GST-Induced dimerization of DNA-binding domains alters characteristics of their interaction with DNA

The steroid hormone 20-hydroxyecdysone (20E) plays a key role in the induction and modulation of morphogenetic events throughout Drosophila melanogaster development. Two members of the nuclear receptor superfamily, the product of the EcR (EcR) and of the ultraspiracle genes (Usp), heterodimerize to form its functional receptor. To study the receptor-DNA interaction, critical for regulating 20E-dependent gene expression, it is necessary to produce large quantities of EcR and Usp DNA-binding domains. Toward this end DNA-binding domains of EcR and Usp (EcRDBD and UspDBD, respectively) were cloned and expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST). However, the results of DNA-binding studies obtained with purified GST-DBDs were found to be questionable because the fused proteins oligomerized in solution due to the presence of GST. Therefore DBDs were released from GST-chimeric proteins by thrombin cleavage and then purified by glutathione-Sepharose 4B chromatography and by gel filtration on Superdex 75 HR. The gel mobility-shift experiments showed that UspDBD exhibited higher affinity than EcRDBD toward a 20-hydroxyecdysone response element from the Drosophila hsp 27 gene (hsp 27pal). Furthermore, formation of the heterodimeric EcRDBD-UspDBD complex was observed to be synergistic when equimolar mixture of both DBDs was incubated with hsp 27pal. Surprisingly, GST-EcRDBD bound hsp 27pal with higher affinity than GST-UspDBD. This difference was accompanied by the impaired ability of the GST-DBDs to interact synergistically with hsp 27pal. This is the first report on expression and purification of the soluble DBDs of the functional ecdysteroid receptor with satisfying yields. Furthermore, our results add to the recent findings which indicate the need for caution in interpreting the activities of GST fusion proteins.     

Mapping of interaction domains between human repair proteins ERCC1 and XPF

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1.     

Direct interaction between amino- and carboxyl-terminal domains of cyclic nucleotide-gated channels

We have examined domain interactions in the rod cyclic nucleotide-gated ion channel using both physiological and biochemical interaction assays. We have found an interaction between two regions of the channel distant in primary structure, the amino-terminal region and the carboxyl-terminal region containing the cyclic nucleotide-binding (CNB) domain. The interaction in functional channels was detected by the formation of a disulfide bond between cysteine residues at position 35 in the amino-terminal region and 481 in the carboxyl-terminal region. The disulfide bond resulted in channel potentiation, which was due, in part, to an increase in availability of C481 to modification when the channels were open. This state dependence is likely to underlie previously reported potentiation of cyclic nucleotide-gated channels by sulfhydryl-reactive compounds. Polypeptides derived from the amino-terminal and carboxyl-terminal regions were shown to interact, even under conditions which precluded disulfide bond formation. These data argue for a previously unknown, direct interaction between disparate regions of channel sequence.     

A site of interaction between pleckstrin's PH domains and G beta gamma

Pleckstrin is a 40 kDa substrate for protein kinase C found in platelets and neutrophils. Based upon its sequence, pleckstrin contains two of the recently-described PH domains that are thought to be binding motifs for phosphatidyl 4,5-bisphosphate (PIP2) and/or G protein beta gamma heterodimers (G beta gamma). In the present studies we have examined the interaction between pleckstrin and G beta gamma by incubating pleckstrin fusion proteins with lysates from human platelets. In this analysis, both the N-terminal and C-terminal PH domains from pleckstrin bound G beta gamma in vitro, as did peptides containing as little as the first 30 residues of the C-terminal pleckstrin PH domain. Introduction of a point mutation into this region, analogous to the mutation in the Btk PH domain that causes X-linked immunodeficiency disease (XID) in mice, dramatically disrupted this interaction. We propose that pleckstrin may interact with G beta gamma, and that one potential site for this interaction involves the first 30 residues of pleckstrin's C-terminal PH domain.     

N-terminal DNA-binding domains contribute to differential DNA-binding specificities of NF-kappa B p50 and p65

We previously reported that either oxidation or alkylation of NF-kappa B in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappa B binding. We now demonstrate that Cys-62 of NF-kappa B p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappa B p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappa B sites. The potential significance of this finding is discussed.     

Mouse beta-globin DNA-binding protein B1 is identical to a proto-oncogene, the transcription factor Spi-1/PU.1, and is restricted in expression to hematopoietic cells and the testis

The hematopoietic-specific DNA-binding protein B1 binds to the DNA consensus sequence AAAGRGGAARYG located twice in intervening sequence 2 of both of the mouse beta-globin genes (D. L. Galson and D.E. Housman, Mol. Cell. Biol. 8:381-392, 1988). B1 was cloned by expression of a murine erythroleukemia (MEL) cell cDNA library in transfected COS cells and screening by electrophoretic mobility shift analysis. B1 is identical to the proto-oncogene Spi-1/PU.1 (Spi-1), an ets family member. Protein-DNA contacts are shown to resemble those of the helix-turn-helix homeodomain proteins. By Northern (RNA) analysis, we found that Spi-1 mRNA is present at low levels during murine CFU-E maturation and is at least 20-fold higher in uninduced MEL, a transformed proerythroblast-like cell line which contains an activating/transforming insertion of spleen focus-forming virus at the Spi-1 locus. Dimethyl sulfoxide-induced MEL cell differentiation decreases Spi-1 mRNA to approximately 20% of the uninduced level before commitment occurs. In addition to erythroid cells, Spi-1 mRNA is present in B cells, myelomonocytes, and mast cells but not in T cells and nonhematopoietic cell types. In situ hybridization demonstrated Spi-1 mRNA expression in bone marrow, spleen, interstitial nonhepatocytes of the liver, and interstitial nontubular cells of the testis. The Spi-1 locus was mapped on human chromosome 11 to the same interval as ACP2 (lysosomal acid phosphatase), between the anonymous DNA markers D11S33 and D11S14. This region has not yet been found to be associated with a human malignancy.     

Regulation of the pAD1 sex pheromone response of Enterococcus faecalis by direct interaction between the cAD1 peptide mating signal and the negatively regulating, DNA-binding TraA protein

Rad52 plays a pivotal role in double-strand break (DSB) repair and genetic recombination in Saccharomyces cerevisiae, where mutation of this gene leads to extreme X-ray sensitivity and defective recombination. Yeast Rad51 and Rad52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in vitro, suggesting that Rad51 and Rad52 act cooperatively. To define the role of Rad52 in vertebrates, we generated RAD52(-/-) mutants of the chicken B-cell line DT40. Surprisingly, RAD52(-/-) cells were not hypersensitive to DNA damages induced by gamma-irradiation, methyl methanesulfonate, or cis-platinum(II)diammine dichloride (cisplatin). Intrachromosomal recombination, measured by immunoglobulin gene conversion, and radiation-induced Rad51 nuclear focus formation, which is a putative intermediate step during recombinational repair, occurred as frequently in RAD52(-/-) cells as in wild-type cells. Targeted integration frequencies, however, were consistently reduced in RAD52(-/-) cells, showing a clear role for Rad52 in genetic recombination. These findings reveal striking differences between S. cerevisiae and vertebrates in the functions of RAD51 and RAD52.     

Distinct Rab-binding domains mediate the interaction of Rabaptin-5 with GTP-bound Rab4 and Rab5

Rabaptin-5 functions as an effector for the small GTPase Rab5, a regulator of endocytosis and early endosome fusion. We have searched for structural determinants that confer functional specificity on Rabaptin-5. Here we report that native cytosolic Rabaptin-5 is present in a homodimeric state and dimerization depends upon the presence of its coiled-coil predicted sequences. A 73 residue C-terminal region of Rabaptin-5 is necessary and sufficient both for the interaction with Rab5 and for Rab5-dependent recruitment of the protein on early endosomes. Surprisingly, we uncovered the presence of an additional Rab-binding domain at the N-terminus of Rabaptin-5. This domain mediates the direct interaction with the GTP-bound form of Rab4, a small GTPase that has been implicated in recycling from early endosomes to the cell surface. Based on these results, we propose that Rabaptin-5 functions as a molecular linker between two sequentially acting GTPases to coordinate endocytic and recycling traffic.     

MUM1/IRF4在滤泡性淋巴瘤中的表达及其临床病理意义

目的 探讨MUM1/IRF4在滤泡性淋巴瘤(FL)中的表达情况及临床病理意义.方法 对96例FL患者标本进行MUM1、CD10、bcl-2、bcl-6、Ki-67免疫组织化学染色,并与患者的临床资料和病理学特征比较.结果 MUM1在96例FL中总的阳性率为59.2%(58/96),其中1～2级组阳性率为36.2%(19/51),3级组阳性率为86.4%(39/45)(x2=24.406,P<0.001).68.9%伴有弥漫成分的FL患者MUM1阳性(x2=8.161,P=0.004).MUM1和CD10的表达呈负相关,83.3%的CD10阴性病例表达MUM1(x1=12.649,P<0.001).MUM1阳性者核分裂和Ki-67标记指数高于MUM1阴性者(t=-3.852、t=-4.610,P<0.001).结论 MUM1可作为FL分型的标志物.MUM1阳性的FL可能为类似非生发中心B细胞分化特征的高度恶性淋巴瘤.