The MUC1-C oncoprotein binds to the BH3 domain of the pro-apoptotic BAX protein and blocks BAX function

The pro-apoptotic BAX protein contains a BH3 domain that is necessary for its dimerization and for activation of the intrinsic apoptotic pathway. The MUC1 (mucin 1) heterodimeric protein is overexpressed in diverse human carcinomas and blocks apoptosis in the response to stress. In this study, we demonstrate that the oncogenic MUC1-C subunit associates with BAX in human cancer cells. MUC1-C·BAX complexes are detectable in the cytoplasm and mitochondria and are induced by genotoxic and oxidative stress. The association between MUC1-C and BAX is supported by the demonstration that the MUC1-C cytoplasmic domain is sufficient for the interaction with BAX. The results further show that the MUC1-C cytoplasmic domain CQC motif binds directly to the BAX BH3 domain at Cys-62. Consistent with binding to the BAX BH3 domain, MUC1-C blocked BAX dimerization in response to (i) truncated BID in vitro and (ii) treatment of cancer cells with DNA-damaging agents. In concert with these results, MUC1-C attenuated localization of BAX to mitochondria and the release of cytochrome c. These findings indicate that the MUC1-C oncoprotein binds directly to the BAX BH3 domain and thereby blocks BAX function in activating the mitochondrial death pathway.     

Induction of G1 cell cycle arrest and P15INK4b expression by ECRG1 through interaction with Miz-1

ECRG1 is a novel candidate of tumor suppressor gene identified from human esophagus. To study the biological role of ECRG1 gene, we performed a GAL4-based yeast two-hybrid screen of a human fetal liver cDNA library. Using the ECRG1 cDNA as bait, we identified two putative clones as associated proteins, Miz-1 and FLNA (Filamin A). The interaction of ECRG1 and Miz-1 was confirmed by glutathione-S-transferase (GST)-pull-down assays in vitro and co-immunoprecipitation experiments in vivo. ECRG1 was co-localized with Miz-1 in nucleus, as shown by confocal microscopy. Transfection of ECRG1 gene into the esophageal cancer (EC) cells inhibited cell proliferation and induced G1 phase arrest of cell cycle. In the co-transfection of ECRG1 and Miz-1 assays, we found inhibition of cell proliferation and G1/S phase in EC cells, but the levels of cell proliferation inhibition and G1/S phase arrest were more strongly compared with the transfection of ECRG1 or Miz-1 alone. In addition, the interaction of ECRG1 and Miz-1 could induce expression of P15(INK4b) gene in esophageal cancer 9706 (EC9706) cells. However, the transfection of ECRG1 or Miz-1 alone was not revealed the expressions of P15(INK4b) gene. When antisense ECRG1 interdicted expression of endogenous ECRG1 in Balb/c-3T3 cells, Transfection of Miz-1 couldn't induce P15(INK4b) expression. The results provide evidences that ECRG1 and Miz-1 in EC cells may be acting as a co-functional protein associated with regulation of cell cycle and induction of P15(INK4b) expression. It suggests that ECRG1 may inhibit tumor cell growth by affecting cell cycle, and that expression of P15(INK4b) may be likely to enhance G1 cell cycle arrest during the interaction of ECRG1 and Miz-1. The physical interaction of ECRG1 and Miz-1 may play an important role in carcinogenesis of EC.     

Keyhole Limpet Hemocyanin: 9-Å CryoEM Structure and Molecular Model of the KLH1 Didecamer Reveal the Interfaces and Intricate Topology of the 160 Functional Units

Hemocyanins are blue copper-containing respiratory proteins in the hemolymph of many arthropods and molluscs. Molluscan hemocyanins are decamers, didecamers, or multidecamers of a 340- to 400-kDa polypeptide subunit containing seven or eight globular functional units (FUs; FU-a to FU-h), each with an oxygen-binding site. The decamers are short 35-nm hollow cylinders, with their lumen narrowed by a collar complex. Our recently published 9-Å cryo-electron microscopy/crystal structure hybrid model of a 3.4-MDa cephalopod hemocyanin decamer [Nautilus pompilius hemocyanin (NpH)] revealed the pathway of the seven-FU subunit (340 kDa), 15 types of inter-FU interface, and an asymmetric collar consisting of five “arcs” (FU-g pairs). We now present a comparable hybrid model of an 8-MDa gastropod hemocyanin didecamer assembled from two asymmetric decamers [isoform keyhole limpet hemocyanin (KLH) 1 of the established immunogen KLH]. Compared to NpH, the KLH1 subunit (400 kDa) is C-terminally elongated by FU-h, which is further extended by a unique tail domain. We have found that the wall-and-arc structure of the KLH1 decamer is very similar to that of NpH. We have traced the subunit pathway and how it continues from KLH1-g to KLH1-h to form an annulus of five “slabs” (FU-h pairs) at one cylinder edge. The 15 types of inter-FU interface detected in NpH are also present in KLH1. Moreover, we have identified one arc/slab interface, two slab/slab interfaces, five slab/wall interfaces, and four decamer/decamer interfaces. The 27 interfaces are described on the basis of two subunit conformers, yielding an asymmetric homodimer. Six protrusions from the cryo-electron microscopy structure per subunit are associated with putative attachment sites for N-linked glycans, indicating a total of 120 sugar trees in KLH1. Also, putative binding sites for divalent cations have been detected. In conclusion, the present 9-Å data on KLH1 confirm and substantially broaden our recent analysis of the smaller cephalopod hemocyanin and essentially solve the gastropod hemocyanin structure.     

Sequence variation in the Mc1r gene for a group of polymorphic snakes

Studying the genetic factors underlying phenotypic traits can provide insight into dynamics of selection and molecular basis of adaptation, but this goal can be difficult for non-model organisms without extensive genomic resources. However, sequencing candidate genes for the trait of interest can facilitate the study of evolutionary genetics in natural populations. We sequenced the melanocortin-1 receptor (Mc1r) to study the genetic basis of color polymorphism in a group of snake species with variable black banding, the genera Sonora, Chilomeniscus, and Chionactis. Mc1r is an important gene in the melanin synthesis pathway and is associated with ecologically important variation in color pattern in birds, mammals, and other squamate reptiles. We found that Mc1r nucleotide sequence was variable and that within our focal Sonora species, there are both fixed and heterozygous nucleotide substitutions that result in an amino acid change and selection analyses indicated that Mc1r sequence was likely under purifying selection. However, we did not detect any statistical association with the presence or absence of black bands. Our results agree with other studies that have found no role for sequence variation in Mc1r and highlight the importance of comparative data for studying the phenotypic associations of candidate genes.     

Solution structure of the first SH3 domain of human vinexin and its interaction with vinculin peptides

Solution structure of the first Src homology (SH) 3 domain of human vinexin (V_SH3_1) was determined using nuclear magnetic resonance (NMR) method and revealed that it was a canonical SH3 domain, which has a typical beta-beta-beta-beta-alpha-beta fold. Using chemical shift perturbation and surface plasmon resonance experiments, we studied the binding properties of the SH3 domain with two different peptides from vinculin hinge regions: P856 and P868. The observations illustrated slightly different affinities of the two peptides binding to V_SH3_1. The interaction between P868 and V_SH3_1 belonged to intermediate exchange with a modest binding affinity, while the interaction between P856 and V_SH3_1 had a low binding affinity. The structure and ligand-binding interface of V_SH3_1 provide a structural basis for the further functional study of this important molecule.     

人干细胞转录因子Nanog和BTB/POZ家族蛋白NAC1的相互作用

目的：研究人干细胞转录因子Nanog和BTB/POZ家族蛋白NAC1的相互作用,并初步确定作用区域。方法：应用免疫共沉淀、GSTpull-down实验验证人Nanog与NAC1的相互作用。结果：人Nanog与NAC1能够相互作用,且NAC1的BTB/POZ结构域对于二者相互作用是必需的。结论：人Nanog和NAC1在体内、外均能形成复合物,二者的相互作用对于人胚胎干细胞的自我更新及肿瘤的发生可能具有重要的作用。     

Two Cooperating Helices Constitute the Lipid-binding Domain of the Bacterial SRP Receptor

Protein targeting by the bacterial signal recognition particle requires the specific interaction of the signal recognition particle (SRP)-ribosome-nascent chain complex with FtsY, the bacterial SRP receptor. Although FtsY in Escherichia coli lacks a transmembrane domain, the membrane-bound FtsY displays many features of an integral membrane protein. Our data reveal that it is the cooperative action of two lipid-binding helices that allows this unusually strong membrane contact. Helix I comprises the first 14 amino acids of FtsY and the second is located at the interface between the A- and the N-domain of FtsY. We show by site-directed cross-linking and binding assays that both helices bind to negatively charged phospholipids, with a preference for phosphatidyl glycerol. Despite the strong lipid binding, helix I does not seem to be completely inserted into the lipid phase, but appears to be oriented parallel with the membrane surface. The two helices together with the connecting linker constitute an independently folded domain, which maintains its lipid binding even in the absence of the conserved NG-core of FtsY. In summary, our data reveal that the two consecutive lipid-binding helices of FtsY can provide a membrane contact that does not differ significantly in stability from that provided by a transmembrane domain. This explains why the bacterial SRP receptor does not require an integral β-subunit for membrane binding.