Prey Plasmids Research Articles

Muscle Develops a Specific Form of Small Heat Shock Protein Complex Composed of MKBP/HSPB2 and HSPB3 during Myogenic Differentiation

Previously, we identified a new mammalian sHSP, MKBP, as a myotonic dystrophy protein kinase-binding protein, and suggested its important role in muscle maintenance (Suzuki, A., Sugiyama, Y., Hayashi, Y., Nyu-i, N., Yoshida, M., Nonaka, I., Ishiura, S., Arahata, K., and Ohno, S. (1998) J. Cell Biol. 140, 1113-1124). In this paper, we develop the former work by performing extensive characterization of five of the six sHSPs so far identified, that is, HSP27, alphaB-crystallin, p20, MKBP/HSPB2, and HSPB3, omitting lens-specific alphaA-crystallin. Tissue distribution analysis revealed that although each sHSP shows differential constitutive expression in restricted tissues, tissues that express all five sHSPs are only muscle-related tissues. Especially, the expressions of HSPB3, identified for the first time as a 17-kDa protein in this paper, and MKBP/HSPB2 are distinctly specific to muscles. Moreover, these sHSPs form an oligomeric complex with an apparent molecular mass of 150 kDa that is completely independent of the oligomers formed by HSP27, alphaB-crystallin, and p20. The expressions of MKBP/HSPB2 and HSPB3 are induced during muscle differentiation under the control of MyoD, suggesting that the sHSP oligomer comprising MKBP/HSPB2 and HSPB3 represents an additional system closely related to muscle function. The functional divergence among sHSPs in different oligomers is also demonstrated in several ways: 1) an interaction with myotonic dystrophy protein kinase, which has been suggested to be important for the maintenance of myofibril integrity, was observed only for MKBP/HSPB2; 2) a myotube-specific association with actin bundles was observed for HSP27 and alphaB-crystallin, but not for MKBP/HSPB2; and 3) sHSPs whose mRNAs are induced by heat shock are alphaB-crystallin and HSP27. Taken together, the results suggest that muscle cells develop two kinds of stress response systems composed of diverged sHSP members, and that these systems work independently in muscle maintenance and differentiation.

The Leukemia-associated Protein Btg1 and the p53-regulated Protein Btg2 Interact with the Homeoprotein Hoxb9 and Enhance Its Transcriptional Activation

BTG1 and BTG2 belong to a family of functionally related genes involved in the control of the cell cycle. As part of an ongoing attempt to understand their biological functions, we used a yeast two-hybrid screening to look for possible functional partners of Btg1 and Btg2. Here we report the physical and functional association between these proteins and the homeodomain protein Hoxb9. We further show that Btg1 and Btg2 enhance Hoxb9-mediated transcription in transfected cells, and we report the formation of a Hoxb9.Btg2 complex on a Hoxb9-responsive target, and the fact that this interaction facilitates the binding of Hoxb9 to DNA. The transcriptional activity of the Hoxb9.Btg complex is essentially dependent on the activation domain of Hoxb9, located in the N-terminal portion of the protein. Our data indicate that Btg1 and Btg2 act as transcriptional cofactors of the Hoxb9 protein, and suggest that this interaction may mediate their antiproliferative function.

Modulating quorum sensing by antiactivation: TraM interacts with TraR to inhibit activation of Ti plasmid conjugal transfer genes.

Conjugal transfer of the Ti plasmid pTiC58 is regulated by a quorum-sensing system involving the transcriptional activator TraR and the acyl homoserine lactone autoinducer N-(3-oxo-octanoyl)-L-homoserine lactone (AAI). Activation of tra gene expression by TraR and AAI is inhibited by TraM, an 11 kDa protein also coded for by the Ti plasmid. Previous studies suggested that TraM interferes with TraR activity by directly interacting with the activator protein. Using the yeast two-hybrid system, constructs of Saccharomyces cerevisiae containing a fusion of traR to the B42 domain of the prey plasmid pJG4.5 and a fusion of traM to the lexA gene of the bait plasmid pEG202 produced beta-galactosidase and grew on medium lacking leucine, both phenotypes indicative of an interaction between the two proteins. Early termination mutants and substitution mutants mapping to the C-terminus of TraM were isolated by screening for alleles unable to interfere with TraR activity in Agrobacterium tumefaciens. These mutants all failed to interact with the TraR fusion in the two-hybrid system. An N-terminal deletion mutant of TraM lacking the first 27 residues weakly interacted with TraR in the two-hybrid system whereas deletions of 48 amino acids or more abolished the interaction. As assessed by Western blot analysis, the mutant fusion proteins were produced at levels indistinguishable from that of the wild-type TraM in the yeast tester strain. Mutants of TraR that were not inhibited by TraM in A. tumefaciens were isolated and fell into two classes. In the first, the mutation resulted in increased expression of wild-type TraR. In the second, a proline residue at position 176 was changed to serine (P176 --> S) or to leucine (P176 --> L). The P176 --> S mutant interacted with wild-type TraM, but at a detectably lower level, in the two-hybrid assay. Mutants of TraR with N-terminal deletions as large as 105 amino acids interfered with the ability of TraM to inhibit wild-type TraR in A. tumefaciens. Two-hybrid assays indicated that these mutants, as well as a C-terminal 49 residue fragment of TraR, can interact with TraM. We conclude that TraM and TraR interact in vivo and that this interaction is responsible for inhibition of TraR-mediated activation. We also conclude that the two proteins interact with each other through domains located at their respective C-termini.

Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300.

Hypoxia-inducible factor 1 alpha (HIF1alpha) and its related factor, HLF, activate expression of a group of genes such as erythropoietin in response to low oxygen. Transfection analysis using fusion genes of GAL4DBD with various fragments of the two factors delineated two transcription activation domains which are inducible in response to hypoxia and are localized in the C-terminal half. Their sequences are conserved between HLF and HIF1alpha. One is designated NAD (N-terminal activation domain), while the other is CAD (C-terminal activation domain). Immunoblot analysis revealed that NADs, which were rarely detectable at normoxia, became stabilized and accumulated at hypoxia, whereas CADs were constitutively expressed. In the mammalian two-hybrid system, CAD and NAD baits enhanced the luciferase expression from a reporter gene by co-transfection with CREB-binding protein (CBP) prey, whereas CAD, but not NAD, enhanced beta-galactosidase expression in yeast by CBP co-expression, suggesting that NAD and CAD interact with CBP/p300 by a different mechanism. Co-transfection experiments revealed that expression of Ref-1 and thioredoxin further enhanced the luciferase activity expressed by CAD, but not by NAD. Amino acid replacement in the sequences of CADs revealed a specific cysteine to be essential for their hypoxia-inducible interaction with CBP. Nuclear translocation of thioredoxin from cytoplasm was observed upon reducing O2 concentrations.

The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15.

Clathrin-mediated endocytosis is a multistep process which requires interaction between a number of conserved proteins. We have cloned two mammalian genes which code for a number of endocytic adaptor proteins. Two of these proteins, termed Ese1 and Ese2, contain two N-terminal EH domains, a central coiled-coil domain and five C-terminal SH3 domains. Ese1 is constitutively associated with Eps15 proteins to form a complex with at least 14 protein-protein interaction surfaces. Yeast two-hybrid assays have revealed that Ese1 EH and SH3 domains bind epsin family proteins and dynamin, respectively. Overexpression of Ese1 is sufficient to block clathrin-mediated endocytosis in cultured cells, presumably through disruption of higher order protein complexes, which are assembled on the endogenous Ese1-Eps15 scaffold. The Ese1-Eps15 scaffold therefore links dynamin, epsin and other endocytic pathway components.

Presenilin 1 Binds to Amyloid Precursor Protein Directly

Mutations in the presenilin genes are associated with early onset familial Alzheimer's disease and lead to accumulation of β-amyloid peptide in the brain of patients, suggesting that presenilin abnormalities induce pathological processing of amyloid precursor protein (APP) in Alzheimer's disease. For the understanding of pathogenesis in this type of familal Alzheimer disease, it is important to know whether presenilins are directly involved in the metabolism of β-amyloid or not. To test whether presenilin 1 (PS1) directly binds to APP, we performed two-hybrid interaction assays between these proteins in yeast cells by using bait plasmids for normal and mutant PS1 and prey plasmids for APP fragments corresponding to the different molecular portions. Positive interaction was observed in any combination between PS1 bait plasmids and APP prey plasmids. Therefore, our results show that PS1 binds to APP directly and suggest that the PS1 protein itself is involved in the metabolism of β-amyloid peptide.