BS69, an adenovirus E1A-associated protein, inhibits the transcriptional activity of c-Myb.

The carboxyl terminus of c-Myb contains a negative regulatory domain that is absent in the v-Myb oncoprotein, but conserved among all the known Myb proteins of animals. This domain inhibits transcriptional activation by c-Myb in animal cells, but not in budding yeast, suggesting that additional protein(s) present in animal cells but not yeast are required for this negative regulatory function. A yeast two-hybrid screen identified BS69, an adenovirus E1A-associated protein, as interacting with the carboxy-terminal region of c-Myb. BS69 contains regions of similarity to the PHD finger, the bromodomain, and the MYND domain, all of which are found in other proteins present in high molecular weight complexes that regulate transcription and/or modify chromatin structure. Further study showed that BS69 inhibited the transcriptional activity of c-Myb, that this inhibition was specific, that it mapped to the carboxyl termini of the two proteins and that it was dose-dependent. A direct interaction between these two proteins was observed in vitro. Furthermore, the 289R E1A protein could inhibit the BS69-mediated decrease in transcriptional activation by c-Myb. By analogy with the inhibition of the Rb/E2F regulatory axis by E1A, we propose that a BS69/Myb regulatory circuit may also be a target of disruption during oncogenesis. Oncogene (2001) 20, 125 - 132.

Isolation and characterization of a hemocyte aggregation inhibitor from hemolymph of Manduca sexta larvae.

A protein that inhibits hemocyte aggregation has been isolated from hemolymph of Manduca sexta larvae and named hemocyte aggregation inhibitor protein (HAIP). HAIP has a M(r) = 50,000, pI = 8.5, and contains 7% carbohydrate. It is present at 230 +/- 20 micrograms/ml in hemolymph of day 3 fifth instar larvae. Antibodies to HAIP do not cross-react with M. sexta hemolin, which is similar in size and charge and also inhibits hemocyte aggregation. HAIP and hemolin have some similarity in amino acid composition and NH2-terminal sequence, but are different in overall secondary structure, as determined by CD spectroscopy. The concentration of HAIP in hemolymph is not affected by injection of larvae with bacteria. A protein of approximately 50,000 daltons that reacts with antibody to M. sexta HAIP is present in hemolymph of Bombyx mori, Heliothis zea, and Galleria mellonella. Although the function of HAIP in vivo is not yet clear, it may have a role in modulating adhesion of hemocytes during defensive responses.

Bacteria-induced protein P4 (hemolin) from Manduca sexta: a member of the immunoglobulin superfamily which can inhibit hemocyte aggregation.

The synthesis of a number of hemolymph proteins is induced in insects in response to bacterial infections. The major induced hemolymph protein in larvae of Manduca sexta is a glycoprotein of Mr = 48,000 known as P4. We have isolated a clone for P4 from a fat body cDNA library constructed from RNA isolated from larvae injected with bacteria. The cDNA has an open reading frame encoding a 411 residue polypeptide with a hydrophobic NH2-terminal sequence, which appears to be a signal peptide. Analysis of the deduced amino acid sequence shows that P4 is a member of the immunoglobulin (Ig) gene superfamily, and is composed largely of four C2 type Ig domains. The M. sexta P4 amino acid sequence is 60% identical with hemolin (P4) from Hyalophora cecropia. The name "hemolin" has also been adopted for the M. sexta P4 protein. Hemolin mRNA levels in fat body begin to increase within 1 h after injection of bacteria into fifth instar larvae and within 4 h after injection of adults. Hemolin associates with the surface of hemocytes and inhibits hemocyte aggregation responses, suggesting a role for the protein in modulating hemocyte adhesion during recognition and response to bacterial infections in insects.

Isolation and characterization of bacteria-induced protein P4 from hemolymph of Manduca sexta.

Insects synthesize several types of hemolymph proteins in response to bacterial infection. The objective of this study was to characterize a 48,000 dalton hemolymph protein induced in larvae of Manduca sexta after injection of bacteria. The protein, isolated by cation exchange and gel filtration chromatography from hemolymph of larvae injected with Micrococcus lysodeikticus, was found to be a glycoprotein with pI = 8.4. The molecular weight, isoelectric point, amino acid composition, and NH2 terminal sequence of the protein are similar to bacteria-induced protein P4 from Hyalophora cecropia, and the M. sexta protein is also designated P4. The hemolymph concentration of M. sexta P4 (35 +/- 7 micrograms/ml in day 3 fifth instar larvae) increases 30- to 45-fold by 48 h after injection of bacteria, but it does not increase in response to injection of distilled water. Lower levels of induction occur after injection of peptidoglycan fragments, zymosan, and lipopolysaccharide. The properties of M. sexta P4 are very similar to those of a previously characterized M. sexta hemolymph protein known as postlarval protein, and antibodies against P4 bind to post-larval protein.