNP containing three additional proteins, and silences Cp mRNA translation. Thus, GluProRS has divergent functions in protein synthesis: in the MSC, its aminoacylation activity supports global translation, but translocation of GluProRS to an inflammation-responsive mRNP causes gene-specific translational silencing.

Amino Acyl-tRNA Synthetases↗

PubMed2003

Regulated release of L13a from the 60S ribosomal subunit as a mechanism of transcript-specific translational control.

Transcript-specific translational control is generally directed by binding of trans-acting proteins to structural elements in the untranslated region (UTR) of the target mRNA. Here, we elucidate a translational silencing mechanism involving regulated release of an integral ribosomal protein and subsequent binding to its target mRNA. Human ribosomal protein L13a was identified as a candidate interferon-Gamma-Activated Inhibitor of Translation (GAIT) of ceruloplasmin (Cp) mRNA by a genetic screen for Cp 3'-UTR binding proteins. In vitro activity of L13a was shown by inhibition of target mRNA translation by recombinant protein. In response to interferon-gamma in vivo, the entire cellular pool of L13a was phosphorylated and released from the 60S ribosomal subunit. Released L13a specifically bound the 3'-UTR GAIT element of Cp mRNA and silenced translation. We propose a model in which the ribosome functions not only as a protein synthesis machine, but also as a depot for regulatory proteins that modulate translation.

3' Untranslated Regions↗

PubMed2003

Translational control by the 3'-UTR: the ends specify the means.

In most cases, translational control mechanisms result from the interaction of RNA-binding proteins with 5'- or 3'-untranslated regions (UTRs) of mRNA. In organisms ranging from viruses to humans, protein-mediated interactions between transcript termini result in the formation of an RNA loop. Such RNA 'circularization' is thought to increase translational efficiency and, in addition, permits regulation by novel mechanisms, particularly 3'-UTR-mediated translational control. Two general mechanisms of translational inhibition by 3'-UTR-binding proteins have been proposed, one in which mRNA closure is disrupted and another in which mRNA closure is required. Experimental evidence for the latter is provided by studies of interferon-gamma-mediated translational silencing of ceruloplasmin expression in monocytic cells. A multi-species analysis has shown that, in most vertebrates, 3'-UTRs are substantially longer than their 5' counterparts, indicating a significant potential for regulation. In addition, the average length of 3'-UTR sequences has increased during evolution, suggesting that their utilization might contribute to organism complexity.

3' Untranslated Regions↗

PubMed2003

Transcript-selective translational silencing by gamma interferon is directed by a novel structural element in the ceruloplasmin mRNA 3' untranslated region.

Transcript-selective translational control of eukaryotic gene expression is often directed by a structural element in the 3' untranslated region (3'-UTR) of the mRNA. In the case of ceruloplasmin (Cp), induced synthesis of the protein by gamma interferon (IFN-gamma) in U937 monocytic cells is halted by a delayed translational silencing mechanism requiring the binding of a cytosolic inhibitor to the Cp 3'-UTR. Silencing requires the essential elements of mRNA circularization, i.e., eukaryotic initiation factor 4G, poly(A)-binding protein, and poly(A) tail. We here determined the minimal silencing element in the Cp 3'-UTR by progressive deletions from both termini. A minimal, 29-nucleotide (nt) element was determined by gel shift assay to be sufficient for maximal binding of the IFN-gamma-activated inhibitor of translation (GAIT), an as-yet-unidentified protein or complex. The interaction was shown to be functional by an in vitro translation assay in which the GAIT element was used as a decoy to overcome translational silencing. Mutation analysis showed that the GAIT element contained a 5-nt terminal loop, a weak 3-bp helix, an asymmetric internal bulge, and a proximal 6-bp helical stem. Two invariant loop residues essential for binding activity were identified. Ligation of the GAIT element immediately downstream of a luciferase reporter conferred the translational silencing response to the heterologous transcript in vitro and in vivo; a construct containing a nonbinding, mutated GAIT element was ineffective. Translational silencing of Cp, and possibly other transcripts, mediated by the GAIT element may contribute to the resolution of the local inflammatory response following cytokine activation of macrophages.

3' Untranslated Regions↗