Smarajit Bandyopadhyay

Thioltransferase (glutaredoxin) reactivates the DNA-binding activity of oxidation-inactivated nuclear factor I.

The reversible oxidative inactivation of transcription factors has been proposed to be important in cellular responses to oxidant stress and in several signal transduction pathways. The nuclear factor I (NFI) family of transcription factors is sensitive to oxidative inactivation due to the presence of a conserved, oxidation-sensitive cysteine residue within the NFI DNA-binding domain. Here we show that restoration of the DNA-binding activity of oxidized NFI-C can be catalyzed in vitro by the cellular enzyme thioltransferase (glutaredoxin) coupled to GSH and GSSG reductase. To test whether GSH-dependent pathways play a role in the maintenance of NFI activity in vivo, we used buthionine sulfoximine, an agent that inhibits GSH synthesis, andN-acetylcysteine, an agent that can replenish intracellular GSH. Pretreatment of HeLa cells with buthionine sulfoximine greatly potentiated the inactivation of NFI by the oxidizing agent diamide. Inclusion of N-acetylcysteine in the culture medium during the recovery period following diamide treatment increased the extent of restoration of NFI activity. These results suggest that maintenance of the DNA-binding activity of NFI proteins during oxidant stress in vivo requires a GSH-dependent pathway, likely involving thioltransferase-catalyzed reduction of the oxidation-sensitive cysteine residue on NFI.

Enzymatic Activity of 2′–5′-Oligoadenylate Synthetase Is Impaired by Specific Mutations that Affect Oligomerization of the Protein

Previous studies from our laboratory have shown that deletion of residues 321 to 344 of the 9-2 isozyme of 2′–5′-oligoadenylate (2–5(A)) synthetase causes a loss of its enzyme activity (Ghosh, S. K., Kusari, J., Bandyopadhyay, S. K., Samanta, H., Kumar, R., and Sen, G. C. (1991) J. Biol. Chem. 266, 15293–15299). Sequence comparison of this region among the different isozymes of 2–5(A) synthetases revealed that the residues at positions 330 to 333 are highly conserved. Alanine-scanning mutagenesis of these residues demonstrated that the residues present at 331, 332, and 333 are important for activity but the proline at position 330 was dispensable. The triple mutant containing Ala residues at 331, 332, and 333 was completely inactive. Different double mutants were slightly active, and the three single mutants were partially active. The triple mutant was further characterized for delineating the nature of its defect. The mutant protein was enzymatically inactive irrespective of whether it was synthesized in rabbit reticulocyte lysate, Escherichia coli or Trichoplusia niinsect cells. It could bind double-stranded RNA and ATP as efficiently as the wild type protein. It was, however, defective in oligomerization. Gel filtration and sedimentation velocity analyses ofin vitro synthesized proteins revealed that the wild type protein, but not the triple mutant, formed tetramers. The tetrameric fraction, but not the monomeric fraction of the wild type protein was enzymatically active. The failure of the triple mutant to participate in homomeric protein-protein interaction was confirmed byin vivo assays in insect cells. These results indicate that tetramerization of the protein is required for the enzymatic activity of the small 2–5(A) synthetases.

Enzymatic characteristics of recombinant medium isozyme of 2'-5' oligoadenylate synthetase.

P69 is an isozyme of the medium size class of human 2′-5′ oligoadenylate synthetases. In this study, recombinant P69 was expressed and used for enzymological and structural investigations. Bacterially expressed P69 was inactive whereas the same protein expressed in insect cells was highly active. Whether this difference could be due to differential post-translational modifications of the protein was investigated. Mutations of appropriate residues showed that myristoylation of the protein was not necessary for enzyme activity. In contrast, inhibition of glycosylation of P69, by tunicamycin treatment of the insect cells, produced an enzymatically inactive protein. Recombinant P69 produced in insect cells was purified by affinity chromatography. It was a dimeric glycoprotein, very stable and completely dependent on double stranded (ds) RNA for activity. The enzyme catalyzed the non-processive synthesis of 2′-5′-linked oligoadenylate products containing up to 30 residues. 2′-O-Methylated dsRNA was incapable of activating P69 and a 25-base pair dsRNA was as effective as larger dsRNA. This expression system will be useful for large scale production of P69 and its mutants for structural studies.

Endothelial Cells Express a Novel, Tumor Necrosis Factor-α-regulated Variant of HOXA9

The expression of the class 1 homeobox (HOX) family of “master control” transcription factors has been studied principally in embryogenesis and neoplasia in which HOX genes play a critical role in cell proliferation, migration, and differentiation. We wished to test whether HOX family members were also involved in a differentiation-like process occurring in normal, diploid adult cells, that is, cytokine-induced activation of endothelial cells (EC). Screening of a human EC cDNA library yielded several members of the A and B groups of HOX transcription factors. One clone represented a novel, alternatively spliced variant of the human HOXA9gene containing a new exon and the expression of which was driven by a novel promoter. This variant termed HOXA9EC appeared restricted to cells of endothelial lineage, i.e. expressed by human EC from multiple sources, but not by fibroblasts, smooth muscle cells, or several transformed cell lines. HOXA9ECmRNA was rapidly down-regulated in EC in response to tumor necrosis factor-α due to an apparent reduction in transcriptional rate. Reporter construct studies showed that the 400 base pairs of genomic DNA directly 5′ to the transcription initiation site ofHOXA9EC contained the information required for both up-regulation in response to cotransfection with a HOXA9ECexpression vector and tumor necrosis factor-α-dependent down-regulation of this gene. These results provide evidence of a novel HOX family member that may participate in either the suppression or the genesis of EC activation.

HOXA9 Participates in the Transcriptional Activation of E-Selectin in Endothelial Cells

ABSTRACT The homeobox gene HOXA9 has recently been shown to be an important regulator of endothelial cell (EC) differentiation and activation in addition to its role in embryonic development and hematopoiesis. In this report, we have determined that the EC-leukocyte adhesion molecule E-selectin is a key target for HOXA9. The depletion of HOXA9 protein in ECs resulted in a significant and specific decrease in tumor necrosis factor alpha (TNF-α)-induced E-selectin gene expression. In addition, HOXA9 specifically activated the E-selectin gene promoter in ECs. Progressive deletional analyses together with site-specific mutagenesis of the E-selectin promoter indicated that the Abd-B-like HOX DNA-binding motif, CAATTTTATTAA, located in the proximal region spanning bp −210 to −221 upstream of the transcription start site was crucial for the promoter induction by HOXA9. Both HOXA9 in EC nuclear extract and recombinant HOXA9 protein bound to this sequence in vitro. Moreover, we showed that HOXA9 binds temporally, in a TNF-α-dependent manner, to the region containing this Abd-B-like element in vivo. We have thus identified a novel and functionally critical cis-regulatory element for TNF-α-mediated transient expression of the E-selectin gene. Further, we provide evidence that HOXA9 acts as an obligate proinflammatory factor by mediating cytokine induction of E-selectin.

Effects of Mutating Specific Residues Present Near the Amino Terminus of 2′–5′-Oligoadenylate Synthetase

In this study, we investigated the role of specific amino acid residues present near the amino terminus of the 9–2 isozyme of 2′–5′-oligoadenylate synthetase. In vitroexpression of deletion mutants showed that residues 1–9 are required for enzyme activity. Within this region, residues 3, 7, and 8 were found to be conserved among all known isozymes of 2′–5′-oligoadenylate synthetase. Mutation of these residues singly or in combination resulted in partial or total loss of enzyme activity. Substitution of the proline residue at position 7 by different residues caused a partial or complete loss of activity. The properties of the inactive P7Q mutant were further explored by expressing the protein in bacteria. The bacterially expressed protein was also enzymatically inactive. The mutant protein could bind the substrate ATP and the activator double-stranded RNA normally. Oligomerization properties of the protein were examined by an affinity-based interaction assay and by glycerol gradient centrifugation; there was no detectable difference between the wild type and the P7Q mutant. These results demonstrated the importance of the proline residue at position 7 in conferring enzyme activity to the protein without affecting its other properties.

Thrombin induces endothelial arginase through AP-1 activation

Arterial thrombosis is a common disease leading to severe ischemia beyond the obstructing thrombus. Additionally, endothelial dysfunction at the site of thrombosis can be rescued by l-arginine supplementation or arginase blockade in several animal models. Exposure of rat aortic endothelial cells (RAECs) to thrombin upregulates arginase I mRNA and protein levels. In this study, we further investigated the molecular mechanism of thrombin-induced arginase changes in endothelial cells. Thrombin strikingly increased arginase I promoter and enzyme activity in primary cultured RAECs. Using different deletion and point mutations of the promoter, we demonstrated that the activating protein-1 (AP-1) consensus site located at -3,157 bp in the arginase I promoter was a thrombin-responsive element. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay further confirmed that upon thrombin stimulation, c-Jun and activating transcription factor-2 (ATF-2) bound to the AP-1 site, which initiated the transactivation. Moreover, loss-of-function studies using small interfering RNA confirmed that recruitment of these two transcription factors to the AP-1 site was required for thrombin-induced arginase upregulation. In the course of defining the signaling pathway leading to the activation of AP-1 by thrombin, we found thrombin-induced phosphorylation of stress-activated protein kinase/c-Jun-NH(2)-terminal kinase (SAPK/JNK or JNK1/2/3) and p38 mitogen-activated protein kinase, which were followed by the phosphorylation of both c-Jun and ATF-2. These findings reveal the basis for thrombin induction of endothelial arginase I and indicate that arginase inhibition may be an attractive therapeutic alternative in the setting of arterial thrombosis and its associated endothelial dysfunction.