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The E-26 transforming specific (ETS)-related gene, TEL, also known as ETV6, encodes a strong transcription repressor that is rearranged in several recurring chromosomal rearrangements associated with leukemia and congenital fibrosarcoma. TEL is a nuclear phosphoprotein that is widely expressed in all normal tissues. TEL contains a DNA-binding domain at the C terminus and a helixloop-helix domain (also called a pointed domain) at the N terminus. The pointed domain is necessary for homotypic dimerization and for interaction with the ubiquitin-conjugating enzyme UBC9. Here we show that the interaction with UBC9 leads to modification of TEL by conjugating it to SUMO-1. The SUMO-1-modified TEL localizes to cell-cycle-specific nuclear speckles that we named TEL bodies. We also show that the leukemia-associated fusion protein TEL͞AML1 is modified by SUMO-1 and found in the TEL bodies, in a pattern quite different from what we observe and report for AML1. Therefore, SUMO-1 modification of TEL could be a critical signal necessary for normal functioning of the protein. In addition, the modification by SUMO-1 of TEL͞AML1 could lead to abnormal localization of the fusion protein, which could have consequences that include contribution to neoplastic transformation. P osttranslational modifications of proteins are important signaling mechanisms during cell cycle, cell development, and cell differentiation. The ubiquitin system is one of the major protein-modification systems required for the highly selective turnover of specific proteins in eukaryotic cells (1). Ubiquitin conjugation involves a protein complex that includes the E1, E2, and E3 enzymes, and it results in formation of an isopeptidic bond between the C terminus of ubiquitin and the -amino group of a lysine in the target protein, leading to degradation of the protein through the 26S proteosome pathway. Eukaryotic cells also express a group of ubiquitin-like proteins, such as SUMO-1 (also known as PIC-1 or UBL1) and RUB-1 (2, 3), that are conjugated to the target protein by an isopeptidic bond similar to that of the ubiquitin system. In contrast to ubiquitination, however, the covalent attachment of ubiquitin-like proteins does not lead to protein degradation. Recently, it was shown that UBC9, a homologue of the E2 enzyme, conjugates SUMO-1 to the target protein (4). Only a few SUMO-1 target proteins have been identified, including PML and SP100, which when modified by SUMO-1 are found in the PML bodies, RanGAP1, IkB␣, HIPK2, and p53 (5-8). The exact function of SUMO-1 modification is not known. In some cases (IkB␣ and p53), it was shown that conjugation of SUMO-1 could lead to protein stabilization and protection from degradation, but in other cases (PML, SP100, RanGAP1, and HIPK2), SUMO-1 conjugation could lead to different subcellular localization of the modified protein (5-8).The TEL gene is also known as ETV6. TEL was originally cloned as one of the two genes rearranged in a t(5;12) chromosomal translocation associated with chronic myelomonocytic leukemia. Thi...

The E-26 transforming specific (ETS)-related gene TEL, also known as ETV6, is involved in a large number of chromosomal rearrangements associated with leukemia and congenital fibrosarcoma. The encoded protein contains two functional domains: a helix-loop-helix (HLH) domain (also known as pointed domain) located at the N terminus and a DNA-binding domain located at the C terminus. The HLH domain is involved in protein-protein interaction with itself and other members of the ETS family of transcription factors such as FLI1. TEL is a transcription factor, and we and others have shown that it is a repressor of gene expression. To understand further the role of TEL in the cell, we have used an in vivo interaction system to identify proteins that interact with TEL. We show that a protein, UBC9, interacts specifically with TEL in vitro and in vivo. UBC9 is a member of the family of ubiquitin-conjugating enzymes. These enzymes usually are involved in proteosomemediated degradation; however, our data suggest that interaction of TEL with UBC9 does not lead to TEL degradation. Our studies show that UBC9 binds to TEL exclusively through the HLH domain of TEL. We also show that TEL expressed as fusion to the DNA-binding domain of Gal4 completely represses a Gal4-responsive promoter, but that the coexpression of UBC9 in the same system restores the activity of the promoter. Targeted point mutation of conserved amino acids in UBC9 essential for enzymatic ubiquitination of proteins does not affect interaction nor transcriptional activity. Based on our data, we conclude that UBC9 physically interacts with TEL through the HLH domain and that the interaction leads to modulation of the transcription activity of TEL.TEL, also known as ETV6, is involved in a large number of chromosomal rearrangements associated with leukemia and congenital fibrosarcoma (1-7). TEL contains two domains: a helix-loop-helix (HLH) domain at the N terminus and a DNA-binding domain [E-26 transforming specific (ETS) domain] at the C terminus. Both domains are conserved in other ETS proteins. The HLH domain is involved in protein-protein interaction, leading to the formation of either a homodimer or heterodimers with other ETS proteins, such as FLI1 (8, 9). TEL is a transcription regulator, and we showed that it is a repressor of gene transcription (6). TEL is expressed in several tissues, but its precise role is not known. The gene often is deleted in patients with leukemia (10). Homozygous deletion of the gene by targeted mutation leads to embryonic death at days E10.5-11.5 because of defective yolk sac angiogenesis (11). Recent studies showed that TEL is the first transcription factor required specifically for hematopoiesis within the bone marrow (12) and support the critical importance of this protein in hematopoiesis.The breakpoints of several chromosomal translocations involving TEL have been cloned, and it was shown that the translocations lead to gene fusion and the expression of chimeric proteins. The chromosomal breakpoints in TEL flank the ...

The enzyme carbonic anhydrase (isoform II) from bovine and human erythrocytes was immobilized using different covalent coupling methods on inert matrices. Immobilized carbonic anhydrase may enable concentration of CO2 for Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase)-catalysed fixation in bioreactors. In the present study the activity of carbonic anhydrase with respect to hydration of CO2 using soluble and immobilized enzymes was determined. The stability of the immobilization matrix, the properties of the immobilized enzymes subjected to a variation in operation variables and the activity profile with respect to storage are reported. Immobilization imparted greater thermal and storage stability and enhanced reusability.

Immobilization of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from spinach leaves is described. This enzyme enables the fixation of carbon dioxide on a five-carbon sugar D-ribulose-1,5-bisphosphate (RuBP). Two different immobilization methods were employed: dicyclohexylcarbodiimide coupling on nylon membrane matrix and dimethylpimelimidate immobilization on protein A agarose. The reusability of immobilized enzymes, coupling efficiency, and temperature-activity relationship of soluble and immobilized Rubisco are presented. The immobilization imparted greater thermal and storage stability. The thermal deactivation rates of the immobilized enzymes were considerably lower than those of the soluble enzyme.