Identification of an heterologous histone complex using reversible crosslinking

Using formaldehyde as a reversible crosslinker, it has been shown that an heterologous dimer is the principal components in a mixture of calf thymus H2A and sea urchin H2B₁. Comparison of the crosslinking ability of formaldehyde with dimethyl suberimidate demonstrates that suberimidate gives a better representation of the free-solution equilibrium of the protein mixture.     

Identification of an antiapoptotic protein complex at death receptors

Stimulation of death receptors activates the extrinsic apoptotic signaling pathway that leads to cell death. Although many steps of this apoptotic signaling cascade are known, few mechanisms that counterbalance the death signal have been described. We identified an antiapoptotic protein complex associated with death receptors that contains glycogen synthase kinase-3 (GSK3), DDX3 and cellular inhibitor of apoptosis protein-1 (cIAP-1). GSK3, DDX3 and cIAP-1 are associated in cells with each other and with death receptors. Blocking the actions of GSK3 or DDX3 potentiated caspase-3 activation induced by stimulation of four different death receptors in several types of cells. GSK3 restrained apoptotic signaling by inhibiting formation of the death-inducing signaling complex and caspase-8 activation. Stimulated death receptors surmount the antiapoptotic complex by causing GSK3 inactivation and cleavage of DDX3 and cIAP-1 to enable progression of the apoptotic signaling cascade, but the antiapoptotic complex remains functional in cancer cells resistant to death receptor stimulation, a resistance that is overcome by GSK3 inhibitors. Thus, an antiapoptotic complex of GSK3, DDX3 and cIAP-1 caps death receptors, providing a checkpoint to counterbalance apoptotic signaling.     

Identification of an IQGAP1/AKAP79 complex in beta-cells

IQGAP1, is a recently discovered scaffold protein proposed to regulate membrane cytoskeleton events through protein-protein interactions with F-actin, E-cadherin, beta-catenin, and CLIP170. The binding of IQGAP1 to its partners is regulated by calcium/calmodulin (Ca(++)/CaM) and the small molecular weight guanine nucleotide triphosphatases (GTPases), Cdc42, and Rac1. Here we identify a novel IQGAP1 scaffolding function by isolating the cyclic AMP dependent kinase (PKA) with IQGAP1. IQGAP1 was co-purified with PKA using 5'-cyclic AMP (cAMP) affinity chromatography and PKA activity was co-immunoprecipitated with IQGAP1 using an anti-IQGAP1 antibody. The association of IQGAP1 with PKA was shown to occur through a direct interaction between A kinase anchoring protein 79 (AKAP79) and the carboxyl-terminal domain of IQGAP1. This suggests that cAMP/PKA may be coupled with Ca(++)/CaM and GTPases through an IQGAP1/AKAP79 complex.     

Identification of GroEL as a constituent of an mRNA-protection complex in Escherichia coli

An RNA-binding activity has been identified in Escherichia coli that provides physical protection of RNA against ribonucleases in an ATP- and Mg²⁺-dependent manner. This binding activity is stimulated under growth conditions known to cause a decrease in the rate of mRNA decay. RNA protection is mediated by a protein complex that contains a modified form of the chaperonin GroEL as an indispensable constituent. These results suggest a new role for GroEL as an RNA chaperone.     

Identification of a myosin VII-talin complex

Myosin VII (M7) plays a role in adhesion in both Dictyostelium and mammalian cells where it is a component of a complex of proteins that serve to link membrane receptors to the underlying actin cytoskeleton. The nature of this complex is not fully known, prompting a search for M7-binding proteins. Co-immunoprecipitation experiments reveal that Dictyostelium M7 (DdM7) interacts with talinA, an actin-binding protein with a known role in cell-substrate adhesion. No additional proteins are observed in the immunoprecipitate, indicating that the interaction is direct. The N-terminal region of the DdM7 tail that lies between the region of predicted coil and the first MyTH4 domain is found to harbor the talinA binding site. Localization experiments reveal that talinA does not serve as a membrane receptor for DdM7 and vice versa. These findings reveal that talinA is a major DdM7 binding partner and suggest that their interaction induces a conformational change in each that, in combination with membrane receptor binding, promotes the assembly of a high avidity receptor complex essential for adhesion of the cell to substrata.     

洋葱伯克氏菌基因型的鉴定及其在苜蓿模型上的毒力分析

[目的]证实来自我国农业和医院环境中部分洋葱伯克氏菌的基因型并通过苜蓿植物模型探测不同基因型对人体的可能毒力.[方法]采用洋葱伯克氏菌基因型的PCR特异性扩增技术对来源于根围、土壤和医院中的57株洋葱伯克氏菌进行了基因型的鉴定,并利用苜蓿植物模型对这些基因型菌株进行了毒力探测.[结果]获得4种不同的基因型,包括基因型Ⅰ、ⅢA、ⅢB、Ⅴ和Ⅸ.来源于医院的基因型Ⅰ和ⅢA菌株以及根围的基因型ⅢB菌株均对苜蓿幼苗有较强的毒力,其对苜蓿幼苗的平均发病率分别达到69%、68%和55%,与农田环境中基因型Ⅴ和Ⅸ对苜蓿幼苗的发病率相比,表现出显著的差异性.[结论]农田环境中洋葱伯克菌的基因型在苜蓿模型上的毒力差异大,根围的基因型ⅢB菌株对苜蓿幼苗具有强毒力,其毒力程度接近于医院致病基因型ⅢA菌.     

Identification of a tetrameric hedgehog signaling complex

Hedgehog (Hh) signal transduction requires a large cytoplasmic multi-protein complex that binds microtubules in an Hh-dependent manner. Here, we show that three members of this complex, Costal2 (Cos2), Fused (Fu), and Cubitus interruptus (Ci), bind each other directly to form a trimeric complex. We demonstrate that this trimeric signaling complex exists in Drosophila lacking Suppressor of Fused (Su(fu)), an extragenic suppressor of fu, indicating that Su(fu) is not required for the formation, or apparently function, of the Hh signaling complex. However, we subsequently show that Su(fu), although not a requisite component of this complex, does form a tetrameric complex with Fu, Cos2, and Ci. This additional Su(fu)-containing Hh signaling complex does not appear to be enriched on microtubules. Additionally, we demonstrate that in response to Hh Ci accumulates in the nucleus without its various cytoplasmic binding partners, including Su(fu). We discuss a model in which Su(fu) and Cos2 each bind to Fu and Ci to exert some redundant effect on Ci such as cytoplasmic retention. This model is consistent with genetic data demonstrating that Su(fu) is not required for Hh signal transduction proper and with the elaborate genetic interactions observed among Su(fu), fu, cos2, and ci.     

Identification of cross-linked peptides from complex samples

We have developed pLink, software for data analysis of cross-linked proteins coupled with mass-spectrometry analysis. pLink reliably estimates false discovery rate in cross-link identification and is compatible with multiple homo- or hetero-bifunctional cross-linkers. We validated the program with proteins of known structures, and we further tested it on protein complexes, crude immunoprecipitates and whole-cell lysates. We show that it is a robust tool for protein-structure and protein-protein-interaction studies.     

Iron homeostasis is dysregulated,but the iron-hepcidin axis is functional,in chronic liver disease

BackgroundPerturbations in iron homeostasis have been reported to be associated with irreversible liver injury in chronic liver disease (CLD). However, it is not clear whether liver dysfunction per se underlies such dysregulation or whether other factors also contribute to it. This study attempted to examine the issues involved.MethodsPatients diagnosed to have chronic liver disease (n = 63), who underwent a medically-indicated upper gastrointestinal endoscopy, were the subjects of this study. Patients with dyspepsia, who underwent such a procedure, and were found to have no endoscopic abnormalities, were used as control subjects (n = 49). Duodenal mucosal samples were obtained to study mRNA and protein levels of duodenal proteins involved in iron absorption. A blood sample was also obtained for estimation of hematological, iron-related, inflammatory and liver function-related parameters.ResultsPatients with CLD had impaired liver function, anemia of inflammation and lower serum levels of hepcidin than control subjects. Gene (mRNA) expression levels of duodenal ferroportin and duodenal cytochrome b (proteins involved in iron absorption) were decreased, while that of divalent metal transporter–1 (DMT-1) was unchanged. Protein expression of DMT-1 was, however, decreased while that of ferroportin was unchanged. In the CLD group, serum hepcidin was predicted independently by serum ferritin and hemoglobin, but not by C-reactive protein (a marker of inflammation). CLD patients with serum ferritin greater than 300 μg/dL had significantly greater liver dysfunction (as indicated by significantly higher serum concentrations of bilirubin, AST and ALT, and MELD scores), higher serum concentrations of CRP and hepcidin, and higher ferroportin protein expression, than those with serum ferritin ≤ 300 μg/dL.ConclusionsIn patients with CLD, anemia of inflammation and low serum hepcidin levels were found to paradoxically co-exist. Expression of duodenal proteins involved in iron absorption were either decreased or unaltered in these patients. The hepcidin response to higher body iron levels and/or inflammation appeared to be functional in these patients, despite the presence of liver disease.     

Identification of gastrin-producing cells in cell cultures and smears: an avidin-biotin-peroxidase complex (ABC) technique

A method is described that uses the avidin-biotin complex (ABC) immunoperoxidase technique to provide a rapid, sensitive, and specific means to quantitate isolated G cells in cultures and suspensions.     

Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex.

HIV-1 integrase protein possesses the 3' processing and DNA strand transfer activities that are required to integrate HIV DNA into a host chromosome. The N-, C-terminal and core domains of integrase are necessary for both activities in vitro. We find that certain pairs of mutant integrase proteins, which are inactive when each protein is assayed alone, can support near wild type levels of activity when both proteins are present together in the reaction mixture. This complementation implies that HIV-1 integrase functions as a multimer and has enabled us to probe the organization of the functional domains within active mixed multimers. We have identified a minimal set of functional integrase domains that are sufficient for 3' processing and DNA strand transfer and find that some domains are contributed in trans by separate monomers within the functional complex.     

The human pre-replication complex is an open complex

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Identification and isolation from breast milk of ceruloplasmin-lactoferrin complex

The presence of a complex of the copper-containing protein ceruloplasmin (Cp) with lactoferrin (Lf) in breast milk (BM) is shown for the first time. In SDS-free polyacrylamide gel electrophoresis (PAGE), electrophoretic mobility of Cp in BM is lower than that of plasma Cp, coinciding with the mobility of the complex obtained upon mixing purified Cp and Lf. Affinity chromatography of delipidated BM on Cp-Sepharose resulted in retention of Lf. SDS-PAGE of the 0.3 M NaCl eluate revealed a single band with M_r ∼ 78,000 that has the N-terminal amino acid sequence of Lf and reacts with antibodies to that protein. Synthetic peptides R-R-R-R (the N-terminal amino acid stretch 2–5 in Lf) and K-R-Y-K-Q-R-V-K-N-K (the C-terminal stretch 29–38 in PACAP 38) caused efficient elution of Lf from Cp-Sepharose. Cp-Lf complex from delipidated BM is not retained on the resins used for isolation of Cp (AE-agarose) and of Lf (CM-Sephadex). Anionic peptides from Cp-(586–597), (721–734), and (905–914)—provide an efficient elution of Cp from AE-agarose, but do not cause dissociation of Cp-Lf complex. When anti-Lf is added to BM flowed through CM-Sephadex, Cp co-precipitates with Lf. Cp-Lf complex can be isolated from BM by chromatography on CM-Sephadex, ethanol precipitation, and affinity chromatography on AE-agarose, yielding 98% pure complex. The resulting complex Cp-Lf(1: 1) was separated into components by chromatography on heparin-Sepharose. Limited tryptic hydrolysis of Cp obtained from BM and from blood plasma revealed identical proteolytic fragments. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 2, pp. 208–215.     

Identification of a DNA processing complex from Deinococcus radiodurans

An efficient DNA strand break repair contributes to the radioresistance of Deinococcus radiodurans, which harbors the DNA repair pathways nearly identical to Escherichia coli. The molecular mechanisms of these proteins functioning in 2 diverse classes of bacteria seem to be different. The macromolecular interactions and formation of multiprotein complexes in vivo have gained significant importance in explaining the mechanism of the complex cellular processes. Here, we report the identification of a novel DNA metabolic protein complex from D. radiodurans. A similar complex has, however, not been found in E. coli. Mass spectrometric analysis showed the presence of a few known DNA repair proteins, molecular chaperones, and a large number of uncharacterized proteins from D. radiodurans R1. Biochemical and immunoblotting results indicated the presence of the protein promoting DNA repair A, DNA polymerase, Mg2+, and (or) Mn2+ -dependent 5'-->3' exonuclease activity along with protein kinase activity and phosphoproteins. DNA ligase activity was completely dependent upon the ATP requirement, as no ligase activity was seen in the presence of NAD as a cofactor. These results suggest the molecular interactions of the known DNA repair proteins with uncharacterized proteins in the macromolecular complex and the regulation of DNA degradation with the involvement of ATP and protein kinase functions.